Abstract
SummaryPlants can provide a cost‐effective and scalable technology for production of therapeutic monoclonal antibodies, with the potential for precise engineering of glycosylation. Glycan structures in the antibody Fc region influence binding properties to Fc receptors, which opens opportunities for modulation of antibody effector functions. To test the impact of glycosylation in detail, on binding to human Fc receptors, different glycovariants of VRC01, a broadly neutralizing HIV monoclonal antibody, were generated in Nicotiana benthamiana and characterized. These include glycovariants lacking plant characteristic α1,3‐fucose and β1,2‐xylose residues and glycans extended with terminal β1,4‐galactose. Surface plasmon resonance‐based assays were established for kinetic/affinity evaluation of antibody–FcγR interactions, and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa; however, the binding characteristics can be restored and even improved with targeted glycoengineering. All plant‐made glycovariants had a slightly reduced affinity to the neonatal Fc receptor (FcRn) compared with HEK cell‐derived antibody. However, this was independent of plant glycosylation, but related to the oxidation status of two methionine residues in the Fc region. This points towards a need for process optimization to control oxidation levels and improve the quality of plant‐produced antibodies.
Highlights
Antibody-based therapeutics comprise a major segment of the fast-growing biopharmaceutical market
VRC01 antibody glycovariants produced in N. benthamiana. cDNA sequences encoding antibody heavy and light chain were cloned into a binary vector (Sainsbury et al, 2009) and transiently expressed in N. benthamiana using agroinfiltration (Kapila et al, 1997)
In order to study the effect of plant N-glycosylation on recombinant antibody Fc-mediated binding activities, the broadly-neutralising HIV antibody, VRC01, was expressed in plants in different glycol formats
Summary
Antibody-based therapeutics comprise a major segment of the fast-growing biopharmaceutical market. There are currently more than 50 monoclonal antibodies approved for clinical use and hundreds are in pre-clinical and clinical development (Ecker et al, 2015). Most of these are IgG antibodies that target cancer and autoimmune disorders, there is growing interest in monoclonal antibodies to tackle infectious diseases (Irani et al, 2015). Whilst binding to cognate antigen is the hallmark of antibody function, engagement of cell-surface Fc receptors can activate immune cells to remove or neutralise antibody-coated targets via processes known collectively as antibody effector functions (Desjarlais and Lazar, 2011). Interaction of the antibody Fc region with FcγRs is influenced by glycans attached to a highly conserved N-glycosylation site within the Fc fragment and their composition can influence binding affinity and receptor-mediated activity (Jefferis, 2009c)
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