Production of a monoclonal antibody in plants with a humanized N‐glycosylation pattern

Abstract

SummaryIn recent years, plants have become an attractive alternative for the production of recombinant proteins. However, their inability to perform authentic mammalian N -glycosylation may cause limitations for the production of therapeutics. A major concern is the presence of β 1,2-xylose and core α 1,3-fucose residues on complex N -linked glycans, as these N -glycan epitopes are immunogenic in mammals. In our attempts towards the humanization of plant N -glycans, we have generated an Arabidopsis thaliana knockout line that synthesizes complex N -glycans lacking immunogenic xylose and fucose epitopes. Here, we report the expression of a monoclonal antibody in these glycan-engineered plants that carry a homogeneous mammalian-like complex N -glycan pattern without β 1,2-xylose and core α 1,3-fucose. Plant and Chinese hamster ovary (CHO)-derived immunoglobulins (IgGs) exhibited no differences in electrophoretic mobility and enzyme-linked immunosorbent specificity assays. Our results demonstrate the feasibility of a knockout strategy for N -glycan engineering of plants towards mammalian-like structures, thus providing a significant improvement in the use of plants as an expression platform.

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“…, 93 These sugars are immunogenic to humans, and serum antibodies against core xylose and core α1,3-fucose have been detected in healthy human blood donors 94 . Strategies to overcome this immunogenicity include use of RNAi knockdown of α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT) in plants 95 , 96 and FucT/XylT-knockout lines 97 98 An afucosylated anti-CD30 monoclonal antibody with G0 structure was produced using glycoengineered aquatic plant Lemna minor and shown to have improved ADCC over the same CHO cell-produced antibody 95 .…”

Improved Virus Neutralization by Plant-produced Anti-HIV Antibodies with a Homogeneous β1,4-Galactosylated N-Glycan Profile

“…4E10 and 2G12 Antigen Binding Assay-4E10 and 2G12 antigen binding specificity was carried out using HIV gp41-specific peptide (KKWNWFDETNWGGG) or gp60 as described recently (9,14).…”

“…The generation of the binary vector used for transient expression of mAb 4E10 (p4E10, Fig. 1) was generated according to 2G12 described by Schähs et al (14).…”

Plant glycans: friend or foe in vaccine development?

“…Genetic knock-out of xylosyltranferases and fucosyltransferases (Figure 1, Structure 5) has been established in the moss Physcomitrella patens via homologous recombination [31] and in the model plant A. thaliana by screening mutant libraries [32]. However, given the presence of gene families in plants, combined with the fact that homologous recombination in plants is very rare, the implementation of these approaches to other plant species is very difficult.…”