title: Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N-glycans.
abstract: The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-α, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1κ-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ- and IgG1κ-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1κ-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, β1,2-xylose and core α1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core α1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.
authors.affiliation: Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: Lotte B
authors.initials: LB
authors.lastname: Westerhof
authors.forename: Ruud H P
authors.initials: RH
authors.lastname: Wilbers
authors.forename: Debbie R
authors.initials: DR
authors.lastname: van Raaij
authors.forename: Dieu-Linh
authors.initials: DL
authors.lastname: Nguyen
authors.forename: Aska
authors.initials: A
authors.lastname: Goverse
authors.forename: Maurice G L
authors.initials: MG
authors.lastname: Henquet
authors.forename: Cornelis H
authors.initials: CH
authors.lastname: Hokke
authors.forename: Dirk
authors.initials: D
authors.lastname: Bosch
authors.forename: Jaap
authors.initials: J
authors.lastname: Bakker
authors.forename: Arjen
authors.initials: A
authors.lastname: Schots
mesh_terms: D000068879:Adalimumab; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis; D000941:Antigens / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007070:Immunoglobulin A / Q000096:biosynthesis*; D007074:Immunoglobulin G / Q000096:biosynthesis*; D007130:Immunoglobulin Idiotypes / Q000378:metabolism; D000069285:Infliximab; D051379:Mice; D059828:Plant Cells / Q000187:drug effects / Q000378:metabolism; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000069549:Ustekinumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000941:Antigens; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007130:Immunoglobulin Idiotypes; D011134:Polysaccharides; D014409:Tumor Necrosis Factor-alpha; D000069285:Infliximab; D000069549:Ustekinumab; D000068879:Adalimumab
keywords: Adalimumab; IgA; Infliximab; N-glycosylation; Ustekinumab; plant