Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N‐glycans

Abstract

Summary 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.

Human Alpha Galactosidases Transiently Produced in Nicotiana benthamiana Leaves: New Insights in Substrate Specificities with Relevance for Fabry Disease

“…Nicotiana benthamiana plants were grown at 21°C and 60–70% humidity in the Unifarm greenhouses of Wageningen University (Westerhof et al, 2014). …”

“…Agrobacterium tumefaciens cultures were grown as previously described (Westerhof et al, 2012, 2014). The constructs were co-expressed or not with the tomato bushy stunt virus silencing inhibitor p19 (Voinnet et al, 2003), by mixing the bacterial cultures 1:1 following 1–2 h incubation at room temperature.…”

“…The inoculated bacterial cultures were used for infiltration of the two youngest leaves of 5–6 weeks old N. benthamiana plants, to ensure optimum protein expression, as previously described (Westerhof et al, 2014). Whole leaves and leaf disks (50 mm) were harvested at different days post-infiltration (dpi), snap-frozen and homogenized in liquid nitrogen.…”

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease

“…Agrobacterium tumefaciens cultures were grown as previously described (56,58). The constructs were co-expressed with the tomato bushy stunt virus silencing inhibitor p19, to ensure optimum expression levels (44,49,59).…”

“…The constructs were co-expressed with the tomato bushy stunt virus silencing inhibitor p19, to ensure optimum expression levels (44,49,59). The inoculated bacterial cultures were used for infiltration of 5-6 weeks old N. benthamiana plants, as previously described (56).…”