Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

Abstract

The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

Maternal and Fetal Mechanisms of B Cell Regulation during Pregnancy: Human Chorionic Gonadotropin Stimulates B Cells to Produce IL-10 While Alpha-Fetoprotein Drives Them into Apoptosis

“…Neither hCG nor the other tested hormones induced a change in the concentration of Igs produced after 12-day culture. Recently, much attention has been put on differentially glycosylated antibodies (24, 37, 38). While no changes in galacosylation, bisection, fucosylation, or sialyation of the Fc region could be observed after in vitro treatment of B cells with pregnancy hormones, we provide evidences that, at least in vitro , hCG increased the production of asymmetrically glycosylated IgG antibodies (AAbs).…”

Glyco-engineering for the production of recombinant IgA1 with distinct mucin-type O-glycans in plants

“…Plants do not show a typical mucin-type O-glycosylation pathway which provides the opportunity for de novo synthesis. However, plants can convert proline residues adjacent to Ser/Thr O-glycosylation sites into hydroxyproline 8 . The hydroxyproline residues can then be further extended with arabinose chains or huge arabinogalactans and neighboring serine residues can be modified with a single galactose residue 24,25 .…”

“…To produce a recombinant human IgA1 with defined mucin-type O-glycans in N. benthamiana we transiently expressed different mammalian glycosyltransferases and proteins required for the biosynthesis and transport of the precursor CMP-sialic acid together with the recombinant glycoprotein 8 . Mucin-type O-glycan formation was efficiently initiated by co-expression of human GalNAc-T2.…”

“…Since expression of human C1GalT1 with its chaperone Cosmc did not result in any detectable core 1 O-glycan formation, 27 we expressed Drosophila melanogaster C1GalT1 which is functional in the absence of a Cosmc-like chaperone. Further expression of human α2,3-sialyltransferase and Mus musculus α2,6-sialyltransferase resulted in the formation of sialylated core 1 O-glycans that are normally found in the hinge region of human IgA1 8 . Our study does not only demonstrate the potential of N. benthamiana for the production of tailored mucin-type O-glycans on therapeutically interesting proteins but raise also the opportunity for the generation of pathologically relevant modifications, like the sialyl Tn antigen found on circulating IgA1 in IgAN patients.…”

“…These engineered glycoproteins display enhanced functions and facilitate the development of novel diagnostic tools for detection of aberrant glycosylation. Recently, we showed that Nicotiana benthamiana plants are a suitable expression host for the generation of distinct O-glycans on recombinant human IgA1 8 . In this study, we provide an addition to our O-glycan engineering repertoire and produce a human IgA1 with galactose-deficient and α2,6-sialylated O-glycans.…”