Expression and glycoengineering of functionally active heteromultimeric IgM in plants

IgM antibodies are an important player of the human's innate defense mechanisms and increasingly have gained interest as therapeutics. Although the expression of IgM antibodies in mammalian cell culture is established, this approach remains costly and alternative methods have not been developed yet. Plants have a proven record for the production of therapeutically relevant recombinant proteins. However, whether they are able to express proteins like IgM antibodies, which range among the most complex human proteins, remains unknown so far. Here we report the in planta generation of the functionally active monoclonal antitumor IgM PAT-SM6 (SM6). SM6 efficiently accumulates in plant leaves and assembles correctly into heterooligomers (pentamers and hexamers). Detailed glycosylation analysis exhibited complex and oligomannosidic N-glycans in a site-specific manner on human-serum IgM and on plant- and human-cell-line-produced SM6. Moreover, extensive in planta glycoengineering allowed the generation of SM6 decorated with sialylated human-type oligosaccharides, comparable to plasma-derived IgM. A glycosylated model of pentameric IgM exhibits different accessibility of the glycosylation sites, explaining site-specific glycosylation. Biochemical and biophysical properties and importantly biological activities of plant-derived SM6 glycoforms are comparable to the human-cell-derived counterparts. The in planta generation of one of the most complex human proteins opens new pathways toward the production of difficult-to-express proteins for pharmaceutical applications. Moreover, the generation of IgMs with a controlled glycosylation pattern allows the study of the so far unknown contribution of sugar moieties to the function of IgMs.