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

Abstract

It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, the specific immunological relevance of individual mAbassociated N-glycan structures is currently largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. Here we report on the generation of a plant-based expression platform allowing the efficient production of mAbs with a homogeneous ␤1,4-galactosylated N-glycosylation structure, the major N-glycan species present on serum IgG. This was achieved by the expression of a highly active modified version of the human ␤1,4-galactosyltransferase in glycoengineered plants lacking plant-specific glycosylation. Moreover, we demonstrate that two anti-human immunodeficiency virus mAbs with fully ␤1,4-galactosylated N-glycans display improved virus neutralization potency when compared with other glycoforms produced in plants and Chinese hamster ovary cells. These findings indicate that mAbs containing such homogeneous N-glycan structures should display improved in vivo activities. Our system, using expression of mAbs in tobacco plants engineered for post-translational protein processing, provides a new means of overcoming the two hurdles that limit the therapeutic use of anti-human immunodeficiency virus mAbs in global health initiatives, low biological potency and high production costs.

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

“…monoclonal IgG antibodies (14), where different glycosylation profiles confer strongly altered biological activities (15)(16)(17)(18). Although the functioning of IgG has been studied intensively over the last decade, knowledge about IgMs (i.e., assembly, functional activities, and the impact of glycosylation on the function) is lagging behind.…”

Manufacturing antibodies in the plant cell

“…The crossing of GalT knocked in plants with ∆FT/XT mutants generated a tobacco line with a humanized glycosylation pattern. These plants were used, in combination with Icon´s magnifection system (see below), for the production of highly homogeneous humanized antibodies [71]. Interestingly, antibodies produced in ∆FT/∆XT/GalT plants were more effective than their CHO-made counterparts in neutralizing HIV infection, apparently due to their homogeneous glycosylation pattern The only human modification that remains to be achieved in plants is the addition of terminal syalic residues.…”

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“…Transient expression protocols provide the required speed for many modern manufacturing processes, as those related to custom production for individualized therapies. The full "humanization" of plant glycosylation patterns is underway, and those steps accomplished so far have produced recombinant mAbs with superior neutralizing activities when compared with competing platforms [71,88]. Finally, as plant recombinant products are gradually available, current restrictions in the regulatory framework may well be relaxed, opening new opportunities.…”

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

“…By using a transgenic line of N. benthamiana (ΔXTFT) lacking plant-specific N-glycan residues (14), mAbs with highly homogenous human-like glycoforms can be generated (9,14,15). Comparing the efficacy of h-13F6 produced aglycosylated and glycosylated in two different production systems may help identify the mechanism(s) of action and a preferred manufacturing system for continued development of the EBOV therapeutic.…”