AUTHOR=Nguyen Kim Dua , Kajiura Hiroyuki , Kamiya Ryo , Yoshida Takahiro , Misaki Ryo , Fujiyama Kazuhito 

TITLE=Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1215580

DOI=10.3389/fpls.2023.1215580

ISSN=1664-462X

ABSTRACT=<p><italic>N</italic>-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two <italic>N</italic>-glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. <italic>Nicotiana benthamiana</italic> has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown <italic>N. benthamiana</italic>, resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type <italic>N. benthamiana</italic> lacked the terminal galactose residues in its <italic>N</italic>-glycan. Therefore, <italic>N</italic>-glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine β1,4-galactosyltransferase (β1,4-GALT) to modify plant <italic>N</italic>-glycan with β1,4-linked Gal residue(s) and <italic>Arabidopsis thaliana</italic> β1,3-galactosylatransferase (β1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of <italic>N</italic>-glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine β1,4-GALT in soil-grown <italic>N. benthamiana</italic> had 42.5% of <italic>N</italic>-glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown <italic>N. benthamiana</italic> plants. Concomitantly, <italic>N-</italic>glycan profile analysis of IgG co-expressed with β1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary β1,4-galactosylated <italic>N</italic>-glycan structures.</p>