Immunological Effect of Glycosylation-Modified Human Colorectal Cancer Antigen GA733-2 Produced from Nicotiana benthaminana Leaves

The GA733-2 antigen, a calcium-independent cell adhesion molecule (CAM) presented in the majority of human epithelia and highly expressed in gastrointestinal and colorectal carcinomas, is used as an immunotherapeutic target in the treatment of gastrointestinal, colorectal and other carcinoma. Recently, recombinant GA733-2 was successfully expressed in plants. Plant-derived recombinant GA733-2 has been proved to induce a humoral immune response. However, plants are not an ideal expression system for producing therapeutic proteins because plant-derived specific N-glycans contain antigenic and/or allergenic β(1,2)-xylose and α(1,3)-fucose, which are absent in mammalian glycans. Here, to reduce β(1,2)-xylose and α(1,3)-fucose glycan structure of recombinant GA733-2 expressed in plants, transgenic Nicotiana benthaminana plants were regenerated after transformation with recombinant Agrobacterium harboring RNA interference construct, pFGC5941/XylT&FucT, expressing hairpin double-stranded RNA for β(1,2)-xylosyltransferase and α(1,3)-fucosyltransferase and inducing sequence-specific RNA silencing. Glycosylation-modified recombinant GA733-2 fused to Fc fragment of antibody (GA733-2-Fc) was produced from transgenic N. benthaminana leaves infiltrated with recombinant Agrobacterium harboring pBCTV/GA733-2-Fc. To add β(1,4)-linked galactose structure, recombinant GA733-2-Fc was also generated from normal N. benthamiana leaves infiltrated with recombinant Agrobacterium harboring pILTAB-GalT expressing β(1,4)-galactosyltrasferase. Glycan structures of glycosylation-modified GA733-2-Fc proteins were determined by MALDI-TOF mass spectroscopy after affinity purification using protein A/G agarose beads. Immunological effects were further determined after intraperitoneal administrations of glycosylation-modified GA733-Fc proteins in mice. This research was supported by a grant (111096-03-2-HD110) from Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries.