title: Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues.
abstract: Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.
authors.affiliation: University of Aachen, Department for Molecular Biotechnology, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Rajan
authors.initials: R
authors.lastname: Sriraman
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Markus
authors.initials: M
authors.lastname: Sack
authors.forename: Carmen
authors.initials: C
authors.lastname: Vaquero
authors.forename: Loïc
authors.initials: L
authors.lastname: Faye
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Ricarda
authors.initials: R
authors.lastname: Finnern
authors.forename: Patrice
authors.initials: P
authors.lastname: Lerouge