Production of monoclonal antibodies with a controlledN‐glycosylation pattern in seeds ofArabidopsis thaliana

Abstract

SummarySeed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDELtagged version of 2G12 exhibited an ER-typical N-glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N-glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

“…Probably, the formation of these compartments does not depend on the amount of recombinant protein produced but on specific properties of the recombinant protein itself (Morandini et al, 2011). In the same study, the 65-kD isoform of Glu decarboxylase and proinsulin were, despite bearing a KDEL tag, both localized in the protein storage vacuoles, a phenomenon reported before (Petruccelli et al, 2006;Van Droogenbroeck et al, 2007;Loos et al, 2011a).…”

Expression of Antibody Fragments with a ControlledN-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis

“…However, the targeting of recombinant proteins in seeds is not yet fully understood. For example, KDEL-tagged as well as secretory proteins have been detected in protein storage vacuoles, in the cell wall, in ER-derived compartments, or in a combination of the three (Herman et al, 1990;Philip et al, 1998Philip et al, , 2001Wright et al, 2001;Reggi et al, 2005;Downing et al, 2006;Petruccelli et al, 2006;Van Droogenbroeck et al, 2007;Abranches et al, 2008;Schmidt and Herman, 2008;Loos et al, 2011). As a consequence of this atypical targeting and deposition, seed-derived recombinant products may carry unusual N-glycosylation profiles (Van Droogenbroeck et al, 2007;Rademacher et al, 2008;Ramessar et al, 2008).…”

Expression of recombinant human anti-TNF-α scFv-Fc in Arabidopsis thaliana seeds

“…In addition, post-translation modifications are also performed as well as they are in mammalian production systems. Because N-glycosylation of peptides was found in TKO mutant A. thaliana seeds (Loos et al, 2011), we selected these mutants for use in antibody expression.…”