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

Abstract

Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custommade human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.Recombinant monoclonal antibodies (mAbs) are of high therapeutic potential and have thus become a major product of the pharmaceutical industry (Aggarwal, 2009). In addition to full-length mAbs, antibodies are being engineered to alter their size, pharmacokinetics, specificity, valency, effector functions, etc. in order to better suit the intended applications (for review, see Filpula, 2007;Harmsen and De Haard, 2007). Of particular interest among these engineered fragments are single-chain variable fragments (scFvs), fusions of variable heavy and variable light domains that retain an antigen-binding function. Due to their smaller size as compared with their full-length counterparts, these molecules penetrate target tissues better and can even bind to intracellular targets. Furthermore, multimerization via disulfide bonds and/or multimerization domains allows for the production of divalent or higher order antibody-like molecules, where increased avidity

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

“…In transformant MBP39, an aberrant localization of the SSPs was observed (Van Droogenbroeck et al, 2007). However, this aberrant localization was not always penetrant and seems dependent of the growth conditions (Loos et al, 2011b), suggesting that there is no direct link between UPR induction and aberrant seed protein localization. It is also unlikely that the format of the antibody itself (VHH-Fc or scFv-Fc) triggers the UPR response, because VHH-Fc fusions do occur in different animal species and there is a high conservation between animal and plant ER folding and assembly.…”

“…Immunolocalization studies on one of these scFv-Fcexpressing lines revealed an aberrant localization of the recombinant scFv-Fc, as well as of endoplasmic reticulum (ER) chaperone binding proteins (BiPs) and calreticulin, and of the endogenous SSP cruciferin, suggesting that the cellular homeostasis was disturbed. However, not all transformants, accumulating scFv-Fc antibodies in ER-derived vesicles, show this phenotype, indicating that also the environmental conditions of seed development and ripening affect the deposition of the recombinant protein (Van Droogenbroeck et al, 2007;Loos et al, 2011b). Similarly, interleukin-10 accumulated in compartments delimited by ribosome-associated membranes, which most likely derive from the ER (Morandini et al, 2011).…”

Proteolytic andN-Glycan Processing of Humanα1-Antitrypsin Expressed inNicotiana benthamiana

“…Aberrant intracellular deposition and as a consequence, incorrect glycosylation of recombinant proteins are often reported. For example, recombinant proteins designed for secretion are frequently also located in the endoplasmic reticulum (ER) and as a consequence, carry oligomannosidic carbohydrates instead of the desired complex-type glycans (Loos et al, 2011;Schneider et al, 2014a). By contrast, KDEL-tagged proteins designed for ER retention are sometimes partially secreted (Van Droogenbroeck et al, 2007;Niemer et al, 2014).…”

Role of plant expression systems in antibody production for passive immunization

“…Alternatively, for parenteral application, the high protein concentration in desiccated seeds facilitates the downstream processing. Different full-length antibody and antibody formats, like ScFv, ScFv-Fc, VHHs and VHH-Fc, have been expressed effectively in seeds of both monocot and dicot plants (De Wilde et al, 2013, Khan et al, 2012, Loos et al, 2011b, Van Droogenbroeck et al, 2009, Virdi et al, 2013.…”