It is widely acknowledged that plant-made pharmaceuticals (PMPs) offer
numerous benefits, including inexpensive production, biological safety and
the facility for production at agricultural scale. At the same time, it is
important to minimize any potential risk associated with this new
technology, including the potential release of bioactive proteins into the
environment. To address this issue, we studied transgenic Nicotiana benthamiana and Nicotiana tabacum plants
expressing two recombinant single-chain variable fragment (scFv) antibodies,
respectively scFvB9 and scFvH10. ScFvB9 was raised against glycoprotein G1
of Tomato spotted wilt virus (TSWV), and scFvH10 was raised against human tumor-associated antigen
tenascin-C. Both antibodies were targeted to the secretory pathway using the
N-terminal signal peptide from Phaseolus vulgaris polygalacturonase-inhibiting protein (PGIP),
and scFvH10 carried in addition a C-terminal KDEL tetrapeptide for retention
in the endoplasmic reticulum (ER). Sterile hydroponic cultures were
established, allowing us to investigate whether scFvB9 and scFvH10 were
present in root exudates. Intercellular fluids extracted from different
plant tissues were analyzed by western blotting revealing the presence of
scFvB9. Successful secretion of scFvB9 in hydroponic medium was also
demonstrated, whereas no scFvH10 could be detected in the leaf, stem or root
apoplast, nor secreted into the hydroponic medium. Our results show
that scFvH10 release or diffusion from the roots of transgenic plants was
not occurring, suggesting that the KDEL signal might contribute to the
environmental biosafety of crops producing PMPs.