Endoplasmic Reticulum Retention Motif Fused to Recombinant Anti-cancer Monoclonal Antibody (mAb) CO17-1A Affects mAb Expression and Plant Stress Response

Abstract

The endoplasmic reticulum (ER) is the main site of protein synthesis, folding, and secretion to other organelles. The capacity of the ER to process proteins is limited, and excessive accumulation of unfolded and misfolded proteins can induce ER stress, which is associated with plant diseases. Here, a transgenic Arabidopsis system was established to express anti-cancer monoclonal antibodies (mAbs) that recognize the tumor-associated antigen GA733-2. The ER retention Lys-Asp-Glu-Leu (KDEL) motif sequence was added to the C-terminus of the heavy chain to retain anti-colorectal cancer mAbs in the ER, consequently boosting mAb production. Agrobacterium-mediated floral dip transformation was used to generate T1 transformants, and homozygous T4 seeds obtained from transgenic Arabidopsis plants expressing anti-colorectal cancer mAbs were used to confirm the physiological effects of KDEL tagging. Germination rates were not significantly different between mAb CO and mAb COK. However, mAb COK primary root lengths were shorter than those of mAb CO plants and non-transgenic Arabidopsis plants in in vitro media. Most ER stress-related genes, with the exception of bZIP28 and IRE1a, were upregulated in mAb COK plants compared to mAb CO plants. Western blot and SDS-PAGE analyses showed that mAb COK plants exhibited up to five-times higher expression and mAb amounts than mAb CO plants. Enhanced expression in mAb COK plants was confirmed by immunohistochemical analyses. mAb COK was distributed across most of the area of leaf tissues, whereas mAb CO was mainly distributed in extracellular areas. Surface plasmon resonance analyses revealed that both mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. These results suggest that the introduction of the KDEL motif is a promising strategy for obtaining enhanced amounts of recombinant therapeutic proteins, but the KDEL sequence may induce ER stress and slightly reduce plant biomass.