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. Monoclonal antibody (mAb) CO17-1A recognize a tumor-associated epitope expressed on the colorectal cancer cell surface. 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 both plants expressing mAb CO without KDEL mAb CO (CO plant) and mAb CO with KDEL mAb COK (COK plant). However, COK plants primary root lengths were shorter than those of 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 COK plants compared to CO plants. Western blot and SDS-PAGE analyses showed that COK plants exhibited up to five times higher expression and mAb amounts than 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 mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. N-glycosylation analysis showed that mAb CO had plant specific residues whereas mAb COK mainly showed an oligo-mannose N-glycan structure without the plant specific glycan residues. In this study, the reduction of plant growth and biomass induced by ER retention signal peptide might be only in in vitro conditions, and thus should be carefully considered for the initial screening for transgenic lines on culture media. Taken together, nevertheless the fusion of ER retention signal peptide is an effective approach for enhancing the yields of recombinant proteins in vivo.

Expression of a Large Single-Chain 13F6 Antibody with Binding Activity against Ebola Virus-Like Particles in a Plant System

“…The higher purification efficiency of both antibodies might be due to the effect of the C-terminal-fused ER retention motif KDEL. Previous studies prove that mAbs were not retained in the ER in the absence of an ER retention motif [ 25 , 26 , 47 , 48 , 49 ]. They indicated that a protein fused with the KDEL motif at the C-terminal end is retained in the ER or is returned to the ER and its production levels are also improved [ 2 , 50 , 51 , 52 ].…”

“…They indicated that a protein fused with the KDEL motif at the C-terminal end is retained in the ER or is returned to the ER and its production levels are also improved [ 2 , 50 , 51 , 52 ]. The ER is a vast subcellular location for the accumulation of proteins that do not require the proteolytic process found in the secretory pathway of intracellular and extracellular spaces [ 23 , 24 , 25 , 26 , 28 , 53 , 54 ]. The figure of Immunoblot analysis to confirm the purified fraction of mAb 13F6-FULL from transgenic N. tabacum plants for Figure 4 A was included as a Figure S2 .…”

“…To make this diagnostic test cost-effective and easily accessible, low cost production of antibodies with a strong binding affinity against the EBOV antigens is crucial. One option to ensure cost-effective production of antibodies is the use of a plant expression system without pathogenic animal contaminants, high-priced manufacturing process, and expensive culture media [ 22 , 23 , 24 , 25 , 26 ]. The advantages of plant expression systems include inexpensive inputs (sunlight, water, and nutrients), the ease of large-scale production by increasing the area located in the seed, and low technology harvest compared with mammalian expression systems [ 22 , 27 , 28 , 29 ].…”

Relationship between ganglioside expression and anti-cancer effects of a plant-derived antibody in breast cancer cells

“…We have previously shown that mAb P COK expressed in tobacco plants suppressed cancer cell proliferation and changed the expression pattern of certain gangliosides, such as GD1a and GM1, in colorectal cancer cells (Ryu et al 2013). A previous study has reported that anti-cancer mAbs produced by transgenic Arabidopsis plants specifically recognize the tumor-associated antigen GA733-2 (Song et al 2018). In this study, we investigated the bioactivity of mAb P COK expressed in transgenic Arabidopsis plants on anti-cancer effects and the expression change of glycosphingolipid in breast carcinoma, not colorectal carcinoma.…”

“…The cDNA fragments encoding a heavy chain (HC) fused to the ER retention signal KDEL (HCK) and light chain (LC) of anti-colorectal cancer mAb CO17-1AK have been successfully cloned to a pBI COK vector ( Fig. 1A) (Song et al 2018). Agrobacterium-mediated plant transformation was conducted to generate transgenic Arabidopsis lines expressing mAb CO17-1AK as previously described (Paris et al 2002).…”