Abstract
In this study, transgenic tobacco plants were generated to express anti-colorectal cancer large single chain (LSC) antibody CO17-1A (LSC CO) and LSC CO tagged with the endoplasmic reticulum (ER) retention signal KDEL (LSC COK). The LSC antibodies were constructed by linking the C-terminus of variable region (VL) of the light chain (LC) to the N-terminus of the heavy chain (HC) of mAb CO17-1A with a linker peptide. Reverse-transcription PCR (RT) and immunoblot analyses showed that the LSC CO17-1AK expression level was higher than LSC CO17-1A in plant. In glycosylation analysis, oligomannose type glycan form was observed in LSC COK and plant-specific α(1,3)-fucose was observed in LSC CO. Binding activity of both LSC CO17-1A and LSC CO17-1AK to human colorectal cancer cell lines SW480 and SW620 were confirmed using indirect cell enzyme-linked immunosorbent assay (ELISA). In indirect cell ELISA, the LSC antibodies had a higher binding activity than full-size mAb CO17-1AK. In surface plasmon resonance (SPR) assay using epidermal cell adhesion molecule (EpCAM) highly expressed on the human colorectal cancer cells, both LSC antibodies showed a similar binding activity to the full-size mAb CO17-1A. These results indicated that LSC antibodies with functional binding activities to human colorectal cancer cells and EpCAM protein were successfully expressed in the transgenic tobacco plant.
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Acknowledgements
This research was supported by the Chung-Ang University Research in 2018. The Bio & Medical Technology Development Program of the National Research Foundation (NRF) and funded by the Korean government (MSIT) (No. 2019M3E5D5067214).
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11816_2020_610_MOESM1_ESM.docx
Comparison of root growth of transgenic seedlings expressing LSC CO17-1AK (LSC COK) and LSC CO17-1A (LSC CO) cultured in vitro MS medium with and without kanamycin antibiotic. A. in vitro cultured transgenic seedlings in non-antibiotic conditions (MS medium, 5 weeks). B. in vitro cultured transgenic seedlings in antibiotic conditions (MS medium containing 100 µg/mL of kanamycin, 5 weeks). NT is non-transgenic seedling. (DOCX 122 kb)
11816_2020_610_MOESM2_ESM.docx
Immunoblot analysis result of purified antibodies from plants using anti-RuBisCO antibody. Western blot with anti-Ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) mAb detected the 50 kDa bands in the purified samples of both LSC CO and LSC COK. CT, column through; COK TB, purified mAb CO17-1AK in tobacco; COK AR, purified mAb CO17-1AK in Arabidopsis; LSC COK, purified LSC CO17-1AK in tobacco; LSC CO, purified LSC CO17-1A in tobacco. (DOCX 121 kb)
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Lee, J.H., Park, S.R., Phoolcharoen, W. et al. Expression, function, and glycosylation of anti-colorectal cancer large single-chain antibody (LSC) in plant. Plant Biotechnol Rep 14, 363–371 (2020). https://doi.org/10.1007/s11816-020-00610-z
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DOI: https://doi.org/10.1007/s11816-020-00610-z