Expression and Functional Evaluation of Recombinant Anti-receptor Activator of Nuclear Factor Kappa-B Ligand Monoclonal Antibody Produced in Nicotiana benthamiana

Wanuttha Boonyayothin; Sirorut Sinnung; Balamurugan Shanmugaraj; Yoshito Abe; Richard Strasser; Prasit Pavasant; Waranyoo Phoolcharoen

doi:10.3389/fpls.2021.683417

Expression and Functional Evaluation of Recombinant Anti-receptor Activator of Nuclear Factor Kappa-B Ligand Monoclonal Antibody Produced in Nicotiana benthamiana

Front Plant Sci. 2021 Jun 23:12:683417. doi: 10.3389/fpls.2021.683417. eCollection 2021.

Authors

Wanuttha Boonyayothin^{1

2}, Sirorut Sinnung³, Balamurugan Shanmugaraj^{1

2}, Yoshito Abe⁴, Richard Strasser⁵, Prasit Pavasant^{3

6}, Waranyoo Phoolcharoen^{1

2}

Affiliations

¹ Research Unit for Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.
² Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
³ Center of Excellence in Regenerative Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
⁴ Department of Pharmaceutical Sciences, School of Pharmacy at Fukuoka, International University of Health and Welfare, Okawa, Japan.
⁵ Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
⁶ Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.

Abstract

Denosumab, an anti-receptor activator of nuclear factor-kappa B ligand antibody (anti-RANKL), is a fully human monoclonal antibody (mAb) available for the treatment of osteoporosis. In the present study, an anti-RANKL mAb was transiently expressed using the geminiviral expression system in Nicotiana benthamiana, and the functional activity of the plant-produced mAb was determined. The highest expression level of the plant-produced mAb was found at 8 days post-infiltration, and it was estimated to be 0.5 mg/g leaf fresh weight. The recombinant mAb from the plant crude extracts was purified by using Protein A affinity column chromatography. The plant-produced mAb demonstrated good in vitro affinity binding with human RANKL, as determined by RANKL-ELISA binding. The function of the plant-produced mAb was evaluated in vitro. CD14-positive cells isolated from human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence of human RANKL and macrophage-colony-stimulating factor (M-CSF) to stimulate osteoclastogenesis. The results demonstrated that plant-produced mAb could significantly decrease the number of osteoclasts compared to commercial denosumab. These results demonstrated that the plant-produced mAb has the potential to inhibit osteoclast differentiation and that it could be considered for osteoporosis treatment.

Keywords: Nicotiana benthamiana; denosumab; osteoclastogenesis; plant-produced monoclonal antibody; receptor activator of nuclear factor kappa-B ligand; transient expression.