Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

Tanapati Phakham; Christine Joy I Bulaon; Narach Khorattanakulchai; Balamurugan Shanmugaraj; Supranee Buranapraditkun; Chatikorn Boonkrai; Sarintip Sooksai; Nattiya Hirankarn; Yoshito Abe; Richard Strasser; Kaewta Rattanapisit; Waranyoo Phoolcharoen

doi:10.3389/fpls.2021.736299

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

Front Plant Sci. 2021 Sep 9:12:736299. doi: 10.3389/fpls.2021.736299. eCollection 2021.

Authors

Tanapati Phakham^{1

2}, Christine Joy I Bulaon^{3

4}, Narach Khorattanakulchai^{3

4}, Balamurugan Shanmugaraj⁵, Supranee Buranapraditkun^{6

7}, Chatikorn Boonkrai^{1

2}, Sarintip Sooksai⁸, Nattiya Hirankarn⁶, Yoshito Abe⁹, Richard Strasser¹⁰, Kaewta Rattanapisit⁵, Waranyoo Phoolcharoen^{3

4}

Affiliations

¹ Interdisciplinary Program of Biomedical Sciences, Graduate School, Chulalongkorn University, Bangkok, Thailand.
² Center of Excellence in Systems Biology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
³ Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
⁴ Plant-Produced Pharmaceutical Research Unit, Chulalongkorn University, Bangkok, Thailand.
⁵ Baiya Phytopharm Co., Ltd., Bangkok, Thailand.
⁶ Department of Microbiology, Faculty of Medicine, Center of Excellence in Immunology and Immune-Mediated Diseases, Chulalongkorn University, Bangkok, Thailand.
⁷ Center of Excellence in Vaccine Research and Development (Chula Vaccine Research Center-Chula VRC), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
⁸ The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand.
⁹ Department of Pharmaceutical Sciences, School of Pharmacy at Fukuoka, International University of Health and Welfare, Okawa, Japan.
¹⁰ Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.

Abstract

The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 μg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda^®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda^®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda^®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

Keywords: Nicotiana benthamiana; Pembrolizumab; anti-PD-1 antibody; cancer immunotherapy; molecular farming; plant-produced Pembrolizumab; transient expression.