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

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

doi:10.3389/fpls.2021.736299

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.

Highlights

Immunotherapy is a form of cancer treatment that utilizes the immune system of a patient to target cancer cells (Pardoll, 2012)
We demonstrated that this rapid transient expression platform could be used for recombinant antibody production within 4 days after infiltration when compared with other expression platforms, as indicated in Figure 1C and Supplementary Table 2
The results indicated that plant-produced Pembrolizumab inhibited the interaction between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) in a dose-dependent manner (Figure 4B) with a half-maximal effective concentration (EC50) of 147.2 ng/ml compared with Keytruda R (EC50 = 146.7 ng/ml)

Summary

Introduction

Immunotherapy is a form of cancer treatment that utilizes the immune system of a patient to target cancer cells (Pardoll, 2012). Among the types of immunotherapies, immune checkpoint inhibitors (ICIs) therapy is based on monoclonal antibody (mAb) to target immune checkpoint molecules on immune cells or cancer cells. These ICIs act by blocking and inhibiting costimulatory molecules between tumor cells and immune cells resulting in the enhanced T-cells activation and revival of anergic tumor-reactive T cells mounted effective antitumor responses (Marin-Acevedo et al, 2018; Wei et al, 2018). One of the most effective ICIs used for the cancer treatment is Pembrolizumab (Keytruda R ) which targets human programmed cell death protein 1 (PD-1). Pembrolizumab was approved by the US Food and Drug Administration (FDA) for more than 15 cancer indications (Paul et al, 2013)

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Conclusion