An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide.

Yuan Lin,Jiahua Xie,Starr Nichols,TinChung Leung,Hafsa Rahimuddin,Farooqahmed S. Kittur,Chayanika Bhattacharya,Chiu-Yueh Hung

doi:10.3389/fpls.2018.01379

Yuan Lin, Jiahua Xie + Show 6 more

Open Access

https://doi.org/10.3389/fpls.2018.01379

Copy DOI

Abstract

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be “self-cleaved” into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd-values of 90.13–149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants.

Highlights

The advent of recombinant DNA technology has launched a revolution in biotechnology by enabling production of a diverse range of biopharmaceuticals in a variety of heterologous hosts
A93 transgenic plants carrying a genetic cassette with heavy chains (HC) and light chains (LC) linked by 2A (Figure 1A) produced similar levels of HC and LC (Figure 4A) and efficiently produced fully assembled Ebola virus (EBOV) monoclonal antibodies (mAbs) (Figures 3A, 5A,B)
A92 transgenic plants carrying HC and LC driven by different promoters and terminators (Figure 1B) could not produce enough LC (Figures 4B, 5C) in comparison with HC (Figure 4B)

Summary

INTRODUCTION

The advent of recombinant DNA technology has launched a revolution in biotechnology by enabling production of a diverse range of biopharmaceuticals in a variety of heterologous hosts. The current study was to investigate the utilizability of F2A to express mAb in plant-based expression system by analyzing the expression levels of HC and LC, the cleavage efficiency of produced polyprotein HC+2A+LC and assembling efficiency of mAb in transgenic tobacco plants. Toward this end, we selected a mAb targeting the surface glycoprotein of Ebola virus (EBOV). These fast growing transgenic tobacco plants are useful for mass production of EBOV mAb

MATERIALS AND METHODS

Findings

CONCLUSION