Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus.

Lei Chen,Da Luo,Weichang Yu,Xiaoyu Yang

doi:10.3389/fpls.2016.01156

Abstract

Bevacizumab, a humanized monoclonal antibody (mAb) targeting to the vascular endothelial growth factor (VEGF), has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme-linked immunosorbent assay (ELISA) analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7–242.8 mg/Kg) in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target human VEGF (hVEGF) antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin) were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

Highlights

Plants including the whole body, specific tissues or organs and plant-derived suspension cells are considered as attractive and competitive platforms for recombinant protein production
Long-term continuous recombinant protein production can be realized in plant platforms because transgenes can be stably integrated into the nuclear genome of host plants, faithfully inherited, and expressed in later generations
The Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were expressed separately in two gene expression cassettes both driven by the maize ubiquitin promoter (Figure 1A)

Summary

Introduction

Plants including the whole body, specific tissues or organs and plant-derived suspension cells are considered as attractive and competitive platforms for recombinant protein production. Plant-derived recombinant proteins may be safer than those from E. coli or mammalian cells because the risk of contamination with human pathogens, which is always a concern when using mammalian cells as a bioreactor, can be well circumvented by plant-based production systems (Thie et al, 2008; Ni and Chen, 2009; Merlin et al, 2014) Because of these properties, various bioactive pharmaceutical proteins have been produced in plants since first expression of a human growth hormone in transgenic tobacco and sunflower callus tissue (Barta et al, 1986), and expression of antibodies, vaccines, hormones, growth factors, and cytokines (De Muynck et al, 2010; Desai et al, 2010; Xu et al, 2011; Huang and McDonald, 2012)

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Results

Conclusion