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Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus
Sunday, 2016/08/28 | 16:16:31

 

 

 

 

 

 

 

 

 

Chen L, Yang X, Luo D, Yu W.

Front Plant Sci. 2016 Aug 9;7:1156. doi: 10.3389/fpls.2016.01156. eCollection 2016.

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.

 

See http://www.ncbi.nlm.nih.gov/pubmed/27555853

 

An external file that holds a picture, illustration, etc.Object name is fpls-07-01156-g001.jpg

Figure 1: T-DNA regions of constructs for rice transformation. (A) pUNBHLC construct with Bevacizumab LC (BLC) and HC (BHC) genes expressed in two expression cassette; (B) pUNBevaHL construct with BLC and BHC expressed as a polyprotein linked with a FMDV 2A self-cleavage peptide; (C) pUNBevaHL-KDEL construct with BLC and BHC expressed as a polyprotein as in (B), but a KDEL ER retention peptide sequence was added to the C-termini of the coding regions of both BLC and BHC genes. Lysogeny broth (LB) and RB denote the left and right borders of the T-DNA region of the binary vector. TNos and T35S are terminators of the Agrobacterium nopaline synthase gene and the CaMV 35S expression cassette, respectively. 2A is the self-cleavage peptide from foot-and-mouth disease virus, and KDEL is the ER retention signal peptide. Two EcoRI (E) restriction enzyme digestion sites are indicated on pUNBevaHL and pUNBevaHL-KDEL constructs in (B,C), and the ∼3 kb fragments flanked by the two EcoRI sites are indicated. The bars under pUNBevaHL construct (B) show the positions of HptII and BLC probes used in Southern blot analysis.

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