Biocomputational analysis to evaluate stacking dual-targeting antibodies in agricultural tobacco plant

Abstract This study explored the application of tobacco plant expression system for production of dual target monoclonal (mAb) proteins and computational analysis to evaluate their biochemical characteristics. The murine anti-colorectal cancer large single-chain antibody (CL) and H-13F6 human anti-Ebola virus full-size monoclonal antibody (EF) or anti-Ebola large single-chain antibody (EL) co-expressed in F1 plant through crossbreeding transgenic plants expressing each specific antibody. The binding activities of these antibodies to their respective target antigens were then examined. PCR analysis confirmed the presence of the CL gene and the heavy (HC) and light chain (LC) genes of EF in F1 plants resulting from the crossbreeding. Additionally, both CL and EL genes were identified in F1 plants produced through the crossbreeding of transgenic plants expressing each CL and EL. RT-PCR and immunoblot analyses confirmed the mRNA and protein expression of all transgenes in F1 plants. CL, EF, and EL were successfully purified from F1 plants. Indirect ELISA analysis revealed that CL × EF proteins exhibited binding activity to Ebola virus-specific antigenic protein, whereas CL × EL lost this binding activity. Conversely, both CL × EF and CL × EL proteins exhibited enhanced binding activity to the colorectal cancer-specific antigenic protein GA733. In addition, computational analyses [Molecular Dynamics (MD) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) approach] were performed for EBOV antigen-antibody complexes and possible three combinations of Fc consistent with experimental results, supporting our assumption and interpretation.