Abstract
Previously, our group engineered a plant-derived monoclonal antibody (MAb) (pHu-E16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed several pHu-E16 variants to improve its efficacy. These variants included a single-chain variable fragment (scFv) of pHu-E16 fused to the heavy chain (HC) constant domains (CH 1-3) of human IgG (pHu-E16scFv-CH 1-3) and a tetravalent molecule (Tetra pHu-E16) assembled from pHu-E16scFv-CH 1-3 with a second pHu-E16scFv fused to the light chain (LC) constant region. pHu-E16scFv-CH 1-3 and Tetra pHu-E16 were efficiently expressed and assembled in plants. To assess the impact of differences in N-linked glycosylation on pHu-E16 variant assembly and function, we expressed additional pHu-E16 variants with various combinations of HC and LC components. Our study revealed that proper pairing of HC and LC was essential for the complete N-glycan processing of antibodies in both plant and animal cells. Associated with their distinct N-glycoforms, pHu-E16, pHu-E16scFv-CH 1-3 and Tetra pHu-E16 exhibited differential binding to C1q and specific Fcγ receptors (FcγR). Notably, none of the plant-derived Hu-E16 variants showed antibody-dependent enhancement (ADE) activity in CD32A+ human cells, suggesting the potential of plant-produced antibodies to minimize the adverse effect of ADE. Importantly, all plant-derived MAb variants exhibited at least equivalent in vitro neutralization and in vivo protection in mice compared to mammalian cell-produced Hu-E16. This study demonstrates the capacity of plants to express and assemble a large, complex and functional IgG-like tetravalent mAb variant and also provides insight into the relationship between MAb N-glycosylation, FcγR and C1q binding, and ADE. These new insights may allow the development of safer and cost effective MAb-based therapeutics for flaviviruses, and possibly other pathogens.
Highlights
The development and implementation of targeted monoclonal antibody (MAb) therapy have provided new opportunities for controlling a wide range of diseases
A. tumefaciens strains containing the pHu-E16scFvCH1-3 construct were co-delivered into N. benthamiana leaves along with the promoter module and an integrase construct through agroinfiltration [14,15]
Western blot analysis after reducing or non-reducing gel electrophoresis confirmed that pHu-E16scFvCH1-3 was produced in leaves with the expected molecular weight (Fig 1B, Lane 5), and that it assembled into a dimer (Fig 1D, Lane 7)
Summary
The development and implementation of targeted monoclonal antibody (MAb) therapy have provided new opportunities for controlling a wide range of diseases. MAbs produced in mammalian cell culture systems have achieved remarkable clinical success, their high cost, long manufacturing time, and restricted production capacity have limited the availability, utility and potential of these drugs. Functional antibody production requires a eukaryotic host cell that can assemble four antibody polypeptides into a heterotetramer and perform complex N-linked glycosylation. Despite this complexity, a MAb was successfully expressed in tobacco plants only three years after the first plant-made biologic [4]. The ability of plants to express and assemble larger or more complex MAb-derived molecules such as tetravalent MAbs or bifunctional MAbs has not been described
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
