Structural and functional characterization of an anti‐West Nile virus monoclonal antibody and its single‐chain variant produced in glycoengineered plants

Summary Previously, our group engineered a plant‐derived monoclonal antibody ( MA b p E 16) that efficiently treated West Nile virus ( WNV ) infection in mice. In this study, we developed a p E 16 variant consisting of a single‐chain variable fragment (sc F v) fused to the heavy chain constant domains ( C H ) of human I g G (p E 16sc F v‐ C H ). pE 16 and pE 16sc F v‐ C H were expressed and assembled efficiently in Nicotiana benthamiana ∆ XF plants, a glycosylation mutant lacking plant‐specific N‐glycan residues. Glycan analysis revealed that ∆ XF plant‐derived p E 16sc F v‐ C H (∆ XF pE16sc F v‐ C H ) and pE 16 (∆ XF p E 16) both displayed a mammalian glycosylation profile. ∆ XF p E 16 and ∆ XF p E 16sc F v‐ C H demonstrated equivalent antigen‐binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell‐produced E16 (m E 16). A single dose of ∆ XF p E 16 or ∆ XF p E 16sc F v‐ C H protected mice against WNV ‐induced mortality even 4 days after infection at equivalent rates as m E 16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MA b and its single‐chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti‐ WNV MA b therapeutic variants that are equivalent in efficacy to p E 16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N‐glycosylation. This platform may lead to a more robust and cost‐effective production of antibody‐based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.