title: Generation and analysis of novel plant-derived antibody-based therapeutic molecules against West Nile virus.
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.
authors.affiliation: The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Junyun
authors.initials: J
authors.lastname: He
authors.affiliation: The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.
authors.forename: Huafang
authors.initials: H
authors.lastname: Lai
authors.affiliation: Department of Medicine, Washington University School of Medicine, St Louis, Missouri, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Engle
authors.affiliation: MacroGenics, Inc, Rockville, Maryland, United States of America.
authors.forename: Sergey
authors.initials: S
authors.lastname: Gorlatov
authors.affiliation: Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Gruber
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
authors.affiliation: Department of Medicine, Washington University School of Medicine, St Louis, Missouri, United States of America; Department of Molecular Microbiology, Washington University School of Medicine, St Louis, Missouri, United States of America; Department of Pathology & Immunology, Washington University School of Medicine, St Louis, Missouri, United States of America.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Diamond
authors.affiliation: The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America; College of Technology and Innovation, Arizona State University, Mesa, Arizona, United States of America.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000096:biosynthesis* / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000008:administration & dosage / Q000096:biosynthesis* / Q000302:isolation & purification; D015922:Complement C1q / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D007116:Immunization, Passive; D018796:Immunoconjugates / Q000737:chemistry / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D030821:Plants, Genetically Modified; D011485:Protein Binding; D017452:Receptors, IgG / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000008:administration & dosage / Q000096:biosynthesis* / Q000302:isolation & purification; D016019:Survival Analysis; D014026:Nicotiana / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology; D014901:West Nile Fever / Q000276:immunology / Q000401:mortality / Q000517:prevention & control* / Q000821:virology; D014902:West Nile virus / Q000276:immunology / Q000472:pathogenicity
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D018796:Immunoconjugates; D007074:Immunoglobulin G; D017452:Receptors, IgG; D057127:Single-Chain Antibodies; D014759:Viral Envelope Proteins; D015922:Complement C1q