title: Significant impact of single N-glycan residues on the biological activity of Fc-based antibody-like fragments.
abstract: Recent studies have demonstrated that IgG-Fc fragments (Fcabs) can be engineered to form antigen-binding sites with antibody properties. Thus they may serve as an attractive alternative to conventional antibodies in therapeutic applications. The critical influence of Fc glycosylation on effector functions of IgGs is well documented; however, whether this applies to Fcabs is not known. Here we used human cells, wild type, and glycoengineered plants to generate four different glycoforms of H10-03-6, an Fcab with engineered HER2/neu-binding sites. Plant-derived H10-03-6 differed in the presence/absence of single oligosaccharide residues, i.e., core fucose and xylose, and terminal galactose. All of the glycoforms had similar binding to HER2/neu expressed on human tumor cells. By contrast, glycoforms that lacked core oligosaccharide modifications (i.e., core α1,3-fucose and β1,2-xylose) showed significantly enhanced binding to the Fcγ receptor IIIa, irrespective of whether plant or human expression systems were used. Consistent with this finding, plant-derived H10-03-6 glycoforms lacking core N-glycan residues mediated higher antibody-dependent cellular cytotoxicity against human tumor cells. No alteration in γ-receptor binding and antibody-dependent cellular cytotoxicity activity was observed upon decoration of N-glycans by terminal galactose. The results point to a significant impact of distinct N-glycan residues on effector functions of Fcabs. Moreover, the outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile. Biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Jakub
authors.initials: J
authors.lastname: Jez
authors.forename: Bernhard
authors.initials: B
authors.lastname: Antes
authors.forename: Alexandra
authors.initials: A
authors.lastname: Castilho
authors.forename: Manuela
authors.initials: M
authors.lastname: Kainer
authors.forename: Susanne
authors.initials: S
authors.lastname: Wiederkum
authors.forename: Josephine
authors.initials: J
authors.lastname: Grass
authors.forename: Florian
authors.initials: F
authors.lastname: Rüker
authors.forename: Max
authors.initials: M
authors.lastname: Woisetschläger
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry*; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014026:Nicotiana / Q000235:genetics
chemical_list: D000906:Antibodies; D007141:Immunoglobulin Fc Fragments; D011134:Polysaccharides; D017452:Receptors, IgG