Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

Abstract

(2015) Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation, mAbs, 7:5, 863-870, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: core fucosylation, sialylation, Nicotiana benthamiana, bisected glycans, glycan modelling, IgG, cetuximab Abbreviations: 3-FucT, Zea maize core a1,3-fucosyltransferase;3-FucT GnTIV, human a1,3-mannosyl-b1,4-N-acetyL-glucosaminyltransferase fused to the CTS region of the Arabidopsis thaliana core a1,3-fucosyltransferase (FUT11); 6-FucT, Mus musculus core a1,6-fucosyltransferase; CH2, constant domain of an IgG heavy chain; CTS, cytoplasmic tail, transmembrane domain and stem region; CxMab cetuximab (Erbitux Ò ); Fab, fragment, antigen-binding; Fc, Fragment crystallizable region of immunoglobulin G; GlcNAc, N-acetylglucosamine; IgG1, Immunoglobulin G subclass 1; LC-ESI-MS, Liquid chromatography-electrospray ionisationmass spectrometry; mAb, monoclonal antibody; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; ST GalT, b1,4-galactosyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; 6-SiaT, a2,6-sialyltransferase;ST GnT-III, b1,4-mannosyl-b1,4-N-acetylglucosaminyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; DXT/FT, Nicotiana benthamiana glycosylation mutants lacking plant specific core b1,2-xylose and a1,3-fucose residuesWe investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (a1,3-and a1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF 3 , and GnGnF 6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core a1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies.

Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

“…Addition of 2FG also resulted in a slight decrease of fucosylation and a slight enhancement of high-mannose and hybrid-type glycans. Recently, it has been demonstrated that decorations of the IgG Fc N-glycan core affect the accessibility and consequently processing of Fc N-glycan antennae50. We speculate that the increased galactosylation observed with expression of the bisecting enzyme, GNTIII, as observed in our study, may be caused by the conformational change induced by bisection.…”

Engineering of complex protein sialylation in plants

“…In contrast to autopolysialylation of polySTs, Ig5FN1 required core fucose for polysialylation. It seems that this core glycan residue induces conformational modifications, thereby influencing the processing of glycan structures, an observation also noticed for the glycosylation modulation of the IgG-Fc N-glycan (19). Selective polysialylation determined by specific core residues has been reported for glycoproteins expressed in CHO cells (30).…”

“…1). Note that, for IgG sialylation, Arabidopsis thaliana α1,3-fucosyltransferase (FUT11) was transiently coexpressed because core α1,3-fucosylation enhances sialylation of the Fc glycans, which are naturally inefficiently sialylated (19). Transgenic ΔXTFT Sia plants stably express rat α2,6-sialyltransferase (ST6) and synthesize α2,6-linked Sia as previously shown using a transient expression approach (14).…”

Reduced paucimannosidic N‐glycan formation by suppression of a specific β‐hexosaminidase from Nicotiana benthamiana

“…Interestingly, the impact of core α1,3‐fucose on GlcNAc trimming shows that specific N ‐glycans on proteins like the Fc domain‐containing Sec‐Fc‐mRFP can be converted into HEXO3 substrates. The impact of core α1,3‐fucose on N ‐glycan modifications has been described recently for the N ‐glycan from the Fc domain of the monoclonal antibody cetuximab (Castilho et al ., ). In addition to the conserved Fc N ‐glycan, cetuximab has a second N ‐glycan in the variable domain of the heavy chain.…”

“…These two N ‐glycosylation sites on the heavy chain allow the comparison of N ‐glycan processing in a given cell in the presence or absence of an additional glycan modification. As a result of this study, it was shown that core α1,3‐fucosylation increases branching, bisecting GlcNAc formation and in particular sialylation of the Fc N ‐glycan presumably by alleviating structural constraints between the Fc N ‐glycan and the IgG1 CH2 domains (Castilho et al ., ).…”