IgG-Fc glycoengineering in non-mammalian expression hosts

Abstract

The remarkable success of therapeutic applications of immunoglobulin G (IgG) in form of monoclonal antibodies and pooled immunoglobulin G preparations has directed attention to this class of glycoproteins. It is commonly appreciated that oligosaccharides attached to the Fc-region play a critical role in the biological activity of IgGs. Thus, glycosylation has been a focus of interest for many scientists and the biopharmaceutical industry and expression hosts have been engineered in order to optimize antibody products. In this review we focus on efforts towards a targeted manipulation of IgG-Fc N-glycans using non-mammalian expression hosts, i.e. yeast, insect cells and plants. Current achievements in generating human-like N-glycan structures will be presented and recent data on the molecular mechanisms that might explain how these potent drugs mediate in vivo activities will be discussed.

Fig. 1

Schematic presentation of selected components of the N-glycosylation pathways in humans, yeast, insect cells and plants. The common ER-resident oligosaccharide precursor Man8 acts as starting point for further modifications along the Golgi apparatus. Och1: α1,6-mannosyltransferase; MnTs: mannosyltransferases; Mns: mannosidase; GnT: N-acetylglucosaminyltransferase; GalT: α1,4-galactosyltransferase; ST: α2,6-sialyltransferase; HEXO: hexosaminidase (N-acetylglucosaminidase); XT: β 1,2-xylosyltransferase; FT: core fucosyltransferase; Fucose can be transferred in α1,3-linkage (plant typical) and α1,6-linkage (mammalian typical). Interestingly, both forms are synthesized in insect cells. Oligosaccharide abbreviations according to www.proglycan.com.

Fig. 2

Fc-Glycoengineering in plants. Overview of mAb glycoforms generated in glycoengineered Nicotiana benthamiana, a tobacco related plant species widely used for recombinant protein production. IgG N-glycans generated (1) in wild-type plants: GnGnXF³; (2) in ΔXT/FT³ plants: GnGn ; (3) in ΔXT/FT + FT⁶: GnGnF⁶; (4) in ΔXTFT + GalT: AA ; (5) in ΔXT/FT along with six mammalian genes of the mammalian sialic acid pathway: NaNa ; (6) in wild-type + GnTIII: GnGnXF³bi . FT⁶: α1,6-fucosyltransferase, GalT: β1,4-galactosyltransferase, ST: α2,6-sialyltransferase, GnTIII: N-acetylglucosaminyltransferase III. Glycan abbreviations according to www.proglycan.com.

Fig. 3

Reconstruction of the human sialylation pathway in plants using the endogenously present metabolite UDP-GlcNAc. Enzymes involved in the process are: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE), N-acetylneuraminic acid phosphate-synthase (NANS), CMP-sialic acid (Neu5Ac) synthetase (CMAS), CMP-Neu5Ac transporter (CST), β1,4-galactosyltransfease (GT) and α2,6-sialyltransferase (ST). In planta protein sialylation was achieved by the coordinated expression and correct subcellular deposition of genes/proteins for (i) biosynthesis (GNE, NANS), (ii) activation (CMAS), (iii) transport (CST), and (iv) transfer of Neu5Ac to terminal galactose (ST) . Glycan abbreviations according to www.proglycan.com.