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

Abstract

Immunoglobulin G (IgG) mediates its immune functions through complement and cellular IgG-Fc receptors (FcγR). IgG contains an evolutionary conserved N-linked glycan at position Asn297 in the Fc-domain. This glycan consists of variable levels of fucose, galactose, sialic acid, and bisecting N-acetylglucosamine (bisection). Of these variations, the lack of fucose strongly enhances binding to the human FcγRIII, a finding which is currently used to improve the efficacy of therapeutic monoclonal antibodies. The influence of the other glycan traits is largely unknown, mostly due to lack of glyco-engineering tools. We describe general methods to produce recombinant proteins of any desired glycoform in eukaryotic cells. Decoy substrates were used to decrease the level of fucosylation or galactosylation, glycosyltransferases were transiently overexpressed to enhance bisection, galactosylation and sialylation and in vitro sialylation was applied for enhanced sialylation. Combination of these techniques enable to systematically explore the biological effect of these glycosylation traits for IgG and other glycoproteins.

A Role for Fc Function in Therapeutic Monoclonal Antibody-Mediated Protection against Ebola Virus

“…Our data points to glycan structures that are associated with enhanced phagocytic activity and NK cell activation (Figure 6). Currently, only a limited number of engineered glycans can be produced on monoclonal Abs (Dekkers et al, 2016). However, efforts to produce Abs with any one of the ~36 naturally occurring Ab glycan substructures are underway and will allow the dissection of the role of specific structures in modulating functional activity (Mimura et al, 2018).…”

Antibody Engineering for Pursuing a Healthier Future

“…The underlined approaches aim at developing glycoproteins with homogeneous N-and O-linked glycans of defined composition (Dicker and Strasser, 2015). Moreover, multi-level glyco-engineering techniques have been investigated to generate IgG with defined Fc-glycans in eukaryotic cells (Dekkers et al, 2016). Additionally, E. coli expression have been successfully employed to produce recombinant human interleukin-2 (IL-2) (Kamionka, 2011).…”

Affinity of human IgG subclasses to mouse Fc gamma receptors

“…All IgGs were produced by transient transfection of HEKfreestyle cells (Thermo Fisher Scientific), as previously described by Vink et al 47 and Dekkers et al 48 After 5 days, IgG-containing cell supernatant from these cells was harvested by spinning twice at maximum speed (> 4000 g) and subsequent filtration with 0.45 nm puradisc syringe filter (Whatmann, GE Healthcare, 10462100).…”