Re-engineering glycosylation in insect cells

The presence of fucose on the N-glycans of IgG antibodies has a major, and negative, impact on their effectiveness in antibody-dependent cellular cytotoxicity; however, most cell lines suitable for production of therapeutic IgG transfer fucose to IgG. Thus, in order to improve the efficacy of IgG, in therapy approaches are required to reduce or abolish the transfer of fucose. On the other hand, insect cells represent a useful tool in the cheaper production of pharmaceutical glycoproteins and, in order to modify their ability to transfer fucose, it is proposed to tackle not specifically the fucosyltransferases, but in a novel approach reduce or abolish the intracellular pool of fucose donors in insect cells by use of knock-out/down strategies; this would have the effect also of eliminating other fucose-containing `foreign` glycan structures such as the immunogenic anti-horseradish peroxidase epitope. Furthermore, the glycans of insect cells tend to be shorter than those of mammals - this is in part due to the action of a hexosaminidase in the secretory pathway, which removes a potential addition site for knocked-in galactosyltransferases and sialyltransferases. Thus, it is also propose to adopt knock-out/down techniques to target this hexosaminidase. Previously, we have examined the GDP-Fuc biosynthesis and hexosaminidase in the fruitfly Drosophila melanogaster and will use this information to identify orthologous enzymes in biotechnologically- relevant insect species. Therefore, this project will bring us closer to having a wider variety of insect cell types with optimised glycosylation suitable for the expression of glycoprotein pharmaceuticals such as IgG.

The presence of fucose on the N-glycans of IgG antibodies has a major, and negative, impact on their effectiveness in antibody-dependent cellular cytotoxicity; however, most cell lines suitable for production of therapeutic IgG transfer fucose to IgG. Thus, in order to improve the efficacy of IgG, in therapy approaches are required to reduce or abolish the transfer of fucose. On the other hand, insect cells represent a useful tool in the cheaper production of pharmaceutical glycoproteins and, in order to modify their ability to transfer fucose, it was proposed to tackle not specifically the fucosyltransferases, but in a novel approach to reduce or abolish the intracellular pool of fucose donors in insect cells by use of knock-out/down strategies; this would have the effect also of eliminating other fucose-containing `foreign` glycan structures such as the immunogenic anti-horseradish peroxidase epitope. Previously, we have examined the GDP-Fuc biosynthesis in the fruitfly Drosophila melanogaster and used this information to identify an orthologous enzyme in a biotechnologically-relevant insect species. We also could successfully knock-out fucosylation in a Drosophila cell line - an approach which may be suitable for other insect cell lines. A relevant patent application is planned.