Glycosylation is the covalent linkage of an oligosaccharide chain to a protein resulting in a glycoprotein. In many glycoproteins, the oligosaccharide chain is attached to the amide nitrogen of an asparagine (Asn) residue and leads to N-glycosylation. Glycosylation represents the most widespread post-translational modification found in natural and biopharmaceutical proteins. For example, more than half of the human proteins are glycosylated and their function frequently depends on particular glycoforms (glycans), which can affect their plasma half life, tissue targeting or even their biological activity. Similarly, more than one-third of approved biopharmaceuticals are glycoproteins and both their function and efficiency are affected by the presence and composition of their N-glycans. The functional activity of therapeutic glycoproteins is also frequently dependent on their glycosylation; this can be the case, for example in blood factors, antibodies and interferons. This requirement for glycosylation explains why many biopharmaceuticals are produced in expression systems with N-glycosylation capability. In recent years plants have emerged as an attractive system for the production of therapeutic proteins, as plants are generally considered to have several advantages, including the lack of animal pathogens such as prions and viruses, low cost and the large-scale production of safe and biologically active valuable recombinant proteins, the case of scale-up, efficient harvesting and storage possibilities. However, N-linked glycans from plants differ in many aspects from those of mammalian cells. In plants, beta(1,2)-xylose and alfa(1,3)-fucose residues have been shown to be linked to the core Man3GlucNAc2-Asn of glycans, whereas they are not detected on mammalian glycans, where sialic acid residues and terminal beta(1,4)-galactosyl structures occur instead. The unique N-glycans added by plants could impact both immunogenicity and functional activity of the protein and, consequently, may represent a limitation for plants to be used as a protein production platform. Indeed, the immunogenicity of beta(1,2)-xylose residues and alfa(1,3)-fucose in mammals has been described (Bardor et. al., 2003, Glycobiology 13: 427). Glyco-engineering with the combined knock-out/knock-in approach of glycosylation-related enzyme genes has been recognized for the avoidance of plant-specific glycan residues as well as the introduction of human glycosylation machinery in plants.
Beta(1,4)-galactose has been introduced in plants by expression of human beta(1,4)-galactosyltransferase I (GalT) (Bakker et. al., 2001, Proc. Natl. Acad. Sci. USA 98: 2899), and chicken and zebrafish beta(1,4)-galactosyltransferase I (WO2008/125972). In several studies, the GalT enzyme was fused to a golgi targeting signal as to alter the localization in Golgi and to improve GalT activity. Bakker et. al. (2006, Proc. Natl. Acad. Sci. USA 103: 7577) and WO2003/078637 describe a fusion of human GalT to the cytoplasmic tail, transmembrane domain, and stem region (CTS domain) of Arabidopsis thaliana xylosyltransferase (XylT). They found that, in tobacco, addition of this CTS domain caused a sharp reduction of N-glycans with core-bound xylose and fucose residues. Vezina et. al. (2009, Plant. Biotechnol. J. 7: 442) and WO2008/151440 fused GalT to the membrane anchorage domain of the N-acetylglucosaminyltransferase I (GNTI) from tobacco, in order to allocate GalT activity in the early plant secretory pathway. Glycans from the N. benthamiana plants expressing the GNTI-GalT fusion comprised galactosylated and non-galactosylated hybrids and immature oligomannose N-glycans, and contained no detectable alfa(1,3)-fucose and beta(1,2)-xylose residues. WO2008/125972 replaced the chicken and zebrafish CTS domain with the CTS of rat sialyltransferase. The zebrafish GalT having substituted its amino-terminal for the CTS region of rat sialyltransferase, produced mainly biantennary, double galactosylated N-glycans in Nicotiana benthamiana. Strasser et. al. (2009, J. Biol. Chem. 284: 20479) fused human GalT to the rat sialyltransferase CTS domain. This fusion protein was expressed in N. benthamiana which lacks plant-specific beta(1,2)-xylosyltransferase and core alfa(1,3)-fucosyltransferase activities and expresses anti-human immunodeficiency virus antibody. The predominant glycoform of the expressed antibodies was the fully galactosylated AA structure, and to some extent incompletely processed and monoantennary galactosylated structures were present. Galactosylated structures represented about 80% of all glycoforms. Moreover, it was observed that the antigen-binding of these plant-derived antibodies was 115-140% as compared to CHO-derived antibodies. Importantly, the fully galactosylated plant-derived antibodies neutralized HIV more efficiently than other glycoforms from plant and CHO cells.
The current invention provides methods and means to improve production of bi-antennary beta(1,4)-galactosylated N-glycan structures and to reduce the production of hybrid-type beta(1,4)-galactosylated N-glycans on glycoproteins in plants and plant cells, as will become apparent from the following description, examples, drawings and claims provided herein.
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