Galactose-extended glycans of antibodies produced by transgenic plants

Abstract

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that ␤1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human ␤1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal ␤1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal ␤1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human ␤1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

“…Numbers indicate the percentage of total peak area represented by Man5-9 peaks (''high-Man''), N-glycans with only one GlcNAc and at least one hexose residue in addition to the trimannosyl core structure (''Hybrids''), and Nglycans containing at least one Xyl or Fuc residue (''Xyl͞Fuc''). much more dramatically compared with WT plants than previously, when the cDNA encoding the full-length human GalT gene was introduced (12). Because MALDI-TOF MS of N-linked glycans using 2,5-dihydroxybenzoic acid can be assumed to provide an adequate reflection of the actual N-glycan makeup, it is very clear that the N-glycan pattern was more akin now to the N-glycans in a tobacco cell culture transformed with a slightly truncated cDNA lacking the first 13 amino acid residues of the cytoplasmic tail of the full-length human GalT (21,22).…”

“…Samsun NN) as described in ref. 12. MGR48 is a mouse monoclonal IgG1 that has been expressed in tobacco plants (41).…”

“…In a previous study, we showed that expression of human ␤-1,4-galactosyltransferase I (GalT) in tobacco plants results in the introduction of terminal ␤-1,4-galactose residues on endogenous plant N-glycans as well as on N-glycans of recombinant antibodies coexpressed in these plants (12). However, the galactose carrying N-glycans still contained the plant-specific ␤-1,2-xylose and ␣-1,3-Fuc residues linked to the core.…”

Cost-effective production of a vaginal protein microbicide to prevent HIV transmission

“…High mannose glycans are the same in plants and mammalian cells, but the processing of complex glycans is distinct, resulting in the inability of plants to incorporate sialic acid on N-linked glycans and the introduction of fucose and xylose linkages that are not found in mammalian cells (40). For injectable products, the presence of plant glycans might result in immunogenic reactions, but because our product is intended for topical application, the potential immunogenicity should not be an issue (21).…”

Manufacturing antibodies in the plant cell

“…This company developed a rapid TMV-based expression system for the production of anti-idiotypic scFv antibodies against nonHodgkin's lymphoma [75]. LSB even successfully began clinical trials, but failed to Deleted: [69] Deleted: [70] Deleted: much…”