RESUMEN
All IgG-type antibodies are N-glycosylated in their Fc part at Asn-297. Typically, a fucose residue is attached to the first N-acetylglucosamine of these complex-type N-glycans. Antibodies lacking core fucosylation show a significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and an increased efficacy of anti-tumor activity. In cases where the clinical efficacy of an antibody is to some extent mediated by its ADCC effector function, afucosylated N-glycans could help to reduce dose requirement and save manufacturing costs. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate here that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-d-lyxo-4-hexulose reductase within the cytosol can efficiently deflect the fucose de novo pathway. Antibody-producing CHO cells that were modified in this way secrete antibodies lacking core fucose as demonstrated by MALDI-TOF mass spectrometry and HPAEC-PAD monosaccharide analysis. Engineering of the fucose de novo pathway has led to the construction of IgGs with a strongly enhanced ADCC effector function. The method described here should have broad practical applicability for the development of next-generation therapeutic antibodies.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteínas Bacterianas/biosíntesis , Inmunoglobulina G/biosíntesis , Oxidorreductasas Actuantes sobre Donantes de Grupos Aldehído u Oxo/biosíntesis , Modificación Traduccional de las Proteínas , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes/biosíntesis , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales Humanizados , Proteínas Bacterianas/genética , Células CHO , Cricetinae , Cricetulus , Glicosilación , Inmunoglobulina G/genética , Oxidorreductasas Actuantes sobre Donantes de Grupos Aldehído u Oxo/genética , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , TrastuzumabRESUMEN
Glycosylation is known to have an impact on pharmacokinetics and pharmacodynamics of therapeutic proteins. While the production of pharmaceutically desirable glycosylation forms of a therapeutic protein can in certain cases be influenced by the upstream process parameters, certain specialized glycan structures can only be produced in large quantities from cell lines that have been genetically engineered.One particular case where a specialized glycostructure has a major impact on pharmacodynamic mode of action is the enhanced ADCC-effector function of afucosylated IgG1-type monoclonal antibodies. Here we describe the methodological details of a powerful yet simple glycoengineering approach targeted at the fucosylation machinery within eukaryotic cells. As an example we demonstrate the modification of the permanent avian cell line AGE1.CR.pIX which is characterized by a unique glycosylation machinery.