Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase.
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.
von Horsten
Hans Henning
HH
ProBioGen AG, Goethestr 54, 13086 Berlin, Germany. henning.von.horsten@probiogen.de
Ogorek
Christiane
C
Blanchard
Véronique
V
Demmler
Christian
C
Giese
Christoph
C
Winkler
Karsten
K
Kaup
Matthias
M
Berger
Markus
M
Jordan
Ingo
I
Sandig
Volker
V
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D061067:Antibodies, Monoclonal, Humanized; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors / Q000096:biosynthesis* / Q000235:genetics; D046188:Protein Modification, Translational; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C115750:GDP-6-deoxy-D-lyxo-4-hexulose reductase; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors; D000068878:Trastuzumab