Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase

Abstract

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.

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“…GDP-rhamnose is a common component of bacterial cell surface glycans. Heterologous expression of bacterial RMD in the cytosol of CHO cells allowed the GDP-fucose de novo pathway to be efficiently bypassed and afucosylated IgG antibodies to be produced 88 . The dead-end product GDP-rhamnose is likely to inhibit the activity of GMD as a competitive inhibitor.…”

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy

“…18,25 The therapeutic anticancer potential of antibodies with decreased fucosylation, however, has been well recognized, and their power is currently being harnessed in clinical trials. 18,[20][21][22][23][24][26][27][28][29][30] In whites, the main antibodies causing FNAIT are the anti-human platelet antigen (HPA) 1a antibodies, found in ;85% of the cases and in ;1:1500 pregnancies. [31][32][33] The HPA-1a epitope resides in the GPIIIa protein of the GPIIb/IIIa complex and is missing in individuals with a single nucleotide polymorphism resulting in an L to P change at position 33 in the mature protein.…”

Cell line development for biomanufacturing processes: recent advances and an outlook

“…Knock-out of endogenous fucosyl transferase resulted in the desired alterations in glycan profile (Lee et al 1989;Yamane-Ohnuki et al 2004). Using a prokaryotic enzyme to divert the metabolic flux from the synthesis of GDP-L-fucose to GDP-D-rhaborthmnose reduced the GDP-L-fucose substrate pool for the fucosylation reaction and led to reduced fucosylation (von Horsten et al 2010). However, it is worth noting that glycosylation pathway is very complex.…”