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  • Modulation of IgG1 immunoeffector function by glycoengineering of the GDP-fucose biosynthesis pathway.

Modulation of IgG1 immunoeffector function by glycoengineering of the GDP-fucose biosynthesis pathway.

Biotechnology and bioengineering (2017-11-19)
Ronan M Kelly, Ronald L Kowle, Zhirui Lian, Beth A Strifler, Derrick R Witcher, Bhavin S Parekh, Tongtong Wang, Christopher C Frye
ABSTRACT

Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG CH 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanosine 5′-diphospho-β-L-fucose sodium salt, ≥85%
Roche
Papain, from Carica papaya
Supelco
Discovery® Glycan SPE Tube, bed wt. 250 mg, volume 3 mL, pk of 54
Supelco
Discovery® Glycan SPE Tube, 5 g, volume 60 mL, pk of 16
Supelco
Discovery® Glycan SPE Bulk Packing, pkg of 50 g
Supelco
Discovery® Glycan SPE Tube, bed wt. 500 mg, volume 6 mL, pk of 30
Supelco
Discovery® Glycan SPE Tube, bed wt. 1 g, volume 12 mL, pk of 20
Sigma-Aldrich
Protein A, Staphylococcus aureus