Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors.

Multimeric fragment crystallizable (Fc) regions and Fc-fusion proteins are actively being explored as biomimetic replacements for IVIG therapy, which is deployed to manage many diseases and conditions but is expensive and not always efficient. The Fc region of human IgG1 (IgG1-Fc) can be engineered into multimeric structures (hexa-Fcs) that bind their cognate receptors with high avidity. The critical influence of the unique -linked glycan attached at Asn-297 on the structure and function of IgG1-Fc is well documented; however, whether the -linked glycan has a similarly critical role in multimeric, avidly binding Fcs, is unknown. Hexa-Fc contains two -linked sites at Asn-77 (equivalent to Asn-297 in the Fc of IgG1) and Asn-236 (equivalent to Asn-563 in the tail piece of IgM). We report here that glycosylation at Asn-297 is critical for interactions with Fc receptors and complement and that glycosylation at Asn-563 is essential for controlling multimerization. We also found that introduction of an additional fully occupied -linked glycosylation site at the N terminus at position 1 (equivalent to Asp-221 in the Fc of IgG1) dramatically enhances overall sialic acid content of the Fc multimers. Furthermore, replacement of Cys-575 in the IgM tail piece of multimers resulted in monomers with enhanced sialic acid content and differential receptor-binding profiles. Thus insertion of additional -linked glycans into either the hinge or tail piece of monomers or multimers leads to molecules with enhanced sialylation that may be suitable for managing inflammation or blocking pathogen invasion.