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

Abstract

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 N-linked glycan attached at Asn-297 on the structure and function of IgG1-Fc is well documented; however, whether the N-linked glycan has a similarly critical role in multimeric, avidly binding Fcs, is unknown. Hexa-Fc contains two N-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 N-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 N-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.

Choice of host cell line is essential for the functional glycosylation of the fragment crystallizable (Fc) region of human IgG1 inhibitors of influenza B viruses

“…The generation of glycan mutants in all combinations has been described previously for the hexa-Fc that contains cysteines at both positions 309 and 575 (23). To make the new mutants described in Fig.…”

“…1 in which Cys-575 was mutated to alanine, PCR overlap extension mutagenesis was used with a pair of internal mismatched primers 5’-ACCCTGCTTGCTCAACTCT-3’ / 3’-GGCCAGCTAGCTCAGTAGGCGGTGCCAGC-5’ for each plasmid vector coding for a designated glycan modification. The parental plasmids used for these new PCR reactions have been described previously (23). The resulting C575A mutants were then further modified to remove Cys-309 using primer pair 5’-TCACCGTCTTGCACCAGGACT-3’ / 3’-AGTCCTGGTGCAAGACGGTGA-5’ to create the panel of double cysteine knockouts described in Fig.…”

“…We took an alternative approach to enhancing the sialic acid content of the Fc of IgG1 (23, 24), by adding the 18 amino-acid tailpiece (tp) from IgM to the C-terminus of the IgG1 Fc, into which a cysteine-to-alanine substitution is made at Cys-575, and including an extra N-glycosylation site to the N-terminus at position Asn-221. The tp also contains a N-glycosylation site at Asn-563.…”

“…The tp also contains a N-glycosylation site at Asn-563. When expressed in CHO-K1 cells, these molecules displayed enhanced binding to the low-affinity Fcγ-receptors (FcγRIIIA and FcγRIIB), and to multiple glycan receptors that control excessive inflammation by IVIG (23–25). Two such hyper-sialylated molecules (D221N/C575A and D221N/C309L/N297A/C575A) also bound recombinant hemagglutinin from influenza A and B viruses, and disrupted influenza A-mediated agglutination of human erythrocytes (24).…”

“…Furthermore, humans have active α2,6-sialyltransferase. As such, CHO derived IgG1 Fcs are only sialylated through α2,3 linkages whereas both α2,3 and α2,6 linkages can be found on human IgG1 Fc (23, 27). Most non-human mammalian cell lines can also attach Neu5Gc.…”