Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus

Abstract

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn 297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn 297 , either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein–Asn 297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn 221 sequon bound influenza virus hemagglutinin and disrupted influenza A–mediated agglutination of human erythrocytes.

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

“…In an earlier study with CHO-K1 cells we demonstrated that a proportion of molecules in which the tailpiece Asn-563 glycan was substituted for alanine ran as multimers in solution when examined by SE-HPLC (24). The loss of the bulky Asn-563 glycan exposes hydrophobic amino acid residues in the tailpiece that facilitate non-covalent interactions in solution.…”

“…(B) The C309L/C575A panel of mutants. Boxed chromatograms represent profiles for equivalent mutants expressed in CHO-K1 cells, as published previously (24).…”

“…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.…”

“…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).…”

“…Human cell lines also carry the risk of contamination and forward transmission of human pathogens, in particular viruses, that may explain why CHO-K1 cells are still the preferred cell line used by the pharmaceutical industry. These issues led us to compare the functional properties of a panel of Fc mutants generated in CHO-K1 cells with the same set of proteins manufactured by HEK 293-F cells (24).…”