Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc

Abstract

It is well established that high doses of monomeric immunoglobulin G (IgG) purified from pooled human plasma [intravenous immunoglobulin (IVIG)] confer anti-inflammatory activity in a variety of autoimmune settings. However, exactly how those effects are mediated is not clear because of the heterogeneity of IVIG. Recent studies have demonstrated that the anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. Here we determine the precise glycan requirements for this anti-inflammatory activity, allowing us to engineer an appropriate IgG1 Fc fragment, and thus generate a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency. This therapeutic molecule precisely defines the biologically active component of IVIG and helps guide development of an IVIG replacement with improved activity and availability.

Figure 1

α2,6 linkages are the predominant sialic acid linkage on IVIG Fc glycans. A. The IgG Fc glycan is a bisecting core of seven sugars (black), and can vary at a number of positions by the addition of fucose (green) to the core, a bisecting GlcNAc (gray), or by addition of galactose (blue) and sometimes sialic acid (red) to the arms. B. Sequential mass spectrometry analysis of SNA-enriched IVIG Fc glycans was performed to determine the sialic acid linkage type and relative proportion of the linkages in the active component of IVIG. The resulting footprint of the B/Y galactosyl fragment monomer derived from the SNA-enriched Fc glycan was compared to the analogous B/Y fragments from (C) 2,3 sialyllactose and (D) 2,6 sialyllactose standards. The spectrum generated from the SNA⁺ IVIG Fc glycan (B) most closely matches that of 2,6 sialyllactose (D), particularly with respect to the m/z 123 and 95 fragments, and the much smaller abundances of m/z 137 and 109 ions. Next, IVIG was treated with linkage-specific sialidases to remove only 2,3 (α2,3 SA) or both 2,3 and 2,6 (α2,3/6 SA) sialic acids. E. The sialidase-treated IVIG preparations were administered to mice prior to K/BxN sera, and footpad swelling was monitored over the next seven days and recorded as clinical scores. Mean and standard deviation of 5 mice per group 5 days post treatment are plotted; *denotes p<0.05 as determined by an Anova test followed by Tukey post hoc test.

Figure 2

In vitro sialylation of IVIG Fcs. A. As shown in the schematic diagram of the sialylation strategy, IVIG Fc fragments were initially treated with 2,3/6 sialidase (2,3/6 SA) to remove all sialic acid residues, galactosylated (β1,4 GT), and finally sialylated with either 2,3 or 2,6 sialyltransferases (α2,3 ST and α2,6 ST, respectively). B. Galactosylation was verified by lectin blotting with ECL (top panel), which recognizes terminal galactose residues. Relative band intensity ratios of ECL to coomassie loading controls are plotted below. The galactosylated Fcs were then sialylated with 2,3 or 2,6 sialyltransferases, and (C) each sialylation reaction was confirmed by lectin blotting for 2,6 linkages with SNA (top panel) and 2,3 linkages with MAL I (middle panel). Coomassie stained loading controls are show below.

Figure 3

Sialic acid-dependent IgG anti-inflammatory activity is linkage specific, although the reduced antibody dependent cytotoxicity is not. A. Anion exchange purified sialylated IVIG Fcs were administered to mice 1 hour prior to K/BxN sera, and paw swelling monitored over the next several days. Means and standard deviations of 4 mice per group are plotted; *denotes p<0.05 as determined by Anova followed by Tukey’s post hoc. To determine whether the reduced ADCC of sialylated antibodies was also dependent on specific linkages, platelet-depleting 6A6-IgG2b antibodies were sialylated as described above. (B) Sialylated 6A6-IgG2b antibodies were administered to mice and platelet counts determined 0, 4, and 24 hours following treatment. Mean and standard deviation of 5 mice per group are plotted; *p<0.05 as determined by Anova followed by Tukey’s post hoc.

Figure 4

Recombinant, sialylated IgG Fc fragments are anti-inflammatory. Recombinant human IgG1 was digested with papain and Fcs were purified by HPLC followed by protein G purification. The recombinant Fcs (rFc) were galactosylated and sialylated in vitro with α2,6 sialyltransferase. A. Glycosylation was confirmed by lectin blotting for terminal galactose with ECL (top panel), α2,6 sialic acid with SNA (middle panel), and coomassie loading controls are shown in the bottom panel. B. Mice were administered 1g/kg IVIG, 0.033g/kg SNA⁺ IVIG Fcs, or 0.33g/kg sialylated rFc (2,6ST rFc) 1 hour prior to K/BxN sera, and footpad swelling was monitored over the next several days. Mean and standard deviation of clinical scores of 4–5 mice per group are plotted; *denotes p<0.05 as determined by Kruskal-Wallis Anova followed by Dunn’s post hoc.