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Recapitulation of IVIG Anti-Inflammatory Activity with a Recombinant IgG Fc

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Science  18 Apr 2008:
Vol. 320, Issue 5874, pp. 373-376
DOI: 10.1126/science.1154315

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.

Although originally used as an antibody-replacement therapy, when given at high doses (1 to 2 g/kg), intravenous immunoglobulin (IVIG) has general anti-inflammatory properties and has been widely used to treat autoimmune diseases, including immune thrombocytopenia (ITP), rheumatoid arthritis, and systemic lupus erythematosus. The anti-inflammatory activity of IVIG has been demonstrated in a variety of animal models of autoimmunity, including autoantibody ITP (1), serum-transfer arthritis (2), and nephrotoxic nephritis (3) and is a property of the Fc fragment and its associated glycan (1, 4, 5). Removal of the terminal sialic acid from IVIG or its papain-derived Fc fragment abrogates the anti-inflammatory activity in these animal models. Conversely, enrichment of the sialylated fraction of IVIG enhances this activity (4).

Because sialic acid can be found in either a 2,3- or 2,6-linkage to the penultimate galactose on the complex, biantennary glycan found at Asn297 in immunoglobulin G (IgG) (Fig. 1A) (6), we set out to determine which linkage type was present in IVIG by analyzing the enzymatically released N-linked glycan from Fc fragments derived from IVIG (Fig. 1, B to D) (7). Sequential mass spectrometric–based glycan analysis revealed a preferential 2,6-linkage in the IVIG preparations that conferred anti-inflammatory activity (Fig. 1, B to D). To confirm this association, we next analyzed IVIG activity after treatment with either 2,3- or 2,3/2,6-sialidase (SA) in the K/BxN serum-transfer arthritis model (8). Consistent with the glycan analysis, we observed complete abrogation of the anti-inflammatory activity when the 2,6-linkages were removed (Fig. 1E and fig. S1). In contrast, no 2,3-linkages were detected on intact IVIG, and the anti-inflammatory activity was unaffected after 2,3-SA treatment. These results are in agreement with previous studies that detected a preferential utilization of the 2,6-linkage of sialic acid–galactose in human IgG preparations and preferential expression of the 2,6-sialyltransferase (2,6ST) in B cells (913).

Fig. 1.

α2,6-linkages are the predominant sialic acid linkages 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 the addition of galactose (blue) and sometimes sialic acid (red) to the arms. Neu5Ac, N-acetylneuraminic acid (sialic acid); Man, mannose. (B) Sequential mass spectrometry (MS) 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 B/Y fragments derived from standards are depicted with the corresponding histogram. The spectrum generated from the SNA+ IVIG Fc glycan (B) most closely matches that of 2,6-sialyllactose (D), particularly with respect to the mass/charge ratio (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 SAs to remove only 2,3- (α2,3SA) or both 2,3- and 2,6- (α2,3/6SA) sialic acids. Me, methyl. (E) The SA-treated IVIG preparations were administered to mice prior to K/BxN sera, and footpad swelling was monitored over the next 7 days and recorded as clinical scores. Means and SDs (error bars) of five mice per group 5 days post-treatment are plotted. *P < 0.05, as determined by an analysis of variance (ANOVA) test, followed by a Tukey post hoc test.

To determine whether the preferential 2,6-sialic acid–galactose linkage was a necessary and sufficient property of the anti-inflammatory activity of the sialylated, N-linked glycan at Asn297, we generated IVIG-derived Fc fragments terminating in either the 2,3- or 2,6-sialic acid linkage. To efficiently sialylate the Fc fragment derived from IVIG, we first converted the population of glycans to the G2 (digalactosylated) (14, 15) form by in vitro treatment with β1,4-galactosyltransferase, resulting in a twofold increase in terminal galactose as determined by Erythrina cristagalli lectin (ECL) binding (Fig. 2, A and B). This galactosylated substrate was then reacted with either 2,3-sialyltransferase (2,3ST) or 2,6ST (Fig. 2A and fig. S2A). In vitro sialylation was evaluated directly by lectin blotting for 2,3- and 2,6-linkages (Fig. 2C) and indirectly by assaying for reduced terminal galactose (ECL binding) (fig. S2, A and B). The 2,6ST quantitatively converted the G2 glycan to a fully sialylated form, as demonstrated by the absence of ECL reactivity (fig. S2). The efficiency of the 2,3ST was estimated to be ∼50%, on the basis of residual ECL binding to these reacted Fc-linked glycans (fig. S2). ECL also bound the Sambucus nigra lectin (SNA)–enriched Fc derived from IVIG (SNA+ Fc), which contained ∼30% fully sialylated Fc glycans (fig. S2), representing a 10-fold enrichment in sialic acid, as compared with unfractionated IVIG-derived Fc fragments. This level of sialylation observed in the SNA-enriched IVIG Fc fragments was sufficient to induce an anti-inflammatory response at a 10-fold reduced dose (as compared with unfractionated IVIG) and was demonstrated to confer enhanced anti-inflammatory activity through a pathway that requires the up-regulation of the inhibitory FcγR on effector inflammatory macrophages (3, 4).

Fig. 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 SA (to remove all sialic acid residues), galactosylated (with β1,4GT), and finally sialylated with either 2,3- or 2,6ST (α2,3ST and α2,6ST, respectively). (B) Galactosylation was verified by lectin blotting with ECL (top), which recognizes terminal galactose residues. Relative band intensity ratios of ECL to coomassie loading controls are plotted (bottom). (C) Galactosylated Fcs were then sialylated with 2,3- or 2,6ST, and each sialylation reaction was confirmed by lectin blotting for 2,6-linkages with SNA (top) and 2,3-linkages with Maackia amurensis lectin I (MAL I) (middle). Coomassie stained loading controls are shown (bottom).

We then administered these preparations to mice (table S1) in the K/BxN serum-induced arthritis model. Joint inflammation was effectively reduced by administering either the SNA+ IVIG Fc fragments, as has been observed previously (4), or the in vitro 2,6ST-treated IVIG Fc fragments (Fig. 3A). Suppression of inflammation was comparable with these two preparations and was 10-fold more active than the total Fc fragments derived from IVIG. In contrast, IVIG Fc fragments sialylated in vitro with 2,3ST were ineffective at reducing the joint inflammation induced by K/BxN serum (Fig. 3A), even at fourfold higher doses (fig. S2).

Fig. 3.

Sialic acid–dependent IgG anti-inflammatory activity is linkage specific, although attenuation of antibody-dependent cytotoxicity is not. (A) In vitro sialylated IVIG Fcs were administered to mice 1 hour before K/BxN sera, and paw swelling was monitored over the next several days. Means and SDs (error bars) of four mice per group are plotted. *P < 0.05, as determined by ANOVA, followed by Tukey's post hoc test. (B) To determine whether the reduced antibody-dependent cell-mediated cytotoxicity of sialylated antibodies was also dependent on specific linkages, platelet-depleting 6A6-IgG2b antibodies were sialylated and administered to mice, and platelet counts were determined at 0, 4, and 24 hours after treatment. Means and SDs (error bars) of five mice per group are plotted; *P < 0.05, as determined by ANOVA, followed by Tukey's post hoc test.

In contrast to the sialic acid–galactose linkage specificity for Fc anti-inflammatory activity, the effect of sialylation of Fc receptor (FcR) binding and the consequent cytotoxicity was linkage-independent. Both terminal 2,3- and 2,6-in vitro sialylated IgG Fc reduced the cytotoxicity of an antiplatelet antibody, 6A6-IgG2b, in an in vivo model of ITP (Fig. 3B), consistent with previous studies (4, 16). Thus, the effect of Fc sialylation on the cytotoxicity of an IgG antibody is not dependent on the specificity of the linkage to the penultimate galactose. In contrast, the anti-inflammatory activity of the sialylated IgG Fc fragment [a property we have previously demonstrated to be independent of the canonical IgG Fc receptors (4, 5)] displayed a clear preference for the 2,6-sialic acid–galactose linkage (Fig. 3A and fig. S2B). These results further support our previous observations that the anti-inflammatory property of IVIG is mediated through a distinct pathway (4, 5) that does not involve binding to canonical FcγRs.

To fully demonstrate that the in vivo anti-inflammatory activity of the 2,6-sialylated IgG Fc is solely a property of the IgG Fc glycan and not the result of other components that might be found in the heterogeneous IVIG Fc preparations, we set out to recapitulate the anti-inflammatory activity of sialylated IVIG Fc using a homogeneous, recombinant human IgG1 Fc substrate (rFc) derived from a cDNA expressed in 293T cells. The purified recombinant human IgG1 Fc fragment was glycan engineered in vitro (as described above) by β1,4-galactosylation, followed by 2,6-sialylation (Fig. 4A). The preparation was purified and characterized by lectin blotting (Fig. 4A) before in vivo analysis. As shown in Fig. 4B, the 2,6-sialylated recombinant human IgG1 Fc fragment demonstrated comparable anti-inflammatory activity to that obtained with either IVIG-derived sialic-enriched Fc fragments (SNA+ IVIG Fc) or in vitro 2,6-sialylated IVIG-derived Fc fragments. Each of these preparations was active at 30 mg/kg, as compared with the 1000 to 2000 mg/kg required for native IVIG (table S1).

Fig. 4.

Recombinant, sialylated IgG Fc fragments are anti-inflammatory and comparable to native IVIG. Recombinant human IgG1 was digested with papain, and Fcs were purified by high-performance liquid chromatography, followed by protein G purification. The rFcs were galactosylated and sialylated in vitro with α2,6ST. (A) Glycosylation was confirmed by lectin blotting for terminal galactose with ECL (top) or α2,6-sialic acid with SNA (middle); coomassie loading controls are shown (bottom). (B) Micewere administered 1 g/kg IVIG, 0.033 g/kg SNA+ IVIG Fcs, or 0.33 g/kg sialylated rFc (2,6ST rFc) 1 hour before K/BxN sera, and footpad swelling was monitored over the next several days. Means and SDs (error bars) of clinical scores of four to five mice per group are plotted. *P < 0.05, as determined by Kruskal-Wallis ANOVA, followed by Dunn's post hoc test.

The exquisite specificity of glycan structures has long been appreciated as providing the structural basis for discrete biological responses, including lectins purified from plants (17), viruses (1820), bacteria (18, 21), and parasites (22, 23). The results reported here demonstrate that the sialic acid–galactose linkage specificity for the N-linked glycan of the IgG Fc confers anti-inflammatory activity on the IgG and is a property of both the glycan and amino acid sequence (4).

The observation that the anti-inflammatory activity of IVIG is dependent on a precise glycan structure on the Fc further supports the model we have previously advanced (4, 5)—one in which a specific receptor for the sialylated Fc, and not a canonical Fc receptor, is involved in this pathway. Our data support a model in which the binding of the 2,6-sialylated Fc to its cognate receptor expressed on a population of splenic macrophages in the mouse results in the trans up-regulation of the inhibitory IgG Fc receptor on effector macrophages, located at sites of inflammation (such as the inflamed joint or glomerulus), thus raising the threshold required for cytotoxic IgGs to engage activation FcRs and trigger inflammatory responses (1, 2, 5, 24). Along these lines, we have observed the preferential binding of 2,6-Fcs to marginal-zone macrophages, implicating a lectin expressed on these cells in this anti-inflammatory activity of IVIG. Furthermore, if our ability to recapitulate this anti-inflammatory property of IVIG in a recombinant molecule at a much reduced dose can be reproduced in the treatment of human autoimmune diseases, the studies described here will facilitate the development of this recombinant alternative as a broadly acting anti-inflammatory drug for the treatment of autoimmune disorders.

Supporting Online Material

www.sciencemag.org/cgi/content/full/320/5874/373/DC1

Materials and Methods

Figs. S1 to S2

Table S1

References

References and Notes

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