Enrichment of Sialylated IgG by Lectin Fractionation Does Not Enhance the Efficacy of Immunoglobulin G in a Murine Model of Immune Thrombocytopenia

Abstract

Intravenous immunoglobulin G (IVIg) is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA (+)) in a murine model of passive immune thrombocytopenia (PIT). We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA) lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA (+) using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA (+) 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01). In contrast, IVIg-SA (+) had no effect on the platelet count. Serum levels of IVIg and IVIg-SA (+) were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model.

The Emerging Importance of IgG Fab Glycosylation in Immunity

“…Recent studies emphasized the importance of glycosylation in the therapeutic effects of IVIg (73). In certain mouse models (74,75), but not in others (13,(76)(77)(78), the anti-inflammatory activity of IVIg depends mainly on a fraction of sialylated Abs. These sialylated Abs may interact with C-type lectins (like DC-SIGN, DCIR) and sialic acid-binding Ig-type lectins, such as CD22, at the surface of immune cells (79,80).…”

“…2B). These studies used Sambucus nigra agglutinin (SNA) lectin-affinity chromatography to fractionate IVIg, thereby predominantly enriching for Fab sialylation rather than Fc sialylation (13,76). Fab-sialylated IVIg downregulated CD54 expression and the MCP-1 secretion of monocytes, whereas neither IVIg without sialylated Fab nor a highly sialylated Fc could achieve these effects (13).…”

Production of α2,6-sialylated IgG1 in CHO cells

“…6,19,20 The impact of the presence of terminal sialic acid residues is also uncertain. [21][22][23][24][25][26][27][28][29][30] Indeed, the discrepancies in the methods used for evaluating biological activity, the variability in the type and level of sialylation, as well as the lack of a systematic in-depth glycan characterization, all contribute to the ambiguity of its role in IgG functions. In humans, sialic acids can be attached to the Fc-glycans either on the C3-or the C6-hydroxyl group of the terminal galactose, through the action of a2,3-sialyltransferases (ST3) or the a2,6-sialyltransferase-1 (ST6).…”

“…22 Contradictory results were also reported on sialylated Fcs' ability to bind FcgRIIIa, but using dissimilar IgG preparations and affinity measurement protocols. 21,28 In other studies assessing the anti-inflammatory properties of a2,6-sialylated IgGs, 21,24,26,30 blood-derived or recombinant antibodies were enriched by affinity chromatography using Sambucus Nigra agglutinin-resins that were shown to catch mainly IgGs bearing Fab-sialylated glycans to the detriment of Fc-sialylated species. 24,32,37 While the specific involvement of Fc's a2,6-sialylation in anti-inflammatory activity was shown by Anthony et al in their model, 35 which receptor the sialylated Fcs are targeting remains controversial.…”

IVIG pluripotency and the concept of Fc-sialylation: challenges to the scientist

“…However, two of the cited articles do not include functional experiments but rather focus on aspects of human IgG sialylation and lectin fractionation. The other articles report that in a human whole blood inflammation assay, the anti-inflammatory activity of IVIG was associated with Fab sialy lation and not Fc sialylation 5 , and that in a murine model of passive-immune thrombo cytopenia no increase in platelet count was observed in response to the administration of IVIG that was enriched for sialylated IgG 6 . Together, the data presented in these publications do not support the proposed Fc-sialylation concept -by contrast, they rather challenge it.…”