Figure 3.

Comparison of recombinant human CD52-Fc variants (I, II, and III) with different immunosuppressive activities. (A) IFN–γ production measured by ELISpot assay from human PBMCs (2 × 10⁶) in 200 μL/well. Samples were incubated with no antigen or tetanus toxoid in the presence of two different preparations of CD52-Fc (CD52 I or CD52 II; 5, 25, and 50 μg/ml). (B) N-linked glycans released from cleaved CD52 I and CD52 II. The abundance of each N-glycan class is the sum of all EICs measured for all glycans in that class relative to the total of all EICs observed for all N-glycans. (C) EIC of m/z 1140.4²⁻ (GlcNAc₅Man₃Gal₂NeuAc₁) demonstrating the PGC-based separation of sialo-glycan isomers observed in CD52 I and CD52 II. (D) Binding of CD52-Fc I and CD52-Fc II (5 and 20 μg/ml) to the α-2,3 sialic acid recognizing lectin MAL-1. (E) ELISpot assay showing activity of CD52-Fc III reconstituted with sialic acid in α2-6, α2-3, and α2-8 linkages with galactose. The data points in (A,D,E) are plotted as mean ± SEM of three independent replicate experiments. Data in B and C are mean ± SDs (n = 3). ANOVA, post-hoc comparisons of pairs and Bonferroni correction were used to test for significant difference between group means.