Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity

Abstract

IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

Figures

Figure 7. Induction of IgG Fc sialylation is associated with disease remission in CIDP.

(A) Serum IgG Fc glycan composition of CIDP patients analyzed by lectin blotting. Relative IgG Fc glycan abundance of CIDP patients before and after a 24-week observation period. Patients undergoing spontaneous disease remission are compared with those with stable or worsening disease (no remission). Statistics were performed by Mann-Whitney U test. (B) IgG Fc glycan composition analysis of CIDP patients (Marburg patient cohort) before and 3–5 weeks after receiving the last IVIG injection. Fold changes in glycan composition are compared with disease activity changes reflected by the INCAT score. Statistics were performed by Spearman test. (C) Complement activation as defined by serum levels of the TCC (SC5b-9) quantified by ELISA before and 3–5 weeks after IVIG therapy (Marburg patient cohort). Statistics were performed by Mann–Whitney U test.

Figure 6. C1q binding to human polyclonal IgG Fc glycovariants.

(A) Lectin immunoblotting of Fc purified from human polyclonal IgG and enriched for sialylated Fc or desialylated (mannose: LCA; terminal galactose: ECL; sialic acid: SNA). (B) C1q binding to polyclonal IgG Fc glycovariants measured by ELISA. Mean and ± SEM of 3 independent experiments. C1q binding was normalized to unmodified Fc at 10% serum. Individual curves were compared with Fc using 2-way ANOVA and Bonferroni post test comparing Fc and desialylated Fc, or Fc and sialic acid–enriched Fc (P values below curve).

Figure 5. C1q binding to IgG Fc glycovariants.

(A) Lectin immunoblotting of RTX glycovariants. (mannose: LCA; terminal galactose: ECL; sialic acid: SNA). (B) Time-course of C1q binding to CD20+ Raji cells in the presence or absence of differentially glycosylated RTX. Mean ± SD of 6 independent experiments. Median fluorescence intensity obtained for the unmodified antibody after 60 minutes was set to 100, and the relative signals were calculated. (C) Quantification of RTX antibody glycovariant-dependent C1q binding to CD20+ Raji and Ramos cells after 30 minutes incubation. Mean ± SD of at least 3 independent experiments. Statistics were performed by 1-way ANOVA and Bonferroni post test. (D) Lectin immunoblotting of hu8-18C5 (anti-MOG) glycovariants. (E) Time-course of C1q binding to MO3.13 MOG cells in the presence or absence of differentially glycosylated variants of hu8-18C5. Mean ± SD of 8 independent experiments. Median fluorescence intensity obtained for the unmodified antibody after 30 minutes was set to 100 and the relative signals were calculated. (F) Quantification of hu8-18C5 antibody glycovariant–dependent C1q binding to MO3.13 MOG cells after 60 minutes. Statistics were performed by 1-way ANOVA and Bonferroni post test. (G) Representative flow cytometry stainings showing C1q binding to the indicated cell type and antibody after 1 hour incubation.

Figure 4. SPR of C1q binding to RTX and tetra-Fc–sialylated RTX.

Sensorgrams and results of global fits (table) from SPR measurements.

Figure 3. Cell-surface antigen expression on target cells and antigen binding properties of antibody glycovariants.

(A) Raji and Ramos cells were analyzed for CD20 expression by flow cytometry. Gray histograms, unstained cells; open histograms, stained cells. (B) MO3.13 cells lentivirally transduced to express human MOG were analyzed for MOG expression by flow cytometry. Gray histograms, unstained cells; open histograms, stained cells. CD20+ Raji cells were incubated with RTX or glycovariants of RTX (C) and MO3.13 MOG cells with hu8-18C5 (anti-MOG) or glycovariants of hu8-18C5 (D) following incubation with a fluorescently labeled anti-Ig antibody and detection of antibody binding by flow cytometry. MFI, median fluorescence intensity.

Figure 2. IgG Fc sialylation impairs complement-dependent but not cell-mediated cytotoxicity.

(A) RTX and tetra-Fc–sialylated RTX mediated lysis of B cells (targets) by autologous NK cells (effectors). Representative experiment and quantification of 3 independent experiments. Statistics were performed by t test. (B) Binding of RTX and tetra-Fc–sialylated RTX to CHO cells either untransfected or transfected with the low-affinity (158F) or high-affinity (158V) variant of FcγRIIIA. (C) Complement-mediated lysis of the CD20+ Raji cells. Representative experiment and quantification of EC50 of 3 independent experiments. Statistics were performed by t test. (D) Complement-mediated lysis of CD20+ Raji cells (left) or MO3.13 MOG cells (right) with RTX and anti-MOG (hu8-18C5) or Fc-galactosylated and sialylated variants of the antibodies, representative of 3 independent experiments. 158F, Phenylalanine 158; 158V, Valine 158.

Figure 1. Characterization of unmodified and tetra-Fc–sialylated RTX.

(A) Immunoblotting of RTX and tetra-Fc–sialylated RTX using lectins specific for individual glycans (mannose: LCA; terminal galactose: ECL; sialic acid: SNA). (B) Liquid chromatography-mass spectrometry (LC/MS) analysis of the tetra-Fc–sialylated RTX after reduction to separate the light chain and heavy chain.