Multi-Angle Effector Function Analysis of Human Monoclonal IgG Glycovariants.

Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies FcγRs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual FcγRs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different FcγRs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fcγ-receptors, with the exception of high affinity FcγRI. The FcγRII and FcγRIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the FcγR binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to FcγRIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the decreased affinity of target bound antibody towards FcRs, showing the importance of the IC-formation for the FcR- mediated effector functions

In vitro glycoengineering of IgG1 and its effect on Fc receptor binding and ADCC activity.

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity

Structures of IgG N-glycans attached to the Asn297 in the Fc domain.

<p>The core (G0) heptasaccharide is highlighted in light blue. GlcNAc, N-acetylglucosamine; Fuc, fucose; Man, mannose; Gal, galactose; NeuAc, N-acetyl neuraminic (sialic) acid.</p

Interaction of target- or F(ab’)₂ -bound IgG with FcγRIIIa.

<p>Prior to interaction analysis IgG was incubated with its monomeric target (A, B) or with F(ab’)₂ (C, D) and loaded onto affinity column (A, C) and examined by SPR (B, D). The IgG: ligand ratio: 1:0 (red), 1:1 (green), 1:2 (blue) and 1:4 (pink).</p

SPR analysis of interaction of IgG1 glycovariants with FcγRs.

<p>Anti His-antibody immobilized on the chip, glycovariants were injected as analytes, after capturing of the respective receptors: A. FcγRI, B. FcγRIIa, C. FcγRIIb and D. FcγRIIIa. Binding of WT antibody was set as 100%. Each graph represents results from at least three independent experiments; data are given as means ± SD.</p

Thermal transitions of IgG1 glycovariants obtained by Thermofluor Stability Assay (TSA).

<p>Melting curves: deglycosylated (black), M3 (blue), G0 (green), G1 (pink), G2 (orange), G1+1SA (light blue), G2+1SA (yellow), G2+2SA (purple), WT (red).</p

Retention profiles of the IgG1 glycovariants obtained by analytical FcγRIIIa affinity chromatography.

<p>The chromatograms, obtained by monitoring signal at 280 nm, show two peaks corresponding to fucosylated and afucosylated antibody fractions (deglycosylated IgG is not shown).</p

Retention profiles of IgG1 glycovariants obtained by analytical FcRn affinity chromatography.

<p>Retention profiles of IgG1 glycovariants obtained by analytical FcRn affinity chromatography.</p

Binding of glycovariants to FcγRIIIa, expressed on living cells.

<p>Unlabeled glycovariants compete for binding to receptor with acceptor-labeled antibody, resulting in decrease of FRET signal: deglycosylated (red diamonds), WT (black circles), G0 (black triangles), G1(black inverted triangles), G2 (black squares). Initial signal was normalized to 1. (For remaining glycovariants refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143520#pone.0143520.s003" target="_blank">S3 Fig</a>).</p

SPR analysis of the interaction of target-bound or F(ab’)₂

<p>Glycovariants were injected to bind antigen (A) or F(ab’)₂ (B), immobilized on the chip, followed by the application of the respective receptors: FcγRI (black), FcγRIIa (grey), FcγRIIb (dark grey) and FcγRIIIa (light grey). Binding of WT antibody was set as 100% Each graph represents results from at least three independent experiments; data are given as means ± SD.</p