Open AccessMulti-Angle Effector Function Analysis of Human Monoclonal IgG Glycovariants
Author(s) -
Tetyana Dashivets,
Marco Thomann,
Petra Rueger,
Alexander Knaupp,
Johannes Büchner,
Tilman Schlothauer
Publication year - 2015
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0143520
Subject(s) - fragment crystallizable region , antibody dependent cell mediated cytotoxicity , antibody , glycosylation , monoclonal antibody , effector , immunoglobulin g , receptor , antigen , fc receptor , cytotoxicity , chemistry , microbiology and biotechnology , context (archaeology) , biology , immunology , biochemistry , in vitro , paleontology
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.