Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality.
Although it is now clear that certain Fc glycan structures on immunoglobulin G (IgG) antibodies (Abs) can have a dramatic influence on binding to selected Fcgamma receptors (FcgammaR) and on Fc-mediated immune functions, the effects of all known Fc glycan structures still have not been exhaustively studied. We report that in vitro analyses of pairs of monoclonal human IgG Abs that differ in the amount of sialic acid in their Fc glycans revealed that, for each of the three Ab pairs we examined, higher levels of sialylation were associated with reduced activity in Ab-dependent cellular cytotoxicity (ADCC) assays. This relationship between sialylation and ADCC activity was observed regardless of whether the differences in the extent of sialylation were derived by different Ab production processes, use of a lectin column to separate monoclonal Ab preparations into differentially sialylated fractions, or use of direct in vitro glycoengineering methods to convert a lesser sialylated Ab into a highly sialylated Ab. Subsequent investigations revealed that, depending on the individual Ab and how the differences in sialylation were derived, the lower ADCC potency of the more sialylated variants was apparently due to lower-affinity binding to FcgammaRIIIa on natural killer (NK) cells and/or, more interestingly, lower-affinity binding to cell-surface antigen. Our data provide the first example of an Fc glycan structure impacting antigen binding and suggest that avoiding Fc glycan sialylation can offer another means of optimizing ADCC activity of Abs.
Scallon
Bernard J
BJ
Discovery Research, Centocor R&D, Inc., Radnor, PA 19087, USA. BScallon@cnyus.jnj.com
Tam
Susan H
SH
McCarthy
Stephen G
SG
Cai
Ann N
AN
Raju
T Shantha
TS
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000941:Antigens / Q000276:immunology; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D011134:Polysaccharides / Q000737:chemistry*; D015202:Protein Engineering; D017452:Receptors, IgG / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; C496124:FCGR3A protein, human; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides; D017452:Receptors, IgG; D019158:N-Acetylneuraminic Acid