Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.
The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA.
['Suzuki|Mami|M|', 'Yamanoi|Ayaka|A|', 'Machino|Yusuke|Y|', 'Kobayashi|Eiji|E|', 'Fukuchi|Kaori|K|', 'Tsukimoto|Mitsutoshi|M|', 'Kojima|Shuji|S|', 'Kohroki|Junya|J|', 'Akimoto|Kazunori|K|', 'Masuho|Yasuhiko|Y|']
D000478:Alkylation D058846:Antibodies, Monoclonal, Murine-Derived D000915:Antibody Affinity D000920:Antibody-Dependent Cell Cytotoxicity D045744:Cell Line, Tumor D018920:Coculture Techniques D004220:Disulfides D053804:Granzymes D006801:Humans D007694:Killer Cells, Natural D054353:Perforin D011485:Protein Binding D059748:Proteolysis D017452:Receptors, IgG D000069283:Rituximab
Interchain disulfide bond Rituximab Rituximab FcγRIIIA Effector function Fcγ receptor RTX A-RTX S-RTX antibody-dependent cellular cytotoxicity ADCC Granzyme B FcγR KHYG-1 cells stably expressing FcγRIIIA KHYG-1/FcγRIIIA S-sulfonated rituximab SDS–PAGE mAb monoclonal antibody reduced and then alkylated rituximab rituximab sodium dodecyl sulfate–polyacrylamide gel electrophoresis