title: Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.
abstract: 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.
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Suzuki
authors.forename: Ayaka
authors.initials: A
authors.lastname: Yamanoi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Machino
authors.forename: Eiji
authors.initials: E
authors.lastname: Kobayashi
authors.forename: Kaori
authors.initials: K
authors.lastname: Fukuchi
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Tsukimoto
authors.forename: Shuji
authors.initials: S
authors.lastname: Kojima
authors.forename: Junya
authors.initials: J
authors.lastname: Kohroki
authors.forename: Kazunori
authors.initials: K
authors.lastname: Akimoto
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Masuho
mesh_terms: D000478:Alkylation; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D000920:Antibody-Dependent Cell Cytotoxicity; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D004220:Disulfides / Q000737:chemistry*; D053804:Granzymes / Q000737:chemistry; D006801:Humans; D007694:Killer Cells, Natural / Q000737:chemistry / Q000276:immunology; D054353:Perforin / Q000737:chemistry; D011485:Protein Binding; D059748:Proteolysis; D017452:Receptors, IgG / Q000737:chemistry* / Q000276:immunology; D000069283:Rituximab
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D004220:Disulfides; C496124:FCGR3A protein, human; D017452:Receptors, IgG; D054353:Perforin; D000069283:Rituximab; C509213:GZMB protein, human; D053804:Granzymes
keywords: A-RTX; ADCC; Effector function; Fcγ receptor; FcγR; FcγRIIIA; Granzyme B; Interchain disulfide bond; KHYG-1 cells stably expressing FcγRIIIA; KHYG-1/FcγRIIIA; RTX; Rituximab; S-RTX; S-sulfonated rituximab; SDS–PAGE; antibody-dependent cellular cytotoxicity; mAb; monoclonal antibody; reduced and then alkylated rituximab; rituximab; sodium dodecyl sulfate–polyacrylamide gel electrophoresis