title: Binding of submaximal C1q promotes complement-dependent cytotoxicity (CDC) of B cells opsonized with anti-CD20 mAbs ofatumumab (OFA) or rituximab (RTX): considerably higher levels of CDC are induced by OFA than by RTX.
abstract: The CD20 mAb ofatumumab (OFA) is more effective than rituximab (RTX) in promoting complement-dependent cytotoxicity (CDC) of B cells via the classical pathway (CP) of complement. CP activation is initiated by C1q binding to cell-bound IgG. Therefore, we examined the role of C1q in the dynamics of complement activation and CDC of B cell lines and primary cells from patients with chronic lymphocytic leukemia, reacted with OFA or RTX. C1q binding, complement activation, and colocalization of C1q with cell-bound mAbs were determined by flow cytometry and high-resolution digital imaging. C1q binds avidly to OFA-opsonized Raji and Daudi cells (K(D) = 12-16 nM) and colocalizes substantially with cell-bound OFA. Cells opsonized with OFA undergo high levels of complement activation and CDC in C1q-depleted serum supplemented with low concentrations of C1q. Under comparable conditions, RTX-opsonized cells bind less C1q; in addition, even when higher concentrations of C1q are used to achieve comparable C1q binding to RTX-opsonized cells, less complement activation and CDC are observed. Greater CDC induced by OFA may occur because C1q is bound in close proximity and with high avidity to OFA, resulting in effective CP activation. Moreover, OFA binds to the small, extracellular CD20 loop, placing the mAb considerably closer to the cell membrane than does RTX. This may facilitate effective capture and concentration of activated complement components closer to the cell membrane, potentially shielding them from inactivation by fluid phase agents and promoting efficient generation of the membrane attack complex.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, 22908, USA.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Pawluczkowycz
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Beurskens
authors.forename: Paul V
authors.initials: PV
authors.lastname: Beum
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Lindorfer
authors.forename: Jan G J
authors.initials: JG
authors.lastname: van de Winkel
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Taylor
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20 / Q000276:immunology*; D016175:B-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D015922:Complement C1q / Q000378:metabolism* / Q000502:physiology; D003179:Complement C3b / Q000378:metabolism; D003171:Complement Pathway, Classical / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D009895:Opsonin Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D000069283:Rituximab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D009895:Opsonin Proteins; D000069283:Rituximab; D015922:Complement C1q; D003179:Complement C3b; C527517:ofatumumab