title: Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.
abstract: Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908;
authors.forename: Erika M
authors.identifier: 0000-0002-6383-1560
authors.initials: EM
authors.lastname: Cook
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908;
authors.forename: Margaret A
authors.identifier: 0000-0003-4203-405X
authors.initials: MA
authors.lastname: Lindorfer
authors.affiliation: Genmab, 3584 CM Utrecht, the Netherlands;
authors.forename: Hilma
authors.initials: H
authors.lastname: van der Horst
authors.affiliation: Genmab, 3584 CM Utrecht, the Netherlands;
authors.forename: Simone
authors.initials: S
authors.lastname: Oostindie
authors.affiliation: Genmab, 3584 CM Utrecht, the Netherlands;
authors.forename: Frank J
authors.identifier: 0000-0002-7165-5771
authors.initials: FJ
authors.lastname: Beurskens
authors.affiliation: Genmab, 3584 CM Utrecht, the Netherlands;
authors.forename: Janine
authors.identifier: 0000-0002-9738-9926
authors.initials: J
authors.lastname: Schuurman
authors.affiliation: Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY 14642;
authors.forename: Clive S
authors.identifier: 0000-0001-6099-3313
authors.initials: CS
authors.lastname: Zent
authors.affiliation: Department of Pathology, University of Rochester Medical Center, Rochester, NY 14642; and.
authors.forename: Richard
authors.initials: R
authors.lastname: Burack
authors.affiliation: Genmab, 3584 CM Utrecht, the Netherlands; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands.
authors.forename: Paul W H I
authors.identifier: 0000-0002-4365-3859
authors.initials: PW
authors.lastname: Parren
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908; rpt@virginia.edu.
authors.forename: Ronald P
authors.identifier: 0000-0003-1578-8382
authors.initials: RP
authors.lastname: Taylor
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000276:immunology / Q000378:metabolism*; D000074323:Alemtuzumab; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity; D000941:Antigens / Q000276:immunology*; D018951:Antigens, CD20 / Q000276:immunology / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology; D001666:Binding Sites, Antibody; D045744:Cell Line, Tumor; D003167:Complement Activation; D003179:Complement C3b / Q000378:metabolism; D003186:Complement C9 / Q000276:immunology / Q000378:metabolism*; D003165:Complement System Proteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000941:Antigens; D018951:Antigens, CD20; D003186:Complement C9; D008562:Membrane Glycoproteins; D000074323:Alemtuzumab; D003179:Complement C3b; D003165:Complement System Proteins; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1