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.

Structure and activation of C1, the complex initiating the classical pathway of the complement cascade

“…Elucidation of structure and function are important for future design of therapeutic intervention strategies aiming at stimulating or inhibiting complement activation. Complement-dependent cytotoxicity inducing antibodies used in cancer therapy rely on their Fc segment being organized in oligomers to which C1 can bind and augmentation of C1 targeting to cancer cells is a proven strategy (26). In combination with the above-mentioned existing experimental data showing that tethering of C1 to a target is sufficient to induce CP activation, our results provide support for IgG-independent targeting of C1 to cancer cells or pathogens as a viable strategy.…”

Novel mechanisms and functions of complement

“…By employing an elegant liposomal surface model and a combination of structural techniques, it was demonstrated that, upon binding to antigen-covered surfaces, IgG molecules arrange in a hexameric form with a distinct conformational arrangement that facilitates binding and activation of the C1 complex 115 . This model not only provides a molecular explanation about the inability of soluble IgG to activate the CP, but also enable the rational design of engineered IgG variants with increased propensity to form hexamers 115,116 ; these ‘hexabodies’ may have broad implications for complement-mediated antibody therapy in cancer, infectious or autoimmune diseases 116,117 .…”

Complement in cancer: untangling an intricate relationship

“…These HexaBodies have been shown to induce strong complement activation and CDC of primary chronic lymphocytic leukaemia cells, even in the presence of limiting amounts of C9 (REF. 98 ). Additionally, dose titration of the therapeutic mAb appears to be critical for optimal CDC.…”