Regulation of antibody-mediated complement-dependent cytotoxicity by modulating the intrinsic affinity and binding valency of IgG for target antigen

Abstract

Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.

Complement-Dependent Activity of CD20-Specific IgG Correlates With Bivalent Antigen Binding and C1q Binding Strength

“…Binding of C1q to OFA opsonized cells was thus clearly stronger than for RTX on all tested cell lines, whereas binding of C1q to OBI opsonized cells could not be detected. Bivalent target engagement therefore positively correlates with strong C1q capture for the investigated anti-CD20 IgGs and expanding this analysis to a larger panel of anti-CD20 IgGs would be of interest, as contrasting observations have been made for other antigens ( 19 , 37 ). The real-time binding assay presented in this study also allowed to resolve the presence of two interactions, as well as their kinetic and affinity values for C1q binding to anti-CD20 IgG opsonized cells.…”

“…For anti-EGFR mAbs it has been shown that monovalent target engagement results in higher CDC efficacy. This could be a consequence of monovalent binding resulting in a higher number of IgGs bound and this, in turn, might increase the likelihood for the formation of multimeric IgG-Fc platforms suitable for C1q binding ( 37 ). For anti-CD20 mAbs, on the other hand, bivalent binding might enhance crosslinking of CD20 and thus lead to more efficient clustering of CD20 and bound mAbs.…”

“…However, the parameters involved in the formation of the optimal antibody platform for C1q binding are not completely understood yet. Contradicting observations have especially been made with respect to functionally monovalent antibodies inducing CDC more efficiently than their counterparts with bivalent binding capability ( 19 , 36 , 37 ). For C1q binding to antibody opsonized cells, affinity values in the low nM range have been reported without resolution of the kinetic binding parameters ( 38 ).…”

“…The degree of bivalent target engagement thus positively correlates with how efficiently these mAbs have been shown to induce CDC in vitro . As the notion that bivalent target engagement is beneficial for CDC stands in contrast to observations made with antibodies targeting EGFR ( 19 ) and HER2 ( 37 ), we set out to develop a real-time binding assay to investigate C1q capture on live cells opsonized with CD20 mAbs. We systematically compared CD20 mAb binding, C1q binding and complement-activation, as well as C3b deposition that can trigger target cell killing independently of MAC formation.…”

Bivalent binding on cells varies between anti-CD20 antibodies and is dose-dependent

“… 25 However, the notion that bivalent binding correlates with CDC efficacy is contradicted by previous studies that found changing one of the Fab arms of anti-epidermal growth factor receptor antibodies to an irrelevant binding epitope, and thereby making them functionally monovalent, enhanced the CDC capacity of these antibodies compared to their parental version. 10 , 30 It is possible that the optimal binding mode for efficient complement activation differs for mAbs targeting different receptors.…”