Abstract 578: Potent complement-dependent cytotoxicity of tumor cells by target bound hexamerization of EGFR antibodies
Authors: Annalina Tammen, Frank J. Beurskens, Stefanie Derer, Ralf Schwanbeck, Paul W.H.I. Parren, Janine Schuurman, Thomas Valerius
Journal: Cancer Research (2016)

Abstract Introduction: Activation of complement (C) constitutes an important effector mechanism of monoclonal antibodies in cancer therapy. Unmodified IgG1 antibodies against the epidermal growth factor receptor (EGFR), a well-validated tumor target, lack the capacity to induce complement-dependent cytotoxicity (CDC) as single agents. Recently, significantly improved CDC was demonstrated after antigen-bound IgG molecules formed hexameric structures through intermolecular Fc-Fc contacts. Single point mutations (E345K or E430G) that enhance hexamerization of IgG molecules conditional on antigen binding have been identified to represent a platform for enhanced C activation (Diebolder et al Science 343:1260, 2014, de Jong et al, PLOS Biology, in press). Alternative strategies to enhance CDC have focused on enhancing C1q binding via affinity-maturation of the C1q binding site (S239D/H268F/S324T/I332E; DFTE) or by generating IgG1/IgG3 isotype chimeras (113F). The aim of the present study was to investigate whether EGFR antibodies optimized for each of these approaches differed in terms of CDC activity. Methods: EGFR antibodies comprising engineered Fc domains that carry mutations to enhance hexamerization (E345K or E430G), or increase C1q affinity (DFTE), or that exist of IgG1/IgG3 chimeras (113F) were constructed. CDC activity was assessed by 51Cr release assays in the presence of human serum. Antibody mediated C1q binding or C4b deposition on tumor cells was analyzed by flow cytometry. Apparent C1q binding affinity was determined by C1q specific ELISA. Results: Here, we demonstrate that affinity maturation of the C1q binding site (DFTE &amp; 113F) increased C1q binding in the absence of antigen, whereas mutations that enhance Fc-Fc interactions (E345K &amp; E430G), and so enhance hexamerization after target binding, did not. However, these latter mutations strongly increased C1q avidity and CDC for EGFR antibodies bound to cells. Notably, the latter strategy translated in superior CDC activity, which was particularly evident for tumor cells with low EGFR expression levels. Furthermore, hexamerization enhanced CDC was particularly sensitive to peptides inhibiting Fc-Fc interactions, supporting their different mode of action compared to other CDC-enhancing strategies. Interestingly, also the EGFR epitope recognized by different antibodies impacted on the CDC activity. Conclusion: Enhancement of hexamer formation by EGFR antibodies conditional on antigen binding is a promising novel approach to improve their anti-tumor activity. Strategies that enhanced complement binding independent of antigen engagement by the antibody appeared less effective. Citation Format: Annalina Tammen, Frank J. Beurskens, Stefanie Derer, Ralf Schwanbeck, Paul W.H.I. Parren, Janine Schuurman, Thomas Valerius. Potent complement-dependent cytotoxicity of tumor cells by target bound hexamerization of EGFR antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 578.