Effect of combinations of EGF-R antibodies on complement-dependent tumor cell lysis

Abstract

14005 Background: Therapeutic EGF-R antibodies have advanced the treatment of colon and head & neck tumors and bear great promise for other cancers. While several methods of action have been identified for EGF-R antibodies, induction of complement depending cytotoxicity (CDC) has so far not been observed. We asked whether antibody combinations might provide benefit for this mechanism. Methods and Results: We investigated 8 EGF-R antibodies of human IgG1 (cetuximab, matuzumab, zalutumumab, 003, 005, 008, 011 and 018) and 1 of human IgG2 isotype (panitumumab). Select combinations led to increased C1q and C4c fixation - determined by immunofluorescence - and correspondingly induced strong CDC of A431 and A1207 cells in 51-Cr release assays. Experiments with Ca++/Mg++ depletion and heat inactivation of serum indicated the classical complement pathway to be responsible. Studies comparing a human IgG1 with its murine parental antibody, respective F(ab’)2 fragments, or its IgA1 variant demonstrated two human IgG1 antibodies to be mandatory for CDC. Immunofluorescence-based cross-competition studies identified two clusters of EGF-R binding epitopes (cluster 1: cetuximab, matuzumab, panitumumab, zalutumumab, 018; cluster 2: 003, 005, 008, 011). In cluster 1, three combinations were non-crossblocking, while the remaining combinations competed with each other. In cluster 2, all combinations were crossblocking. None of the combinations of cluster 1 and cluster 2 was crossblocking. All possible dual combinations of the 8 IgG1 antibodies were investigated for complement activation. Strong synergism was observed for all non-crossblocking combinations, whereas none of the crossblocking combinations triggered CDC. Conclusions: Here, we demonstrate a remarkable synergy for EGF-R antibodies. Antibody combinations were identified resulting in potent complement activation via the classical pathway and effective CDC of tumor cells. Studies on a large antibody panel indicated that the observed synergy is a general mechanism which can be activated by combining human IgG1 antibodies recognizing different, non-overlapping epitopes. Our findings demonstrate an unexpected quality of EGF-R antibodies which may be exploited to develop novel and more effective treatments for solid cancers. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Genmab