Most clinical anti-EGFR antibodies do not neutralize both wtEGFR and EGFRvIII activation in glioma

BACKGROUND: Although the epidermal growth factor receptor (EGFR) and its truncated, autoactive mutant EGFRvIII are bona fide drivers of tumorigenesis in some gliomas, therapeutic antibodies developed to neutralize this axis have not improved patient survival in a limited number of trials. Previous studies using cells transduced to exogenously express EGFRvIII may have compromised mechanistic studies of anti-EGFR therapeutics. Therefore, we re-assessed the activity of clinical EGFR antibodies in patient-derived gliomaspheres that endogenously express EGFRvIII. METHODS: The anti-tumor efficacy of antibodies was assessed using in vitro proliferation assays and intracranial orthografts. Receptor activation status, antibody engagement, oncogenic signaling and mechanism of action after antibody treatment were analyzed by immunoprecipitation and western blotting. Tracking of antibody•receptor complexes was conducted using immunofluorescence. RESULTS: The EGFR domain III-targeting antibodies, cetuximab, necitumumab, nimotuzumab and matuzumab did not neutralize EGFRvIII activation. Chimeric mAb806 (ch806) neutralized EGFRvIII, but not wtEGFR activation. Panitumumab was the only antibody that neutralized both EGFRvIII and wtEGFR, leading to reduction of p-S6 signaling and superior in vitro and in vivo anti-tumor activity. Mechanistically, panitumumab induced recycling of receptor but not degradation as previously described. Panitumumab, via its unique avidity, stably crosslinked EGFRvIII to prevent its activation while ch806 induced a marked reduction in the active EGFRvIII disulphide-bonded dimer. CONCLUSIONS: We discovered a previously unknown major resistance mechanism in glioma in that most EGFR domain III-targeting antibodies do not neutralize EGFRvIII. The superior in vitro and in vivo anti-tumor activity of panitumumab support further clinical testing of this antibody against EGFRvIII-stratified glioma.