Abstract 2514: Most clinically approved anti-EGFR antibodies fail to neutralize EGFRvIII, explaining their lack of efficacy in high-grade glioma
Authors: Greenall Sameer A. , Adams Timothy E. , Johns Terrance G. 
Published: August 2018

Abstract
The epidermal growth factor receptor (EGFR), a cell-surface receptor tyrosine kinase, is amplified or overexpressed in 60% of gliomas. Furthermore, in 30% of gliomas, an autoactive, disulphide-bonded truncation mutant, EGFRvIII, is co-expressed. Both the wild-type (wt) and EGFRvIII are central to driving gliomagenesis. Multiple anti-EGFR antibodies have been developed to neutralise these receptors; however, despite promising preclinical results, their efficacy against glioma in the clinic has been disappointing for reasons that are not understood. We have tested the efficacy and mechanism of action of the majority of known FDA-approved or preclinical anti-EGFR antibodies [panitumumab, cetuximab, necitumumab, nimotuzumab, matuzumab and chimeric 806 (ch806)] against five primary EGFRvIII-bearing glioma lines. We find that cetuximab, necitumumab, nimotuzumab and matuzumab all fail to fully neutralize EGFRvIII activity, leading to sustained downstream signalling and modest antiproliferative effects. ch806 neutralized EGFRvIII activity but did not engage and neutralize wtEGFR. Only panitumumab was able to completely neutralize both wtEGFR and EGFRvIII activation, resulting in a superior antiproliferative response. Not all EGFRvIII-bearing cell lines were responsive to panitumumab (3 out of 5), suggesting that the expression of EGFRvIII is not indicative of sensitivity to anti-EGFR therapy and that additional mechanisms of resistance are present when EGFRvIII is expressed. Only when cetuximab or necitumumab was combined with ch806, to neutralize both wtEGFR and EGFRvIII, was an antiproliferative response similar to panitumumab achieved. Mechanistically, panitumumab neutralized p-ERK but not p-AKT or p-STAT3 signaling. Panitumumab, cetuximab and ch806 induced EGFRvIII internalization and recycling by Rab11-positive endosomes back to the cell surface, while wtEGFR was degraded. Subsequent wtEGFR translation replaced the degraded wtEGFR, ensuring receptor pool replenishment. Finally, panitumumab does not exert its effects by breaking the active EGFRvIII disulphide-bonded dimer; rather, it may work by trapping EGFRvIII into an inactive high molecular weight receptor cluster, an effect that was not observed for cetuximab. Animal trials to compare the efficacy of these antibodies against one another in intracranial orthografts of patient-derived cell lines are ongoing. Collectively, our results challenge the existing premise that EGFRvIII is neutralized and degraded by EGFR antibodies and demonstrate that both wtEGFR and EGFRvIII must be neutralized for maximum antitumor effect. These results may help explain why existing antibodies such as cetuximab and nimotuzumab have failed in clinical trials in glioma and provide rationale that panitumumab may offer a superior and effective candidate for a further clinical trial in glioma.
Citation Format: Sameer A. Greenall, Timothy E. Adams, Terrance G. Johns. Most clinically approved anti-EGFR antibodies fail to neutralize EGFRvIII, explaining their lack of efficacy in high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2514.