Therapeutic anti-EGFR antibody 806 generates responses in murine de novo EGFR mutant-dependent lung carcinomas

Abstract

Activating EGFR mutations occur in human non-small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and approximately 10%-30% of lung adenocarcinomas having EGFR kinase domain mutations. An EGFR-targeting monoclonal antibody, mAb806, recognizes a conformational epitope of WT EGFR as well as the truncated EGFRvIII mutant. To explore the anticancer spectrum of this antibody for EGFR targeted cancer therapy, mAb806 was used to treat genetically engineered mice with lung tumors that were driven by either EGFRvIII or EGFR kinase domain mutations. Our results demonstrate that mAb806 is remarkably effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, resulting in dramatic tumor regression in the EGFRvIII-driven murine lung cancers. Another EGFR-targeting antibody, cetuximab, failed to show activity in these lung tumors. Furthermore, treatment of murine lung tumors driven by the EGFR kinase domain mutation with mAb806 also induced significant tumor regression, albeit to a less degree than that observed in EGFRvIII-driven tumors. Taken together, these data support the hypothesis that mAb806 may lead to significant advancements in the treatment of the population of NSCLC patients with these 2 classes of EGFR mutations.

Figure 1. Murine lung tumors driven by EGFRvIII expression are sensitive to mAb806 and ch806 antibody treatment but resistant to cetuximab treatment.

Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf^–/– mice were treated with either mAb806 or ch806 at 0.5 mg per dose or cetuximab at 1 mg per dose through daily i.p. injection. Antibodies were given every 2 days at the same dose after the first week of treatment. Serial MRI was performed at the indicated time points, and corresponding sections of representative mice in each treatment group are shown. Data (expressed as mean ± SD) illustrate the tumor regression measured by MRI, and statistical analyses were performed using 2-tailed unpaired Student’s t test. All mice were kept on a doxycycline diet throughout the experiment. H indicates the area of the heart.

Figure 2. Histopathological features of EGFRvIII-driven lung adenocarcinomas in mice treated with mAb806.

(A) Lung adenocarcinoma driven by EGFRvIII expression for more than 8 weeks (top row). After 1 week of treatment with mAb806, tumors became smaller and had increased fibrosis (middle row). Lung specimens were grossly normal when mAb806 treatment ended at 5 weeks (bottom row). Arrows show a fibrotic nodule, consisting of fibroblasts and macrophages. No tumor cells were found in this particular fibrosis area. Original magnification, ×100 (left column), ×800 (right column). (B) Similar patterns and intensities of immunohistological staining of total EGFR can be observed in control mice and mice treated with mAb806 for 1 week (top- and bottom-left panels, respectively); intensity of phospho-EGFR staining of tumor cells decreased after 1 week of treatment (bottom-right panel) compared with that in untreated tumors (top-right panel). Representative photos were taken under ×200 magnification. (C) TUNEL staining shows an increase in the number of apoptotic nuclei (red arrows) in EGFRvIII-driven lung tumors after 1 week of treatment with mAb806 (bottom-left panel) compared with untreated tumors (top-left panel). Representative photos were taken under ×200 magnification. Data (expressed as mean ± SD) represent the apoptotic indices in lung tumors before and after 1 week of mAb806 treatment, determined from at least 200 high-power fields (HPF). Statistical analyses were performed using 2-tailed unpaired Student’s t test (right panel).

Figure 3. Western blot analysis of whole lung lysates from mAb806-treated Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf^–/– mice.

Whole lung lysates from mice at different time points of mAb806 treatment were analyzed. Inhibition of EGFR phosphorylation was observed after 1 week of treatment, while total EGFR levels decreased after 5 weeks of treatment. Erk1/2 phosphorylation was inhibited throughout the mAb806 administration; Akt phosphorylation decreased at 1 week of treatment and slightly increased after 5 weeks of treatment when compared with the 1-week time point. β-Actin served as a loading control.

Figure 4. EGFR kinase domain mutation L858R–driven mouse lung adenocarcinoma responds to ch806 treatment.

(A) Tet-op-EGFR L858R-IRES-Luciferase/CCSP-rtTA mice were treated with ch806 at 0.5 mg per dose by daily i.p. injection for 4 weeks. MRI showed decreased tumor volume after 2 and 4 weeks of treatment. Data (expressed as mean ± SD) illustrate the tumor regression measured by MRI, and statistical analyses were performed using 2-tailed unpaired Student’s t test. (B) Histopathological analysis shows shrinkage of tumors and marked macrophage infiltration in ch806-treated Tet-op-EGFR L858R-IRES-Luciferase/CCSP-rtTA mice (right 2 panels), when compared with untreated Tet-op-EGFR L858R-IRES-Luciferase/CCSP-rtTA control mice (left 2 panels). Arrows show foci of residual tumors.