Targeting a unique EGFR epitope with monoclonal antibody 806 activates NF-kappaB and initiates tumour vascular normalization

Abstract

Monoclonal antibodies (mAbs) and tyrosine kinase inhibitors targeting the epidermal growth factor receptor (EGFR), which is often pathogenetically overexpressed or mutated in epithelial malignancies and glioma, have been modestly successful, with some approved for human use. MAb 806 was raised against de2-7EGFR (or EGFRvIII), a constitutively active mutation expressed in gliomas, but also recognizes a subset (<10%) of wild-type (wt) EGFR when it is activated by autocrine loop, overexpression or mutation. It does not bind inactive EGFR in normal tissues like liver. Glioma xenografts expressing the de2-7EGFR treated with mAb 806 show reduced receptor autophosphorylation, increased p27(KIP1) and reduced cell proliferation. Xenografts expressing the wtEGFR activated by overexpression or autocrine ligand are also inhibited by mAb 806, but the mechanism of inhibition has been difficult to elucidate, especially because mAb 806 does not prevent wtEGFR phosphorylation or downstream signalling in vitro. Thus, we examined the effects of mAb 806 on A431 xenograft angiogenesis. MAb 806 increases vascular endothelial growth factor (VEGF) and interleukin-8 production by activating NF-kappaB and normalizes tumour vasculature. Pharmacological inhibition of NF-kappaB completely abrogated mAb 806 activity, demonstrating that NF-kappaB activation is necessary for its anti-tumour function in xenografts. Given the increase in VEGF, we combined mAb 806 with bevacizumab in vivo, resulting in additive activity.

Figure 1

Effect of mAb 806 treatment on tumour angiogenesis (a) Bar graphs showing the mean change in H-score between mAb 806-treated and control tumours for the biological parameters shown. (b) Representative (250×) images of CD31⁺ blood vessels in the mAb 806-treated or control tumours, stratified by intratumoral position. scale bar: 25 μM. (c) Bar graphs showing the mean change in the MVD, MSA and total vessel area in the mAb 806-treated or control tumours.

Figure 2

Effect of mAb 806 treatment on blood vessel function. (a) Blood vessels with continuous pericyte coverage (arrowhead) or incomplete pericyte coverage (arrow), scale bar: 20 μM. Bar graphs showing the mean change in the density of (b) vessels with any pericyte coverage and (c) vessels with complete pericyte coverage in the mAb 806-treated or control tumours. Bar graphs showing the effects of mAb 806 treatment on (d) the mean oxygenation at day 17, (e) the prevalence of severe hypoxia (less than 2.5 mmHg) and (f) the mean oxygenation in case-control analysis (i.e. matched for tumour volume).

Figure 3

In vivo function of NF-κB and VEGF on mAb 806 anti-tumour activity. Treatment with mAb 806 significantly increases nuclear NF-κB DNA (a) as measured by ELISA or (b) Western blot of the p65 subunit. (c) Survival curves using a combined end-point of moribund condition or tumour volume reaching 1000 mm³ in mice treated with vehicle, mAb 806 (1 mg), BAY 11–7085 (100 μg) or a combination of both on the days indicated. (d) Survival curves using a combined end-point of moribund condition or tumour volume reaching 1500 mm³ in mice treated with vehicle, mAb 806 (1 mg), bevacizumab (400 μg) or a combination of both on the days indicated.