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

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 p27KIP1 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-κB and normalizes tumour vasculature. Pharmacological inhibition of NF-κB completely abrogated mAb 806 activity, demonstrating that NF-κB 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.


Introduction
Amplification of the EGFR gene was the first reported genetic alteration described in glioma and is associated with gene rearrangements [1]. The first rearrangement to be described in detail was an extracellular domain deletion known as the de2-7 EGFR (epidermal growth factor receptor) (or EGFRvIII) [2,3]. Numerous subsequent studies have shown this to be the most common mutation in glioma, occurring in about 50% of cases where the EGFR gene is amplified [4]. This cancer-specific EGFR mutant has a specific deletion between exons 2 and 7 of the EGFR. The truncation of exons 2-7 leads to the elimination of 267 amino acids from the extracellular domain and the insertion of a novel glycine at the fusion junction and renders the mutant EGFR unable to bind any known ligand [5,6]. Despite this, the de2-7 EGFR is capable of a low level of constitutive signalling that is augmented by the receptor's impaired internalization and down-regulation [7].
Monoclonal antibody (MAb) 806 is a novel EGFR antibody that was generated by immunizing with cells expressing the de2-7 EGFR, but unexpectedly, it also bound a small proportion of the wild-type (wt) EGFR in tumours and cell lines that overexpress the receptor [8]. Not surprisingly then, mAb 806 does not bind the unique junctional peptide found at the site of the de2-7 EGFR mutation; instead it binds to a short cysteine loop in the extracellular domain of the EGFR that is only transiently exposed as the wt EGFR moves from its inactive to active conformation [9]. The loop is constitutively exposed in the de2-7 EGFR, consistent with our

Results and discussion
Treatment with mAb 806 inhibited A431 xenograft growth at day 14 after inoculation ( Fig. S1), at which time tumours were collected for immunohistochemistry (Fig. S2) To determine if mAb 806 might influence tumour angiogenesis, we examined the effect of mAb 806 on two representative angiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). These two factors were chosen following a qPCR screen of A431 cells designed to identify the soluble pro-angiogenic factors expressed by this cell line (Table S1). Expression of VEGF and IL-8 inA431 cells was determined by qPCR and ELISA ( Fig. S3) and both were up-regulated by hypoxia (Table S1). Overall, VEGF expression was higher in the mAb 806 group than the control group (P Ͻ 0.03, Fig.1a). However, VEGF expression was also influenced by intratumoral location (periphery versus interior; ANOVA P Ͻ 0.002). In the control group, the interior expressed significantly less VEGF than the periphery (P Ͻ 0.01, Fig. 1a). MAb 806 treatment did not increase VEGF expression in the periphery of the tumour relative to control (P ϭ 0.7, Fig.1a) but substantially increased VEGF expression in the interior of the tumours (P Ͻ 0.0001, Fig.1a). MAb 806 treatment resulted in a large and significant increase in IL-8 expression in all parts of the tumour (P Ͻ 0.0001, Fig.1a).
Given that mAb 806 increased the expression of two proangiogenic factors VEGF and IL-8, we analysed the effect of mAb 806 of blood vessel density and size. Mean vessel density (MVD) was significantly influenced by both mAb 806 treatment and intratumoral location ( Fig. 1b and c, ANOVA, P Ͻ 0.0001). Analysis of the entire tumour showed that control xenografts had a MVD of 6.7 vessels/field and a mean surface area (MSA) of 270 m 2 (Fig. 1c). The interior of these control tumours were significantly less vascularized than the periphery (5.3 versus 7.7 vessels/field, P Ͻ 0.01) and the vessels in the interior were also significantly larger (MSA 400 versus 185 m 2 , P Ͻ 0.05). Across the whole tumour MVD was significantly higher in mAb 806 treated xenografts compared to the control xenografts (Fig. 1c, 10 versus 6.7 vessels/field, respectively, P Ͻ 0.0001). Although the MVD of the periphery of the mAb 806-treated xenografts was higher than the control group (10.2 versus 7.7 vessels/field, P Ͻ 0.0005), the increase was even more marked in the interior of mAb 806-treated tumours (9.9 versus 5.3 vessels/field in, P Ͻ 0.0001). Similar results were obtained when MVD was assessed on morphological criteria using haematoxylin and eosin stained sections (images not shown, 14.2 in mAb 806 treated xenografts versus 10.6 vessels/field in control treated xenografts, P Ͻ 0.0001).
Both treatment with mAb 806 and intratumour location affected vessel size ( Fig. 1b and c, ANOVA P Ͻ 0.001). There was an overall decrease in MSA in the mAb 806-treated xenografts compared to the control xenografts (Fig. 1c, 155 versus 270 m 2 , respectively, P Ͻ 0.001). Although vessels in the periphery of the mAb 806-treated tumours were smaller than those in the control tumours (142 versus 185 m 2 , respectively, P Ͻ 0.05), the reduction in MSA was more marked in the interior of the tumour (170 versus 400 m 2 , P Ͻ 0.001). The combined effect of these opposing changes in MVD and MSA results in no significant change in total vessel area (Fig. 1c, 4.18% versus 3.42%, P ϭ 0.16). Lyve-1 staining for lymphatic vessels was very sparse, confirming that CD31 ϩ structures were predominantly blood vessels (Fig. S4). These changes seen following treatment with mAb 806 are consistent with the model of 'vascular normalization' which has been described with anti-angiogenic agents including bevacizumab [15,16] (Fig. 2a, arrowhead) and incomplete pericyte coverage (Fig. 2a, arrow). MAb 806 significantly increased the total number of pericyte-covered vessels compared to control (Fig. 2b, 6.6 versus 4.5 vessels/field, respectively; P ϭ 0.03) and also significantly increased the number of vessels with complete pericyte coverage compared to control (Fig. 2c, 5.2 versus 3.1 covered vessels/field, P Ͻ 0.005).
Secondly, intratumoral oxygen was measured using the Oxford Oxylite Probe (Oxford, UK). A431 xenograft oxygenation was measured on day 17 following treatment with vehicle or mAb 806 (mean volume 1374 and 930 mm 3 , respectively). Mean oxygenation was significantly improved in mAb 806-treated xenografts compared to control (Fig. 2d, 5.8 Ϯ1.3 versus 9.6 Ϯ1.0 mmHg; P ϭ 0.038). However, this understates the improvement in oxygenation as vehicle treated tumours had substantially more areas of severe hypoxia compared to mAb 806 treated xenografts (Fig. 2e, 47% and 0.3%, respectively, P Ͻ 0.0001). As differences in tumour volume may confound the measurement of oxygenation, we undertook a case-control analysis where tumours from the above control group were matched with tumours of equivalent volume from the mAb 806 treated group. This latter group comprised all the mice killed for oxygen measurement on day 17 as well as a cohort of mice that had were deliberately treated for an additional week for the express purposes of performing this analysis. The volumes of the matched vehicle and mAb 806 treatment groups were statistically equivalent: (1110 and 1009 mm 3 , respectively). This analysis showed that whilst the control group was hypoxic, the mAb 806-treated group was normoxic (Fig. 2f, 6.2 versus 10.2 mmHg, P ϭ 0.031), demonstrating that this effect was independent of tumour volume. Taken together our data clearly show that mAb 806 mediates normalization of the tumour vasculature.
Other anti-EGFR therapeutics such as cetuximab [20][21][22] and gefitinib [23,24] do not cause vascular normalization. Indeed, they mediate decreases in MVD and reduced expression of angiogenic factors such as VEGF and IL-8. To fully validate our mAb 806 analysis we also determined the effect of mAb 528, a prototypical ligand inhibitory EGFR antibody, on several of the key angiogenesic indicators described above. Treatment of A431 xenografts with mAb 528 (Fig. S5) resulted in a reduction in proliferation, a reduction in MVD, no change in MSA, a reduction in total vessel area and no change in IL-8 expression (Table 1), results which differ from those obtained following treatment with mAb 806 but consistent with the literature [25]. Thus our method of analysis provided the expected outcome for a previously described EGFR therapeutic antibody. The ability of mAb 806 to influence both VEGF and IL-8 expression led us to examine its effect on the NF-B pathway, as its activation can induce both of these angiogenic factors [26]. Vehicle or mAb 806-treated A431 xenografts were harvested on day 14 (treatment schedule per Fig. S1) and NF-B p65 DNA binding activity assayed by ELISA for NF-B activity. There was significantly increased NF-B p65 DNA binding activity in the nucleus of A431 xenografts treated with mAb 806 (Fig. 3a, P Ͻ 0.002). There was also significantly more p65 subunit of NF-B in the nucleus of the mAb 806 treated group when analysed by Western blotting (Fig. 3b, 109% increase, P Ͻ 0.001). This suggests that mAb 806 activates the NF-B pathway, thereby increasing the expression of VEGF and IL-8. The role of VEGF in angiogenesis is well established and the significance of IL-8 to this process has been recently underscored [27]. Although the changes in these two factors are almost certainly not the only ones associated with vascular normalization, they probably typify the changes in the balance of angiogenic factors related to the normalization process. Indeed, the expression of at least 10 soluble angiogenic factors by A431 cells highlights the complexity of this process (Table S1). Interestingly, vascular normalization was still seen after 14 days of treatment, suggesting that mAb 806 causes sustained normalization compared to the transient phenomenon previously reported [15][16][17][18].
We then examined the broader role of NF-B activation with respect to the anti-tumour effect of mAb 806 by co-administering mAb 806 with BAY 11-7085, a specific inhibitor of the NF-B pathway that prevents the phosphorylation and degradation of IB␣ [28]. Before using it in our xenograft model we demonstrated   (Fig. S3), confirming previous reports of this compounds capacity to inhibit IL-8 and VEGF [29,30]. A431 xenografts were treated with mAb 806, BAY 11-7085 or combination therapy (Fig. 3c). Survival analysis showed significant differences across the four groups (Fig. 3c, P Ͻ 0.002) [31][32][33], the inhibitor of Cyclin Kinase 2.

Importantly, this induction of p21 inA431 cells is dependent on activation NF-B [31], firmly establishing a anti-proliferative role for NF-B in these cells. Furthermore, a more general role for NF-B as a tumour suppressor in some instances is now well established [34]. Interestingly, a microarray analysis of the mRNA modulated by a small molecule weight inhibitor specific to the EGFR (PD153035) showed that it reduces the invasiveness of cervical carcinoma cells and this was associated with the activation of NF-B [35]. The in vivo consequences were not examined in this study. Finally, while seemingly unlikely, it would be interesting to determine if mAb 806 retained its vascular normalization activity in the presence of BAY11-708.
Given the increase in VEGF seen with mAb 806 therapy, we treated A431 xenografts with vehicle, mAb 806, bevacizumab (an anti-VEGF antibody) or combination therapy (Fig. 3d). Survival analysis showed a significant difference across the 4 groups ( Fig. 3d, P Ͻ 0.0001 log rank). The median survivals were 21 days (control), 24 days (mAb 806), 34 days (bevacizumab) and not reached (combination). The control group did significantly worse than all other groups. The combination group did significantly better than either of the single agent's arms (P Ͻ 0.05 for both comparisons). Hence, there is additional therapeutic benefit in combining mAb 806 with a conventional anti-angiogenic agent that potently reduces the level of VEGF and MVD [36].

Antibodies
The mAb 806 (IgG2b) which recognizes a sub-set of the wt EGFR expressed on the cell surface has been described previously [8,9,40]  After staining with the primary antibodies described above, bound antibodies were detected with the appropriate secondary antibody. Where the primary antibody was of rat origin, slides were rinsed before application of biotinylated anti-rat secondary (ab-6851, Abcam) followed by application of Streptavidin-HRP (Dakocytomation, Carpinteria, CA, USA). Where the primary antibody was of rabbit origin, sections were rinsed before application of HRP-labelled anti-rabbit antibodies (Envisionϩ System, Dakocytomation). Bound antibodies were detected with AEC substrate solution (0.1 mol/l acetic acid, 0.1 mol/l sodium acetate, 0.02 mol/l AEC, and 0.03% H2O2). Slides were then counterstained in haematoxylin (BDH Laboratory, Poole, UK) and mounted. Appropriate species and isotype control staining was performed for all slides.

Analysis of vessel density and immunohistochemical staining
For each section, up to 17 images were captured by light microscopy at the 250ϫ magnification. Images were obtained in a systematic manner to ensure homogenous and representative sampling of non-overlapping areas of viable tumour. Fifty percent of images were captured from the tumour peripheral and the rest from the interior of the tumour. Quantification of CD31 ϩ and Lyve-1 ϩ vessel density involved counting the number of CD31 ϩ vessels in each (250ϫ) image. Vessels density was also counted in haematoxylin and eosin sections. Again, up to 17 images were captured by light microscopy as described above but at 400ϫ magnification. Rounded or tubular spaces, especially if lined by endothelial cells, were considered to be blood vessels. The cross-sectional area of CD31 ϩ vessels (defined as all CD31 ϩ regions and any hollow structure contained therein) was also determined by morphometric analysis using Leica QWin (North Ryde, Australia) image analysis software and expressed as a percentage of the total viable tumour area in each image [42].
For analysis of immunohistochemical staining, up to 17 images of viable tumour were captured by light microscopy at 400ϫ magnification. For each image, an H-score for prevalence and intensity of staining was generated using Leica Qin images analysis software [43]. In brief, a composite score was obtained by multiplying each level of intensity staining (0-3) by the percentage area that it comprised (0-100%) and then adding up the resultant products. The minimum score was 0 (0 staining in 100% of the image) and the maximum scare was 300 (3ϩ staining in 100% of the image). Artefactual, acellular and non-viable areas were excluded from the scoring procedure.

Measurement of intratumoral oxygenation in anaesthetized mice
Intratumoral oxygen was determined in anaesthetized mice using the Oxford Oxylite Probe [44]. In brief, mice were anaesthetized then kept warm on a temperature-regulated pad. Up to three tracks were created through the equatorial plan of the tumour tissue using a 21-gauge needle. The probe was then drawn through the track(s), taking multiple oxygen measurements (in mmHg) as it traversed the track(s). The initial 5% of readings just after entry in to the tumour and the final 5% of readings just prior to exit from the tumour were discarded because of the risk of contamination by external air. Mice were killed at the end of the experiment.

Supporting Information
Additional Supporting Information may be found in the online version of this article:   Fig. S4 A431 xenografts from the experiment described in Fig. S1 where stained with the lymph vessel marker lyve-1. Representative images are shown. Fig. S5