Targeting a unique EGFR epitope with monoclonal antibody 806 activates NF-κB and initiates tumour vascular normalization
Hui K. Gan
Oncogenic Signalling Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorMartha Lappas
Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Australia
Search for more papers by this authorDiana X. Cao
Tumour Targeting Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorAnna Cvrljevdic
Oncogenic Signalling Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorAndrew M. Scott
Tumour Targeting Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorCorresponding Author
Terrance G. Johns
Oncogenic Signalling Laboratory, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
Correspondence to: Terrance JOHNS, Monash Institute of Medical Researcc, C/- Monash Medical Centre, 246 Clayton Rd, Clayton 3168 Victoria, Australia.Tel.: +61 3 9594 7247Fax: +61 3 9594 7114E-mail: [email protected]Search for more papers by this authorHui K. Gan
Oncogenic Signalling Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorMartha Lappas
Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Australia
Search for more papers by this authorDiana X. Cao
Tumour Targeting Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorAnna Cvrljevdic
Oncogenic Signalling Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorAndrew M. Scott
Tumour Targeting Laboratory, Ludwig Institute for Cancer Research, Austin Health, Heidelberg, Australia
Search for more papers by this authorCorresponding Author
Terrance G. Johns
Oncogenic Signalling Laboratory, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
Correspondence to: Terrance JOHNS, Monash Institute of Medical Researcc, C/- Monash Medical Centre, 246 Clayton Rd, Clayton 3168 Victoria, Australia.Tel.: +61 3 9594 7247Fax: +61 3 9594 7114E-mail: [email protected]Search for more papers by this authorAbstract
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.
Supporting Information
Fig. S1 Treatment of established A431 xenografts with mAb 806. Mice (n = 4–5 mice) were treated on the days shown (arrows) with 1 mg of mAb 806 (▿) or vehicle (▪). Mean tumor volume was 41 mm3 on day 3 when treatment started. Data shown in all cases are mean tumor volume ± S.E. The mAb 806 group was significantly smaller than the control group on day 14 (mean volumes 285 mm3 and 714 mm3, respectively, P ∇ 0.00012).
Fig. S2 Representative images (400x) of staining for the biological parameters indicated in the A431 xenografts treated with mAb 806 or vehicle, scale bar: 20 μM.
Fig. S3 BAY 11-7085 inhibits IL-8 production in A431 cells in a dose-dependent manner. A431 cells were treated O/N in serum-free media with the concentration of BAY 11-7085 indicated. Next morning the Bay 11-7085 was removed and cells placed in fresh media for an additional 24 hrs, after which time the media was collected and assayed for IL-8. Data expressed as pg/ml of IL-8.
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 Treatment of established A431 tumors with mAb 528. Mice (n ∇ 5 mice) were treated on the days shown (arrows) with 1 mg of mAb 528 (▿) or vehicle control (▪). Mean tumor volume was 75 mm3 on day 4 when treatment started. Data shown in all cases is mean tumor volume ± S.E. The mAb 528 group was significantly smaller than then control group on day 13 (mean volumes 241 mm3 and 709 mm3, respectively, P ∇ 0.0017). Note that the tumor volumes in this experiment were equivalent to that in Fig. S1.
Table S1 Cultured cells, grown to 95–100% confluency levels, were incubated under serum free conditions at 37°C for 24 hrs, either un-treated (normoxia) or within BD GasPak™ EZ Anaerobe Pouch System bags, which produced a hypoxic environment consisting of less than 1% oxygen (hypoxia). Following treatment, mRNA were isolated from cells and cDNA prepared. The PCR reaction occurred following 40 cycles consisting of the following run conditions: 50°C (2 min.), 95°C (10 min.), 95°C (15 sec.) and 60°C (1 min.), using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Scoresby, VIC, Australia). The 18S house keeping gene and DNAse free ddH2O were used as controls in all cases.
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