De2-7 Epidermal Growth Factor Receptor Research Articles

MAb 806 Enhances the Efficacy of Ionizing Radiation in Glioma Xenografts Expressing the de2-7 Epidermal Growth Factor Receptor

Mutations of the epidermal growth factor receptor (EGFR) are common in glioma. The most frequent mutation, de2-7 EGFR/EGFRvIII, occurs in approximately 40% of high-grade gliomas and confers resistance to ionizing radiation (IR). We have previously shown that mAb 806, a novel EGFR-specific antibody, is able to inhibit the growth of U87MG.Δ2-7 glioma xenografts expressing the de2-7 EGFR and may have potential as a therapeutic. Nude mice bearing U87MG.Δ2-7 xenografts were treated with mAb 806 and/or IR. Comparison of tumor volumes, the effect of treatment on angiogenesis as determined by mean vessel density, and expression changes in prosurvival protein pAkt between treatment groups were undertaken. Treatment of mice bearing U87MG.Δ2-7 xenografts with mAb 806 and IR resulted in schedule-dependent radiosensitization. Maximal benefit was obtained when antibody treatment was given before irradiation, with the greatest inhibition of both tumor angiogenesis and tumor growth. Combination treatment mediated radiosensitization by selectively blocking the phosphorylation of the prosurvival protein Akt at serine 473, a process that is independent of DNA-dependent protein kinase catalytic subunit. Our results provide a rationale for the use of mAb 806 in combination with IR for the treatment of glioma and potentially other solid tumors bearing the de2-7 EGFR.

Immuno-PET Quantitation of de2-7 Epidermal Growth Factor Receptor Expression in Glioma Using 124I-IMP-R4–Labeled Antibody ch806

Overexpression, activation, and mutations of the epidermal growth factor receptor (EGFR) are commonly found in solid tumors. The aim of this study was to develop a PET-based method for detecting the constitutively active mutant de2-7 EGFR, which is associated with disease progression and resistance to chemotherapy and radiotherapy in glioma. The chimeric antibody ch806, which selectively binds an epitope of the EGFR that is exposed only on overexpressed, mutant, or ligand-activated forms of the receptor, was conjugated to the radiohalogen (124)I via the residualizing ligand IMP-R4, and in vitro properties were characterized. In vivo biodistribution and small-animal PET studies were performed in BALB/c nude mice bearing U87MG.de2-7 glioma xenografts. Imaging results were correlated with measured tumor uptake of the radioconjugate. (124)I-IMP-R4-ch806 had an immunoreactivity of 78.3% and was stable for 7 d when incubated in serum in vitro. The biodistribution analysis of (124)I-IMP-R4-ch806 demonstrated a maximal uptake of 30.95 +/- 6.01 percentage injected dose per gram (%ID/g) in U87MG.de2-7 xenografts at 48 h after injection, with prolonged tumor retention (6.07 +/- 0.80 %ID/g at 216 h after injection). The tumor-to-blood ratio increased from 0.44 at 4 h after injection to a maximum of 4.70 at 168 h after injection. PET of (124)I-IMP-R4-ch806 biodistribution was able to clearly detect the U87MG.de2-7 tumors at 24 h after injection and for at least 168 h after injection. Correlation between tumor PET image quantitation of (124)I-IMP-R4-ch806 and %ID/g determined from resected tissues (r = 0.9350) was excellent. These results show that immuno-PET with (124)I-IMP-R4-ch806 is feasible and allows noninvasive quantitation of de2-7 EGFR expression in vivo.

Abstract B234: Dual targeting of the tyrosine kinase receptors EGFRvIII and c-Met in glioblastoma multiforme

Abstract The most common mutation of the epidermal growth factor receptor (EGFR) in glioblastoma multiforme (GBM) is an extracellular truncation of 267 amino acids known as EGFRvIII (or de2-7 EGFR). While the EGFRvIII is unable to bind any known ligand, it displays a low level of constitutive activation that is enhanced by its inability to undergo downregulation. HGF/SF is the ligand for the receptor tyrosine kinase c-Met, both of which are often co-expressed in GBM. The presence of the HGF/c-Met axis or expression of EGFRvIII each independently enhances GBM growth, angiogenesis capacity, migration and invasiveness. Using tyrosine kinase array technology, we now show that expression of EGFRvIII in U87MG glioblastoma cells (U87MG. 2-7 cells) leads to co-activation of several receptor tyrosine kinases, including PDGFR and c-Met. A fully human neutralizing antibody directed to HGF (AMG 102) did not inhibit this EGFRvIII mediated activation of c-Met in vitro, demonstrating that it is ligand independent. We examined if this transactivation of c-Met influenced the efficacy of therapies targeting the HGF:c-Met axis. Treatment of parental U87MG xenografts with low doses of AMG 102 resulted in significant inhibition of tumor growth (see Burgess et al. 2006), while U87MG. 2-7 xenografts (see Johns et al 2007) were profoundly resistant to AMG 102 treatment. Treatment with Panitumumab, a fully human antibody directed to the EGFR and EGFRvIII, was able to inhibit the EGFRvIII mediated activation of c-Met and significantly reversed the resistance to AMG 102 observed in U87MG. 2-7 xenografts. Indeed, combination therapy with Panitumumab and AMG 102 resulted in a marked increase in tumor cell apoptosis, especially in the central parts of the xenograft. An EGFRvIII molecule with an active kinase but incapable of autophosphorylation at the five major autophosphorylation sites (tyrosine changed to phenylalanine) involved in signal transduction was also able to mediate resistance to AMG 102, suggesting that the activation of c-Met by EGFRvIII occurs via a direct interaction rather than through these docking sites. These studies show that expression of EGFRvIII leads to the promiscuous activation of several kinases, causing resistance to ligand inhibitory based strategies. Since Panitumumab can reverse this process, the combination of this antibody with AMG 102 may be an effective treatment for GBM patients positive for EGFRvIII. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B234.

The Plasticity of Oncogene Addiction: Implications for Targeted Therapies Directed to Receptor Tyrosine Kinases

A common mutation of the epidermal growth factor receptor (EGFR) in glioblastoma multiforme (GBM) is an extracellular truncation known as the de2-7 EGFR (or EGFRvIII). Hepatocyte growth factor (HGF) is the ligand for the receptor tyrosine kinase (RTK) c-Met, and this signaling axis is often active in GBM. The expression of the HGF/c-Met axis or de2-7 EGFR independently enhances GBMgrowth and invasiveness, particularly through the phosphatidylinositol-3 kinase/pAkt pathway. Using RTK arrays, we show that expression of de2-7 EGFR in U87MG GBM cells leads to the coactivation of several RTKs, including platelet-derived growth factor receptor β and c-Met. A neutralizing antibody to HGF (AMG102) did not inhibit de2-7 EGFR-mediated activation of c-Met, demonstrating that it is ligand-independent. Therapy for parental U87MG xenografts with AMG 102 resulted in significant inhibition of tumor growth, whereas U87MG.Δ2-7 xenografts were profoundly resistant. Treatment of U87MG.Δ2-7 xenografts with panitumumab, an anti-EGFR antibody, only partially inhibited tumor growth as xenografts rapidly reverted to the HGF/c-Met signaling pathway. Cotreatment with panitumumab and AMG 102 prevented this escape leading to significant tumor inhibition through an apoptotic mechanism, consistent with the induction of oncogenic shock. This observation provides a rationale for using panitumumab and AMG 102 in combination for the treatment of GBM patients. These results illustrate that GBM cells can rapidly change the RTK driving their oncogene addiction if the alternate RTK signals through the same downstream pathway. Consequently, inhibition of a dominant oncogene by targeted therapy can alter the hierarchy of RTKs resulting in rapid therapeutic resistance.

The Efficacy of Epidermal Growth Factor Receptor–Specific Antibodies against Glioma Xenografts Is Influenced by Receptor Levels, Activation Status, and Heterodimerization

Factors affecting the efficacy of therapeutic monoclonal antibodies (mAb) directed to the epidermal growth factor receptor (EGFR) remain relatively unknown, especially in glioma. We examined the efficacy of two EGFR-specific mAbs (mAbs 806 and 528) against U87MG-derived glioma xenografts expressing EGFR variants. Using this approach allowed us to change the form of the EGFR while keeping the genetic background constant. These variants included the de2-7 EGFR (or EGFRvIII), a constitutively active mutation of the EGFR expressed in glioma. The efficacy of the mAbs correlated with EGFR number; however, the most important factor was receptor activation. Whereas U87MG xenografts expressing the de2-7 EGFR responded to therapy, those exhibiting a dead kinase de2-7 EGFR were refractory. A modified de2-7 EGFR that was kinase active but autophosphorylation deficient also responded, suggesting that these mAbs function in de2-7 EGFR-expressing xenografts by blocking transphosphorylation. Because de2-7 EGFR-expressing U87MG xenografts coexpress the wild-type EGFR, efficacy of the mAbs was also tested against NR6 xenografts that expressed the de2-7 EGFR in isolation. Whereas mAb 806 displayed antitumor activity against NR6 xenografts, mAb 528 therapy was ineffective, suggesting that mAb 528 mediates its antitumor activity by disrupting interactions between the de2-7 and wild-type EGFR. Finally, genetic disruption of Src in U87MG xenografts expressing the de2-7 EGFR dramatically enhanced mAb 806 efficacy. The effective use of EGFR-specific antibodies in glioma will depend on identifying tumors with activated EGFR. The combination of EGFR and Src inhibitors may be an effective strategy for the treatment of glioma.

Epidermal Growth Factor Receptor and Epidermal Growth Factor Receptor Variant III Gene Expression in Metastatic Colorectal Cancer

The epidermal growth factor receptor (EGFR) variant type III (variously called EGFRvIII, de2-7 EGFR, or triangle upEGFR) has an in-frame deletion of the extracellular domain and is found in numerous types of human tumors. Because EGFRvIII has been reported to be tumor specific and has oncogenic potential, it is being investigated as a potential therapeutic target, but to our knowledge, there is only 1 previous report about EGFRvIII by immunohistochemistry in colorectal cancer. Our aim was to indicate the frequency of gene expressions of EGFRvIII and EGFR in metastatic colorectal cancer (mCRC). Forty-five patients with mCRC who received the chemotherapy for metastatic disease were analyzed for the EGFRvIII variant. Paraffin-embedded tumor tissues were dissected using laser-captured microdissection and analyzed for the EGFR and EGFRvIII messenger RNA expression using a quantitative real-time reverse-transcriptase polymerase chain reaction method. Gene expression values (relative messenger RNA levels) are expressed as ratios (differences from the cycle threshold values) between the target gene and internal reference gene (beta-actin). Twenty-five women and 20 men with a median age of 55 years (range, 25-76 years) were included in this study. We did not find any expression of EGFRvIII in these 45 patients except for control cell lines as U87.EGFRvIII. However, EGFR gene expression was found in 43 of 45 (95.6%) with a range of 0.38-2.83. Our results demonstrate that the expression of EGFRvIII is rare, but most colon cancer demonstrates EGFR gene expression. We conclude that EGFRvIII does not play an important role in mCRC.

Treatment of Human Tumor Xenografts with Monoclonal Antibody 806 in Combination with a Prototypical Epidermal Growth Factor Receptor–Specific Antibody Generates Enhanced Antitumor Activity

Monoclonal antibody (mAb) 806 is a novel epidermal growth factor receptor (EGFR) antibody with significant antitumor activity that recognizes a mutant EGFR commonly expressed in glioma known as delta2-7 EGFR (de2-7 EGFR or EGFRvIII) and a subset of the wild-type (wt) EGFR found in cells that overexpress the receptor. We have used two human xenograft mouse models to examine the efficacy of mAb 806 in combination with mAb 528, a prototypical anti-EGFR antibody with similar specificity to cetuximab. Treatment of nude mice, bearing s.c. or i.c. tumor human xenografts expressing the wt or de2-7 EGFR, with mAbs 806 and 528 in combination resulted in additive and in some cases synergistic, antitumor activity. Interestingly, mAb 528 was also effective against xenografts expressing the ligand independent de2-7 EGFR when used as a single agent, showing that its antitumor activity is not merely mediated through inhibition of ligand binding. When used as single agents, neither mAbs 806 or 528 induced down-regulation of the de2-7 EGFR either in vitro or in vivo. In contrast, the combination of antibodies produced a rapid and dramatic decrease in the total cell surface de2-7 EGFR both in vitro and in xenografts. Consistent with this decrease in total cell surface de2-7 EGFR, we observed up-regulation of the cell cycle inhibitor p27(KIP1) and a decrease in tumor cell proliferation as measured by Ki-67 immunostaining when the antibodies were used in combination in vivo. Thus, mAb 806 can synergize with other EGFR-specific antibodies thereby providing a rationale for its translation into the clinic.

Engineering and characterisation of chimeric monoclonal antibody 806 (ch806) for targeted immunotherapy of tumours expressing de2-7 EGFR or amplified EGFR

We report the generation of a chimeric monoclonal antibody (ch806) with specificity for an epitope on the epidermal growth factor receptor (EGFR) that is different from that targeted by all other anti-EGFR therapies. Ch806 antibody is reactive to both de2-7 and overexpressed wild-type (wt) EGFR but not native EGFR expressed in normal tissues at physiological levels. Ch806 was stably expressed in CHO (DHFR −/−) cells and purified for subsequent characterisation and validated for use in preliminary immunotherapy investigations. Ch806 retained the antigen binding specificity and affinity of the murine parental antibody. Furthermore, ch806 displayed enhanced antibody-dependent cellular cytotoxicity against target cells expressing the 806 antigen in the presence of human effector cells. Ch806 was successfully radiolabelled with both iodine-125 and indium-111 without loss of antigen binding affinity or specificity. The radioimmunoconjugates were stable in the presence of human serum at 37°C for up to 9 days and displayed a terminal half-life (T1/2β) of approximately 78 h in nude mice. Biodistribution studies undertaken in BALB/c nude mice bearing de2-7 EGFR-expressing or amplified EGFR-expressing xenografts revealed that 125I-labelled ch806 failed to display any significant tumour retention. However, specific and prolonged tumour localisation of' 111In-labelled ch806 was demonstrated with uptake of 31%ID g−1 and a tumour to blood ratio of 5 : 1 observed at 7 days postinjection. In vivo therapy studies with ch806 demonstrated significant antitumour effects on established de2-7 EGFR xenografts in BALB/c nude mice compared to control, and both murine 806 and the anti-EGFR 528 antibodies. These results support a potential therapeutic role of ch806 in the treatment of suitable EGFR-expressing tumours, and warrants further investigation of the potential of ch806 as a therapeutic agent.

Targeting the Transition State

Because epidermal growth factor receptor (EGFR) hyperactivity is associated with many epithelial cancers, it is a popular target for the development of cancer therapies. Some tumors contain a variant form of EGFR (de2-7 EGFR) that has a deletion mutation in the extracellular domain, resulting in low constitutive activity. Johns et al. investigated the mechanism by which a monoclonal antibody mAb 806 binds the de2-7 EGFR effectively, but binds wild-type EGFR, when at normal concentrations on the cell surface, minimally. This antibody has been therapeutically effective against de2-7 EGFR-expressing tumors and tumors with amplified wild-type EGFR that have increased abundance of cell surface EGFR. The epitope for mAb 806 was identified to be residues 287 to 302 of the extracellular domain by immunoblotting of EGFR fragments and yeast surface display. The binding of mAb 806 to a peptide consisting of residues 287 to 302 was analyzed by surface plasmon resonance and the measured affinity (30 nM) correlated with the affinity for mAb 806 to de2-7 EGFR on cells. Analysis of the published crystal structures of the EGFR suggests that the receptor exists in three conformations: (i) a tethered form that is inactive and represents 95% of the unliganded receptor; (ii) an untethered form that is in equilibrium with the tethered state; and (iii) the dimerized, active form, which is abundant in the presence of ligand. Because mAb 806 binds to only a small fraction of wild-type EGFR on cells, the authors suggest that it is unlikely that this antibody binds the tethered form of the receptor. The dimerized receptor also does not have additional portions of the mAb 806 epitope exposed compared with the tethered form. However, in the untethered conformation, more of the mAb 806 epitope is exposed. Analysis of the binding of mAb 806 to cells expressing EGFR mutants that cannot form the tethered or dimerized conformations, which would be similar to de2-7 EGFR, indicated that mAb 806 does recognize the untethered conformation of the receptor. The authors propose that selectivity for the de2-7 EGFR tumors comes from the presence of this mutant receptor in the untethered form. The ability of mAb 806 to inhibit EGFR in cells with amplified EGFR compared to cells with normal concentrations of EGFR may arise because an increased proportion of the amplified EGFR is in the untethered conformation, thus providing selectivity for the effects of mAb 806. T. G. Johns, T. E. Adams, J. R. Cochran, N. E. Hall, P. A. Hoyne, M. J. Olsen, Y.-S. Kim, J. Rothacker, E. C. Nice, F. Walker, G. Ritter, A. A. Jungbluth, L. J. Old, C. W. Ward, A. W. Burgess, K. Dane Wittrup, A. M. Scott, Identification of the epitope for the epidermal growth factor receptor-specific monoclonal antibody 806 reveals that it preferentially recognizes an untethered form of the receptor. J. Biol. Chem. 279 , 30375-30384 (2004). [Abstract] [Full Text]

Identification of the Epitope for the Epidermal Growth Factor Receptor-specific Monoclonal Antibody 806 Reveals That It Preferentially Recognizes an Untethered Form of the Receptor

The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers, an observation often correlated with poor clinical outcome. Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2-7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain. Monoclonal antibody (mAb) 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2-7 EGFR and a subset of the wild type (wt) EGFR when overexpressed but does not bind the wt EGFR expressed in normal tissues. Despite only binding to a low proportion of the wt EGFR expressed in A431 tumor cells (approximately 10%), mAb 806 displays robust antitumor activity against A431 xenografts grown in nude mice. To elucidate the mechanism leading to its unique specificity and mode of antitumor activity, we have determined the EGFR binding epitope of mAb 806. Analysis of mAb 806 binding to EGFR fragments expressed either on the surface of yeast or in an immunoblot format identified a disulfide-bonded loop (amino acids 287-302) that contains the mAb 806 epitope. Indeed, mAb 806 binds with apparent high affinity (approximately 30 nm) to a synthetic EGFR peptide corresponding to these amino acids. Analysis of EGFR structures indicates that the epitope is fully exposed only in the transitional form of the receptor that occurs because EGFR changes from the inactive tethered conformation to a ligand-bound active form. It would seem that mAb 806 binds this small proportion of transient receptors, preventing their activation, which in turn generates a strong antitumor effect. Finally, our observations suggest that the generation of antibodies to transitional forms of growth factor receptors may represent a novel way of reducing normal tissue targeting yet retaining antitumor activity.

The tumor-specific de2-7 epidermal growth factor receptor (EGFR) promotes cells survival and heterodimerizes with the wild-type EGFR.

Mutations of the epidermal growth factor receptor (EGFR) gene are found at a relatively high frequency in glioma, with the most common being the de2-7 EGFR (or EGFRvIII). This mutation arises from an in-frame deletion of exons 2-7, which removes 267 amino acids from the extracellular domain of the receptor. Despite being unable to bind ligand, the de2-7 EGFR is constitutively active and imparts a significant in vivo growth advantage to glioma cells. In order to examine the signalling pathways activated by the de2-7 EGFR and its biological effects in an in vitro system, the de2-7 EGFR gene was transfected into the murine IL-3-dependent pro-B-cell line BaF/3. Expression of the de2-7 EGFR enhanced the survival of BaF/3 cells in the absence of IL-3 by reducing apoptosis in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. Interestingly, while de2-7 EGFR also enhanced proliferation of BaF/3 cells in low levels of IL-3, this effect was independent of PI3-K. Survival and proliferation were further enhanced when BaF/3 cells were cotransfected with the de2-7 and wt EGFR. This was due to heterodimerization between the de2-7 and wt EGFR leading to trans-phosphorylation of the wt EGFR. This observation is directly relevant to glioma where de2-7 and wt EGFR appear to be coexpressed. Thus, expression of de2-7 EGFR in BaF/3 cells provides an in vitro model for evaluating the signalling pathways activated by this receptor.

Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478.

Blockade of epidermal growth factor receptor (EGFR) signaling with specific inhibitors of the EGFR tyrosine kinase retards cellular proliferation and arrests the growth of tumor xenografts. AG1478, an inhibitor of the EGFR tyrosine kinase, is used in laboratory studies; however, its therapeutic potential has not been elucidated. Therefore, we evaluated an aqueous form of AG1478 for its antitumor activity in mice bearing human xenografts expressing the WT EGFR or a naturally occurring ligand-independent truncation of the EGFR [delta2-7 (de2-7) EGFR or EGFRvIII]. Parenteral administration of soluble AG1478 blocked phosphorylation of the EGFR at the tumor site and inhibited the growth of A431 xenografts that overexpress the WT EGFR and glioma xenografts expressing the de2-7 EGFR. Strikingly, even subtherapeutic doses of AG1478 significantly enhanced the efficacy of cytotoxic drugs, with the combination of AG1478 and temozolomide displaying synergistic antitumor activity against human glioma xenografts. AG1478 was also examined in combination with mAb 806, an anti-EGFR antibody that was raised against the de2-7 EGFR but unexpectedly also binds a subset of the EGFR expressed in cells exhibiting amplification of the EGFR gene. The combination of AG1478 and mAb 806 displayed additive, and in some cases synergistic, antitumor activity against tumor xenografts overexpressing the EGFR. Here, we demonstrate that different classes of inhibitors to the EGFR can have synergistic antitumor activity in vivo. These results establish the antitumor efficacy of the EGFR inhibitor AG1478 and provide a rationale for its clinical evaluation in combination with both chemotherapy and other EGFR therapeutics.

Expression of constitutively activated EGFRvIII in non-small cell lung cancer.

The epidermal growth factor receptor (EGFR) variant type III (variously called EGFRvIII, de2-7 EGFR or deltaEGFR) has an in-frame deletion of the extracellular domain and is found in numerous types of human tumors. Since EGFRvIII has been reported to be tumor-specific and has oncogenic potential, it is being investigated as a potential therapeutic target. Because the cell-specific expression of EGFRvIII in lung has not been well documented, we examined the expression of EGFRvIII in 76 non-small cell lung cancers (NSCLCs) and 10 non-neoplastic lung tissues by immunohistochemistry using a new monoclonal antibody specific for this variant receptor. We found a higher incidence (30 of 76, 39%) of enhanced EGFRvIII expression in NSCLC than previously described. Interestingly, the presence of EGFRvIII was also observed in several normal tissue components of lung (e.g., normal bronchial epithelium). Given the high prevalence of EGFRvIII in NSCLC, a newly developed phospho-specific (activated) EGFR antibody was employed for immunohistochemical analysis that permitted visualization of activated EGFR and/or EGFRvIII in tumors. This study presents evidence, for the first time, that EGFRvIII expressed in human tumors is phosphorylated and hence activated. Our results suggest that the sustained activation of EGFRvIII is implicated in the pathogenesis of NSCLC and thus EGFRvIII is a potential therapeutic target in this challenging disease.

Novel monoclonal antibody specific for the de2-7 epidermal growth factor receptor (EGFR) that also recognizes the EGFR expressed in cells containing amplification of the EGFR gene.

In some respects, the EGFR appears to be an attractive target for tumor-targeted antibody therapy: it is overexpressed in many types of epithelial tumor and inhibition of signaling often induces an anti-tumor effect. The use of EGFR specific antibodies, however, may be limited by uptake in organs that have high endogenous levels of the wild type EGFR such as the liver. The de2-7 EGFR (or EGFRvIII) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. Antibodies directed to this tumor specific variant of the EGFR provide an alternative targeting strategy, although the lower proportion of tumors that express the de2-7 EGFR restricts this approach. We describe a novel monoclonal antibody (MAb 806) that potentially overcomes the difficulties associated with targeting the EGFR expressed on the surface of tumor cells. MAb 806 bound to de2-7 EGFR transfected U87MG glioma cells (U87MG.Delta 2-7) with high affinity (approximately 1 x 10(9) M(-1)), but did not bind parental cells that express the wild type EGFR. Consistent with this observation, MAb 806 was unable to bind a soluble version of the wild type EGFR containing the extracellular domain. In contrast, immobilization of this extracellular domain to ELISA plates induced saturating and dose response binding of MAb 806, suggesting that MAb 806 can bind the wild type EGFR under certain conditions. MAb 806 also bound to the surface of A431 cells, which due to an amplification of the EGFR gene express large amounts of the EGFR. Interestingly, MAb 806 only recognized 10% of the total EGFR molecules expressed by A431 cells and the binding affinity was lower than that determined for the de2-7 EGFR. MAb 806 specifically targeted U87MG.Delta 2-7 and A431 xenografts grown in nude mice with peak levels in U87MG.Delta 2-7 xenografts detected 8 h after injection. No specific targeting of parental U87MG xenografts was observed. Following binding to U87MG.Delta 2-7 cells, MAb 806 was rapidly internalized by macropinocytosis and subsequently transported to lysosomes, a process that probably contributes to the early targeting peak observed in the xenografts. Thus, MAb 806 can be used to target tumor cells containing amplification of the EGFR gene or de2-7 EGFR but does not bind to the wild type EGFR when expressed on the cell surface.

Aberrant receptor signaling in human malignant gliomas: mechanisms and therapeutic implications

Alterations of the epidermal growth factor receptor (EGFR) occur frequently in malignant gliomas through gene amplification or rearrangement, especially in a large fraction of de novo type glioblastomas. The most common of these mutant EGFRs (variously named de2-7 EGFR, ΔEGFR or EGFRvIII) lacks a portion of the extracellular ligand-binding domain. Here, we review the evidence that shows that expression of ΔEGFR bestows in vivo growth advantages to human glioma cells through its constitutively active tyrosine kinase activity. Thus, ΔEGFR may provide a novel therapeutic target for the most aggressive type of glioblastoma.

The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers Is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named DeltaEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active DeltaEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of DeltaEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.