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

International Journal of Cancer. Journal International Du Cancer. Mar, 2002  |  Pubmed ID: 11920591

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.

The Crystal Structure of Myelin Oligodendrocyte Glycoprotein, a Key Autoantigen in Multiple Sclerosis

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2003  |  Pubmed ID: 12960396

Myelin oligodendrocyte glycoprotein (MOG) is a key CNS-specific autoantigen for primary demyelination in multiple sclerosis. Although the disease-inducing role of MOG has been established, its precise function in the CNS remains obscure. To gain new insights into the physiological and immunopathological role of MOG, we determined the 1.8-A crystal structure of the MOG extracellular domain (MOGED). MOGED adopts a classical Ig (Ig variable domain) fold that was observed to form an antiparallel head-to-tail dimer. A dimeric form of native MOG was observed, and MOGED was also shown to dimerize in solution, consistent with the view of MOG acting as a homophilic adhesion receptor. The MOG35-55 peptide, a major encephalitogenic determinant recognized by both T cells and demyelinating autoantibodies, is partly occluded within the dimer interface. The structure of this key autoantigen suggests a relationship between the dimeric form of MOG within the myelin sheath and a breakdown of immunological tolerance to MOG that is observed in multiple sclerosis.

Generation of Anti-idiotype Antibodies for Application in Clinical Immunotherapy Laboratory Analyses

Hybridoma and Hybridomics. Aug, 2003  |  Pubmed ID: 14511567

The chimeric monoclonal antibody ch806 specifically targets the tumor-associated mutant epidermal growth factor receptor (de 2-7EGFR or EGFRVIII) and is currently under investigation for its potential use in cancer therapy. The humanised monoclonal antibody hu3S193 specifically targets the Lewis Y epithelial antigen and is currently in Phase I clinical trials in patients with advanced breast, colon, and ovarian carcinomas. To assist the clinical evaluation of ch806 and hu3S193, laboratory assays are required to monitor their serum pharmacokinetics and quantitate any immune responses to the antibodies. Mice immunized with ch806 or hu3S193 were used to generate hybridomas producing antibodies with specific binding to ch806 or hu3S193 and competitive for antigen binding. These anti-idiotype antibodies (designated Ludwig Melbourne Hybridomas, LMH) were investigated as reagents suitable for use as positive controls for HAHA or HACA analyses and for measuring hu3S193 or ch806 in human serum. Anti-idiotypes with the ability to concurrently bind two target antibody molecules were identified, which enabled the development of highly reproducible, sensitive, specific ELISA assays for determining serum concentrations of hu3S193 and ch806 with a 3 ng/mL limit of quantitation using LMH-3 and LMH-12, respectively. BIAcore analyses determined high apparent binding affinity for both idiotypes: LMH-3 binding immobilized hu3S193, Ka = 4.76 x 10(8) M(-1); LMH-12 binding immobilised ch806, Ka = 1.74 x 10(9) M(-1). Establishment of HAHA or HACA analysis of sera samples using BIAcore was possible using LMH-3 and LMH-12 as positive controls for quantitation of immune responses to hu3S193 or ch806 in patient sera. These anti-idiotypes could also be used to study the penetrance and binding of ch806 or hu3S193 to tumor cells through immunohistochemical analysis of tumor biopsies. The generation of anti-idiotype antibodies capable of concurrently binding a target antibody on each variable domain provides reagents with high sensitivity for the assessment of safety and pharmacokinetic profiles of target antibodies administered clinically.

Antitumor Efficacy of Cytotoxic Drugs and the Monoclonal Antibody 806 is Enhanced by the EGF Receptor Inhibitor AG1478

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2003  |  Pubmed ID: 14676326

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.

The Tumor-specific De2-7 Epidermal Growth Factor Receptor (EGFR) Promotes Cells Survival and Heterodimerizes with the Wild-type EGFR

Oncogene. Aug, 2004  |  Pubmed ID: 15221011

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.

CR1/CR2 Interactions Modulate the Functions of the Cell Surface Epidermal Growth Factor Receptor

The Journal of Biological Chemistry. May, 2004  |  Pubmed ID: 15016810

Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr(246) is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr(246) impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation.

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 Journal of Biological Chemistry. Jul, 2004  |  Pubmed ID: 15075331

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.

Immunological Effects of Chimeric Anti-GD3 Monoclonal Antibody KM871 in Patients with Metastatic Melanoma

Cancer Immunity : a Journal of the Academy of Cancer Immunology. Feb, 2005  |  Pubmed ID: 15723450

We conducted an open label dose-escalation phase I trial of chimeric anti-GD3 mAb KM871 in patients with metastatic melanoma. Patients were entered into one of five dose levels (1, 5, 10, 20, and 40 mg/m2) and received three infusions of KM871 at 2-wk intervals. A metastatic melanoma site was biopsied at day 7-10. Pharmacokinetics, immune function, and mechanism of action of KM871 were analysed. A total of 17 patients were entered into the trial; 15 were evaluable. KM871 had a serum half-life (T1/2-beta) based on ELISA of 10.39 +/- 1.12 d (mean +/- SD). Trough levels >1.0 microg/mL KM871 at 2 wk postinfusion were seen with the 10 mg/m2 and higher dose levels. There were no significant changes in white blood cell subsets or serum complement levels during KM871 treatment. KM871 was stable in vivo and maintained binding affinity and complement-dependent cytotoxicity (CDC) function up to 2 wk postinfusion. No significant trends in CDC or antibody-dependent cellular-cytotoxicity (ADCC) activity in patients were observed during treatment. Analysis of tumour biopsies demonstrated a significant increase in CD4+ T cell infiltrates compared to control patient tumours (P = 0.010), and in patients with either stable disease (2 patients) or a clinical partial response (1 patient) at restaging, a significant increase in CD3 and CD4 infiltrates in tumour over nonresponding patients was observed. The favourable immune properties of KM871, combined with this preliminary clinical data, indicate that KM871 has potential for the treatment of metastatic melanoma.

The Antitumor Monoclonal Antibody 806 Recognizes a High-mannose Form of the EGF Receptor That Reaches the Cell Surface when Cells Over-express the Receptor

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. May, 2005  |  Pubmed ID: 15774576

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. 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 EGFR expressed in normal tissues. Recently, we demonstrated that the mAb 806 epitope is restricted to a short cysteine loop of the EGFR (amino acids 287-302) that is only available for antibody binding in a transitional form of the receptor, which occurs as the receptor changes from its inactive tethered conformation to a dimeric untethered form. The truncation associated with the de2-7 EGFR mutation renders this receptor constitutively untethered, leading to increased binding of mAb 806. We now show that mAb 806 preferentially binds the immature high-mannose wt and de2-7 EGFR precursors normally located in the endoplasmic reticulum, indicating that this form of the wt EGFR is also constitutively untethered. Using the unique specificity of mAb 806, we clearly demonstrated the presence of these high-mannose EGFR precursors on the cell surface. Given that the high-mannose forms of the wt EGFR must be untethered they may contribute to the spontaneous EGFR signaling reported in cells overexpressing the receptor. These precursor forms of the EGFR thus represent novel tumor targets and contribute to the exceptional selectivity of mAb 806 for EGFR when overexpressed in cancer cells. As our observations are likely to apply to other receptors overexpressed in cancer, they suggest a strategy for developing antitumor antibodies even when the target receptor is expressed in normal tissue.

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

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Sep, 2005  |  Pubmed ID: 16144944

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.

Enhanced Efficacy of Radioimmunotherapy with 90Y-CHX-A''-DTPA-hu3S193 by Inhibition of Epidermal Growth Factor Receptor (EGFR) Signaling with EGFR Tyrosine Kinase Inhibitor AG1478

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Oct, 2005  |  Pubmed ID: 16203806

Monoclonal antibodies and tyrosine kinase inhibitors specific for the epidermal growth factor receptor (EGFR) have been shown to enhance the effect of external beam radiation on EGFR-positive tumors. The effect of EGFR signaling abrogation by EGFR tyrosine kinase inhibitor on the efficacy of radioimmunotherapy has not been reported previously. This study investigated the effect of EGFR tyrosine kinase inhibition on the efficacy of radioimmunotherapy in a human cancer xenograft model.

Functional Effects of Glycosylation at Asn-579 of the Epidermal Growth Factor Receptor

Biochemistry. Nov, 2005  |  Pubmed ID: 16274239

We have investigated functional effects of glycosylation at N(579) of the epidermal growth factor receptor (EGFR). Our previous study showed that the population of cell-surface expressed EGFRs in A431 cells, a human epidermoid carcinoma cell line, is composed of two subpopulations that differ by glycosylation at N(579) [Zhen et al. (2003) Biochemistry 42, 5478-5492]. To characterize the subpopulation of receptors not glycosylated at N(579), we established a 32D cell line expressing a point mutant of the EGFR (N579Q), which cannot be glycosylated at this position. Analysis of epitope accessibility suggests that the lack of glycosylation at N(579) weakens auto-inhibitory tether interactions, and cross-linking experiments suggest a somewhat elevated level of preformed N579Q-EGFR dimers in the absence of ligand relative to wild-type EGFR (WT-EGFR). However, ligand drives the majority of N579Q-EGFR dimerization, suggesting that untethering, while necessary, is not sufficient to drive dimerization. Ligand-binding experiments reveal a much greater fraction of N579Q-EGFRs in a high-affinity state compared to the fraction of WT-EGFRs in a high-affinity state. However, differences in the kinetic association and dissociation rates indicate that the high-affinity states of the WT and the N579Q receptors are distinct. EGF-stimulated phosphorylation in cells expressing N579Q-EGFRs results in notable differences in the pattern of tyrosine phosphorylated proteins compared with that obtained in cells expressing WT-EGFRs. Moreover, although WT-EGFRs confer cell survival in 32D cells in the absence of interleukin-3 and EGF, we found that receptors lacking glycosylation at N(579) do not. This is the first study of which we are aware to show that selective glycosylation of a specific N-glycosylation site can produce two functionally distinct receptors.

An Automated Peptide and Protein Thiazolidine Coupling Chemistry for Biosensor Immobilization Giving a Unique N-terminal Orientation

Analytical Biochemistry. Jan, 2006  |  Pubmed ID: 16310754

Epidermal Growth Factor Receptor Activation: an Upstream Signal for Transition of Quiescent Astrocytes into Reactive Astrocytes After Neural Injury

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2006  |  Pubmed ID: 16837601

Modulating the behaviors of reactive astrocytes is a potential therapeutic strategy for neurodegenerative diseases. We found that upregulation and activation of the epidermal growth factor receptor (EGFR) occur in astrocytes after different injuries in optic nerves in vivo. Activation of EGFR regulates genes and cellular processes representing most major markers of reactive astrocytes and genes related with glaucomatous optic neuropathy and other neural disorders. These results suggest that activation of EGFR is a common, regulatory pathway that triggers quiescent astrocytes into reactive astrocytes in response to neural injuries in the optic nerve, and perhaps other parts of the CNS. Targeting EGFR activation using an EGFR tyrosine kinase inhibitor prevents the loss of retinal ganglion cells in a model of glaucomatous optic neuropathy. Because these inhibitors are currently used clinically, our results present an approach to reactive astrocytes as a potential new target for the treatment of neurodegenerations.

The Efficacy of Epidermal Growth Factor Receptor-specific Antibodies Against Glioma Xenografts is Influenced by Receptor Levels, Activation Status, and Heterodimerization

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Mar, 2007  |  Pubmed ID: 17363548

Factors affecting the efficacy of therapeutic monoclonal antibodies (mAb) directed to the epidermal growth factor receptor (EGFR) remain relatively unknown, especially in glioma.

Internalization, Intracellular Trafficking, and Biodistribution of Monoclonal Antibody 806: a Novel Anti-epidermal Growth Factor Receptor Antibody

Neoplasia (New York, N.Y.). Dec, 2007  |  Pubmed ID: 18084617

Overexpression of the epidermal growth factor receptor (EGFR) in epithelial tumors is associated with poor prognosis and is the target for a number of cancer therapeutics. Monoclonal antibody (mAb) 806 is a novel anti-EGFR antibody with significant therapeutic efficacy in tumor models when used as a single agent, and displays synergistic antitumor activity in combination with other EGFR therapeutics. Unlike other EGFR antibodies, mAb 806 is selective for tumor cells and does not bind to normal tissue, making it an ideal candidate for generation of radioisotope or toxin conjugates. Ideally, antibodies suited to these therapeutic applications must bind to and actively internalize their cognate receptor. We investigated the intracellular trafficking of fluorescently tagged mAb 806 in live cells and analyzed its biodistribution in a tumor xenografted nude mouse model. Following binding to EGFR, mAb 806 was internalized through dynamin-dependent, clathrin-mediated endocytosis. Internalized mAb 806 localized to early endosomes and subsequently trafficked to and accumulation in lysosomal compartments. Furthermore, biodistribution analysis in nude mice showed specific uptake and retention of radiolabeled mAb 806 to human tumor xenografts. These results highlight the potential use of mAb 806 for generation of conjugates suitable for diagnostic and therapeutic use in patients with EGFR-positive malignancies.

The Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor AG1478 Increases the Formation of Inactive Untethered EGFR Dimers. Implications for Combination Therapy with Monoclonal Antibody 806

The Journal of Biological Chemistry. Feb, 2007  |  Pubmed ID: 17092939

The epidermal growth factor receptor (EGFR) has at least two fundamental conformations: an inactive tethered conformation and an active untethered, ligand-bound "back-to-back" dimer, which may be part of an oligomeric complex. Monoclonal antibody (mAb) 806 is an EGFR-specific antibody that only binds a transitional form of the receptor after it untethers but before forming the back-to-back, ligated, active oligomer. We have shown that AG1478, a tyrosine kinase inhibitor of the EGFR, synergistically inhibits the growth of tumors overexpressing EGFR when used in combination with mAb 806 but the mechanism for this was not elucidated (Johns, T. G., Luwor, R. B., Murone, C., Walker, F., Weinstock, J., Vitali, A. A., Perera, R. M., Jungbluth, A. A., Stockert, E., Old, L. J., Nice, E. C., Burgess, A. W., and Scott, A. M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15871-15876). We now show that AG1478 increases binding of mAb 806 to the cell surface through two distinct mechanisms: an immediate effect on the conformation of EGFR and a longer term increase in cell surface under-glycosylated EGFR, an event known to increase mAb 806 reactivity. Cross-linking studies demonstrated the presence of spontaneously occurring mAb 806-reactive dimers on the surface of cells overexpressing EGFR, which are rapidly increased by AG1478. Because they react with mAb 806, these dimers must exist in a conformation distinct from the ligated back-to-back dimer. Indeed, we detected similar dimers in 293T cells expressing the EGFR lacking the small dimerization/activation arm essential to the formation of the back-to-back dimer. Thus, some of the EGFR on the cell surface of cancer cells must exist as an untethered dimer that adopts a previously unreported conformation that is inactive. This information was used to optimize the therapeutic synergy between mAb 806 and AG1478 in a xenograft model.

A Phase I Clinical Trial with Monoclonal Antibody Ch806 Targeting Transitional State and Mutant Epidermal Growth Factor Receptors

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2007  |  Pubmed ID: 17360479

An array of cell-surface antigens expressed by human cancers have been identified as targets for antibody-based therapies. The great majority of these antibodies do not have specificity for cancer but recognize antigens expressed on a range of normal cell types (differentiation antigens). Over the past two decades, our group has analyzed thousands of mouse monoclonal antibodies for cancer specificity and identified a battery of antibodies with limited representation on normal human cells. The most tumor-specific of these antibodies is 806, an antibody that detects a unique epitope on the epidermal growth factor receptor (EGFR) that is exposed only on overexpressed, mutant, or ligand-activated forms of the receptor in cancer. In vitro immunohistochemical specificity analysis shows little or no detectable 806 reactivity with normal tissues, even those with high levels of wild-type (wt)EGFR expression. Preclinical studies have demonstrated that 806 specifically targets a subset of EGFR expressed on tumor cells, and has significant anti-tumor effects on human tumor xenografts, primarily through abrogation of signaling pathways. The present clinical study was designed to examine the in vivo specificity of a chimeric form of mAb 806 (ch806) in a tumor targeting/biodistribution/pharmacokinetic analysis in patients with diverse tumor types. ch806 showed excellent targeting of tumor sites in all patients, no evidence of normal tissue uptake, and no significant toxicity. These in vitro and in vivo characteristics of ch806 distinguish it from all other antibodies targeting EGFR.

Unligated Epidermal Growth Factor Receptor Forms Higher Order Oligomers Within Microclusters on A431 Cells That Are Sensitive to Tyrosine Kinase Inhibitor Binding

Biochemistry. Apr, 2007  |  Pubmed ID: 17381163

Characterization of the association states of the unligated epidermal growth factor receptor (EGFR) is important in understanding the mechanism of EGFR tyrosine kinase activation in a tumor cell environment. We analyzed, in detail, the association states of unligated, immunotagged EGFR on the surface of intact epidermoid carcinoma A431 cells, using AlexaFluor488 and AlexaFluor546 anti-EGFR antibody, mAb528, as probes. Image correlation microscopy revealed the presence of unligated EGFR in submicron scale clusters containing an average of 10-30 receptors (mean cluster density = 32 +/- 9 clusters per square micron). Lifetime-based Förster resonance energy transfer (FRET) techniques as a function of acceptor:donor labeling ratio disclosed a clustering of the unligated EGFR in clusters containing an average of four receptors on the nanometer (<10 nm) scale. The relationship between the nanoscale and submicron scale associations was determined using a new analysis that combines nanoscale information from lifetime-detected FRET imaging with submicron scale information obtained with image correlation microscopy. This analysis revealed the presence of monomers (or small oligomers) and larger clusters containing 15-30 receptors that were partially associated on the sub-10 nm scale. Pretreatment of the cells with the tyrosine kinase inhibitor AG1478 caused a partial dispersal of the submicron clusters (mean cluster density = 85 +/- 15 clusters per square micron; mean degree of association = 4-10 receptors per cluster) and reduced the level of FRET down to our limit of detection. These results are consistent with a higher order nanoscale receptor organization of the unligated receptor population that is partially controlled by the kinase domains. The ramifications of the results to mechanisms of EGFR activation in a tumor cell environment are discussed.

Heparin-binding Epidermal Growth Factor-like Growth Factor Signaling in Flow-induced Arterial Remodeling

Circulation Research. May, 2008  |  Pubmed ID: 18436796

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is activated by reduced endothelial shear stress and stimulates smooth muscle cell proliferation in vitro. Moreover, HB-EGF is augmented at sites of intimal hyperplasia and atherosclerosis, conditions favored by low/disturbed shear stress. We thus tested whether HB-EGF contributes to low flow-induced negative hypertrophic remodeling (FINR) of a mouse carotid artery. Blood flow was surgically decreased in the left and increased in the right common carotid arteries. After 21 days, the left carotid artery exhibited lumen narrowing, thickening of intima-media and adventitia, and increased circumference that were inhibited by approximately 50% in HB-EGF(+/-) and approximately 90% in HB-EGF(-/-) mice. FINR was also inhibited by the EGF receptor inhibitor AG1478. In contrast, eutrophic outward remodeling of the right carotid artery was unaffected in HB-EGF(+/-) and HB-EGF(-/-) mice, nor by AG1478. FINR-induced proliferation and leukocyte accumulation were reduced in HB-EGF(-/-). FINR was associated with increased reactive oxygen species, increased expression of pro-HB-EGF and tumor necrosis factor alpha-converting enzyme (pro-HB-EGF sheddase), increased phosphorylation of EGF receptor and extracellular signal-regulated kinase 1/2, and increased nuclear factor kappaB activity. Apocynin and deletion of p47(phox) inhibited FINR, whereas deletion of HB-EGF abolished nuclear factor kappaB activation in smooth muscle cells. These findings suggest that HB-EGF signaling is required for low flow-induced hypertrophic remodeling and may participate in vascular wall disease and remodeling.

Antibodies Specifically Targeting a Locally Misfolded Region of Tumor Associated EGFR

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2009  |  Pubmed ID: 19289842

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

A Truncated Soluble Epidermal Growth Factor Receptor-Fc Fusion Ligand Trap Displays Anti-tumour Activity in Vivo

Growth Factors (Chur, Switzerland). Jun, 2009  |  Pubmed ID: 19333814

A number of therapeutic strategies including small molecule tyrosine kinase inhibitors and monoclonal antibodies have been developed to target the epidermal growth factor receptor (EGFR) signalling axis for the treatment of cancer. To date, the focus of therapeutic intervention has been the EGFR itself. In the current study, we have assembled and expressed in mammalian cells a soluble, EGFR ligand trap comprising the first 501 amino acids of the mature EGFR sequence fused in-frame with a human IgG Fc domain. The fusion protein, designated sEGFR501.Fc, was secreted as a 220 kDa disulphide-linked homodimer that exhibited high affinity (0.4-8 nM) in competition assays for a number of EGFR ligands including EGF and transforming growth factor-alpha (TGF-alpha). sEGFR501.Fc inhibited EGF-stimulated tyrosine phosphorylation of the EGFR of the lung cancer cell lines A549 and H1437, and inhibited and blocked the proliferation of H1437 cells. Administration of sEGFR501.Fc to mice bearing human tumour xenografts derived from A431 (epidermoid carcinoma) and DU145 (androgen-independent prostate cancer) tumour cell lines resulted in modest retardation of tumour growth. These results provide proof-in-principle that using high affinity soluble receptors is a viable method for inhibiting multi-ligand systems, and the impetus to optimize this approach and develop reagents with greater affinity and broader specificity.

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

Neoplasia (New York, N.Y.). May, 2009  |  Pubmed ID: 19412429

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 GBM growth 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 beta 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.Delta 2-7 xenografts were profoundly resistant. Treatment of U87MG.Delta 2-7 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.

Targeting a Unique EGFR Epitope with Monoclonal Antibody 806 Activates NF-kappaB and Initiates Tumour Vascular Normalization

Journal of Cellular and Molecular Medicine. Sep, 2009  |  Pubmed ID: 19432811

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.

Fyn and SRC Are Effectors of Oncogenic Epidermal Growth Factor Receptor Signaling in Glioblastoma Patients

Cancer Research. Sep, 2009  |  Pubmed ID: 19690143

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.

Immuno-PET Quantitation of De2-7 Epidermal Growth Factor Receptor Expression in Glioma Using 124I-IMP-R4-labeled Antibody Ch806

Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine. Jun, 2010  |  Pubmed ID: 20484439

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.

MAb 806 Enhances the Efficacy of Ionizing Radiation in Glioma Xenografts Expressing the De2-7 Epidermal Growth Factor Receptor

International Journal of Radiation Oncology, Biology, Physics. Oct, 2010  |  Pubmed ID: 20638193

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.

Tumor Heterogeneity is an Active Process Maintained by a Mutant EGFR-induced Cytokine Circuit in Glioblastoma

Genes & Development. Aug, 2010  |  Pubmed ID: 20713517

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.

Activation of Src Induces Mitochondrial Localisation of De2-7EGFR (EGFRvIII) in Glioma Cells: Implications for Glucose Metabolism

Journal of Cell Science. Sep, 2011  |  Pubmed ID: 21878501

A common mutation of the epidermal growth factor receptor in glioma is the de2-7EGFR (or EGFRvIII). Glioma cells expressing de2-7EGFR contain an intracellular pool of receptor with high levels of mannose glycosylation, which is consistent with delayed processing. We now show that this delay occurs in the Golgi complex. Low levels of de2-7EGFR were also seen within the mitochondria. Src activation dramatically increased the amount of mitochondrial de2-7EGFR, whereas its pharmacological inhibition caused a significant reduction. Because de2-7EGFR is phosphorylated by Src at Y845, we generated glioma cells expressing a Y845F-modified de2-7EGFR. The de2-7EGFR(845F) mutant failed to show mitochondrial localisation, even when co-expressed with constitutive active Src. Low levels of glucose enhanced mitochondrial localisation of de2-7EGFR, and glioma cells expressing the receptor showed increased survival and proliferation under these conditions. Consistent with this, de2-7EGFR reduced glucose dependency by stimulating mitochondrial oxidative metabolism. Thus, the mitochondrial localisation of de2-7EGFR contributes to its tumorigenicity and might help to explain its resistance to some EGFR-targeted therapeutics.

Phosphorylation of Dedicator of Cytokinesis 1 (Dock180) at Tyrosine Residue Y722 by Src Family Kinases Mediates EGFRvIII-driven Glioblastoma Tumorigenesis

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2012  |  Pubmed ID: 22323579

Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.