The propeptides of VEGF-D determine heparin binding, receptor heterodimerization, and effects on tumor biology.

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

A high-affinity ErbB4Fc fusion protein is a potent antagonist of heregulin-mediated receptor activation.

Ligand-mediated activation of ErbB3 and ErbB4 is implicated in the pathogenesis of several human malignancies including cancer of the ovary and melanoma. We have used the broad ErbB ligand specificity of ErbB4 to assemble and express an ErbB4 fusion protein comprising the first 497 amino acids of the mature ErbB4 ectodomain fused to the human IgG Fc constant region. The purified fusion protein, designated sErbB4.497.Fc, binds the ErbB receptor ligands betacellulin and heregulin-ß1 (HRG-ß1) with high affinity (K(D) = 130 pM), an increase in affinity of 10- to 20-fold, respectively, compared with sErbB4.615.Fc. sErbB4.497.Fc inhibited ligand-stimulated phosphorylation of epidermal growth factor receptor and ErbB2, and blocked HRG-ß1 activation of the IKB/MAP/JNK/AKT signalling pathways. sErbB4.497.Fc inhibited HRG-ß1-stimulated proliferation in MCF7 cells. In a mouse tumour xenograft model, sErbB4.497.Fc as a monotherapy modestly inhibited the growth of MDA-MB-231 breast cancer cells. sErbB4.497.Fc may be useful in an adjuvant setting in combination with conventional therapeutic agents.

Ligand binding induces a conformational change in epidermal growth factor receptor dimers.

The activation of the epidermal growth factor receptor (EGFR) kinase requires ligand binding to the extracellular domain (ECD). Previous reports demonstrate that the EGFR-ECD can be crystallized in two conformations - a tethered monomer or, in the presence of ligand, an untethered back-to-back dimer. We use Biosensor analysis to demonstrate that even in the monomeric state different C-terminal extensions of both truncated (EGFR(1-501))-ECD and full-length EGFR(1-621)-ECD can change the conformation of the ligand-binding site. The binding of a monoclonal antibody mAb806, which recognizes the dimer interface, to the truncated EGFR(1-501)-Fc fusion protein is reduced in the presence of ligand, consistent with a change in conformation. On the cell surface, the presence of erythroblastosis B2 (erbB2) increases the binding of mAb806 to the EGFR. The conformation of the erbB2: EGFR heterodimer interface changes when the cells are treated with epidermal growth factor (EGF). We propose that ligand induces kinase-inactive, pre-formed EGFR dimers and heterodimers to change conformation leading to kinase-active tetramers, where kinase activation occurs via an asymmetric interaction between EGFR dimers.

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR.

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.

Conformational dynamics in a truncated epidermal growth factor receptor ectodomain.

Structural studies have revealed two forms of the monomeric epidermal growth factor receptor (EGFR) ectodomain: a compact (tethered) form stabilized by interdomain interactions and an extended (untethered) form in the presence of ligand. An important question is whether the ligand induces a conformational transition from a tethered to untethered form or whether there is a preexisting conformational equilibrium between tethered and untethered states. To distinguish between these two possibilities, we investigated a truncated receptor, EGFR501 (spanning residues 1-501), that contains the minimal elements required for high-affinity ligand binding in solution. Conformational transitions and dynamics were inferred by means of fluorescence from five internal tryptophan residues that are located within or close to the ligand-binding domains of EGFR501. A preexisting conformational equilibrium between tethered and untethered states in EGFR501 was deduced from (1) the nonlinear Arrhenius temperature dependence of fluorescence and (2) fluorescence polarization showing independently mobile domains. In contrast, the ligand-EGFR501 complex revealed a linear Arrhenius temperature dependence of fluorescence and increased fluorescence polarization due to a lack of significant interdomain motions. The data suggest that the role of the ligand is to trap the EGFR501 in the untethered state that is transiently formed in solution through a preexisting conformational equilibrium.

Use of multiple reaction monitoring for multiplex analysis of colorectal cancer-associated proteins in human feces.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide with an annual incidence of almost a million cases and an annual mortality around 500,000. The fecal occult blood test is currently the first line method for CRC screening, but has unacceptably low sensitivity and specificity. Improved screening tests are therefore urgently required for early-stage CRC screening when therapy is most likely to be effective. We describe a discovery-based proteomics hypothesis using orthogonal multi-dimensional fractionation (1-D SDS-PAGE, RP-HPLC, size exclusion chromatography) to mine deep into the fecal proteome for the initial discovery process, which generated a library containing 108 human fecal proteins with the associated peptide and MS/MS data. These data were then used to develop and optimize a multiplex multiple reaction monitoring assay for 40 non-redundant human proteins present in the feces. To show proof of principal for clinical analysis, multiplex screening of these 40 proteins was carried out on fecal samples from eight CRC patient and seven normal volunteers. We identified 24 proteins consistently found in all samples and nine proteins found only in the CRC patients, showing the potential of this approach for the analysis of potential CRC biomarkers. Absolute quantitation using C-terminal isotopically labeled synthetic peptides corresponding to hemoglobin and carcinoembryonic antigen 5 was also performed.

Creation and biophysical characterization of a high-affinity, monomeric EGF receptor ectodomain using fluorescent proteins.

X-ray structural studies revealed two conformations of the epidermal growth factor receptor (EGFR) ectodomain (ECD): a compact, tethered conformation in the absence of EGF and an untethered or extended conformation in the presence of EGF. An EGFR-ECD derivative with a monomeric red fluorescent protein (mRFP) at the N-terminus and an enhanced green fluorescent protein (eGFP) at the C-terminus (dual-tag-EGFR-ECD) was created and characterized. The dual-tag-EGFR-ECD construct was shown to have high affinity (nanomolar range) for both EGF and EGFR monoclonal antibody (mAb528). The dual-tag-EGFR-ECD was further characterized by fluorescence-detected analytical ultracentrifugation, lifetime FRET, and fluorescence anisotropy. We found no evidence of a tethered unliganded conformation, nor did we observe a large shape change upon ligand binding as predicted by the crystal models. Increases in steady-state anisotropy upon binding of EGF to the dual-tag-EGFR-ECD were observed and interpreted as changes in the protein flexibility and dynamics. We conclude the fluorescent protein tags perturb the EGFR-ECD structure, making it extended with a 50-fold higher affinity for EGF relative to that of the nontagged EGFR-ECD.

A truncated soluble epidermal growth factor receptor-Fc fusion ligand trap displays anti-tumour activity in vivo.

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.

Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2.

Vascular endothelial growth factor (VEGF)-D is a secreted glycoprotein that induces angiogenesis and lymphangiogenesis. It consists of a central domain, containing binding sites for VEGF receptor-2 (VEGFR-2) and VEGFR-3, and N- and C-terminal propeptides. It is secreted from the cell as homodimers of the full-length form that can be proteolytically processed to remove the propeptides. It was recently shown, using adenoviral gene delivery, that fully processed VEGF-D induces angiogenesis in vivo, whereas full-length VEGF-D does not. To better understand these observations, we monitored the effect of VEGF-D processing on receptor binding using a full-length VEGF-D mutant that cannot be processed. This mutant binds VEGFR-2, the receptor signaling for angiogenesis, with approximately 17,000-fold lower affinity than mature VEGF-D, indicating the importance of processing for interaction with this receptor. Further, we show that members of the proprotein convertase (PC) family of proteases promote VEGF-D processing, which facilitates the VEGF-D/VEGFR-2 interaction. The PCs furin and PC5 promote cleavage of both propeptides, whereas PC7 promotes cleavage of the C-terminal propeptide only. The finding that PCs promote activation of VEGF-D and other proteins with roles in cancer such as matrix metalloproteinases, emphasizes the importance of these enzymes as potential regulators of tumor progression and metastasis.

Murine fecal proteomics: a model system for the detection of potential biomarkers for colorectal cancer.

Tumor related products shed into the feces offer a potential source of biomarkers for the detection of colorectal cancer (CRC). Using SDS-PAGE followed by nanoflow reversed-phased LC-MS/MS to analyse fecal samples from Apc(Min/+) mice (that develop spontaneous multiple intestinal neoplasia with age) we have identified 336 proteins (115 proteins of murine origin, 201 from fecal bacteria, 18 associated with food intake and 2 of apparent parasitic origin). 75% of the murine proteins identified in this study are predicted to be extracellular or associated with the cell plasma membrane. Of these proteins, a number of the murine homologues of colorectal cancer associated proteins (CCAP) such as hemoglobin, haptoglobin, hemopexin, alpha-2-macroglobulin and cadherin-17 have been identified, demonstrating the potential of fecal proteomics for detecting potential biomarkers and paving the way for subsequent MS/MS based biomarker studies on similar human samples.

Experimental determination of quantum dot size distributions, ligand packing densities, and bioconjugation using analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) was used to characterize the size distribution and surface chemistry of quantum dots (QDs). AUC was found to be highly sensitive to nanocrystal size, resolving nanocrystal sizes that differ by a single lattice plane. Sedimentation velocity data were used to calculate the ligand packing density at the crystal surface for different sized nanocrystals. Dihydrolipoic acid poly(ethylene glycol) was found to bind between 66 and 60% of the surface cadmium atoms for CdSe nanocrystals in the 1.54-2.59 nm radius size regime. The surface ligand chemistry was found to affect QD sedimentation, with larger ligands decreasing the sedimentation rate through an increase in particle volume and increase in frictional coefficient. Finally, AUC was used to detect and analyze protein association to QDs. Addition of bovine serum albumin (BSA) to the QD sample resulted in a reduced sedimentation rate, which may be attributed to an associated frictional drag. We calculated that one to two BSA molecules bind per QD with an associated frictional ratio of 1.2.

A novel magnetic bead-based assay with high sensitivity and selectivity for analysis of telomerase in exfoliated cells from patients with bladder and colon cancer.

Telomerase activity is elevated in more than 85% of cancer cells and absent in most of the normal cells and thus represents a potential cancer biomarker. We report its measurement in colon and bladder cancer cells captured using antibody-coated magnetic beads. The cells are lysed and telomerase activity is detected using a biosensor assay that employs an oligonucleotide containing the telomerase recognition sequence also covalently coupled to magnetic beads. Telomerase activity is measured by the incorporation of multiple biotinylated nucleotides at the 3'-end of the oligonucleotide strands during elongation which are then reacted with streptavidin-conjugated horseradish peroxidase. A luminescent signal is generated when hydrogen peroxidase is added in the presence of luminol and a signal enhancer. LOD experiments confirm sensitivity down to ten cancer cell equivalents. The telomerase assay reliably identified patient samples considered by an independent pathological review to contain cancer cells. Samples from normal healthy volunteers were all telomerase negative. The assay, which is amenable to automation, demonstrated high sensitivity and specificity in a small clinical cohort, making it of potential benefit as a first line assay for detection and monitoring of colon and bladder cancer.

Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis.

The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. For many years it has been believed that receptor activation occurs via a monomer-dimer transition that is associated with a conformational change to activate the kinase. However, little is known about the quaternary state of the receptor at normal levels of expression (<10(5) receptors/cell). We employed multidimensional microscopy techniques to gain insight into the state of association of the human EGFR, in the absence and presence of ligand, on the surface of intact BaF/3 cells (50,000 receptors/cell). Image correlation microscopy of an EGFR-enhanced green fluorescent protein chimera was used to establish an average degree of aggregation on the submicron scale of 2.2 receptors/cluster in the absence of ligand increasing to 3.7 receptors/cluster in the presence of ligand. Energy transfer measurements between mixtures of fluorescein isothiocyanate-EGF and Alexa 555-EGF were performed using fluorescence lifetime imaging microscopy as a function of the donor: acceptor labeling ratio to gain insight into the spatial disposition of EGFR ligand binding sites on the nanometer scale. In the context of a two-state Förster resonance energy transfer (FRET)/non-FRET model, the data are consistent with a minimum transfer efficiency of 75% in the FRET population. The microscopy data are related to biophysical data on the EGFR in the A431 cell line and the three-dimensional structure of the ligated EGFR extracellular domain. In the context of a monomer-dimer-oligomer model, the biophysical data are consistent with a significant fraction of ligated EGFR tetramers comprising two dimers juxtaposed in a side-by-side (or slightly staggered) arrangement. Our data are consistent with a specific higher order association of the ligand-bound EGFR on the nanometer scale and indicate the existence of distinct signaling entities beyond the level of the EGFR dimer which could play an important role in receptor transactivation.

Fluorescence and analytical ultracentrifugation analyses of the interaction of the tyrosine kinase inhibitor, tyrphostin AG 1478-mesylate, with albumin.

Quantifying the interaction of drugs with carrier proteins in plasma is of importance for understanding effective drug delivery to disease-affected tissues. In this study, we employed analytical ultracentrifugation and steady-state fluorescence spectroscopy to characterize the interaction of a potential new anticancer drug, AG 1478-mesylate, with plasma proteins in a suspension of normal serum albumin (NSA). We found that mesylate salt of AG 1478, an epidermal growth factor receptor kinase inhibitor, sediments in 0.1%(w/v) NSA as a complex with a sedimentation coefficient of 3.8 S. This is consistent with the size of human serum albumin. This interaction was quantitated by meniscus depletion sedimentation and fluorescence titration analyses. AG 1478-mesylate binds to albumin with an apparent single-site affinity (K(d)) of 120 microM. In this article, we show that the cyclodextrin carrier molecule, Captisol, increases the apparent affinity of the hydrophobic AG 1478-mesylate for albumin (K(d)=4-6 microM), and we propose that the AG 1478-mesylate-Captisol (1:1) complex binds to albumin with at least 10-fold higher affinity than does AG 1478-mesylate ligand alone. A fluorenylmethoxycarbonyl-sulfonic acid (FMS) derivative of the 6-aminoquinazoline analog of AG 1478, which was designed to have improved serum-binding properties, was shown by fluorescence analysis to bind with approximately 100-fold greater affinity than the parent compound. This has significant implications in the effective delivery of therapeutic agents in vivo.

The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF).

A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric a-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and (1)H-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblastsAs we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.

Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

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.