A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases

A. Shrivastava1,2, M.A. von Wronski1, A.K. Sato3, D.T. Dransfield3, D. Sexton3, N. Bogdan1, R. Pillai1, P. Nanjappan1, B. Song1, E. Marinelli1, D. DeOliveira3, C. Luneau3, M. Devlin3, A. Muruganandam3, A. Abujoub3, G. Connelly3, Q.L. Wu3, G. Conley3, Q. Chang3, M.F. Tweedle1, R.C. Ladner3, R.E. Swenson1 and A.D. Nunn1

1Ernst Felder Laboratories, Bracco Research USA, 305 College Road East, Princeton, NJ 08540 and 3Dyax Corp., 300 Technology Square, Cambridge, MA 02139, USA

2 To whom correspondence should be addressed. E-mail: ajay.shrivastava{at}bru.bracco.com


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with KDs below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Keywords: c-MET/HAPs/heterodimer/peptide/VEGFR-2


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The receptor tyrosine kinase (RTK) family of proteins is one of the most studied families of mammalian proteins (Ullrich and Schlessinger, 1990Go; Schlessinger, 2000Go) because of their involvement in many diseases, including cancer (Vlahovic and Crawford, 2003Go) and neurodegenerative diseases (Connor and Dragunow, 1998Go). RTKs have three common features: an extracellular ligand binding domain, a single transmembrane helix and a cytoplasmic tyrosine kinase domain (Schlessinger, 1988Go; Lemmon and Schlessinger, 1994Go; Jiang and Hunter, 1999Go). Most efforts to develop RTK agonist/antagonists have targeted either the extracellular domain or the cytoplasmic kinase domain (Zwick et al., 2002Go; Abou-Jawde et al., 2003Go; Bennasroune et al., 2004Go). Binders to the cytoplasmic kinase domain often are small molecules that can penetrate the cell membrane and block tyrosine kinase activity via the adenosine triphosphate (ATP) binding site or kinase substrate binding site and often require high concentrations, with the risk of systemic toxicity. On the other hand, binders to the extracellular domain of RTKs most often include monoclonal antibodies (mAbs), single-chain Fvs (scFvs), Fab antibody fragments or small peptides. Among this group of targeted therapeutics, small peptides are desirable because of their ability to penetrate solid tumors owing to their small size (Aina et al., 2002Go; Mori, 2004Go).

Peptides may also be used as delivery vehicles for carrying radioactive molecules or toxins to the internal core of tumors. There have been many attempts to deliver cytotoxic drugs selectively using mAbs and other targeting molecules (Allen, 2002Go; Pastan and Kreitman, 2002Go; Thorpe, 2004Go). Owing to their large size, the use of antibody-based targeting agents has again been limited largely to either vascular targets present on the luminal side of the tumor endothelium (Thorpe, 2004Go) or hematological malignancies (Reff et al., 2002Go). In this application, peptides would have the additional advantage of rapid renal clearance (owing to their smaller size) and therefore lower expected bone-marrow toxicity as compared with labeled antibodies or scFvs. Despite these advantages, the use of peptides to a great extent has also been limited by low affinity, reduced ability to compete with the natural ligands of the RTKs and instability in serum (Adams and Schier, 1999Go; Szardenings, 2003). There are several examples of multimerization to improve the affinity of peptides (Mourez et al., 2001Go; Sadler and Tam, 2002Go). However, this strategy has not been routinely used to improve the efficacy of peptides in cancer therapy because multimers of peptides (tetramer and higher) can become as large as scFvs in some cases and therefore do not provide a significant size advantage for tumor therapy.

Here we report a novel and general method of generating high-affinity peptide (HAP) binders to RTKs and demonstrate its utility by using it to produce HAPs to vascular endothelial growth factor receptor (VEGFR-2) and c-Met. Vascular endothelial growth factor receptors (VEGFRs) are receptors of the VEGF family of ligands (Ferrara et al., 2003Go). The VEGF family of ligands is believed to be one of the main mediators of angiogenesis, a process of development and growth of new capillary blood vessels from pre-existing vessels that was recognized 30 years ago (Folkman, 1971, 2003Go; Ferrara et al., 2003Go). Among VEGFRs, VEGFR-2 expression is mainly restricted to proliferating endothelial cells and is present on both the luminal and abluminal sides of tumor endothelial cells (Terman et al., 1991Go; Quinn et al., 1993Go; Feng et al., 2000Go). On the other hand, C-Met binds to hepatocyte growth factor, HGF or scatter factor (Bottaro et al., 1991Go) and its activation is often associated with cancer metastasis. Activation of c-Met is usually accomplished through over-expression, co-expression with HGF or through somatic/inherited point mutations, and is often associated with a poorer prognosis in cancer (Danilkovitch-Miagkova and Zbar, 2002Go). Our approach to HAPs generation is based on a multi-step strategy involving peptide phage display to generate submicromolar affinity binders, an in vitro assay based on the avidin/biotin system to select non-competitive and synergistic peptide pairs suitable for heterodimerization and chemical synthesis of heterodimers. Our experimental work focused on RTKs, but we suggest that this strategy may also be extended to generate HAPs against other families of proteins.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phage library selection

Multiple cyclic peptide, or ‘constrained loop’, phage display libraries were prepared using standard phage methods (Sato et al., 2002Go; Fleming et al., 2004Go) and pooled for initial screening. The pooled cyclic libraries and a linear library (Lin20) were depleted against Trail R4/Fc fusion (an irrelevant Fc fusion protein from R&D System, Cat. No. 633-TR) and then selected against human VEGFR-2/Fc fusion (R&D System, Cat. No. 357-KD) or c-Met/Fc (R&D System, Cat. No. 358-MT) using protein A magnetic beads. The pooled cyclic libraries for c-MET/FC consisted of Tn-6 and Tn-7 libraries in addition to Tn-8, Tn-9, Tn-10, Tn-11 and Tn-12 libraries (used for VEGFR-2/Fc). Phage were eluted with VEGF (R&D System, Cat. No. 293-VE) for VEGFR-2/Fc or HGF (R&D System, Cat. No. 294-HG) for c-Met/Fc, amplified and tested as described in Supplementary data available at PEDS Online.

Affinity maturation library construction and selection

In a secondary TN-8 library, the phage isolate sequence PKWCEEDWYYCMIT (P-2) was used as a template to construct a library that allowed one-, two- and three-base mutations to the parent sequence at each variable codon using a method of peptide optimization by soft randomization (Fairbrother et al., 1998Go). Once the library had been constructed, the selection was conducted in the same manner as in the initial selections, except that VEGF was replaced with P-2 (parent peptide) at 50 µM (details in the Supplementary data).

Identification of P-4 peptide

P-4 peptide was identified by synthesizing several truncated P-1 peptides with a fluorescein label and measuring KD values of those truncated peptides by FP assay (Sato et al., 2002Go; Burke et al., 2003Go; Fleming et al., 2004Go).

Chemical synthesis of peptides

Monomeric peptides and dimers were synthesized as reported (Pillai et al., 2004Go). More details can be found in the Supplementary data.

SPR and FP assay

For FP affinity determinations of KD, VEGFR-2/Fc (or c-Met/Fc) fusion affinities to fluorescein-labeled peptide were determined as described previously (Sato et al., 2002Go; Burke et al., 2003Go; Fleming et al., 2004Go). For SPR determinations of KD, VEGFR-2/Fc (or c-Met/Fc) fusion was cross-linked to the dextran surface of a CM5 sensor chip by the standard amine coupling procedures. For association, peptides (25–400 nM in HBS-P) were injected at 20 µl/min for 1 min using the Kinject program (details in the Supplementary data). Sensorgrams were analyzed using the simultaneous ka/kd fitting program in the BIAevaluation software 3.1.

Human VEGFR-2 cloning and transfection in 293H cells

Full-length VEGFR-2 amplified from human umbilical vein endothelial cells (HUVEC), using polymerase chain reaction (PCR) with pfu polymerase (Stratagene, Cat. No. 600135) and cloned into pcDNA6/V5-HisC vector to produce full-length receptor expression vector pf-VEGFR-2 (details in the Supplementary data). 293H cells (Invitrogen, Cat. No. 11631) were grown in 96-well plates coated with poly-D-lysine according to the supplier's recommendations. Transfection was done according to the manufacturer's instructions using Lipofectamine 2000 reagent (Invitrogen, Cat. No. 11668–019).

Preparation of peptide/neutravidin HRP solutions

To prepare peptide/NA-HRP complexes, 5.34 µl of 250 µM biotinylated peptide (4 equiv.) stock solution and 10 µl of 33 µM NA-HRP (1 equiv.) were added to 1 ml of M199 binding medium. Binding medium was prepared by adding one M199 medium packet (GIBCO, Cat. No. 31100-035), 20 ml of 1 mM HEPES (GIBCO, Cat. No.15630-080) and 2 g of DIFCO gelatin (DIFCO, Cat. No. 0143-15-1) to 950 ml of H2O (pH adjusted to 7.4 with 1 M NaOH). This mixture was incubated on a rotator at 4°C for 60 min, followed by addition of 50 µl of soft release avidin–Sepharose (50% slurry in doubly distilled water) to remove excess peptides and another incubation for 30 min on a rotator at 4°C. Finally, the soft release avidin–Sepharose was pelleted by centrifuging at 12 000 r.p.m. for 5 min at room temperature and the resulting supernatant was diluted to the desired concentration for the assays using M199 binding medium. Peptide mixtures for the competition assay were prepared with 4P-2'/NA HRP (3.33 nM) solution plus 1.25 µM of uncomplexed peptide P-1, P-2 or P-X (not complexed with avidin HRP). To prepare peptide/neutravidin HRP complexes for synergy assays, 5.34 µl of 250 µM biotinylated peptide stock solutions (or mixture of peptides, to give peptide molecules four times the number of avidin HRP molecules) and 10 µl of 33 µM neutravidin HRP were added to 1 ml of M199 medium. These solutions were diluted to a single concentration of 4P'/NA HRP (5.55 nM) solution to be added to individual wells.

Assay to detect the binding of peptide/NA HRP complex

Twenty-four hours after transfection, mock and pf-VEGFR-2 transfected 293H cells were washed 1x with 100 µl of M199 medium and incubated with 80 µl of blocking solution (0.5 mg/ml neutravidin, Pierce, Cat. No. 31000) at 37°C. After 1 h, cells were washed 2x with 100 µl of M199 medium and incubated with 70 µl of peptide/neutravidin HRP dilutions (prepared as described earlier) for 2.5 h at room temperature. After incubation at room temperature, the plates were cooled to 4°C for 30 min incubation. Subsequently, cells were washed 5x with ice-cold M199 medium and 1x with ice-cold PBS (in that order). After the final wash, 100 µl of ice-cold TMB solution (KPL, Cat. No. 50-76-00) were added to each well and each plate was incubated for at 37°C 30 min in an air incubator. Finally, the HRP enzyme reaction was stopped by adding 50 µl of 1 N phosphoric acid to each well and binding was quantitated by measuring the absorbance at 450 nm using a microplate reader (Bio-Rad Model 3550).

Competition of peptides with 125I-labeled VEGF for binding to HUVEC cells

HUVEC (Cambrex, Cat. No. CC-2519) were grown according to the supplier's recommendations. Binding of 125I-VEGF165 (Amersham, 1000–2500 Ci/mmol) to confluent HUVEC was carried out as described earlier (Bikfalvi et al., 1991Go) except that the 125I-VEGF165 was held constant at 300 pM and the concentration of unlabeled competing compounds was varied to generate an IC50 for each.

Competition of peptides with VEGF to block VEGFR-2 phosphorylation

The ability of compounds to block the VEGF-induced phosphorylation of VEGFR-2 was carried out using an immunoprecipitation–western blotting procedure as described earlier (Lin et al., 1998Go), except that the target cells used were HUVEC and the anti-phosphotyrosine antibody used was PY20 (BD Biosciences, Cat. No. 610000).

Competition of peptides with VEGF to block HUVEC migration

The ability of compounds to block VEGF-induced migration was tested with HUVEC using the BD Bio-Coat Angiogenesis system for Endothelial Cell Migration (BD Biosciences, Cat. No. 354143) according to the manufacturer's instructions.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identification of peptide binders against human VEGFR-2 using phage display

Peptide phage display is a common technique for identifying peptide binders to various targets (Hetian et al., 2002; Sato et al., 2002Go; Fleming et al., 2004Go). In order to identify peptide binders to human VEGFR-2 (VEGFR-2), several M13 phage libraries of cyclic (TN-8, TN-9, TN-10, TN-11 and TN-12) and linear (Lin-20) peptides were screened using recombinant VEGFR-2/Fc fusion protein as shown in Figure 1A. Phage particles specific for binding to VEGFR-2/Fc were identified using standard phage display techniques (Sato et al., 2002Go; Fleming et al., 2004Go). Subsequently, individual phage isolates were tested for binding to VEGFR-2/Fc and TRAIL/Fc (negative control obtained from R&D System) in ELISA format (data not shown). After sequencing, a panel of recurring peptide sequences was chemically synthesized as unlabeled and as fluorescein-labeled peptides using standard solid-phase synthesis methods (Sato et al., 2002Go). The affinities of these peptides (KD values) were determined by fluorescence polarization (FP) assays (Sato et al., 2002Go; Burke et al., 2003Go; Fleming et al., 2004Go) with fluorescein-labeled peptides and/or surface plasmon resonance (SPR) measurements (Malmqvist, 1999Go) with unlabeled peptides. Using these assays, we identified peptide binders of VEGFR-2/Fc with affinities between 69 nM and 1 µM (Figure 1B). Different KD values were observed for some peptides with FP and SPR assays due to fluorescein addition (Figure 1B).



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Fig. 1. Identification of low affinity peptide binders of VEGFR-2 using phage display. (A) Schematic of the phage display libraries selected against VEGFR-2/Fc; library variation and diversity indicated in figure. (B) VEGFR-2/Fc binding peptide sequences identified using phage display. Binding of chemically synthesized peptides was confirmed and KD values were obtained using FP assay and (or) SPR (BIAcore) assay with human VEGFR-2/Fc. Ac represents the acetyl protecting groups. *P-3 was identified using affinity maturation of P-2 peptide. **P-4 was identified through P-1 peptide truncation studies.

 
Out of many sequences and initial leads from these libraries, we selected one peptide (P-2; Figure 1B) for affinity maturation (Fairbrother et al., 1998Go). This library was selected as in the previous selection, except that free P-2 peptide was used in place of VEGF for phage elution. After sequence analysis, a panel of peptides was synthesized and tested using FP and SPR. As a result, we identified a higher affinity peptide P-3 (KD {approx} 3.2 nM) versus the parental peptide P-2 (KD {approx} 280 nM).

In addition, we identified P-4 peptide by synthesizing different truncated versions of P-1 peptide with a fluorescein label and measuring the affinity using FP assay (Sato et al., 2002Go; Burke et al., 2003Go; Fleming et al., 2004Go).

Identification of peptides for heterodimerization

We used a novel assay based on the avidin–biotin interaction (Figure 2A) to identify non-competitive and synergistic peptide pairs suitable for heterodimerization. This assay exploits the ability of a single avidin molecule to bind four different biotin molecules with high affinity and specificity (Bratthauer, 1999Go). In our assay, we prepared a tetrameric complex (4P'/NA-HRP) of biotinylated VEGFR-2/Fc binding peptide (where P' denotes biotinylated peptide) with neutravidin HRP (NA-HRP) and evaluated its binding to 293 H cells that were transiently transfected with pf-VEGFR-2 (full-length human VEGFR-2 c-DNA cloned into the pcDNA6C vector). As a negative control, we tested the binding of 4P'/NA-HRP to 293 H cells that had been transfected either with no DNA (mock-transfected) or vector DNA (pcDNA6C transfected). There was no difference in the binding of 4P'/NA-HRP to mock or vector-transfected cells, therefore all of the subsequent control experiments were carried out with mock-transfected cells. As another negative control, we tested the binding of complexes containing a non-binding biotinylated peptide (P-X'). We used neutravidin-HRP instead of streptavidin or avidin because it has lower non-specific binding to molecules other than biotin (Hiller, et al., 1987Go; Alon et al., 1990Go).



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Fig. 2. Identification of peptides suitable for heterodimerization. (A) Schematic of an assay to evaluate the effects of peptide multimerization on binding to pf-VEGFR-2 transfected cells. (B) P-1' (2nd pair of bars) and P-2' (3rd pair of bars) complexed with NA-HRP specifically bound to pf-VEGFR-2 transfected cells. No specific binding was observed with P-X' (negative control, 1st pair of bars)/NA-HRP complex. (C) Cross-competition studies: specific binding (binding to pf-VEGFR-2 transfected cells minus mock transfected cells) P-2'/N-HRP (1.67 nM) complex is competed with 1.25 µM of P-2 (bar 4) and not competed with same concentration of P-X (bar 2) or P-1(bar 3). In bar 1, no peptide was added as a competitor. (D) Synergy between P-1 and P-2: mixed tetrameric complexes of P-1 and P-2 with NA-HRP (bars 3, 4 and 5) were better than pure tetrameric complexes (bars 1 and 2).

 
In our assay, 4P-1'/NA-HRP and 4P-2'/NA-HRP tetrameric complexes bound specifically to pf-VEGFR-2-transfected cells and did not bind to mock-transfected cells (Figure 2B). The control 4P-X'/NA-HRP tetrameric complex did not bind to either pf-VEGFR-2 or mock-transfected cells (Figure 2B). If the assay was carried out in two steps, incubation of biotinylated peptide followed by incubation with neutravidin-HRP, no binding was detected with moderate-affinity peptides such as P-1, P-2 and P-4, indicating that multivalency was critical to achieve detectable binding in the assay (data not shown). We also used this assay to evaluate the ability of peptides to compete (Figure 2C) and to exhibit binding synergy (Figure 2D). In the cross-competition assay, the specific binding (binding to pf-VEGFR-2 transfected cells minus binding to mock-transfected cells) of 4P-2'/NA-HRP was competed with P-2 peptide and was not competed with P-1 or P-X peptide (Figure 2C). Similarly, the specific binding of 4P-1'/NA-HRP complex was competed with P-1 peptide and was not competed with P-2 or P-X peptide (data not shown). This suggested that P-1 and P-2 bind to two different epitopes of VEGFR-2.

To determine whether P-1 and P-2 bind VEGFR-2 synergistically, we evaluated the binding of mixed tetramers of P-1 and P-2 peptides (2P-1'/2P-2'/NA-HRP) along with pure tetrameric complexes of P-1 or P-2 (4P-1'/NA-HRP or 4P-2'/NA-HRP) as shown in Figure 2D. Mixed tetramers were created by adding 2 mol equiv. of P-1' and 2 mol equiv. of P-2' to 1 mol equiv. of NA-HRP. We found that the mixed tetramer showed much higher binding than pure tetrameric complexes (Figure 2D). Mixed tetramers of P-1 and P-2 in different ratios (3:1, 1:3) also gave higher binding than either pure tetramer (Figure 2D). This suggested that P-1 and P-2 are able to bind synergistically (at least on NA-HRP) to VEGFR-2 and, based on the fact that synergism is seen in each of the 3:1 complexes where only one copy of a peptide is present, may be suitable candidate sequences to use in preparing a synthetic heterodimer. This strategy was also used to identify additional peptide pairs suitable for heterodimerization.

Chemical synthesis and testing of heterodimers

We used standard solid-phase peptide synthesis to prepare the monomers and a novel linking strategy using glutaric acid bis-N-hydroxysuccinimide to prepare dimers (Pillai et al., 2004Go). In this method, a monomeric peptide was reacted with an excess of glutaric acid bis-N-hydroxysuccinimidyl ester, which produced complete monoacylation. After concentration, the excess bis-NHS ester was removed by trituration with ethyl acetate. The monoacylated peptide was coupled to the second peptide and proceeded to completion. We prepared two heterodimers composed of promising peptide pairs (identified using the neutravidin-HRP assay) and two homodimers (Figure 3A). When the binding of these dimers to human VEGFR-2/Fc was measured by SPR, D-1 with a KD of 0.6 nM (a heterodimer of P-1 and P-2) showed >100-fold greater affinity for VEGFR-2/Fc than the best monomer (P-1 with a KD of 69 nM) used in synthesizing D-1. Similarly, D-2 with a KD of 0.5 nM (a heterodimer of P-3 and P-4) bound with about a 6-fold lower KD than the best component peptide (P-3 with a KD of ~3 nM). At the same time, neither of two homodimers (D-3 and D-4, Figure 3A) showed any improved binding over their monomeric counterparts (P-3 and P-4, Figure 1B). These data suggest that heterodimers comprising non-competitive and synergistic peptide pairs will often be high affinity peptides (HAPs).



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Fig. 3. Evaluation of heterodimers and homodimers in multiple binding and functional assays. (A) Synthetic heterodimers (D-1 and D-2) and homodimers (D-3 and D-4) with their KD values. (B) Competition of peptides with 125I-VEGF for binding to human VEGFR-2 in HUVEC cells. (C) Competition of peptides with human VEGF to block VEGF-dependent phosphorylation of VEGFR-2 in HUVEC cells. (D) Competition of peptides with VEGF to block VEGF dependent migration of HUVEC cells.

 
To assess further the utility of these HAPs, we compared the heterodimer (D-2) with its corresponding homodimers (D-3 and D-4) and monomers (P-3 and P-4) for their ability to compete with VEGF (a natural ligand of VEGFR-2) in several binding and functional assays (Figure 3B–D). In the first assay, we tested the ability of D-2 to compete with 125I-labeled VEGF for binding to HUVECs. In this assay, D-2 with an IC50 of 3 nM was at least 130-fold more potent in its ability to compete with 125I-labeled VEGF than the best monomer (P-3 with an IC50 of 400 nM) or the best homodimer (D-4 with an IC50 of 450 nM) (Figure 3B). It was observed that all of the 125I-VEGF counts could not be competed off, probably owing to the presence of VEGF receptors other than VEGFR-2 (e.g. VEGFR-1 and neuropilin-1) in HUVECs. The IC50 values in Figure 3B are based on 125I-VEGF counts (CPM) that could be specifically competed with the peptides. It is interesting that homodimer D-4 was better than monomer P-4, whereas D-3 was not significantly different from P-3. In the NA-HRP assays, we observed a similar multivalency advantage for P-4 peptide but no advantage for P-3 peptide (data not shown). Based on these results, the NA-HRP assay may also be used to predict the behavior of homodimers before chemical synthesis. This difference in the homodimers of P-3 and P-4 is probably a result of how they interact with receptor molecules.

Similarly, we found that D-2 was more potent than the corresponding homodimers in its ability to block VEGF-dependent phosphorylation of VEGFR-2 in HUVECs (Figure 3C) and the IC50 of 0.5 nM for D-2 vs 100 nM for the best homodimer suggest that the difference in potency is at least 200-fold (data not shown). Lastly, we compared the ability of D-2 and the highest affinity monomeric peptide, P-3, to block VEGF-dependent endothelial cell migration. In this assay, the IC50 for the heterodimer was about 1 nM, while 800 nM of the monomeric peptide was required to produce the same inhibition (Figure 3D). Similar results were obtained using the D-1 heterodimer (data not shown). This suggests that HAPs (D-1 and D-2) are not only better binders of VEGFR-2 than either of their corresponding monomers or homodimers, but are also better inhibitors of VEGF binding and the VEGF-induced activation of VEGFR-2. The enhanced ability of the HAPs to block VEGF binding and receptor activation was of a much greater magnitude than the enhancement in binding affinity. This may relate to their slower off-rates and/or an increased ability to hinder sterically VEGF binding by virtue of their larger size.

In addition to the experiments presented here, we observed that VEGFR-2-targeted HAPs potently inhibit HUVEC in vitro tubule formation and proliferation (data not shown).

HAPs against c-Met

To test the generality of our approach, we created HAPs against another RTK, c-Met. Using peptide phage display, we first identified a number of low-affinity phage binders (p-1 to p-7) to the human c-Met/Fc fusion protein (Figure 4A). Subsequently we chemically synthesized the binding peptides coded for by the phage and determined their KD values by FP (Figure 4A). This was followed by cross-competition studies (data not shown) and the final generation of a high-affinity heterodimer (Dcm comprising p-2 and p-4) against c-Met as shown in Figure 4B. In this case, Dcm had at least a 250-fold lower KD than the best monomeric component peptide (p-2) used in synthesizing Dcm. This suggests that our strategy can be used as a general approach to generate HAPs to the extracellular domains of RTK proteins.



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Fig. 4. Identification of HAPs binder of c-MET. (A) Human c-Met/Fc binding peptide sequences (with KD below 1 µM) identified using phage display. Binding of chemically synthesized peptides was confirmed and KD values were obtained using FP assay with human c-Met/Fc. (B) Based on cross-competition studies, Dcm (a heterodimer of p-2 and p-4) was synthesized with a KD value of 0.8 nM in an SPR assay.

 

    Discussion
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 Abstract
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 Materials and methods
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 Discussion
 References
 
We have described a general strategy to create low molecular weight high-affinity peptides (HAPs) against the extracellular domains of two RTKs, VEGFR-2 and c-Met (Figure 5A). Significantly, we found that HAPs against VEGFR-2 were much better competitors of the natural ligand than either of the starting monomeric peptides or homodimers of those peptides in various competitive binding and functional assays. HAPs against VEGFR-2 and c-Met were also very specific in their binding. No non-specific binding of HAPs to several receptors, including the VEGF receptors VEGFR-1 and neuropilin-1, were observed (A.Shrivastava, N.Bogdan and M.A.von Wronski, unpublished results). Similarly, HAPs against c-Met did not bind to two unrelated Fc fusion proteins VEGFR-3/Fc and TRAIL-R4/Fc (A.Shrivastava, unpublished results). These HAPs are much smaller than antibodies or antibody fragments and possess an affinity comparable to that of the natural ligands (or antibodies) of RTKs. Therefore, HAPs offer significant advantages over antibodies or scFvs in RTK targeting. More specifically, HAPs against VEGFR-2 would be useful in blocking VEGFR-2 on both the luminal and abluminal sides of tumor endothelial cells (Feng et al., 2000Go). HAPs against VEGFR-2 carrying radiotherapeutic isotopes or toxins would also likely be more effective in blocking tumor angiogenesis and destroying tumor blood vessels than similarly conjugated antibodies. HAPs against c-Met, on the other hand, would bind directly to the surface of c-Met positive tumor cells and therefore could be useful in directly damaging the tumors and inhibiting their metastasis to other organs (Danilkovitch-Miagkova and Zbar, 2002Go). In addition, we found that HAPs were much more resistant to serum effects than monomeric peptides. We found that 50% of monomeric P-3'/NA-HRP (1 equiv. of P-3' per NA-HRP) complex binding was lost in the presence of serum as compared with no change in monomeric D-2'/NA-HRP complex binding (A.Shrivastava, unpublished results). To explore further the serum sensitivity of HAPs, we synthesized a 125I-labeled D-2 dimer analog and found no difference in its binding to pf-VEGFR-2 transfected 293 H cells with or without 40% rat serum (A.Shrivastava and M.A.von Wronski, unpublished results).



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Fig. 5. Strategy for HAPs identification and possible mechanisms of HAPs interaction with target. (A) Multistep process to develop HAPs involves identification of low-affinity peptide binders using phage display, identification of appropriate pairs of peptides that are non-competing and show synergistic binding and finally chemical synthesis of the heterodimers. (B) Two possible mechanism for interaction of HAPs with a target: (i) two HAPs interact with a dimer of target molecules; (ii) single HAPs interact with a single target molecule.

 
There are at least two possible mechanisms for HAPs to interact with target RTKs (Figure 5B). First, each HAP molecule may interact with two different epitopes on two target receptors (Figure 5B, i). However, this mechanism does not explain the increased potency of the heterodimers over the homodimers in the VEGFR-2 functional assays. In addition, this mechanism does not explain how HAPs may be interacting with VEGFR-2 present on HUVEC cell membranes (which is mostly monomeric in the absence of VEGF ligand). Thus, an alternative mechanism with a single HAP molecule interacting with two different epitopes of a single target molecule is more likely (Figure 5B, ii).

In addition to our findings, two different studies utilizing heterodimerization to generate high-affinity binding molecules (Schaffer et al., 2003Go; Melkko et al., 2004Go) have recently been reported. In the first report, agonists and antagonists of the insulin receptor (IR) were created by homo- and heterodimerization of two previously reported peptide binders to the IR (Schaffer et al., 2003Go). The authors proposed, however, that their constructs cross-link separate receptor molecules. In the second report, Melkko et al. identified a heterodimeric compound with a 12 nM KD from monomeric non-peptide structures that had 430 nM and non-measurable KDs, respectively (Melkko et al., 2004Go). All these approaches point to the tremendous potential benefits of generating multivalent ‘heteromolecular’ binding compounds as a new generation of therapeutic and diagnostic reagents.

In conclusion, our approach of generating heterodimers of peptides with affinity for two distinct, non-competing epitopes on a single target molecule allowed us to create high-affinity peptide binders to two different RTKs (VEGFR-2 and c-Met), indicating that the approach has broad utility. The large extracellular domains of the two receptors may have facilitated this strategy. Binding to adjacent, but non-overlapping, epitopes on a single target molecule provides the benefits of multivalent binding without the strong dependence on target density that limits multimers of a single binding moiety. We would expect that for some highly abundant target molecules heterodimers might have somewhat less of an advantage over homodimers (or higher order multimers), which can bind in a multivalent manner to closely spaced receptor molecules. In general, we believe, our approach will improve the usefulness of peptides, at least as RTK-targeting therapeutics, and likely for other target types also.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received March 4, 2005; revised July 13, 2005; accepted July 16, 2005.

Edited by Laurent Jespers