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
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Abstract |
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Keywords: c-MET/HAPs/heterodimer/peptide/VEGFR-2
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Introduction |
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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, 2002; Pastan and Kreitman, 2002
; Thorpe, 2004
). 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, 2004
) or hematological malignancies (Reff et al., 2002
). 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, 1999
; Szardenings, 2003). There are several examples of multimerization to improve the affinity of peptides (Mourez et al., 2001
; Sadler and Tam, 2002
). 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., 2003). 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, 2003
; Ferrara et al., 2003
). 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., 1991
; Quinn et al., 1993
; Feng et al., 2000
). On the other hand, C-Met binds to hepatocyte growth factor, HGF or scatter factor (Bottaro et al., 1991
) 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, 2002
). 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.
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Materials and methods |
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Multiple cyclic peptide, or constrained loop, phage display libraries were prepared using standard phage methods (Sato et al., 2002; Fleming et al., 2004
) 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., 1998). 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., 2002; Burke et al., 2003
; Fleming et al., 2004
).
Chemical synthesis of peptides
Monomeric peptides and dimers were synthesized as reported (Pillai et al., 2004). 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., 2002; Burke et al., 2003
; Fleming et al., 2004
). 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 (25400 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. 11668019).
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 avidinSepharose (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 avidinSepharose 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, 10002500 Ci/mmol) to confluent HUVEC was carried out as described earlier (Bikfalvi et al., 1991) 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 immunoprecipitationwestern blotting procedure as described earlier (Lin et al., 1998), 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.
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Results |
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Peptide phage display is a common technique for identifying peptide binders to various targets (Hetian et al., 2002; Sato et al., 2002; Fleming et al., 2004
). 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., 2002
; Fleming et al., 2004
). 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., 2002
). The affinities of these peptides (KD values) were determined by fluorescence polarization (FP) assays (Sato et al., 2002
; Burke et al., 2003
; Fleming et al., 2004
) with fluorescein-labeled peptides and/or surface plasmon resonance (SPR) measurements (Malmqvist, 1999
) 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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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., 2002; Burke et al., 2003
; Fleming et al., 2004
).
Identification of peptides for heterodimerization
We used a novel assay based on the avidinbiotin 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, 1999). 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., 1987
; Alon et al., 1990
).
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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., 2004). 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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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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Discussion |
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In addition to our findings, two different studies utilizing heterodimerization to generate high-affinity binding molecules (Schaffer et al., 2003; Melkko et al., 2004
) 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., 2003
). 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., 2004
). 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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References |
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Received March 4, 2005; revised July 13, 2005; accepted July 16, 2005.
Edited by Laurent Jespers