A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelial growth factor to its kinase domain receptor.

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis. The VEGF receptor KDR/Flk-1 (a kinase domain receptor) mediates various biological activities of VEGF related to proliferation, differentiation, and migration of endothelial cells. Here we present a novel peptide designated K237-(HTMYYHHYQHHL), which was isolated from a phage-displayed peptide library, binding to KDR with high affinity and specificity. By interfering with the VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF(165) in a dose-dependent and cell type-specific manner. The peptide also exerted an anti-angiogenesis activity in vivo as revealed using the chick embryo chorioallantoic membrane angiogenesis assay. Moreover, the peptide K237 significantly inhibited the growth of solid tumors implanted beneath the breasts and their metastases to lungs in severe combined immunodeficient mice. Taken together, these findings suggest that the peptide K237 can functionally disrupt the interaction between VEGF and the KDR receptor and cause potent biological effects that include the inhibition of angiogenesis and tumor growth. As a consequence, this peptide (and its future derivatives) may have use as a potential cancer therapy.

Neovascularization is critical for supporting the rapid growth of solid tumors beyond 1-2 mm in diameter (1) and for tumor metastasis (2). The generation of new capillaries involves a multistep process involving dissolution of the membrane of the originating vessel, endothelial cell migration and proliferation, and formation of a new vascular tube (3)(4)(5). Suppression of any one of these steps would inhibit the formation of new vessels and would therefore affect tumor growth and metastasis.
Tumor angiogenesis appears to be achieved by the expres-sion of angiogenic agents within solid tumors that stimulate host vascular endothelial cell mitogenesis and possibly chemotaxis. So far, several angiogenic factors have been identified (6) including the particularly potent vascular endothelial growth factor (VEGF), 1 which acts as an endothelial cell-specific mitogen and plays important roles during tumor angiogenesis (7)(8)(9). Inhibition of VEGF using specific antagonists such as VEGFspecific monoclonal antibodies can therefore result in the blockade of tumor growth and metastasis in experimental animal model systems (10 -12). Inhibition of tumor implantation and growth is also achieved by the expression of antisense to VEGF (13)(14)(15) and by the intracellular expression of dominant negative KDR receptors (16). Receptor-blocking monoclonal antibodies (11) or suppression of VEGF expression (12) can also result in the regression of established tumors. As a result, VEGF antagonists have been believed to have significant potential in the context of cancer therapy. The screening of phage-displayed libraries is a powerful technique for identifying peptides with desirable biological or physical properties, particularly when it is combined with iterative cycles of phage selection and amplification (17). New agonists and antagonists for cell membrane receptors have been successfully identified using this process (18), and examples include the RGD-containing peptides that bind to specific cell surface integrins and inhibit integrin-mediated cell adhesion (19,20). Peptide display libraries have also been used to derive a peptide-(ATWLPPR), which specifically inhibited human endothelial cell proliferation in vitro and totally abolished VEGFinduced angiogenesis in vivo (21). Thus, phage-displayed technology has been shown to be an effective to the identification of novel peptides that may inhibit cell adhesion or angiogenesis.
In this study, we attempted to identify peptides that might block the binding of VEGF to KDR. A random 12-mer peptide library displayed on the surface of filamentous phage (M13) was screened by biopanning against the extracellular domain of KDR. This led to the isolation of peptide K237-(HTMYYH-HYQHHL), which not only competed with VEGF binding to KDR but also specifically inhibited human endothelial cell proliferation in vitro. Moreover, this peptide inhibited angiogenesis in the chick embryo choroallantoic membrane (CAM) angiogenesis assay and significantly reduced the tumor growth and metastasis in severe combined immunodeficient (SCID) mice. for amplifying KDR cDNA coding the immunoglobulin (Ig) similar to domains I-IV (sense, 5Ј-AAGGGATCCCCCAG-GCTCAGCATACAA-3Ј, and antisense, 5Ј-GGCGAATTCGGGTGGGA-CATACACAAC-3Ј)  Expression and Purification of KDR-The cDNA coding the Ig-like domains I-IV of the VEGF receptor KDR (22,23) was obtained from human umbilical vein endothelial cells (HUVECs) by reverse transcription-PCR according to manufacturers' instructions (Invitrogen). The amplified cDNA was then cloned into glutathione S-transferase (GST) expression vector pGEX-2T (Amersham Biosciences) and sequenced. Escherichia coli JM109 competent cells were transformed with pGEX-2T-KDR, cultured in Luria-Bertani (LB) medium supplemented with 60 g/ml ampicillin, and induced with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside at 37°C to allow expression of fusion protein GST-KDR. The pellets from 200 ml of induced bacteria were ultrasonicated on ice in 2 ml of TE buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA) and then centrifuged at 12,000 rpm for 10 min at 4°C. The resulting precipitate was resuspended in 2 ml of 2% sodium deoxycholate and incubated for 30 min at room temperature followed by centrifugation at 10,000 rpm for 10 min at 4°C. The pellet was then resuspended in 1 ml of denaturation buffer (10 mM dithiothreitol, 50 mM Tris-HCl, pH 8.5, 8 M urea) and shaken gently for 3 h at room temperature. Soluble GST-KDR (sKDR) was obtained by gradually diluting the denatured protein with 8 -10 ml of refolding buffer (5 mM reduced glutathione, 2 mM oxidized glutathione, 5 mM EDTA, 50 mM Tris-HCl, pH 8.5) and shaking for 4 h at room temperature. The supernatant containing the sKDR was analyzed by SDS-PAGE and shown to correspond to a single detectable protein species as determined by Coomassie Brilliant Blue staining.

Materials-Primers
Assay of VEGF Binding to sKDR-For direct binding of [ 125 I]VEGF, the sKDR (0.2 g/liter, 0.01 M phosphate buffer, pH 6.2) was coated on 96-well plates (50 l/well) and kept at 4°C overnight. The wells were washed with washing buffer (0.9% NaCl, 0.01 M phosphate buffer, pH 6.2) and then blocked with 0.3% bovine serum albumin, 25 mM HEPES, pH 7.6, for 3 h at room temperature. [ 125 I]VEGF at different concentrations (0, 2, 4, 6, and 8 ng/ml) was added to wells followed by incubation at room temperature for 2 h. After washing with the blocking buffer, bound radioactivity was measured by a ␥-counter (Beckman 2000, Fullerton, CA).
The competitive binding of [ 125 I]VEGF to both immobilized and free sKDR was also determined. sKDR was coated onto 96-well plates as described above. [ 125 I]VEGF (8 ng/ml, determined as the saturation point from the direct binding assay) was then mixed with sKDR at different concentrations and added to the sKDR-coated wells. After 3 h at room temperature, the wells were washed and [ 125 I]VEGF bound to coated KDR was measured.
Screening a 12-mer Phage Display Library with Soluble KDR-The procedure for screening the phage-displayed library was modified according to instructions of the manufacturer of the kit (New England Biolabs, Beverly, MA). The cell culture dishes (60-mm-diameter) were coated with GST-p21, GST-VEGF, GST-Flt-1 (these fusion proteins were produced by our laboratory), or GST-KDR. All proteins were added at 100 g/ml in 3-ml volume/dish and incubated at room temperature for 2 h prior to blocking with phosphate buffer containing 1% bovine serum albumin overnight at 4°C. The phage library containing 10 12 clones was sequentially added to non-bait-coated dishes (those coated with GST-p21, GST-VEGF, and GST-Flt-1) for preabsorption. In each case, the library was shaken gently at room temperature for 1 h. Finally the preabsorbed library was applied to sKDR-coated dishes for specific screening. After thorough washing, plate-bound phage clones were eluted with elution buffer (0.22 M Gly-HCl, pH 2.2) and neutralized immediately. Four rounds of selection were performed, after which individual plaque was picked up at random and subjected to analysis by phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing following amplification in E. coli ER2537.
Phage ELISA Using HUVEC-Human umbilical cords were digested with 0.1% collagenase II, and the HUVECs were collected and grown in RPMI 1640 medium supplemented with 20% FCS. The control cells (NIH-3T3) were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS. For phage ELISA assays, HUVECs and NIH 3T3 cells were seeded in 96-well plates at a density of 3 ϫ 10 3 cells/well. After an overnight incubation, cells were fixed with ice-cold glutaraldehyde (0.125%) in phosphate-buffed saline for 10 min at room temperature and then washed with phosphate-buffed saline. Phosphate-buffed saline containing 3% bovine serum albumin was used to block the plates by overnight incubation at 4°C. After blocking, phages (5 ϫ 10 12 pfu/ml) were added to the plates and incubated for 2 h at room temperature. Wells were then washed 10 times with Tris-buffered saline, pH 7.5, containing 0.1% Tween 20, and bound phage were detected by ELISA using a horseradish peroxidase-conjugated anti-M13 monoclonal antibody.
Competition Assay of Positive Phages and Synthesized Peptides-sKDR was immobilized on 96-well plates and blocked as described above. Seven different phage clones, which were selected by the above phage ELISA, were then incubated with the sKDR-coated plates for 1 h at room temperature at a concentration of 10 13 pfu/ml. After this preincubation, [ 125 I]VEGF (2 g/liter, 50 l) was added directly to the wells without the removal of the phage. An additional 2-h incubation at room temperature was then performed followed by thorough washing. Bound radioactivity was then counted using a liquid scintillation counter (Wallac Co., Turku, Finland). For the two phage clones, which exhibited the most promising results, synthetic peptides were generated that corresponded to the encoded displayed peptide motifs. These peptides were then used in the competition assay with [ 125 I]VEGF (see above).
HUVEC Proliferation Assay-HUVECs were seeded into 24-well plates at a density of 3 ϫ 10 4 cells/well in RPMI 1640 medium supplemented with 20% FCS (NIH 3T3 cells were seeded at a density of 1 ϫ 10 4 cells/well in Dulbecco's modified Eagle's medium supplemented with 10% FCS). After 24 h, the medium was replaced with RPMI 1640 containing 4% FCS (for NIH 3T3 cells, the medium was replaced with Dulbecco's modified Eagle's medium containing 0.4% FCS). Cells were cultured for another 24 h and then incubated in the presence of rhVEGF 165 (2 ng/ml) together with peptide K237 or control peptide at various concentrations (0, 50, 100, 200, and 300 M). 48 h later, [ 3 H]thymidine was added to the cells, and after a 6-h incubation at 37°C, the cells were washed, harvested, and analyzed for their incorporation of [ 3 H]thymidine using a scintillation counter. NIH 3T3 cells were used as the control in this experiment.
Angiogenesis Assay-The CAM angiogenesis assay was performed as described previously with some modifications (24,25). After the fertilized eggs had been incubated for 3 days and then opened, the embryos were incubated in Petri dishes (100-mm-diameter) at 37°C with 100% humidity. After 3 days, round glass cellulose filters (3-mm-diameter) soaked with either 10 l of rhVEGF 165 (2 ng/ml) alone or with 10 l of rhVEGF 165 (2 ng/ml) plus 20 l of peptide K237 or control peptide at various concentrations (0.1, 0.5, 1.0, and 5 M) were placed on the surface of CAM. Filters were replaced daily with new filters with the fresh rhVEGF 165 and peptide until angiogenic response peaked. CAMs were examined and documented by photomicroscope.
Experiments of Solid Tumor Growth and Metastasis in Mice-A SCID mouse model was used to further investigate the effects of K237 on tumor growth and metastasis. For this purpose, BICR-H1 cells (1 ϫ 10 7 ) were injected beneath the breasts of SCID mice (BICR-H1 cells correspond to a breast carcinoma cell line, which has the ability to generate spontaneous lung metastasis). On the next day, the peptide K237 and control peptide were injected (60 l, 0.5 mM, respectively), around tumor sites. Peptide injections continued every 2 days for a period of 3 weeks, and tumor size was measured every 3 days with Vernier caliper. Tumor volume was calculated by the following formula: /6 ϫ (long diameter) ϫ (short diameter) 2 . On the 48th day of post-cell injection, mice were sacrificed. Nodules formed by implanted tumors at injection sites were isolated and then weighed. The lungs were also removed, fixed with 10% formalin, embedded in paraffin, cross-sectioned, stained with hematoxylin and eosin, and analyzed microscopically for the presence of tumor metastasis. Statistical significance between test and control groups was determined using the Student's t test (26).

RESULTS
Binding of sKDR to [ 125 I]VEGF-Total RNA was extracted from HUVECs, and a cDNA fragment encoding the extracellular Ig-like domains I-IV of KDR was amplified by reverse transcription-PCR and cloned into vector pGEX-2T. After sequence analysis of the inserted cDNA, the GST-KDR fusion protein was expressed in E. coli JM109. After denaturation and refolding, the purity of the prepared sKDR was estimated to be 85-90% as determined by SDS-PAGE (data not shown).
sKDR was then examined for its ability to bind to [ 125 I]VEGF using a solid-phase binding assay. This analysis revealed that the binding of sKDR became saturated when [ 125 I]VEGF was at the concentration of 8 ng/ml, indicating a specific interaction between the soluble receptor and the ligand (Fig. 1A). The binding of [ 125 I] VEGF to immobilized sKDR was reduced when free sKDR was added in competition binding experiments (Fig.  1B).
The Isolated Clones Binding to KDR-The sKDR was used to develop receptor antagonists. To do this, a random 12-mer peptide library composed of 2 ϫ 10 9 independent phage clones was screened through biopanning against plate-bound sKDR. The sKDR-binding phage population became enriched over the course of four cycles of biopannings, and after the fourth round of selection, roughly 40% of the phage clones analyzed exhibited sKDR binding activity (125 of 300 clones that were ana-lyzed; data not shown). Of the 125 sKDR-binding clones that were identified, eight clones were able to bind specifically to plate-immobilized sKDR and HUVECs ( Fig. 2A). Sequences of the displayed peptides that were encoded by these phage clones were determined through sequencing of the phage DNA (the DNA sequence of peptide K93 was not available because of the quality of the sequencing gel) (Table I). A multiple alignment analysis on all selected sequences was performed using the DNAStar sequence analysis package (DNAStar, Inc. Madison, WI) to identify possible consensus motifs, which might be responsible for binding to KDR. No such consensus sequences could be identified, but it was noted that all of the selected peptides contained one or multiple histidines and that all of the peptides were rather hydrophobic.
In light of the ability of the selected peptides to bind sKDR, we examined the ability of these peptides to block the interaction between KDR and its ligand, VEGF. To do this, a fixed amount of phage particles (10 13 pfu/ml) was added to sKDRcoated wells, and its ability to block the binding of [ 125 I]VEGF was then examined. This analysis revealed that phage-K237 could inhibit the binding of [ 125 I]VEGF to sKDR (Fig. 2B), whereas phage-K93 inhibited the interaction to a somewhat FIG. 1. sKDR binds to VEGF specifically. A, solid-phase binding assay of [ 125 I]VEGF binding to sKDR. Purified sKDR or GST alone (GST as control) was absorbed onto the surface of 96-well plates (50 l/well, 2 g/ml), and various amounts of [ 125 I]VEGF were then added to the wells (as indicated). The plates were incubated for 3 h at room temperature and washed extensively, and bound radioactivity was then quantified using a ␥-counter. The data presented are the means of three independent experiments, and the error bars indicate mean Ϯ S.D. B, competition binding assay using [ 125 I]VEGF and free sKDR. Plates were coated with sKDR as described in A, and [ 125 I]VEGF (8 ng/ml) was then mixed with various concentrations of sKDR prior to addition to wells and incubation for 3 h at room temperature. After extensive washing, the bound radioactivity was then measured. The data represent the mean Ϯ S.D. of three independent assays.

FIG. 2. Characterization of binding activity of selected phagedisplayed clones.
A, individual phage clones (5 ϫ 10 12 pfu/ml), which had been selected on the basis of their ability to bind to immobilized recombinant sKDR, were added to HUVEC monolayers and incubated for 2 h at room temperature. After washing, the cell-bound phage was then detected using a horseradish peroxidase-conjugated anti-M13 monoclonal antibody (wild-type M13 phage particles were used as negative control in this assay). The data presented are the mean Ϯ S.D. of three independent experiments. B, individual sKDR-binding phage clones were tested for their ability to compete with [ 125 I]VEGF for binding to immobilized sKDR. Phages were added to sKDR-coated wells in a 96-well plate at a concentration of 10 13 pfu/ml. After 1-h incubation with phage, [ 125 I]VEGF was added to the wells, and bound radioactivity was then measured after an additional 2-h incubation and extensive washing. The data are the mean Ϯ S.D. of three independent experiments. lesser extent; the other clones did not interfere with [ 125 I]VEGF binding to sKDR.
Peptide K237 Blocks the Interaction Between VEGF and KDR-Based on the results of the competition assay, phage clone K237 was chosen for further study. The phage-encoded peptide was synthesized and used in an in vitro competition assay as described above. This analysis revealed that the peptide K237 could antagonize the binding of [ 125 I]VEGF to sKDR in a dose-dependent manner (Fig. 3).
Peptide K237 Specifically Inhibits the Proliferation of HUVEC-It is well known that VEGF stimulates the proliferation of endothelial cells through the KDR receptor. Because peptide K237 had the ability to block interaction between VEGF and KDR, we therefore decided to test whether peptide K237 might also suppress endothelial cell proliferation in response to rhVEGF 165 . As shown in Fig. 4, peptide K237 significantly suppressed the mitogenic response of HUVEC to rhVEGF 165 in a dose-dependent manner. Cellular proliferation was reduced by Ͼ90% when peptide K237 was present at a concentration of 300 M, whereas an equivalent concentration of the control peptide had no significant effect on the cell growth (Fig. 4).
Using the same in vitro proliferation assay, we also examined the effect of K237 on NIH 3T3 cells to confirm the specificity of the growth inhibition that we observed in HUVECs treated with rhVEGF 165 . This analysis revealed that the proliferation of NIH 3T3 cells was not modified by peptide K237 (data not shown).
Peptide K237 Inhibits Vascularization of CAM-The effect of peptide K237 on angiogenesis was tested by a CAM vascularization assay. For this experiment, a round glass cellulose filter was impregnated with rhVEGF 165 alone or with rhVEGF 165 plus either peptide K237 or the control peptide at varying concentrations. These filters were then directly applied to the CAM, and angiogenesis was measured 3 days later. This experiment revealed that vascularization around the treated sites was significantly reduced in CAMs that received rh-VEGF 165 plus peptide K237 (5 M) versus CAMs that received rhVEGF 165 plus the control group or rhVEGF 165 alone (Fig. 5).
Peptide K237 Suppressed Tumor Growth and Metastasis in Vivo-To determine the effect of peptide K237 on tumor growth and metastasis in vivo, SCID mice were injected subcutaneously with human breast carcinoma cells (BICR-H1 cell line). The tumor sites were subcutaneously subjected to injections with peptide K237 or control peptide every 2 days, and the growth of the tumors was then assessed over time. The results of this analysis showed that there was a significant (70%) reduction in the weight of implanted tumors (p Ͻ 0.05) that were injected with peptide K237 versus the control group whose five-tumor weight was 1.56 g as measured at sacrifice (the 48th day following implantation of the tumor cells) (Fig. 6, panels 1  and 2).
The SCID mouse lungs were also examined at sacrifice by histological staining and microscopic inspection. The mean numbers of metastasized nodules per mouse were calculated from three inspected cross-lung sections. The results revealed that all animals injected with the control peptide developed intrapulmonary metastatic tumor nodules (identified as clusters of over five tumor cells), and there were 16 tumor nodules in total (Fig. 6, panel 3, C and D). In contrast, only three of the five animals injected with the peptide K237 developed detectable pulmonary nodules, and seven nodules could be observed in total (Fig. 6, panel 3, A and B). Based on the number of the pulmonary nodules per mouse in the two treatment groups, peptide K237 was determined to reduce the total number of metastasis by 53% (p Ͻ 0.05) as compared with the control peptide.

DISCUSSION
Previous studies have demonstrated that antiangiogenic therapy is a promising approach for the treatment of cancer (1,2), in part because solid tumor growth and metastasis are dependent on tumor vascularization. Furthermore, it has been shown that blocking the interaction between VEGF and its receptor can result in the regression of murine and human tumors (8). We report here the identification of a VEGF peptide The phage-display peptide library was subjected to four rounds of biopanning against plate-immobilized recombinant GST-KDR fusion protein (sKDR). Individual phage clones selected by this procedure were then analyzed for their ability to bind to immobilized sKDR and to HUVEC monolayers in a phage-ELISA assay. This resulted in the identification of eight individual phage clones, which scored positively in all of these assays. The sequences of seven of these clones are shown below. antagonist that we identified by screening a phage-displayed peptide library.
Direct screening of phage-displayed peptide libraries with soluble receptors is a relative new area of research, and there are only limited reports of such analysis in the literature. A major challenge is the removal of clones with nonspecific binding properties. In this study, we performed three rounds of negative selection to eliminate nonspecific binding clones using three recombinant fusion proteins (GST-p21, GST-VEGF, and GST-Flt-1), which contained the GST moiety that was fused to our bait protein (GST-KDR). This resulted in the depletion of non-KDR-specific binding clones from the phage library. To isolate specific VEGF antagonists, the phage-displayed library was then subjected to four rounds of biopanning against platebound sKDR. 125 individual clones, which exhibited the ability to bind to sKDR, were then tested in a phage ELISA assay using HUVECs as targets. This resulted in the identification of eight individual phage clones, which could bind specifically both to recombinant sKDR and to primary HUVECs. Sequence analysis revealed that eight clones were histidine-rich and hydrophobic, but no clear conservative motif was identified. Furthermore, attempts to align the individual selected peptide sequences with the primary sequence of the VEGF did not identify any homology. However, it remains possible that one or more of the peptides might be a structural mimic of a discontinuous or conformational epitope with VEGF. This is potentially important because Muller et al. (27) have recently solved the crystal structure of VEGF and have mapped the region responsible for KDR binding to a series of discontinuous residues.
When the eight selected phage clones were analyzed for their ability to successfully compete with [ 125 I]VEGF for binding to immobilized sKDR, only two clones showed detectable activity. The most efficient one of these, peptide K237 HTMYYH-HYQHHL was selected for further analysis. This peptide was not only capable of specific binding to sKDR and to HUVECs, but it also inhibited the VEGF-induced proliferation of HUVECs. However, it did not modify the growth of a fibroblast cell line NIH 3T3, indicating that its inhibitory effect on HUVEC proliferation is not a result of nonspecific cellular cytotoxicity but an effect of K237 blocking the interaction between VEGF and its receptor KDR. Finally, peptide K237 was able to inhibit the growth and metastasis of a breast carcinoma cell line in SCID mice. This suggests that this peptide as a VEGF antagonist might have potential application for the treatment of a variety of cancers. The small size of this peptide also offers the possibility of generating structurally similar molecules via standard organic synthesis. This could result in the production of inexpensive orally available drugs analogous to the RGD-peptidomimetic compound SCH221153, which binds to both ␣ v ␤ 3 and ␣ v ␤ 5 integrin receptor and inhibits angiogenesis and tumor growth (28).
In summary, our results demonstrate that the peptide K237 FIG. 6. K237 inhibits tumor growth and metastasis in vivo. Panel 1, BICR-H1 breast cancer cells were injected beneath the breasts of SCID mice (5 mice/experimental group). Starting on the following day, peptide K237 or control peptide was introduced into the tumor site by subcutaneous injection. These peptide injections continued every other day for 3 weeks. The mice were sacrificed on the 48th day following initial implantation of the tumor cells, and tumor nodules were isolated and weighed (tumor size was measured every 3 days with Vernier caliper). The lung metastases were also examined at sacrifice. A, photographs of mice treated with the control peptide (images were recorded at the time of sacrifice). B, photographs of mice treated with peptide K237 (images were recorded at the time of sacrifice). Panel 2, the tumor size in SCID mice treated with K237 or control peptide was measured every 3 days, and the volume was calculated. The data shown are the mean Ϯ S.D. of five individual mice. Data from the two treatment groups were also compared using Student's t test (p Ͻ 0.05). Panel 3, paraffin-embedded tissue sections from the lungs of sacrificed mice were stained with hematoxylin and eosin and examined microscopically. Images of representative lungs are shown. A and B, a normal lung (A) and a tiny metastatic nodule (B) from mouse treated with K237. C and D, various metastatic nodules in the lungs treated with control peptide.
FIG. 5. K237 inhibits VEGF-induced angiogenesis in chorioallantoic membranes. Chick embryos were monitored following the implantation of the CAM with glass cellulose filter papers that had been impregnated either with 10 l of rhVEGF 165 alone (2 ng/ml) (A) or with 10 l of rhVEGF 165 plus 20 l of control peptide (5 M) (B) or with VEGF plus the K237 peptide (C). Images are representative of results obtained using five similar chick embryos.
is an effective VEGF antagonist. It functioned as an inhibitor of angiogenesis in vivo and reduced tumor growth and metastasis in a small animal model system. Thus, this molecule (or its derivatives) may have utility in clinical applications that might include cancer biotherapy, a treatment of diabetic retinopathy and other angiogenic or proliferative disorders that involve endothelial cells.