MXPA98009945A - Useful methods and compositions for angiogene inhibition - Google Patents

Abstract

The present invention describes methods for the inhibition of angiogenesis in tissues using alphavbeta3 vitronectin antagonists and particularly for inhibiting angiogenesis in inflamed tissues and in tumor tissues and metastases using therapeutic compositions containing alphavbe antagonists.

Description

METHODS AND USEFUL COMPOSITIONS FOR THE INHIBITION OF ANGIOGENESIS FIELD-LCO-FIELD The present invention relates generally to the field of medicine, and relates specifically to methods and compositions for inhibiting tissue angiogenesis, which use avr3 receptor antagonists of vitronectin. Background Integrins are a class of cellular receptors known to bind extracellular matrix proteins, and therefore mediate cell-cell and cell-extracellular matrix interactions, referred to as adhesion events. However, although many integrins and ligands that bind to an integrin are described in the literature, the biological function of many of the integrins remains elusive. The integrin receptors constitute a family of proteins with shared structural characteristics of heterodimeric, non-covalent glycoprotein complexes, formed of OI and β subunits. It is now known that the vitronectin receptor, called by its original characteristic of preferential fixation to vitronectin, refers to three different integrins, designated vß., Vv3 and av5. Horton, Int. J. Exp. Pathol., 71: 741-759 (1990). avßi binds to fibronectin and vitronectin. c-vß3 binds to a wide variety of ligands, including fibrin, fibrinogen, laminin, thrombospondin, vitronectin, Willebrand factor, osteo-spinalin, and bone sialoprotein I. or; vß5 is fixed to vitronectin. The specific cell adhesion roles that these three integrins play in the many cell interactions in tissues are still under investigation, but it is clear that there are different integrins with different biological functions. One important recognition site in the ligand for many integrins is the tripeptide sequence of arginine-glycine-aspartic acid (RGD). The RGD is found in all ligands identified above for the vitronectin receptor integrins. This RGD recognition site can be mimicked by polypeptides ("peptides") that contain the RGD sequence, and these RGD peptides are known inhibitors of integrin function. It is important to note, however, that depending on the sequence and structure of the RGD peptide, the specificity of the inhibition can be altered to target specific integrins. For discussions of the RGD recognition site, see Pierschbacher et al., Nature, 309: 30-33 (1984), and Pierschbacher et al., Proc. Nati Acad. Sci., USA, 81: 5985-5988 (1984). Different RGD polypeptides of variable integrin specificity have also been described, in Grant et al., Cell, 58: 933-943 (1989), Cheresh et al., Cell, 58: 945-953 (1989), Aumailley et al. , FEBS Letts. , 291: 50-54 (1991), and Pfaff et al., J. Biol. Chem., 269: 20233-20238 (1994), and in U.S. Patents 4,517,686, 4,578,079, 4,589,881, 4,614,517, 4,661,111, 4,792,525 , 4,683,291, 4,879,237, 4,988,621, 5,041,380, and 5,061,693. Angiogenesis is a process of vascularization of tissue, which involves the growth of blood vessels in recent development within a tissue, and is also referred to as neovascularization. The process is mediated by the infiltration of endothelial cells and smooth muscle cells. It is believed that the process proceeds in any of three ways: vessels may sprout from previously existing vessels, de novo development of vessels may arise from precursor cells (vasculogenesis), or existing small vessels may enlarge in diameter. Blood and collaborators, Bioch. Biophvs. Acta., 1032: 89-118 (1990). It is known that vascular endothelial cells contain at least five integrins dependent on RGD, including the receptor (c.vß3 or? Fvß5) of vitronectin, the receptor (vßx) of collagen types I and IV, the receptor (o-aßx) of laminin, fibronectin-laminin / collagen receptor (oc3ß?) and fibronectin receptor (? 5ß?). Davis and collaborators, J. Cell. Biochem., 51: 206-218 (1993). It is known that the smooth muscle cell contains at least six RGD-dependent integrins, including Q-sßi, avß3 and o ^ -ßs- Angiogenesis is an important process in neonatal growth, but it is also important in the healing of lesions and in the pathogenesis of a wide variety of clinical diseases, including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, macular degeneration and retinal neovascularization and similar conditions. Reference is made to these clinical manifestations associated with angiogenesis as angiogenic diseases. Folkman et al., Science, 235: 442-447 (1987). Angiogenesis is generally absent in adult or mature tissues, although it does occur in the healing of lesions and in the corpeus leuteum growth cycle. See, for example, Moses et al., Science, 248: 1408-1410 (1990). It has been proposed that the inhibition of angiogenesis would be a useful therapy for restricting the growth of tumors. The inhibition of angiogenesis has been proposed by (1) inhibiting the release of "angiogenic molecules" such as bFGF (basic fibroblast growth factor), (2) the neutralization of angiogenic molecules, such as by the use of anti-ßbFGF antibodies, and (3) the inhibition of the response of endothelial cells to angiogenic stimuli. This latter strategy has received attention, and Folkman et al., Cancer Biolosy, 3: 89-96 (1992), have described many inhibitors of endothelial cell response, including the collagenase inhibitor, the total basic membrane movement inhibitors. , angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D3 analogues, alpha-interferon, and the like which can be used to inhibit angiogenesis. For additional proposed inhibitors, see- Blood et al., Bioch. Biophys. Acta., 1032: 89-118 (1990), Moses et al., Science, 248: 1408-1410 (1990), Ingber et al., Lab. Invest. , 59: 44-51 (1988), and U.S. Patents 5,092,885, 5,112,946, 5,192,744, and 5,202,352. None of the angiogenesis inhibitors described in the above references are directed to the inhibition of vß3. Peptides containing RGD that inhibit the? Vß3 receptor of vitronectin have also been described. Aumailley et al., FEBS Letts. , 291: 50-54 (1991), Choi et al., J. Vasc. Surs , 19: 125-134 (1994), Smith et al., J. Biol. Chem., 265: 12267-12271 (1990), and Pfaff et al., J. Biol. Chem., 269: 20233-20238 (1994). . However, the role of the avß3 integrin in angiogenesis has never been suggested or identified until the present invention. For example, Hammes et al., Nature Med., 2: 529-53 (1996) confirms the findings of the present invention. Specifically, the document shows that cyclic peptides including cyclic RGDfv, the structure and function of the latter of which has been previously described in the priority applications on which the present application is based, inhibited retinal neovascularization in a model of mouse retinal neovascularization induced by hypoxia. In a separate study that also supports the present invention, as well as priority requests, Luna et al., Lab. Invest. , 75: 563-573 (1996) described two peptides containing particular cyclic methylated RGDs, which were partially effective in inhibiting retinal neovascularization in the mouse model of ischemic oxygen-induced retinopathy. In contrast, the peptides of the present invention exhibit almost complete inhibition of neovascularization in the systems of the models described herein. Inhibition of cell adhesion in vitro, using immunospecific monoclonal antibodies for different OI or ß integrin subunits, has implicated avß3 in cell adhesion of a variety of cell types, including microvascular endothelial cells. Davis et al., J. Cell. Biol. , 51: 206-218 (1993). In addition, Nicosia et al., Am. J. Pathol., 138: 829-833 (1991), described the use of the RGD peptide GRGDS to inhibit in vitro the formation of "microvessels" of rat aorta cultured in collagen gel. . However, the inhibition of the formation of "microvessels" in vitro in collagen gel cultures is not a model for the inhibition of angiogenesis in a tissue, because the structures of the microvessels have not been shown to be the same as the shoots. capillaries, nor that the formation of microvessels in collagen gel culture is the same as neovascular growth within intact tissue, such as arthritic tissue, tumor tissue or diseased tissue where inhibition of angiogenesis For angiogenesis to occur, the endothelial cells must first be degraded and cross the basic membrane of the blood vessels in a manner similar to that used by the tumor cells during the invasion and formation of the metastasis. The inventors have previously reported that angiogenesis depends on the interaction between vascular integrins and extracellular matrix proteins. Brooks et al., Science, 264: 569-571 (1994). On the other hand, it was reported that programmed cell death (apoptosis) of angiogenic vascular cells is initiated by interaction, which would be inhibited by certain antagonists of the vascular avß3 integrin. Brooks et al., Cell, 79: 1157-1164 (1994). More recently, the inventors have reported that the binding of matrix metalloproteinase-2 (MMP-2) to the vitronectin receptor (? V5) can be inhibited by using the v5 antagonists, and thereby inhibiting the enzymatic function of the proteinase . Brooks et al., Cell, 85: 683-693 (1996). Apart from the studies reported in the present, the Applicants are not informed of any other demonstration that angiogenesis can be inhibited in tissue using cell adhesion inhibitors. In particular, others have never previously shown that the o-vß3 function is required for angiogenesis in a tissue, or that o-vß3 antagonists can inhibit angiogenesis in a tissue. Brief Description of the Invention The statement of the present invention demonstrates that tissue angiogenesis requires the avß3 integrin, and that avß3 inhibitors can inhibit angiogenesis. The statement also demonstrates that antagonists of other integrins, such as o-IIbβ3, or oívβ1, do not inhibit angiogenesis, presumably because these other integrins are not essential for angiogenesis to occur. The invention, therefore, describes methods for the inhibition of angiogenesis in a tissue, which comprises administering to the tissue a composition comprising an angiogenesis inhibiting amount of an avß3 antagonist. The tissue to be treated can be any tissue in which the inhibition of angiogenesis is desirable, such as diseased tissue where neovascularization is occurring. Exemplary tissues include inflamed tissue, solid tumors, metastases, tissues undergoing restenosis, and similar tissues. An av3 antagonist for use in the present methods is capable of binding to avß3, and competitively inhibiting the ability of c_vß3 to bind to a natural ligand. Preferably, the antagonist exhibits specificity for vß3 over other integrins. In a particularly preferred embodiment, the vß3 antagonist inhibits the binding of fibrinogens or other ligands containing RGD to vß3, but does not substantially inhibit fibrinogen binding to αβvβ3. A preferred avß3 antagonist may be a fusion polypeptide, a cyclic or linear polypeptide, a derivatized polypeptide, a monoclonal antibody that immunoreacts with avβ3, an organic vß3 mimetic or a functional fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS In the drawings forming a portion of the statement: Figures 1A-1D illustrate the tissue distribution of the integrin, β3 and βx subunits in normal skin and skin that is undergoing wound healing , designated as granulation tissue. Immunohistochemistry was performed with antibodies to β3 and I r as described in Example 3A. Figures IA and IB respectively illustrate the anti-ß3 immunoreactivity in normal skin and granulation tissue. Figures 1C and ID illustrate respectively the immunoreactivity of anti-ßi in normal skin and in granulation tissue. Figures 2A-2D illustrate the tissue distribution of von Illebrand factor and laminin ligands that bind respectively to the integrin β3 and βx subunits, in normal skin and in skin that is undergoing healing of a wound, designated as tissue of granulation. Immunohistochemistry was performed with antibodies to Von ille-brand (anti-vWF) and laminin (anti-laminin) as described in Example 3B. Figures 2A and 2B illustrate respectively the immunoreactivity of anti-vWF in normal skin and in granulation tissue. Figures 2C and 2D respectively illustrate the anti-laminin immunoreactivity in normal skin and in granulation tissue. Figures 3A-3D illustrate the tissue distribution of the vitronectin integrin receptor, avß3, in tissue biopsies of bladder cancer, colon cancer, breast cancer, and lung cancer, respectively. Immunohistochemistry was performed with the antibody LM609 against avß3, as described in Example 3C. Figure 4 illustrates a typical photomicrograph of a chick chorioallantoic membrane of this invention, devoid of blood vessels, in an untreated 10-day-old chick embryo. The preparation is described in Example 5B. Figures 5A-5C illustrate the tissue distribution of the ßx and avß3 integrins in the chick chorioallantoic membrane preparation of this invention. Figure 5A shows the distribution of the ßx subunit in an untreated chicken chorioallantoic membrane preparation of 10 days of age, as detected by immunofluorescence immunoreactivity with CSAT, an anti-β-antibody. Figure 5B shows the distribution of the vß3 receptor in an untreated chicken chorioallantoic membrane preparation of 10 days of age, as detected by immunofluorescence immunoreactivity with LM609, an anti-c-vß3 antibody. Figure 5C shows the distribution of the c-vß3 receptor in a 10-day-old chicken chorioallantoic membrane preparation treated with bFGF, as detected by immunofluorescence immunoreactivity with LM609, an anti-o-vß3 antibody. The treatments and results are described in Example 5C. Figure 6 illustrates the quantification in a bar graph of the relative expression of avß3 and ßx in chicken chorioallantoic membranes aged 10 days untreated and treated with bFGF, as described in Example 6. Intensity is plotted fluorescence on the Y-axis, with the integrin profiles traced on the X-axis. Figures 7A-7C illustrate the appearance of an untreated chicken chorioallantoic membrane 10 days old, a chicken chorioallantoic membrane treated with bFGF, and a chicken chorioallantoic membrane treated with TNFo-, respectively, whose procedures and results are described in Example 6A. Figures 8A-8E illustrate the effect of local antibody treatment on angiogenesis induced by bFGF in a 10-day chicken chorioallantoic membrane, as described in Example 7A1). Figure 8A shows a chorioallantoic membrane preparation of untreated chicken that is devoid of blood vessels. Figure 8B shows the infiltration of new vasculature into an area previously devoid of vasculature, induced by treatment with bFGF. Figures 8C, 8D and 8E respectively, show the effects of antibodies against ßx (anti-ß-L, - CSAT), avß5 (anti-c.vß5; P3G2) and vß3 (anti-avß3; LM609). Figures 9A-9C illustrate the effect of intravenous injection of synthetic peptide 66203 on tumor-induced angiogenesis, as described in Example 7E2). Figure 9A shows the lack of inhibitory effect of intravenous treatment with a control peptide (control peptide tumor) on angiogenesis resulting from tumor induction. Figure 9B shows the inhibition of such angiogenesis by intravenous injection of peptide 66203 (cyclic RGD tumor). Figure 9 C shows the lack of inhibitory effects or cytotoxicity in previously existing mature vessels, after the intravenous infusion of peptide 66203 in an area adjacent to the treated area of the tumor (chicken chorioallantoic membrane adjacent to the cyclic RGD). Figures 10A-10C illustrate the effect of intravenous application of monoclonal antibodies to angiogenesis induced by the growth factor, as described in Example 7B1). Figure 10A shows the angiogenesis induced by bFGF not exposed to antibody treatment (control). No inhibition of angiogenesis resulted when a similar preparation was treated with the P3G2 anti-v5 antibody, as shown in Figure 10B. The inhibition of angiogenesis resulted in the treatment of the LM609 anti-? Vß3 antibody, as shown in Figure 10C. Figures 11A-11C illustrate the effect on embryonic angiogenesis after local application of anti-integrin antibodies, such as it is described in Example 7C. Angiogenesis was not inhibited by the treatment of a 6-day chicken chorioallantoic membrane with the anti-β-L and anti-avß5 antibodies, which are shown respectively in Figures HA and 11B. In contrast, treatment with anti-o.vβ3 antibody LM609 resulted in the inhibition of blood vessel formation, as shown in Figure 11C. Figure 12 illustrates the quantification of the number of vessels entering a tumor in a chick chorioallantoic membrane preparation, as described in Example 7D1). The graph shows the number of vessels as traced on the Y axis, which result from the local application of either CSAT (anti-ß-t), LM609 (anti-avß3) or P3G2 (anti-avß5). Figures 13A-13D illustrate a comparison between the weights of the wet tumors 7 days after treatment and the initial weights of the tumors, as described in example 9Al) a. Each bar represents the average ± S.E. of 5-10 tumors per group. Tumors were derived from chorioallantoic membrane preparations of human melanoma chicken (M21-L) (Figure 13A), pancreatic carcinoma (Fg) (Figure 13B), lung carcinoma (UCLAP-3) (Figure 13C), and laryngeal carcinoma (HEp3) (Figure 13D), and were treated intravenously with PBS, CSAT (anti-ßx), or LM609 (anti-ßvß3). The graphs show the weight of the tumor, as plotted on the Y axis, which resulted from the intravenous application of either CSAT (anti-ß, LM609 (anti-avß3) or PBS, as indicated on the X axis. The Figures 14A and 14B illustrate the histological sections of tumors treated with P3G2 (anti-o-v5) (Figure 14A) and LM609 (anti-av3) (Figure 14B), and stained with hematoxylin and eosin, as described in Example 9Al) b. As shown in Figure 14A, tumors treated with the control antibody (P3G2) showed numerous viable tumor cells and actively dividing, as indicated by the mitotic figures. (arrow heads), as well as through multiple blood vessels (arrows) along the tumor stroma. In contrast, few, if any, tumor cells or blood vessels were detected in the tumors treated with LM609 (anti-avß3) in Figure 14B. Figures 15A-15E correspond to M21L tumors treated with peptides, as described in Example 9A2) and are as follows: Figure 15A, control cyclic RAD peptide (69601); Figure 15B, cyclic RGD peptide (66203); Figure 15C, adjacent chicken chorioallantoic membrane tissue taken from the same embryos treated with cyclic RGD peptide (66203) and high amplification (13x) of tumors treated with the control RAD (69601) in Figure 15D or the RGD peptide cyclical (66203) in Figure 15E. Figure 15D illustrates the normal vessels of the tumor treated with the RAD control peptide (69601). Figure 15E illustrates examples of broken tumor blood vessels (arrows) treated with the cyclic RGD peptide (66203). Figures 16A-16E depict the inhibition of angiogenesis by the angiogenesis antagonists in the in vivo rabbit eye model assay, as described in Example 10. Figures 16A and 16B illustrate the angiogenesis of the rabbit eye in the presence of bFGF and mAb LM609 (anti-cevß3). Figures 16C, 16D and 16E illustrate the inhibition of angiogenesis of the rabbit eye in the presence of bFG and mAb LM609 (anti-avß3). Figure 17 depicts a flowchart of how the mouse chimeric mouse: human in vivo model was generated, as described in Example 11. A portion of the skin of a SCID mouse was replaced with a human neonatal foreskin, and He will heal for 4 weeks. After the graft had healed, the human prepuce was inoculated with human tumor cells. During the next 4 week period, a measurable tumor was established, which comprised a human tumor with human vasculature growing from human skin within the human tumor. Figure 18 illustrates the percentage of individual cells derived from chicken chorioallantoic membranes treated with mAb and treated with peptide, and stained with Apop Tag, as determined by FACS analysis and described in Example 12. Black bars and dotted cells represent embryo cells treated 24 hours and 48 hours before the test, respectively. Each bar represents the average ± S.E. of three replicas. Chicken chorioallantoic membranes were treated with mAb LM609 (anti-arvß3), or CSAT (anti-β].), Or PBS. Chicken chorioallantoic membranes were also treated with the cyclic peptide 66203 (cyclo-RGDfV, indicated as Peptide 203) or the cyclic control peptide 69601 (cyclo-RADfV, indicated as Peptide 601). Figures 19A and 19B illustrate the combined results of individual cell suspensions of chicken chorioallantoic membranes from embryos treated with either CSAT (anti-ßj.) (Figure 19A) or LM609 (anti-avß3) (Figure 19B), stained with Apop Tag and propidium iodide, and analyzed by FACS, as described in Example 12C. The Y axis represents spotting of Apop Tag in cell numbers (Apoptosis), the X axis represents the spotting of propidium iodide (DNA content). The horizontal line represents the negative gateway for Apop Tag spotting. The left and right panels indicate chicken chorioallantoic membrane cells from CSAT-treated embryos (anti-ß (Figure 19A) and LM609 (anti-avß3) (Figure 19B), respectively.) Cell cycle analysis was performed by analysis of approximately 8,000 events per condition.

Figures 20A-20C depict chicken chorioallantoic membrane tissue from embryos treated with CSAT, and Figures 20D-20F depict chicken chorioallantoic membrane tissue from embryos treated with LM609 (anti-oívß3) prepared as described in Example 12C. Figures 20A and 20D illustrate tissues stained with Apop Tag and visualized by fluorescence (FITC) superimposed on a D.I.C. Figures 20B and 20E illustrate the same tissues stained with mAb LM609 (anti-c_vß3) and visualized by fluorescence (rhodamine). Figures 20C and 20F represent the combined images of the same tissues stained with both Apop Tag and LM609, where the yellow spotting represents the co-location. The bar represents 15 and 50 μm in the left and right panels, respectively. Figure 21 shows the result of an inhibition of the cell binding assay with peptide 85189, as described in Example 4A. The effects of the peptide antagonist were evaluated over a dose range of .001 to 100 μM, as plotted on the X axis. The cell junction is plotted on the Y axis measured at an optical density (O.D.) of 600 nm. Cell binding was measured on surfaces coated with vitronectin- (broken lines) against laminin- (full lines). Figures 22A and 22B show the consecutive sequence of chicken MMP-2 cDNA together with the deduced amino acid sequence shown in the second line. The third and fourth lines respectively show the deduced amino acid sequence of human and mouse MMP-2, as described in Example 4A. The chicken cDNA sequence is listed in SEQ ID NO 29 together with the encoded amino acid sequence which is also represented separately as SEQ ID NO 30. The numbering of the first nucleotide of the 5 'untranslated region and the region that encodes the sequence of proenzymes shown in Figure 22A as a negative number, is in fact represented as the number 1 in the Sequence Listing, making the latter appear longer than the figure; however, the sequence of nucleotides in the figure is identical in length and sequence to that, as presented in the list with the exception of the numbering. In accordance with the above, references to the position of nucleotides for chicken and human MMP-2 in the specification, such as in primers for use in the amplification of MMP-2 fragments, are based on the position of the nucleotide, as it is indicated in the figure, and not as it is listed in the Sequence Listing. Figure 23 shows the results in bar graph form of a solid-phase receptor binding assay of MMP-2 treated with iodine to bind to avß3, with and without the presence of inhibitors, as further described in Example 4B . The data are plotted as CPM fixed on the Y axis against the different potential inhibitors and controls. Figure 24 shows the specificity of compositions of chicken-derived MMP-2 for the integrin receptors either vß3 and IIBß3 in the presence of MMP-2 inhibitors, as described further in Example 4B. The data are presented as described in the legend in Figure 23. Figure 25 shows the effect of the GST fusion protein of chicken MMP-2 (410-637) on angiogenesis induced by bFGF, as described in FIG. Example 7A3). Figures 25A-B and 25C-D respectively show the effects of the control (a non-MMP-2 fragment containing fusion protein) and the GST fusion protein of the MMP-2 fragment. Figures 26 and 27 illustrate in the form of bar graphs the index of angiogenesis (a measurement of branch points) of the effects of the fusion protein GST (labeled CTMMP-2) of chicken MMP-2 (410-637) against control (RAP-GST or GST-RAP) in chicken chorioallantoic membranes treated with bFGF, as described in Example 7A3). The angiogenic index is plotted on the Y axis against the treatments separated on the X axis. Figure 28 shows the effects of peptides and organic compounds on the angiogenesis induced by bFGF, as measured by the effect on branching points plotted on the Y axis against the different treatments on the X axis, including bFGF alone, and chicken chorioallantoic membranes treated with bFGF with peptides 69601 or 66203 and organic compounds 96112, 96113 and 96229, as described in Examples 7B and 14. Figure 29 graphically shows the dose response of peptide 85189 in the inhibition of angiogenesis induced by bFGF as further described in Example 7B2), where the number of branch points are plotted on the Y axis against the amount of peptide administered to the embryo on the X axis. Figure 30 shows the inhibitory activity of the 66203 peptides (labeled 203) and 85189 (labeled 189) on the angiogenesis s induced by bFGF in the chicken chorioallantoic membrane assay, as described in Example 7B2). The controls did not include any peptides in chicken chorioallantoic membranes treated with bFGF and peptide 69601 (labeled 601). The data is plotted as described in the legend of Figure 27. Figures 31A-L show the effect of different treatments against chicken chorioallantoic membrane preparations not treated during a time course starting in 24 hours and ending in 72 hours. , as further described in Example 7B3). In Figures 31A-C, 31D-F, 31G-I, and 31J-L, photographs are shown for the untreated labeled categories, bFGF, bFGF + MAID (treated with bFGF followed by exposure to the GST fusion protein of MMP-2 (2-4) chicken) and bFGF + control (treatment with bFGF followed by MMP-2 (10-1) chicken), respectively.

Figures 32, 33 and 34 respectively show the reduction in tumor weight for UCLAP-3, M21-L and FgM tumors after intravenous exposure to control peptide 69601 and antagonist 85189, as further described in Example 9A. The data are plotted with the tumor weight on the Y axis against peptide treatments on the X axis. Figure 35 illustrates the effect of the peptides and antibodies on tumor growth in melanoma in the chimeric mouse model: human, as Additional description is described in Example 11B. The peptides evaluated included the control 69601 (labeled 601) and the antagonist 85189 (labeled 189). The antibody tested was LM609. The volume of the tumor in cubic millimeters is plotted on the Y axis against the different treatments on the X axis. Figures 36A and B respectively show the effect of antagonist 85189 (labeled 189) compared to control peptide 69601 (labeled 601) in reducing the volume and wet weight of M21L tumors over a dose range of 10, 50 and 250 μg / injection, as further described in Example 11C. Figures 37A and 37B show the effectiveness of the 85189 antagonist peptide (labeled 189 with a solid line and filled circles) against the control 69601 peptide (labeled 601 in a dotted line and open frames) in the inhibition of M21L tumor volume in the mouse model: human with two different treatment regimens, as described further in Example 11C. The volume of the tumor in cubic millimeters is plotted on the Y axis against the days on the X axis. Figures 38 to 42 schematically illustrate the different chemical syntheses of the antagonists of the organic avß3 molecule, as described further in Example 13. Figures 43 and 44 show the effects of different organic molecules on the angiogenesis induced by bFGF in a chick chorioallantoic membrane assay, as further described in Example 14. The branching points are plotted on the Y axis against the different compounds used at 250 μg / milliliter on the X axis in Figure 43, and 100 μg / milliliter in Figure 44. Detailed Description of the Invention A. Definitions Amino acid residue: An amino acid formed after chemical digestion (hydrolysis) of a polypeptide in its peptide bonds. The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any amino acid residue L, as long as the polypeptide retains the desired functional property. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present in the carboxy terminus of a polypeptide. In harmony with the standard polypeptide nomenclature (described in J. Biol. Chem., 243: 3552-59 (1969), and adopted in 37 CFR §1.822 (b) (2)), in the following Correspondence Table Abbreviations for amino acid residues are shown: Table of Correspondences Sími 3? L? Amino Acid I-Letter 3-Letters Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala alanine S Serine I He isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline K Lys lysine H His histidine Q Gln glutamine E Glu glutamic acid Z Glx Glu and / or Gln Trp tryptophen R Arg arginine D Asp aspartic acid N Asn asparagine B Asx Asn and / or Asp C Cys cysteine X xaa unknown / other In addition, the following have the meanings below: BOC tert-butyloxycarbonyl DCCI dicyclohexylcarbodiimide DMF dimethylformamide OMe methoxy HOBt 1-hydroxybenzotriazole It should be noted that all sequences of amino acid residues are represented herein by formulas whose left and right orientation is in the conventional direction of amino terminus to carboxy terminus. On the other hand, it should be noted that a hyphen at the beginning or end of a sequence of amino acid residues indicates a peptide bond to another sequence of one or more amino acid residues. Polypeptide: refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino group and the carboxy group of contiguous amino acid residues. Peptide: as used herein, refers to a linear series of no more than about 50 amino acid residues connected to one another as in a polypeptide. Cyclic peptide: refers to a compound having a heteroatom ring structure that includes many amide bonds, as in a typical peptide. The cyclic peptide may be a "head-to-tail" cyclized linear polypeptide in which a n-terminus of the linear peptide has formed an amide bond with the terminal carboxylate of the linear peptide, or it may contain a ring structure in which the The polymer is homodietic or heterodétic, and comprises amide bonds and / or other bonds to close the ring, such as disulfide bridges, thioesters, thioamides, guanidino, and the like. Protein: refers to a linear series of more than 50 amino acid residues connected to one another as in a polypeptide. Fusion protein: refers to a polypeptide that contains at least two different polypeptide domains, operably linked by a typical ("fused") peptide bond, wherein the two domains correspond to peptides that are not fused in nature . Synthetic peptide: refers to a chain of chemically produced amino acid residues, linked together by peptide bonds, which is free of naturally occurring proteins and fragments thereof. B. General Considerations The present invention is generally related to the discovery that angiogenesis is mediated by the specific vitronectin receptor? Vß3, and that the inhibition of c-vß3 function inhibits angiogenesis. This discovery is important because of the role that angiogenesis plays in a variety of disease processes. By inhibiting angiogenesis, one can intervene in the disease, alleviate the symptoms, and in some cases cure the disease.

Where the growth of new blood vessels is the cause of, or contributes to, the pathology associated with a disease, the inhibition of angiogenesis will reduce the ill effects of the disease. Examples include rheumatoid arthritis, diabetic retinopathy, inflammatory diseases, restenosis, and the like. Where the growth of new blood vessels is required to support the growth of a noxious tissue, the inhibition of angiogenesis will reduce the blood supply to the tissue and, thereby, will contribute to the reduction in tissue mass based on the blood supply requirements. Examples include the growth of tumors in which neovascularization is a continuous requirement, in order that the tumor grows beyond a few millimeters in thickness, and for the establishment of solid tumor metastases. The methods of the present invention are effective in part because the therapy is highly selective for angiogenesis and not other biological processes. As shown in the Examples, only the growth of new vessels contains substantial vß3, and therefore, therapeutic methods do not adversely affect mature vessels. On the other hand, vß3 is not widely distributed in normal tissues, but rather is selectively found in new vessels, ensuring, by the same token, that the therapy can be selectively directed to the growth of new vessels. The discovery that β1-β3 inhibition alone will effectively inhibit angiogenesis allows the development of therapeutic compositions with potentially high specificity, and therefore relatively low toxicity. Therefore, although the invention discloses the use of peptide-based reagents, which have the ability to inhibit one or more integrins, one can design other reagents that more selectively inhibit avß3 Y- P ° r 1 ° both, do not have the secondary effect of inhibiting other biological processes apart from those mediated by avß3. For example, as shown by the present teachings, it is possible to prepare highly selective monoclonal antibodies for avß3 immunoreaction, which are also selective for the inhibition of vß3 function. In addition, peptides containing RGD can be designed to be selective for the inhibition of ovv3, as is further described herein. Prior to the discoveries of the present invention, it was not known that angiogenesis, and any of the processes dependent on angiogenesis, could be inhibited in vivo by the use of reagents that antagonize the biological function of? Fvß3. C. Methods for Inhibition of Angiogenesis The invention provides a method for the inhibition of angiogenesis in a tissue, and therefore, the inhibition of tissue events that depend on angiogenesis. Generally, the method comprises administering to the tissue a composition comprising an inhibitory amount of angiogenesis of an avß3 antagonist. As described above, angiogenesis includes a variety of processes that involve the neovascularization of a tissue, including "germination", vasculogenesis, or enlargement of the vessel, processes of angiogenesis that are all mediated by, and depend on the expression of? Ívß3. With the exception of traumatic wound healing, Corpus leuteum formation and embryogenesis, it is believed that most processes of angiogenesis are associated with disease processes, and therefore, the use of the present therapeutic methods are selective for the disease, and they do not have harmful side effects. There are a variety of diseases in which angiogenesis is believed to be important, which are referred to as angiogenic diseases, including, but not limited to, inflammatory disorders such as immune and non-immune inflammation, chronic joint rheumatism and psoriasis., disorders associated with inappropriate or untimely invasion of vessels such as diabetic retinopathy, neovascular glaucoma, restenosis, capillary proliferation in atherosclerotic plaques and osteoporosis, and disorders associated with cancer, such as solid tumors, solid tumor metastases, angiofibromas, retrolental fibroplasia, hemangiomas, Kaposi's sarcoma and similar cancers that require neovascularization to support tumor growth. Therefore, methods that inhibit angiogenesis in a diseased tissue alleviate the symptoms of the disease and, depending on the disease, can contribute to the cure of the disease. In one embodiment, the invention contemplates the inhibition of angiogenesis, by itself, in a tissue. The extent of angiogenesis in a tissue, and hence the extent of the inhibition achieved by the present methods, can be assessed by a variety of methods, such as those described in the Examples for detecting immature vessel structures and nascent vß3-immunopositive immunohistochemistry. As described herein, any of a variety of tissues, or organs formed by organized tissues, can support angiogenesis in disease conditions, including skin, muscles, intestines, connective tissue, joints, bones and similar tissues in which blood vessels can invade after angiogenic stimuli. Therefore, in a related embodiment, a tissue to be treated is an inflamed tissue and the angiogenesis to be inhibited is angiogenesis of inflamed tissue, where there is neovascularization or inflamed tissue. In this class the method contemplates the inhibition of angiogenesis in arthritic tissues, such as in a patient with chronic joint rheumatism, in inflamed immune or non-immune tissues, in psoriatic tissue and the like. The patient treated in the present invention in its many modalities is desirably a human patient, although it will be understood that the principles of the invention indicate that the invention is effective with respect to all mammals, which are intended to be included in the term " patient". In this context, it is understood that a mammal includes any mammalian species in which the treatment of diseases associated with angiogenesis is desirable, particularly agricultural and domestic mammalian species. In another related embodiment, a tissue to be treated is a retinal tissue from a patient with a retinal disease such as diabetic retinopathy, macular degeneration or neovascular glaucoma, and the angiogenesis to be inhibited is retinal tissue angiogenesis where there is neovascularization of retinal tissue. In a further related embodiment, a tissue to be treated is a tumor tissue of a patient with a solid tumor, a metastasis, a skin cancer, a breast cancer, a hemangioma or angiofibroma and similar cancers, and the angiogenesis that It is going to inhibit is angiogenesis of tumor tissue where there is a neovascularization of a tumor tissue. Typical solid tumor tissues that can be treated by the present methods include lung, pancreas, breast, colon, laryngeal, ovarian, and similar tissues. In the Examples, the angiogenesis of exemplary tumor tissue, and the inhibition thereof, are described. Inhibition of tumor tissue angiogenesis is a particularly preferred modality due to the important role that neovascularization plays in tumor growth. In the absence of neovascularization of tumor tissue, the tumor tissue does not obtain the required nutrients, it slows down in growth, stops further growth, recedes and finally becomes necrotic, resulting in the elimination of the tumor. Stated in other words, the present invention provides a method of inhibiting tumor neovascularization, by inhibiting tumor angiogenesis, in accordance with the present methods. Similarly, the invention provides a method of inhibiting tumor growth by practicing methods of inhibiting angiogenesis. The methods are also particularly effective against the formation of metastasis because (1) their formation requires the vascularization of a primary tumor, in such a way that the metastatic cancer cells can leave the primary tumor and (2) its establishment in a secondary site it requires neovascularization to support the growth of the metastasis.

In a related embodiment, the invention contemplates the practice of the method in conjunction with other therapies, such as conventional chemotherapy directed against solid tumors and for the control of the establishment of metastasis. Administration of the angiogenesis inhibitor is typically conducted during or after chemotherapy, although it is preferred to inhibit angiogenesis after a chemotherapy regimen at times when the tumor tissue is responding to toxic aggression, by inducing that angiogenesis recover by providing a supply of blood and nutrients to the tumor tissue. In addition, it is preferred to administer methods of inhibiting angiogenesis after surgery, where solid tumors have been removed as a prophylaxis against metastasis. While the present methods apply to the inhibition of tumor neovascularization, the methods can also be applied to the inhibition of tumoral ramus growth, to the inhibition of the formation of tumor metastasis, and to the regression of established tumors. The Examples demonstrate the regression of a tumor established after a single intravenous administration of an αvβ3 antagonist of this invention. Restenosis is a process of migration and proliferation of smooth muscle cells (SMC) in the percutaneous transluminal coronary angioplasty site that hinders the success of angioplasty. The migration and proliferation of SMCs during restenosis can be considered an angiogenesis process that is inhibited by the present methods. Therefore, the invention also contemplates the inhibition of restenosis by means of the inhibition of angiogenesis according to the present methods, in a patient after the angioplasty procedures. For the inhibition of restenosis, the vß3 antagonist is typically administered after the angioplasty procedure for from about 2 to about 28 days, and more typically for about the first 14 days after the procedure. The present method for the inhibition of angiogenesis in a tissue, and therefore also for practicing methods for the treatment of diseases related to angiogenesis, comprises contacting a tissue in which angiogenesis is occurring, or which is in risk of occurring, with a composition comprising a therapeutically effective amount of a vß3 antagonist capable of inhibiting the binding of o-vß3 to its natural ligand. Therefore, this method comprises administering to a patient a therapeutically effective amount of a physiologically tolerable composition containing an o.vβ3 antagonist of the invention. The dose ranges for administration of the c-vß3 antagonist depend on the form of the antagonist, and its potency, as further described herein, and are large enough to produce the desired effect in which angiogenesis is relieved. and the symptoms of the disease mediated by angiogenesis. The dose should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dose will vary with the age, condition, sex and degree of the disease in the patient, and one of experience in the art can determine it. The dose can also be adjusted by the individual doctor in case of any complication. A therapeutically effective amount is an amount of a sufficient antagonist of ovv3 to produce an inhibition of angiogenesis, which can be measured, in the tissue being treated, i.e., an inhibiting amount of angiogenesis. Inhibition of angiogenesis can be measured in situ by immunohistochemistry, as described herein, or by other methods known to one skilled in the art. While an antagonist of ocvß3 can take the form of a vβ3 mimetic, a peptide containing RGD, an anti-vβ3 monoclonal antibody, or a fragment thereof, it will be noted that the power, and therefore an expression of a "therapeutically effective" amount may vary. However, as shown by the present test methods, one skilled in the art can quickly assess the potency of an antagonist of orvβ3 candidate of this invention. The potency of an ovv3 antagonist can be measured by a variety of means, including the inhibition of angiogenesis in the chick chorioallantoic membrane assay, in the rabbit eye test in vivo, in the chimeric mouse assay: human in vivo, and by measuring the inhibition of natural ligand binding to αβvβ3, all as described herein, and similar assays. A preferred v 3 antagonist has the ability to substantially inhibit the binding of a natural ligand such as a fibrinogen or α-v β 3 vitronectin in solution at antagonist concentrations of less than 0.5 micromolar (μm), preferably less than 0.1 μm, and more preferably less than 0.05 μm. By "substantially" it is meant that at least a 50 percent reduction in fibrinogen binding is observed by inhibition in the presence of the α-v β 3 antagonist, and is referred to as a 50 percent inhibition in the present. as an IC50 value. A more preferred vß3 antagonist exhibits avß3 selectivity over other integrins. Therefore, a preferred c-vß3 antagonist substantially inhibits the binding of fibrinogen to c-vß3, but does not substantially inhibit the binding of fibrinogen to another integrin such as o-vβi, βvβ5 or βIIbβ3. Particularly preferred is an ovv3 antagonist which exhibits 10-fold to 100-fold lower IC50 activity in the inhibition of fibrinogen binding to avß3 compared to IC50 activity in the inhibition of fibrinogen binding to another integrin. Exemplary assays for measuring IC 50 activity in the inhibition of fibrinogen binding to an integrin are described in the Examples. A therapeutically effective amount of an avß3 antagonist of this invention, in the form of a monoclonal antibody, is typically such an amount that when administered in a physiologically tolerable composition, is sufficient to achieve a plasma concentration of from about 0.01. microgram (μg) per milliliter (ml) to about 100 microgram / milliliter, preferably from about 1 microgram / milliliter to about 5 microgram / milliliter, and usually about 5 microgram / milliliter. Stated otherwise, the dose may vary from about 0.1 milligram / kilogram to about 300 milligrams / kilogram, preferably from about 0.2 milligrams / kilogram to about 200 milligrams / kilogram, more preferably from about 0.5 milligrams / kilogram to about 20 milligrams / kilogram, in one or more dose administrations daily, for one or many days. Where the antagonist is in the form of a fragment of a monoclonal antibody, the amount can be adjusted rapidly based on the mass of the fragment relative to the mass of the total antibody. A preferred plasma concentration in molarity is from about 2 micromolar (μM) to about 5 millimolar (mM), and preferably about 100 μM to 1 mM antibody antagonist. A therapeutically effective amount of an avß3 antagonist of this invention, in the form of a polypeptide, or other similarly sized small molecule avß3 mimetic, is typically such an amount of polypeptide that when administered in a physiologically tolerable composition, it is sufficient to achieve a plasma concentration of from about 0.1 microgram (μg) per milliliter (ml) to about 200 microgram / milliliter, preferably from about 1 microgram / milliliter to about 150 microgram / milliliter. Based on a polypeptide having a mass of about 500 grams per mole, the concentration of plasma preferred in morality is from about 2 micromolar (μM) to about 5 millimolar (mM), and preferably about 100 μM to 1 mM antibody antagonist. Stated otherwise, the dose per body weight may vary from about 0.1 milligram / kilogram to about 300 milligrams / kilogram, and preferably from about 0.2 milligrams / kilogram to about 200 milligrams / kilogram, in one or more dose administrations daily, for one or many days. The monoclonal antibodies or polypeptides of the invention can be administered parenterally by injection or by gradual infusion over time. Although typically the tissue to be treated in the body can be accessed by systemic administration, and therefore, can be treated more frequently by intravenous administration of the therapeutic compositions, other tissues and delivery elements are contemplated where there is the probability that the target tissue contains the target molecule. Therefore, the monoclonal antibodies or polypeptides of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be delivered by peristaltic elements. Therapeutic compositions containing a monoclonal antibody or a polypeptide of this invention are conventionally administered intravenously, such as by injection of a single dose, for example, the term "single dose" when used with reference to a therapeutic composition. of the present invention, refers to physically discrete units suitable as a unit dose for the subject, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in association with the required diluent; that is, the carrier, or vehicle. In a preferred embodiment, as shown in the Examples, the avß3 antagonist is administered in a single dose intravenously.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The amount to be administered and the time depend on the subject to be treated, the ability of the subject's system to use the active ingredient, and the degree of therapeutic effect desired. The precise amounts of active ingredient that is required to be administered depends on the judgment of the medical specialist, and are peculiar to each individual. However, the dose ranges suitable for systemic application are described herein, and depend on the route of administration. Suitable regimens for administration are also variable, but are typified by an initial administration followed by repeated doses at intervals of one hour or more by a subsequent injection or other administration. Alternatively, a sufficient continuous intravenous infusion is contemplated to maintain blood concentrations in the ranges specified for in vivo therapies. As demonstrated by the present Examples, inhibition of angiogenesis and tumor regression occurs as early as 7 days after initial contact with the antagonist. The additional or prolonged exposure to the antagonist is preferably for 7 days to 6 weeks, preferably approximately 14 to 28 days. In a related embodiment, the Examples demonstrate the relationship between the inhibition of vß3 and the induction of apoptosis in the neovasculature cells carrying avß3. Therefore, the invention also contemplates methods for the inhibition of apoptosis in the neovasculature of a tissue. The method is practiced substantially as described herein, for the inhibition of angiogenesis in all tissues and conditions described therefor. The only noticeable difference is one of the effect time, which is that apoptosis manifests rapidly, typically approximately 48 hours after contact with the antagonist, while inhibition of angiogenesis and regression of the tumor manifests itself more slowly, as is described in the present. This difference affects the therapeutic regimen in terms of time of administration, and desired effect. Typically, administration for apoptosis of the neovasculature can be from 24 hours to approximately 4 weeks, although it is preferred from 48 hours to 7 days. D. Therapeutic Compositions The present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. The therapeutic compositions of the present invention contain a physiologically tolerable carrier together with an ovv3 antagonist as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the anti-ßvß3 antagonist therapeutic composition is not immunogenic when administered to a mammalian or human patient for therapeutic purposes. As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and the grammatical variations thereof, since they refer to compositions, carriers, diluents and reagents, are used interchangeably, and represent that the materials they are capable of administration to or on a mammal, without the production of undesirable physiological effects such as nausea, dizziness, gastric discomfort and the like. The preparation of a pharmacological composition containing the active ingredients dissolved or dispersed therein is well known in the art, and should not be limited on the basis of the formulation. Typically those compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, may also be prepared in liquid before use. The presentation can also be emulsified. The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting agents or emulsifiers, pH regulating agents and the like, which improve the effectiveness of the active ingredient. The therapeutic composition of the present invention may include pharmaceutically acceptable salts of the components therein. The pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, tartaric, mandelic and the like. Salts that are formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Particularly preferred are the TFA salts and HCl, when used in the preparation of avß3 cyclic polypeptide antagonists. In the Examples, representative salts of the peptides are described. Physiologically tolerable carriers are well known in the art. Examples of liquid carriers are sterile aqueous solutions which do not contain any material in addition to the active ingredients and water, or contain a pH regulator such as sodium phosphate at the physiological pH value, physiological saline or both, such as saline. regulated with phosphate. Still further, the aqueous carriers may contain more than one pH regulator salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. The liquid compositions may also contain liquid phases in addition to, and for the exclusion of water. Examples of those additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. A therapeutic composition contains an inhibitory amount of angiogenesis of an o-vß3 antagonist of the present invention, typically formulated to contain an amount of at least 0.1 weight percent antagonist by weight of total therapeutic composition. A percentage by weight is a weight ratio of inhibitor to the total composition. Therefore, for example, 0.1 weight percent is 0.1 gram of inhibitor per 100 grams of total composition. E. Antagonists of a-tß3 Integrin The avß3 antagonists are used in the present methods to inhibit angiogenesis in tissues, and can take a variety of forms including compounds that interact with vß3 in a manner that interferes with functional interactions with the natural ligands of avß3. Exemplary antagonists include analogs of o-vß3 derived from the ligand binding site in vß3, mimetics of either avß3 or a natural ligand of o-vß3, which mimic the framework region involved in the binding interactions of avß3-ligand, polypeptides having a sequence corresponding to a functional binding domain of the natural ligand specific for λβ3, which corresponds particularly to the RGD-containing domain of a natural ligand of avβ3, and antibodies that immunoreact with either av av3 or the natural ligand, all of which exhibit antagonist activity as defined herein. 1. Polypeptides In one embodiment, the invention contemplates antagonists of vß3 in the form of polypeptides. A polypeptide (peptide)? Vß3 antagonist may have the sequence characteristics of either the natural ligand of? Vß3 or avß3 itself in the region involved in the v3-ligand interaction, or exhibits antagonistic activity towards v3 as described herein . An antagonist peptide of -xvß3 contains the tripeptide RGD and corresponds in sequence to the natural ligand in the region containing RGD. Preferred RGD-containing polypeptides have a sequence corresponding to the amino acid residue sequence of the RGD-containing region of a natural ligand of βvß3 such as fibrinogen, vitronectin, von Willebrand factor, laminin, thrombospondin, and similar ligands. . The sequence of these ligands of vß3 is well known. Therefore, the avß3 antagonist peptide can be derived from any of the natural ligands, although fibrinogen and vitronectin are preferred. A particularly preferred o; vß3 antagonist peptide preferentially inhibits? Fvß3 binding to its natural ligand (s) when compared to other integrins, as described above. These peptides specific for vß3 are particularly preferred at least because the specificity for αββ3 reduces the incidence of undesirable side effects such as the inhibition of other integrins. The identification of avß3 antagonist peptides that have selectivity for oívß3, can be quickly identified in a typical binding inhibition assay, such as the enzyme-linked immunosorbent assay described in the Examples. A polypeptide of the present invention typically comprises no more than about 100 amino acid residues, preferably no more than about 60 residues, more preferably no more than about 30 residues. The peptides may be linear or cyclic, although the particularly preferred peptides are cyclic. Where the polypeptide is greater than about 100 residues, it is typically provided in the form of a fusion protein or protein fragment, as described herein. The preferred cyclic and linear peptides and their designations are shown in Table 1 in the Examples. It should be understood that an object polypeptide need not be identical to the amino acid residue sequence of the natural ligand of o: vß3, as long as it includes the required sequence and is capable of functioning as an avß3 antagonist in an assay such as those that are described in the present. An object polypeptide includes any analogue fragment or chemical derivative of a polypeptide whose amino acid residue sequence is shown herein, so long as the polypeptide is a β-vß3- antagonist. Therefore, a polypeptide present may be subject to different changes, substitutions, insertions, and deletions, where such changes provide certain advantages in their use. In this regard, the avß3 antagonist polypeptide of this invention corresponds to, rather than being identical to, the sequence of a cited peptide wherein one or more changes are made and this retains the ability to function as a c-vß3 antagonist. in one or more of the tests, as described herein. Thus, a polypeptide can be in any of a variety of forms of peptide derivatives, that includes amides, conjugates with proteins, cyclic peptides, polymerized peptides, analogs, fragments, chemically modified peptides, and similar derivatives. The term "analogue" includes any polypeptide having a sequence of amino acid residues substantially identical to a sequence that is specifically shown herein, wherein one or more residues have been substituted conservatively with a functionally similar residue, and which displays the activity antagonist for vß3, as described herein. Examples of conservative substitutions include the substitution of a polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of a basic residue such as lysine, arginine or histidine, on the other, or the replacement of an acidic residue, such as aspartic acid or glutamic acid, by another. The phrase "conservative substitution" also includes the use of a chemically derivatized residue instead of a non-derivatized residue, with the proviso that that polypeptide displays the required inhibitory activity. A "chemical derivative" refers to an object polypeptide having one or more chemically derivatized residues by the reaction of a functional side group. In addition to the side group derivates, a chemical derivative may have one or more modifications of the base structure, including a-amino substitutions such as N-methyl, N-ethyl, N-propyl and the like, and a-carbonyl substitutions such as thioester, thioamide, guanidino and the like. These derivatized molecules include, for example, those molecules in which the free amino groups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, or formyl groups. The free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. The free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-imbenzysthistidine. Also included as chemical derivatives are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline can be replaced by proline; 5-hydroxylysine can be replaced by lysine; 3-methylhistidine can be replaced by histidine; homoserin can be replaced by serine; and ornithine can be replaced by lysine. The polypeptides of the present invention also include any polypeptide having one or more additions and / or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, as long as the required activity is maintained. A particularly preferred derivative is a cyclic peptide according to the formula cyclo (Arg-Gly-Asp-D-Phe-NMeVal), abbreviated c (RGDf-NMeV), in which there is a c-amino group substituted with N- methyl in the valine residue of the peptide, and the cyclization has bound the primary and carboxy amino termini of the peptide. The term "fragment" refers to any subject poly-peptide having a shorter amino acid residue sequence than that of a polypeptide whose amino acid residue sequence is shown herein. When a polypeptide of the present invention has a sequence that is not identical to the sequence of a natural ligand of avß3, this is typically due to the fact that one or more conservative or non-conservative substitutions have been made, usually no more than about 30 substitutes are substituted. percent in number, and preferably no more than 10 percent in number of amino acid residues. Additional residues can also be added at any term of a polypeptide, for the purpose of providing a "linker" by which the polypeptides of this invention can be conveniently fixed to a label or solid matrix, or carrier. The labels, solid matrices and carriers that can be used with the polypeptides of this invention are described hereinbelow. The linkers of amino acid residues are usually at least one residue and can be 40 or more residues, more often from 1 to 10 residues, but do not form epitopes of avβ3 ligand. Typical amino acid residues that are used to bind are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. In addition, an object polypeptide may differ, unless otherwise specified, from the natural sequence of an avß3 ligand by modification of the sequence by acylation of terminal NH2, eg, acetylation, or thioglycolic acid amidation , by terminal carboxylation, for example, with ammonia, methylamine, and similar terminal modifications. Terminal modifications are useful, as is well known, to reduce susceptibility to proteinase digestion, and thus serve to prolong the half-life of polypeptides in solutions, particularly biological fluids where proteases may be present. . In this regard, the cyclization of the polypeptide is also a useful terminal modification, and is also particularly preferred because of the stable structures formed by cyclization, and in view of the biological activities observed for those cyclic peptides, as described herein. Any peptide of the present invention can be used in the form of a pharmaceutically acceptable salt. Suitable acids which are capable of forming salts with the peptides of the present invention include inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, sulfonic acid methane, acetic acid, acetic acid phosphoric acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid , or similar. Particularly preferred are the salts of hydrochloric acid and trifluoroacetic acid. Suitable bases capable of forming salts with the peptides of the present invention include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like.; and organic bases such as mono-, di- and tri-alkyl and arylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, and the like). In addition, a peptide of this invention can be prepared as described in the Examples, without including a free ionic salt in which the acid or charged base groups present in the side groups of amino acid residues (e.g., Arg, Asp, and the like) associate and neutralize each other to form an "internal salt" compound. A peptide of the present invention also referred to herein as an object polypeptide, can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solid phase Merrifield synthesis, are preferred for reasons of purity, antigenic specificity, exemption from undesirable side products, ease of production and the like. An excellent compendium of the many techniques available in Steward et al., "Solid Phase Peptide Synthesis", W.H. Freeman Co. , San Francisco, 1969; Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976; J. Meienhofer, "Hormonal Proteins and Peptides", Volume 2, page 46, Academic Press (New York), 1983; Merrifield, Adv. Enzymol., 32: 221-96, 1969; Fields et al., Int. J. Peptide Protein Res., 35: 161-214, 1990; and in U.S. Patent No. 4,244,946 for the synthesis of the solid phase peptide, and Schroder et al., "The Peptides", Volume 1, Academic Press (New York), 1965 for the synthesis of classical solution, each of which is incorporated herein by reference. Suitable protecting groups that can be used in those syntheses are described in the above texts and in J.F.W. McOmie, "Protective Groups in Organic Chemistry," Plenum Press, New York, 1973, which is incorporated herein by reference. In general, the solid phase synthesis methods contemplated comprise the sequential addition of one or more amino acid residues, or appropriately protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable protecting group, which can be removed selectively. A different protecting group is used, which can be removed selectively for amino acids that contain a reactive side group such as lysine. Using a solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support, through its carboxyl or amino-deprotected group. The protecting group of the amino or carboxyl group is then selectively removed, and the next amino acid is combined in the sequence having the suitably protected complementary group (amino or carboxyl), and reacted under suitable conditions to form the amide bond with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from its newly added amino acid residue, and then the next amino acid (suitably protected) is added, and so on. After all the desired amino acids have been linked in the proper sequence, and the protective groups of the remaining terminal and side groups are removed sequentially or concurrently, to produce the final linear poly-peptide. The resulting linear polypeptides prepared for example, as described above, can be reacted to form their corresponding cyclic peptides. Zimmer et al., Peptides 1992, pages 393-394, ESCOM Science Publishers, B.V., 1993 describe an exemplary method for preparing a cyclic peptide. Typically, the methyl ester of the peptide protected by terbutoxycarbonyl in methanol is dissolved, and a solution of sodium hydroxide is added, and the mixture is reacted at 20 ° C (20 ° C) to hydrolytically remove the methyl ester protecting group. After evaporating the solvent, the protected peptide is extracted by terbutoxycarbonyl, with ethyl acetate of acidified aqueous solvent. The protecting group terbutoxycarbonyl is then removed under slightly acidic conditions in dioxane cosolvent. The unprotected linear peptide with amino and free carboxy terms, thus obtained, is converted to its corresponding cyclic peptide by the reaction of a diluted solution of linear peptide, in a mixture of dichloromethane and dimethylformamide, with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole and N-methylmorpholine. The resulting cyclic peptide is then purified by chromatography. Alternative methods for the synthesis of the cyclic peptide are described in Gurrath et al., Eur. J. Biochem. , 210: 911-921 (1992), and are described in the Examples. In addition, the ovv3 antagonist can be provided in the form of a fusion protein. Fusion proteins are proteins produced by recombinant DNA methods, as described herein, in which the subject polypeptide is expressed as a fusion with a second carrier protein such as a glutathione sulfhydryl transferase (GST) or other carrier well known. Preferred fusion proteins comprise an MMP-2 polypeptide described herein. In the Examples it describes the preparation of an MMP-2 fusion protein. Peptides and derivative peptides particularly preferred for use in the present methods are c- (GrGDFV) (SEQ ID NO 4), c- (RGDfV) (SEQ ID NO 5), c- (RADfV) (SEQ ID NO 6), c- (RGDFv) (SEQ ID NO 7), c- (RGDf-NMeV) (SEQ ID NO 15) and linear peptide YTAECKPQVTRGDVF (SEQ ID NO 8), where "c-" indicates a cyclic peptide, the uppercase letters are single-letter codes for an amino acid L- and lowercase letters are single-letter codes for amino acids D. The sequence of amino acid residues of these peptides are also shown in SEQ ID NOs 4, 5, 6, 7, 15 and 8, respectively. Also preferred are the MMP-2 derived polypeptides described herein, having the sequences shown in SEQ ID NOs 17-28 and 45. 2. Monoclonal Antibodies The present invention describes, in one embodiment, avß3 antagonists in the form of monoclonal antibodies that immunoreact with avß3 and inhibit the binding of avß3 to its natural ligand, as described herein. The invention also describes cell lines that produce the antibodies, methods for producing the cell lines, and methods for producing the monoclonal antibodies. A monoclonal antibody of this invention comprises the antibody molecules that 1) immunoreact with isolated βνβ3, and 2) inhibit the binding of fibrinogen to ovvβ3. Preferred monoclonal antibodies that preferentially bind to o-vß3 include a monoclonal antibody having the immunoreaction characteristics of mAb LM 609, secreted by the hybridoma cell line ATCC HB 9537. The hybridoma cell line ATCC HB 9537 was deposited in accordance with the requirements of the Budapest Treaty with the American Type Culture Collection (ATCC), 1301 Parklawn Drive, Rockville, Maryland, United States, on September 15, 1987. The term "antibody or antibody molecule" in the different Grammatical forms are used herein as a collective noun that refers to a population of immunoglobulin molecules and / or immunologically active portions of the immunoglobulin molecules, ie, molecules that contain an antibody or paratope combining site. An "antibody combining site" is that structural portion of an antibody molecule formed by variable and hypervariable regions of light chain that bind specifically to the antigen. Exemplary antibodies for use in the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of an immunoglobulin molecule containing the paratope, including those portions known in the art as Fab, Fab ', F (ab') 2 and F (v), and to which also it is referred to as antibody fragments. In another preferred embodiment, the invention contemplates a truncated immunoglobulin molecule comprising a Fab fragment derived from a monoclonal antibody of this invention.

The Fab fragment, which lacks the Fe receptor, is soluble, and provides therapeutic advantages in the serum half-life, and the diagnostic advantages in the modes of use of the soluble Fab fragment. The preparation of a soluble Fab fragment is generally known in the immunological art, and can be achieved by a variety of methods. For example, the Fab and F (ab ') 2 portions (fragments) of the antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, in substantially intact antibodies by methods that are well known. See, for example, U.S. Patent No. 4,342,566 to Theofilopolous and Dixon. The Fab 'antibody portions are also well known, and are produced from the F (ab') 2 portions followed by the reduction of the disulfide bonds that link the two heavy chain portions as with mercaptoethanol, and followed by the alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact immunoglobulin molecules is preferred, and are used herein as illustrative. The phrase "monoclonal antibody" in its different grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site, capable of immunoreacting with a particular epitope. A monoclonal antibody typically displays, therefore, a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody can, therefore, contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, for example, a bispecific monoclonal antibody. A monoclonal antibody is typically composed of antibodies produced by the clones of a single cell called a hybridoma, which secretes (produces) only one kind of antibody molecule. The hybridoma cell is formed by the fusion of an antibody-producing cell and a myeloma or other self-perpetuating cell line. The preparation of these antibodies was first described by Kohier and Milstein, Nature, 256: 495-497 (1975), whose description is incorporated by reference. Zola, Monoclonal Antibodies: A Manual of Technicians, CRC Press, Inc. (1987) describes additional methods. The hybridoma supernatants thus prepared can be screened for the presence of antibody molecules that immunoreact with avß3, and for the inhibition of avß3 binding to natural ligands. Briefly, to form the hybridoma from which the composition of monoclonal antibodies is produced, a myeloma or other self-perpetuating cell line is fused with the lymphocytes obtained from the spleen of a mammal hyperimmunized with a source of avß3, such as avß3 isolated from M21 human melanoma cells, as described by Cheresh et al., J. Biol. Chem., 262: 17703-17711 (1987). It is preferred that the myeloma cell line that is used to prepare a hybridoma be from the same species as the lymphocytes. Typically, the preferred mammal is a mouse of strain 129 G1X +. Mouse myelomas suitable for use in the present invention include the P3X63-Ag8.653, and hypoxanthine-aminopterin-thymidine-sensitive Sp2 / 0-Agl4 cell lines (HAT), which are available from the American Type Culture Collection, Rockville , Maryland, United States, under the designations CRL 1580 and CRL 1581, respectively. Splenocytes typically fuse with myeloma cells using polyethylene glycol (PEG) 1500. The fused hybrids are selected for their sensitivity to HAT. Hybridomas that produce a monoclonal antibody of this invention are identified using the enzyme-linked immunosorbent assay (ELISA) described in the Examples. A monoclonal antibody of the present invention can also be produced by the initiation of a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of appropriate specificity. The crop is maintained under conditions, and for a period of time sufficient for the hybridoma to secrete the antibody molecules into the medium. The medium containing the antibody is then collected. The antibody molecules can then be further isolated by well-known techniques. Useful media for the preparation of these compositions are well known in the art and are commercially available, and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is the minimum essential medium of Dulbecco (DMEM; Dulbecco et al., Virol. 8: 396, 1959) supplemented with 4.5 grams / 1 glucose, 20 mM glutamine, and 20 percent fetal bovine serum. An exemplary endogenous mouse strain is Balb / c. Other methods for producing a monoclonal antibody, a hybridoma cell, or a hybridoma cell culture are also well known. See, for example, the method of isolation of monoclonal antibodies from an immunological repertoire, as described by Sastry et al., Proc. Nati Acad. Sci. USA, 86: 5728-5732 (1989); and Huse et al., Science, 246: 1275-1281 (1989). This invention also contemplates the hybridoma cell, and the cultures that contain a hybridoma cell that produce a monoclonal antibody of this invention. Particularly preferred is the hybridoma cell line secreting monoclonal antibody mAb LM 609 designated ATCC HB 9537. mAb LM609 was prepared as described by Cheresh et al., J. Biol. Chem., 262: 17703-17711 (1987), and their preparation is also described in the Examples. The invention contemplates, in one embodiment, a monoclonal antibody having the immunoreaction characteristics of mAb LM609. It is also possible to determine, without undue experimentation, whether a monoclonal antibody has the same (i.e., equivalent) specificity (immunoreaction characteristics) as a monoclonal antibody of this invention, by means of ascertaining whether the former prevents the latter from binding to a target molecule previously selected. If the monoclonal antibody being tested competes with the raono-clonal antibody of the invention, as shown by a decrease in binding by the monoclonal antibody of the invention, in standard competition assays for binding to the target molecule when it is present in the solid phase, then it is likely that the two monoclonal antibodies bind to it, or to a closely related epitope. Yet another way of determining whether a monoclonal antibody has the specificity of a monoclonal antibody of the invention is to pre-incubate the monoclonal antibody of the invention with the target molecule with which it is normally reactive, and then add the monoclonal antibody that is is testing to determine if the monoclonal antibody being tested is inhibited in its ability to fix the target molecule. If the monoclonal antibody being tested is inhibited, then, in all likelihood, it has the same, or functionally equivalent, epitope specificity as the monoclonal antibody of the invention. A further way to determine whether a monoclonal antibody has the specificity of a monoclonal antibody of the invention is to determine the amino acid residue sequence of the CDR regions of the antibodies in question. Antibody molecules that have identical, or functionally equivalent, sequences of amino acid residues in their CDR regions have the same binding specificity. Methods for sequencing polypeptides are well known in the art. The immunospecificity of an antibody, its binding capacity to the target molecule, and the concomitant affinity exhibited by the antibody for the epitope, are defined by the epitope with which it immunoreacts the antibody. The epitope specificity is defined at least in part by the amino acid residue sequence of the variable region of the heavy chain of the antibody immunoglobulin, and in part by the amino acid residue sequence of the light chain variable region. The use of the term "having the binding specificity" of india that the equivalent monoclonal antibodies exhibit the same, or similar immunoreaction (binding) characteristics, and compete for binding to a previously selected target molecule. Humanized monoclonal antibodies offer advantages over murine monoclonal antibodies, particularly in that they can be used therapeutically in humans. Specifically, human antibodies are not cleared from the circulation as rapidly as "foreign" antigens, and do not activate the immune system in the same way as foreign antigens and foreign antibodies. Methods for preparing "humanized" antibodies are generally well known in the art, and can be rapidly applied to the antibodies of the present invention. Therefore, the invention contemplates, in one embodiment, a monoclonal antibody of this invention being humanized by means of grafting to introduce components of the human immune system without substantially interfering with the ability of the antibody to bind the antigen. 3. Specific Mimetics of α-, β3 The present invention demonstrates that o-vβ3 antagonists can generally be used in the present invention, antagonists which may include polypeptides, antibodies, and other molecules, designated "mimetics", which have the ability to to interfere with the function of avß3. Antagonists that specifically interfere with the function of ovv3, and that do not interfere with the function of other integrins, are particularly preferred. In this context, it is noted that a variety of reagents may be suitable for use in the present methods, as long as these reagents possess the required biological activity. Generically, these reagents are referred to as mimetics because they possess the ability to "mimic" a binding domain either in avß3 or in the ligand of? Ívß3 involved in the functional interaction of the receptor and the ligand, and to interfere with the same with ( that is, interfere) the normal function. A mimetic of? Vss3 is any molecule, other than an antibody or peptide derived from the ligand, which exhibits the properties described above. This can be a synthetic peptide, an analogue or derivative of a peptide, a compound that is shaped like the binding pocket of the binding domain described above, such as an organic mimic molecule, or another molecule. A preferred mimetic of this invention is a molecule with an organic base, and therefore, it is referred to as an organic mimetic. Compounds 7, 9, 10, 12, 14, 15, 16, 17 and 18 are the particularly preferred organic mimetic molecules which function as avß3 antagonists by being a mimetic for a βIV3 ligand, as described in Example 10. The design of an avß3 mimetic can be driven by any of a variety of structural analysis methods for drug design known in the art, including molecular modeling, two-dimensional nuclear magnetic resonance (2-D NMR) analysis , X-ray crystallography, random tracking of peptides, analog of peptides or other libraries of chemical polymers or compounds, and similar drug design methodologies. In view of the broad structural evidence presented in the present specification, which shows that a? Vß3 antagonist can be a fusion polypeptide (e.g., an MMP-2 fusion protein), a small polypeptide, a cyclic peptide, a derived peptide, an organic mimic molecule, or a monoclonal antibody, which are variously different chemical structures that share the functional property of selective inhibition of or; vß3, the structure of an antagonist of o; vß3 object useful in the present methods should not be so limited, but includes any mimetic of? £ vß3, as defined herein. F. Methods for Identifying Q-Antagonists. The invention also discloses test methods for identifying candidate avß3 antagonists, to be used in accordance with the present methods. In these test methods, the molecules are evaluated to see their potency in the inhibition of the binding of o-vß3 to the natural ligands, and in addition they are evaluated to see their potency in the inhibition of angiogenesis in a tissue. The first assay measures the inhibition of direct binding of the natural ligand to avß3, and a preferred embodiment is described in detail in the Examples. The assay typically measures the degree of inhibition of the binding of a natural ligand, such as fibrinogen, to avß3 isolated on the solid phase, by enzyme-linked immunosorbent assay. The assay can also be used to identify compounds that exhibit specificity by immunosorbent assay, and not inhibit natural ligands from binding to other integri-nies. The specificity assay is conducted by performing parallel enzyme-linked immunosorbent assays, wherein both β1β3 and other integrins are screened concurrently in separate test chambers to see their respective abilities to bind to a natural ligand, and to view the compound candidate to inhibit the respective abilities of integrins to bind to a previously selected ligand. The preferred trace assay formats are described in the Examples. The second test measures angiogenesis in the chick chorioallantoic membrane (CAM) and is referred to as the CAM assay. Others have described the CAM assay in detail, and it has also been used to measure both angiogenesis and neovascularization of tumor tissues. See Ausprunk et al., Am. J. Pathol. , 79: 597-618 (1975) and Ossonski et al., Cancer Res. 40: 2300-2309 (1980). The chicken chorioallantoic membrane assay is a well-recognized test model for angiogenesis in vivo because whole tissue neovascularization is occurring, and the current chicken embryo blood vessels are growing within the chicken chorioallantoic membrane or within the tissue that grew in the chicken chorioallantoic membrane. As demonstrated herein, the chicken chorioallantoic membrane assay illustrates the inhibition of neovascularization based on both the amount and extent of new vessel growth. On the other hand, it is easy to monitor the growth of any tissue transplanted on the chicken chorioallantoic membrane, such as a tumor tissue. Finally, the assay is particularly useful because there is an internal control for the toxicity in the assay system. The chicken embryo is exposed to any test reagent, and therefore the health of the embryo is an indication of toxicity. The third assay measures angiogenesis in the rabbit eye model in vivo, and is referred to as the rabbit eye test. Others have described the rabbit eye test in detail, and have also been used to measure both angiogenesis and neovascularization in the presence of angiogenic inhibitors such as thalidomide. See D 'Amato et al., Proc. Nati Acad. Sci., USA, 91: 4082-4085 (1994). The rabbit eye test is a well-recognized test model for angiogenesis in vivo because the process of neovascularization, exemplified by the rabbit's blood vessels growing from the edge of the cornea inside the cornea, is easily visualized through the naturally transparent cornea of the eye. Additionally, both the extent and amount of stimulation or inhibition of neovascularization or regression of neovascularization can be easily monitored over time. Finally, the rabbit is exposed to any test reagent, and therefore the health of the rabbit is an indication of the toxicity of the test reagent. The fourth assay measures angiogenesis in the mouse model of chimeric mouse: human, and is referred to as the chimeric mouse assay. Others have described the test in detail, and have also been described herein to measure angiogenesis, neovascularization, and regression of tumor tissues. See Yan, et al., J. Clin. Invest., 91: 986-996 (1993). The chimeric mouse assay is a useful assay model for viewing angiogenesis in vivo, because transplanted skin grafts closely resemble normal human skin histologically, and neovascularization of the entire tissue is occurring, where the human blood vessels Current are growing from grafted human skin, within human tumor tissue on the surface of grafted human skin. The origin of neovascularization within the human graft can be determined by immunohistochemical staining of the neovasculature with markers of human-specific endothelial cells. As demonstrated herein, the chimeric mouse assay demonstrates the regression of neovascularization based on both the amount and extent of regression of new vessel growth. On the other hand, it is easy to monitor the effects on the growth of any transplanted tissue on the grafted skin, such as a tumor tissue. Finally, the test is useful because there is an internal control for toxicity in the test system. The chimeric mouse is exposed to any test reagent, and therefore the health of the mouse is an indication of toxicity. G. Manufacturing Article The invention also contemplates an article of manufacture, which is a container labeled to provide an avß3 antagonist of the invention. A manufacturing article comprises packaging material and a pharmaceutical agent contained within the packaging material. The pharmaceutical agent in an article of manufacture is any of the ovv3 antagonists of the present invention, formulated in a pharmaceutically acceptable form, as described herein, in accordance with the indications described. The article of manufacture contains a quantity of pharmaceutical agent sufficient to be used in the treatment of a condition indicated herein, either in single or multiple doses. The packaging material comprises a label indicating the use of the pharmaceutical agent contained therein, for example, to treat conditions assisted by the inhibition of angiogenesis, and similar conditions described herein. The label may also include instructions for use, and related information that may be required for marketing. The packaging material may include container (s) for storage of the pharmaceutical agent. As used herein, the term "packaging material" refers to a material such as glass, plastic, paper, aluminum foil, and the like, capable of holding a pharmaceutical agent within fixed elements. Thus, for example, the packaging material can be plastic or glass jars, laminated envelopes and similar containers that are used to contain a pharmaceutical composition, including the pharmaceutical agent. In preferred embodiments, the packaging material includes a label that is a tangible expression describing the content of the article of manufacture, and the use of the pharmaceutical agent contained therein. EXAMPLES The following examples related to this invention are illustrative and, of course, should not be considered as specifically limiting the invention. On the other hand, variations of the invention, now known or further developed, which would be within the scope of one skilled in the art, will be considered to fall within the scope of the present invention claimed hereinbelow. 1. Preparation of Synthetic Peptides a. Synthesis Procedure The linear and cyclic polypeptides listed in Table 1 were synthesized, using standard solid phase synthesis techniques such as, for example, those described by Merri-field, Adv. Enzvmol., 32: 221-96, (1969), and Fields, G.B. and Noble, R.L., Int. J. Peptide Protein Res., 35: 161-214, (1990). First, two grams (g) of BOC-Gly-D-Arg-Gly-Asp-Phe-Val-OMe (SEQ ID NO 1) was dissolved in 60 milliliters (ml) of methanol, to which 1.5 milliliters of 2 N of sodium hydroxide solution was added, to form a mixture. The mixture was then stirred for 3 hours at 20 degrees C (20C). After evaporation, the residue was taken up in water, acidified to a pH of 3 with dilute HCl, and extracted with ethyl acetate. The extract was dried over Na 2 SO 4, evaporated again, and the resultant BOC-Gly-D-Arg-Gly-Asp-Phe-Val-OH (SEQ ID NO 2) was stirred at 20 C for 2 hours, with 20 milliliters of water. N of HCl in dioxane. The resulting mixture was evaporated to obtain H-Gly-D-Arg-Gly-Asp-Phe-Val-OH (SEQ ID NO 3), which was subsequently dissolved in a mixture of 1800 milliliters of dichloromethane and 200 milliliters of dimethylformamide ( DMF) followed by cooling to 0C. After that, 0.5 grams of dicyclohexylcarbodiimide (DCCI), 0.3 grams of 1-hydroxybenzotriazole (HOBt) and 0.23 milliliters of N-methylmorpholine were successively added, with stirring. The resulting mixture was stirred for another 24 hours at OC, and then at 20C for another 48 hours. The solution was concentrated and treated with a mixed bed ion exchanger to rid it of salts. After the resulting resin was removed by filtration, the clarified solution was evaporated and the residue was purified by chromatography, resulting in cycle recovery (-Gly-D-Arg-Gly-Asp-Phe-Val) (SEQ ID. NO 4). The following peptides, listed in Table 1 were analogously obtained using the abbreviations of amino acid residues of single-letter code, and identified by a peptide number designation: cyclo (Arg-Gly-Asp-D-Phe-Val) ( SEQ ID NO 5); Cycle (Arg-Ala-Asp-D-Phe-Val) (SEQ ID NO: 6); Cyclo (Arg-D-Ala-Asp-Phe-Val) (SEQ ID NO 9); Cyclo (Arg-Gly-Asp-Phe-D-Val) (SEQ ID NO 7); and cycle (Arg-Gly-Asp-D-Phe-NMeVal) (methylation is in the alpha-amido nitrogen of the amide bond of the valine residue) (SEQ ID NO 15). A peptide designated as 66203, which had a sequence identical to that of peptide 62184, only differed from the latter by containing the HCl salt rather than the TFA salt present in 62184. The same is true for peptides 69601 and 62185 and for 85189 and 121974. b. Alternative Synthesis Procedure i. Cycle Synthesis (Arg-Glv-Asp-DPhe-NmeVal), TFA Salt Fmoc-Arg (Mtr) -Gly-Asp (OBut) -DDPhe-NMeVal-ONa was synthesized, using Merrifield solid-phase procedures, by addition sequence of NMeVal, DPhe, Asp (OBut), Gly and Fmoc-Arg (MTr) in a stepwise fashion, to a 4-hydroxymethyl-phenoxymethyl-polystyrene resin (Wang type resin) (the usual Merrifield-type methods of the Peptide synthesis is applied as described in Houben-Weyl, Ic, Volume 15/11, Pages 1 to 806 (1974). Polystyrene resin and amino acid residue precursors are commercially available with the chemical companies Aldrich, Sigma or Fluka ). After the completion of the sequential addition of amino acid residues, the resin is removed from the peptide chain using a 1: 1 mixture of trifluoroacetic acid / dichloromethane, which provides the product Fmoc-Arg (Mtr) -Gly-Asp ( OBut) -DPhe-NMeVal-OH. The Fmoc group is then removed with a 1: 1 mixture of piperidine / dimethylformamide, which provides the precursor Arg (Mtr) -Gly-Asp (OBut) -DPhe-NMeVal-OH, which is then purified by liquid chromatography of high performance in the usual way. For cyclization, a solution of 0.6 grams of Arg (Mtr) -Gly-Asp (OBut) -DPhe-NMeVal-OH (synthesized above) in 15 milliliters of DMF (dimethylformamide; Aldrich) is diluted with 85 milliliters of dichloromethane (Aldrich ), and 50 milligrams of NaHCO3 are added. After cooling in a dry ice / acetone mixture, 40 μl of diphenylphosphoryl azide (Aldrich) is added. After standing at room temperature for 16 hours, the solution is concentrated. The concentrate is filtered by gel (Sephadex G10 column in isopropanol / water 8: 2) and then purified by high performance liquid chromatography in the customary manner. Treatment with TFA (trifluoroacetic acid) / H20 (98: 2) gives cyclo- (Arg-Gly-Asp-DPhe-NMeVal) x TFA, which is then purified by high performance liquid chromatography in the usual manner; RT = 19.5; FAB-MS (M + H): 589. ii. Synthesis of "Internal Salt" The salt of trifluoroacetic acid is removed from the cyclic peptide produced above, by suspending the cycle- (Arg-Gly-Asp-DPhe-NMeVal) x TFA in water, followed by evaporation under vacuum, to remove trifluoroacetic acid.

Reference is made to the cyclic peptide formed as an "inner salt", and is designated cyclo- (Arg-Gly-Asp-DPhe-NMeVal). The term "internal salt" is used because the cyclic peptide contains two oppositely charged residues which counterbalance intra-electronically to each other to form a global non-charged molecule. One of the charged residues contains an acid fraction, and the other charged residue contains an amino fraction.

When the acid fraction and the amino fraction are in close proximity to one another, the acid fraction can be deprotonated by the amino fraction, which forms a kind of carboxylate / ammonium salt with a global neutral charge. iii. Treatment of HCl to give cyclo- (Arg-Gly-Asp-DPhe-NMeVal) x HCl 80 milligrams of cyclo- (Arg-Gly-Asp-DPhe-NMeVal) were dissolved in 0.01 M HCl five to six times, and it was dried by freezing after each dissolution operation. Subsequent purification by high performance liquid chromatography gave cyclo- (Arg-Gly-Asp-DPhe-NMeVal) x HCl; FAB-MS (M + H): 589. iv. Treatment of methanesulfonic acid to give cyclo- (Arq-Gly-Asp-DPhe-NMeVal) x MeSO.H 80 milligrams of cyclo- (Arg-Gly-Asp-DPhe-NMeVal) were dissolved in 0.01 M of MeS03H (methanesulfonic acid) five to six times, and dried by freezing after each dissolution operation. Subsequent purification by high performance liquid chromatography gave cyclo- (Arg-Gly-Asp-DPhe-NMeVal) x MeS03H; RT = 17.8; FAB-MS (M + H): 589. Alternative methods of cyclization include derivatization of the side-chain chains of an acyclic peptide precursor with sulfhydryl fractions, and when exposed to slightly higher pH conditions than the conditions Normal physiological pH (pH 7.5), form intramolecularly disulfide bonds with other sulfhydryl groups present in the molecule, to form a cyclic peptide. Additionally, the C-terminal carboxylate moiety of an acyclic peptide precursor can be reacted with a free sulfhydyl moiety present within the molecule to produce cyclized thioester peptides. In the inhibition of angiogenesis assays, as described in Example 7, where the synthetic peptides were used, peptide 66203 in HCl was slightly more effective in inhibiting angiogenesis than the identical peptide in trifluoroacetic acid.

Table 1 Designation of the Sequence of Amino Acids SEO ID NO Peptide 62181 cycle (GrGDFV) 4 62184 (66203 *) cycle (RGDfV) 5 62185 (69601 *) cycle (RADfV) 6 62187 cycle (RGDFv) 7 62880 YTAECKPQVTRGDVF 8 62186 cycle (rADFV) 9 62175 cycle (ARGDfL) 10 62179 cycle (GRGDfL) 11 62411 TRQVVCDLGNPM 12 62503 GVVRNNEALARLS 13 62502 TDVNGDGRHDL 14 121974 (85189 *) cycle (RDGf-NH2Me -V) 15 112784 cycle (RGEf-NH2Me-V) 16 huMMP-2 (410-631) ** 17 huMMP-2 (439-631) ** 18 huMMP-2 (439-512) ** 19 huMMP-2 (439- 546) ** 20 huMMP-2 (510-631) ** 21 huMMP-2 (543-631) ** 22 chMMP-2 (410-637) *** 23 chMMP-2 (445-637) *** 24 chMMP-2 (445-518) *** 25 chMMP-2 (445-552) *** 26 chMMP-2 (516-637) *** 27 chMMP-2 (549-637) *** 28 * The peptides designated with an asterisk are prepared in hydrochloric acid and are identical in sequence to the designated peptide in the same line; Peptides without an asterisk are prepared in trifluoroacetic acid. The lowercase letters indicate an amino acid D; the uppercase letters indicate an amino acid L. "The amino acid residue sequences of human MMP-2 for the synthetic peptides are indicated by the corresponding residue positions shown in Figures 22A and 22B. (MMP-2 refers to a member of the matrix metalloproteinase family of enzymes.) The human MMP-2 sequences are listed with the natural cysteine residues, but are not listed with the designed cysteine residues, as described for fusion peptides. The unnatural cysteine residues were replaced by the natural amino acid residue at the indicated residue positions, in order to facilitate the solubility of the synthetic as well as expressed fusion proteins, and to ensure proper folding for the presentation of the site. Fixation. *** The amino acid residue sequences of chicken MMP-2 for synthetic peptides are indicated by the residue positions or corresponding ones that are shown in Figures 22A and 22B. The chicken MMP-2 sequences are enlisted with the natural cysteine residues, but not with the designed cysteine residues, as described for the fusion peptides as described above. 2. Monoclonal Antibodies The monoclonal antibody LM609 secreted by the hybridoma ATCC HB 9537 was produced using standard hybridoma methods, by immunization with isolated avß3, absorbed on Sepharose-lentillectin beds. Avß3 has been isolated from human melanoma cells designated M21, and the anti-body was produced as described by Cheresh et al., J. Biol. Chem., 262: 17703-17711 (1987). The M21 cells were provided by Dr. DL Morton (University of California, Los Angeles, CA) and grown in suspension cultures in RPMI 1640 culture medium containing 2 mM L-glutamine, 50 milli-grams / milliliter of gentamicin sulfate. and 10 percent fetal bovine serum. It has been shown that monoclonal antibody LM609 specifically immunoreacts with the avß3 complex, and does not immunoreact with the ocv subunit, the β3 subunit, or other integrins. 3. Characterization of the Tissue Distribution of the Expression of a "ß? A. Immunofluorescence with Anti-Inteqrin Receptor Antibodies During wound healing, the basic membranes of blood vessels express many adhesive proteins, including von Willebrand factor, fibronectin, and fibrin. In addition, many members of the integrin family of the adhesion receptors are expressed on the surface of smooth muscle culture and endothelial cells. See, Cheresh, Proc.

Nati Acad. Sci., USA, 84: 6471 (1987); Janat et al., J. Cell Phvsiol. , 151: 588 (1992); and Cheng et al., J. Cell Phvsiol. , 139: 275 (1989). Among these integrins is avß3, the endothelial cell receptor for von Wille-brand factor, fibrinogen (fibrin), and fibronectin, as described by Cheresh, Proc. Nati Acad. Sci., USA, 84: 6471 (1987). This integrin initiates a pathway of calcium-dependent signaling that leads to the migration of endothelial cells, and therefore appears to play a fundamental role in vascular cell biology, as described by Leavelsey et al., J. Cell Biol., 121: 163 (1993). To investigate the expression of avß3 during angiogenesis, wounded granulation tissue was obtained from human or adjacent normal skin of patients who consented, washed with 1 milliliter of phosphate-buffered saline, and embedded in O.C medium. (Tek fabric). The embedded tissues were instantly frozen in liquid nitrogen for about 30 to 45 seconds. Sections of six microns thick of the frozen blocks were cut in a cryostat microtome for subsequent immunoperoxidase staining with antibodies specific for either β3 integrins (β1β3 or β1ββ3) or the ββ subfamily of the integrins. The results of staining of normal human skin and wounded granulation tissue are shown in Figures 1A-1D. The AP3 and LM534 monoclonal antibodies, directed to the β3 and β1A integlines respectively, were used for the immunohistochemical analysis of the frozen sections. The tissue experiments of four different human donors yielded identical results. The photomicrographs are shown at an enlargement of 300x. The integrin? Vss3 was abundantly expressed in the blood vessels in the granulation tissue (Figure IB), but was not detectable in the dermis or in the normal skin epithelium of the same donor (FIG. IA). In contrast, the ßx integrins were they expressed abundantly in the blood vessels and stromal cells in both normal skin (Figure IC) and in granulation tissue (FIG. ID) and, as previously shown as described by Adams et al., Cell, 63: 425 (1991), in the basic cells inside the epithelium. B. Immunofluorescence with Anti-Liane Antibodies The additional sections of normal skin and human granulation tissues, prepared above, were also examined to see the presence of ligands for ß3 integrins and ßl r von Willebrand factor and laminin , respectively. The von Willebrand factor was localized to the blood vessels in normal skin (Figure 2A) and granulation tissue (Figure 2B), while laminin was localized to all blood vessels as well as to the epithelial basement membrane in both tissue preparations (Figures 2C and 2D).

C. Distribution of Anti-oi "ß3 Antibodies in Cancer Tissue In addition to the previous analyzes, biopsies of cancerous tissue from human patients were also examined to see the presence and distribution of avß3. The tissues were prepared as described in Example IA, with the exception that they were stained with the monoclonal antibody LM609 prepared in Example 2, which is specific for the integrin receptor complex, avß3. In addition, tumors were also prepared for microscopic histological analysis by fixing representative examples of tumors in Fixing Buns for 8 hours and serial sections cut t stained with H & E. Figures 3A-3D show the results of immunoperoxidase staining of cancerous tissues of the bladder, colon, chest, and lung, respectively. avß3 was abundantly expressed only in the blood vessels present in the four cancer biopsies analyzed, and not in any other cell present in the tissue. The results described herein thus show that the avß3 integrin receptor is selectively expressed in specific tissue types, namely granulates, metastatic tissues and other tissues in which angiogenesis is occurring, and not in normal tissue in where the formation of new blood vessels has stopped. These tissues, therefore, provide an ideal objective for the therapeutic aspects of this invention. 4. Identification of Specific Synthetic Peptides a a "ß ?. Detected by Inhibition of Cellular Binding, and by a Ligand Receptor Binding Assay. Inhibition of Cell Binding As a means to determine the specificity of the integrin receptor of the antagonists of this invention, cell binding assays were performed as described later. Briefly, CS-1 hamster melanoma cells lacking expression of β1β3 and cβv5 were first transfected with a plasmid to express the β3 subunit as previously described by Filardo et al., J. Cell Biol. , 130: 441-450 (1995). The specificity of the potential β1β3 antagonists was determined by the ability to block the binding of CS-1 cells expressing avß3 to VN or laminin coated plates. As an example of a typical assay, the wells were first coated with 10 microgram / milliliter substrate overnight. After rinsing and blocking with 1 percent heat denatured BSA in PBS at room temperature for 30 minutes, peptide 85189 (SEQ ID NO 15) was mixed separately over a concentration range of 0.0001 uM to 100 uM, with CS-1 cells for application to wells with a cell number of 50,000 cells / well. After an incubation of 10-15 minutes at 37 ° C, the solution containing the cells and the peptides was discarded. The number of cells bound after staining with 1 percent crystal violet was then determined. The crystal violet associated with the cells was leached by the addition of 100 microliters (μl) of 10 percent acetic acid. Cell adhesion was quantified by measuring the optical density of leached crystal violet at a wavelength of 600 nm. Figure 21 shows the result of a typical assay with a? Vß3 antagonist, here the peptide 85189. No inhibition was detected with the peptide on surfaces coated with laminin. In contrast, complete inhibition of binding was obtained on surfaces coated with VN with a peptide concentration of 10 μM or more, as shown by the dose-response curve. Similar assays were performed with fusion proteins containing different regions of the MMP-2 protein. The polypeptides derived from MMP-2 include regions of the C terminus of MMP-2 active in the binding interaction with oívß3 and, thereby, are capable of inhibiting the activation of MMP-2 and associated activities. These polypeptides are prepared either as synthetic polypeptides having a sequence derived from the C-terminal domain of MMP-2, as described in Example 1, or as fusion proteins that include all or a portion of the C-terminal domain of MMP-2, prepared as described below. C-terminal molecules of MMP-2 are presented for specific sequences of both chicken and human. The C-terminal domain of chicken-derived MMP-2, also referred to as the hemopexin domain, immediately continuous to the hinge region, comprises amino acid residues 445-637 of MMP-2. Subsequently, the complete nucleotide and the encoded amino acid sequence of chicken MMP-2 are described. Subsequently, the nucleotide and the encoded amino acid sequence of human MMP-2 are also described. The C-terminal domain in the human MMP-2 corresponding to the chicken region of 445-637 starts at amino acid residue 439 and ends with 631 due to six missing residues of the human sequence, as shown in Figures 22A and 22B. Table 1 lists the synthetic C-terminal MMP-2 peptides derived from either human or chicken, for use in the practice of the methods of this invention. The amino acid residue sequences of the synthetic peptides are the same as those generated by the counterparts of the recombinant fusion protein, but without the GST fusion component. C-terminal MMP-2 fusion proteins, derived from both chicken and human, were prepared as described below. An MMP-2 fusion protein is a polypeptide having a C-terminal domain sequence of MMP-2 or a portion thereof fused (operably linked by the covalent peptide bond) to a carrier protein (fusion), such as glutathsulfhydryl transferase (GST). To amplify different regions of chicken and human MMP-2, the primer sequences were designed based on the known cDNA sequences of chicken and human MMP-2. Figures 22A and 22B show the complete upper strand of the unprocessed chicken MMP-2 cDNA nucleotide sequence, also referred to as progelati-nase, together with the deduced amino acid sequence shown in the second line (Aimes et al., Biochem. J., 300: 729-736, 1994). The third and fourth lines of the figure respectively show the deduced amino acid sequence of human MMP-2 (Collier et al., J. Biol. Chem., 263: 6579-6587 (1988)) and mouse (Reponen et al. J. Biol. Chem., 267: 7856-7862 (1992)). Identical residuals are indicated with points, while different residuals are given by their one-letter IUPAC inscription. The missing waste is indicated by a dash. The numbering of the amino acid residues starts from the first proenzyme residue, giving the signal peptide residues negative numbers. The nucleotide sequence is numbered according to the above. The putative translation initiation (ATG) is marked with three forward arrowheads, and the translation termination signal (TGA) is indicated by an asterisk. The amino-terminal sequences for the proenzyme and the active chicken enzyme are contained with diamonds and individual arrowheads. The nucleotide sequences of progelatinase and chicken amino acid residues are listed together as SEQ ID NO 29, while the encoded amino acid residue sequence is listed separately as SEQ ID NO 30. The templates for generating amplified regions of MMP- 2 chicken were either a cDNA encoding the full-length mature chicken MMP-2 polypeptide, provided by Dr. JP Quigley of the State University of New York at Stoney Brook, New York, or a cDNA generated from a sample excised from chicken chorioallantoic membrane tissue. For the latter, the cDNA was obtained with MuLV reverse transcriptase and a downstream primer specific for nucleotides with 3 'terminal., 5 'ATTGAATTCTTCTACAGTTCA3' (SEQ ID NO 31), whose 5 'and 3' ends were respectively complementary to nucleotides 1932-1912 of the published chicken MMP-2 sequence. The reverse transcriptase polymerase chain reaction (RT-PCR) was performed in accordance with the manufacturer's specifications for the GeneA p RNA PCR kit (Perkin Elmer). The primer was also designed to contain an internal EcoRI restriction site. From any of the cDNA templates described above, many C-terminal regions of chicken MMP-2 were obtained, each having the natural cysteine residue at position 637 at the carboxy terminus, by polymerase chain reaction with the 3 'primer listed above (SEQ ID NO 31), paired with one of many 5' primers listed below. The amplified regions encoded the following MMP-2 fusion proteins, which have sequences corresponding to amino acid residue positions, as shown in Figures 22A and 22B, and also listed in SEQ ID NO 30: 1) 203- 637; 2) 274-637; 3) 292-637; 4) 410-637; 5) 445-637. Upstream or 5 'primers to amplify each of the nucleotide regions to encode the MMP-2 fusion proteins listed above were designed to encode the 3' sites of initiation of the polypeptide to an internal BamHl restriction site. designed, that is, introduced by polymerase chain reaction, to allow directional ligation within the expression vectors either pGEX-l? T or pGEX-3X. The 5 'primers included the following sequences, whose 5' and 3 'ends correspond to the positions of the indicated 5' and 3 'nucleotides of the chicken MMP-2 sequence, as shown in Figures 22A and 22B (also the position start sites of the amino acid residue for each primer are indicated): 1) Nucleotides 599-619, which encode a 203 start site 5 'ATGGGATCCACT-GCAAATTTC3' (SEQ ID NO 32); 2) Nucleotides 809-830, which encode a start site 274 5 'GCCGGATCCATGACCA-GTGTA3' (SEQ ID NO 33); 3) Nucleotides 863-883, which encode a start site 292 5 'GTGGGATCCCTGAAG-ACTATG3' (SEQ ID NO 34); 4) Nucleotides 1217-1237, which encode a 410 5 'start site GGGGATCCTTAAGG-GGATTC3' (SEQ ID NO 35); and 5) Nucleotides 1325-1345, which encode a 445 start site 5 'CTCGGATCCTCTGCA-AGCACG3' (SEQ ID NO 36). The indicated nucleotide regions of the template cDNA were subsequently amplified by 35 cycles (tempering temperature 55C), in accordance with the manufacturer's instructions for the Expand High Fidelity PCR System (Boehringer Mannheim). The resulting polymerase chain reaction products were gel purified, digested with the restriction enzymes BamH1 and EcoRI, and re-purified before ligation into their vector either pGEX-1ΔT or pGEX-3X (Pharmacia Biotech, Uppsala, Sweden) that have been digested in the same manner, as well as were dephosphorylated before the ligation reaction. The choice of plasmid was based on the required reading frame of the amplification product. The BSJ72 or BL21 cells of the E strain were transformed. competent coli, with constructions separated by heat shock. The resulting colonies were screened for incorporation of the respective MMP-2 fusion protein coding plasmid by polymerase chain reaction, for the dideoxy sequencing of positive clones, to verify the integrity of the introduced coding sequence. In addition, verification of incorporation of the plasmid was confirmed by expression of the appropriately sized GST-MMP-2 fusion protein. Purification of each of the recombinant GST-MMP-2 fusion proteins was performed using IPTG-induced phase-order cultures of magnitude, essentially as described by the manufacturer for the GST System Gene Fusion System (Pharmacia Biotech). Briefly, the recovered bacteria were destroyed by the action of the plants by sonification, and were incubated with detergent before clarification and immobilization of the recombinant protein in glutathione 4B-coupled with sepharose (Pharmacia Biotech). After extensive washing, the immobilized fusion proteins of the affinity matrix were leached separately with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, and extensively dialyzed against PBS to remove the residual glutathione before its use. Previous attempts to produce the fusion proteins between chicken MMP-2 residues 445 and 637 that only had one cysteine residue encoded resulted in insoluble products. Therefore, in order to generate additional soluble MMP-2 fusion proteins, derived from the C-terminal region that did not include an endogenous terminal cysteine residue, as is present in the fusion protein described previously, introduced nucleotide sequences within the amplified MMP-2 regions, to encode a cysteine residue would be necessary, depending on the particular fusion protein. A cysteine residue is naturally present in the sequence of chicken MMP-2 at position 446 and position 637. In the human sequence, these positions correspond respectively to 440 and 631. Therefore, the fusion proteins to contain terminal cysteine residues designed at the amino- or carboxy-terminus of the chicken MMP-2 sequences of interest, in order to allow disulfide binding with the naturally occurring cysteine in the other term, such as the construction requires it. The oligonucleotide primers were designed in accordance with the above, to allow amplification of the C-terminal regions of chicken MMP-2, for the expression of the soluble MMP-2 / GST fusion proteins. The C-terminal regions of amplified chicken MMP-2 included those for coding amino acid residue positions 445-518, 445-552, 516-637 and 549-637. For the fusion proteins containing the 517 residue, the tyrosine residue naturally encoded by a cysteine was replaced, to allow disulfide binding with cysteine at the residue position either 446 or 637. For the fusion proteins containing the residue 551, the tryptophan residue naturally encoded by a cysteine was replaced to allow disulfide binding with the naturally encoded cysteine at the residue position either 446 or 637. Briefly, the construction of the plasmid pGEX-3X encoding the protein of Recombinant GST / MMP-2 fusion (410-637), prepared above, was used as a template for amplification, in accordance with the manufacturer's protocol for the Expand High Fidelity PCR Kit (Boehringer Mannheim), using a set of primers of oligonucleotide whose design was based on the published chicken MMP-2 sequence (which is also shown in Figures 22A and 22B). An upstream primer, designed to encode a starting site of the chicken MMP-2 protein at position 445, after an internally designed BamHl endonuclease restriction site, for insertion into the vector pGEX-3X, had the sequence of nucleotides (5'CTCGGATCCTTCTGCAAGCACG3 '(SEQ ID NO 37)). The 5 'and 3 * ends of the primer corresponded respectively to positions 1325-1345 of the chicken MMP-2 sequence in the figure. Another upstream primer, designed to encode a starting site of the chicken MMP-2 protein at position 516, after an internally designed BamHl restriction site, for insertion into the pGEX-1? T GST vector, and for encoding a cysteine residue at position 517, had the nucleotide sequence (5 'GCAGGATCCGAGTGCTGGGTTTATAC3' (SEQ ID NO 38)). The 5 'and 3' ends of the primer corresponded respectively to positions 1537-1562 of the chicken MMP-2 sequence. A third upstream primer, designed to encode a starting site of the chicken MMP-2 protein at position 549, after an internal designed EcoRI endonuclease restriction site, for insertion into the pGEX-1? T GST vector , and to encode a cysteine residue at position 551, had the nucleotide sequence (5 * GCAGAATTCAACTGTGGCAGAAACAAG3 '(SEQ ID NO 39)). The 5 'and 3' ends of the primer corresponded respectively to positions 1639-1665 of the chicken MMP-2 sequence. These upstream primers were used separately with one of the following downstream primers listed below, to produce the above described regions of the C-terminal domain of chicken MMP-2. A first primer downstream (anti-sense), designed to encode a terminus site of the chicken MMP-2 protein at position 518, to encode a cysteine residue at position 517, and to contain a restriction site of internal EcoRI endonuclease for insertion into a GST vector, had the nucleotide sequence (5 'GTAGAATTCCAGCACTCAT-TTCCTGC3' (SEQ ID NO 40)). The 5 'and 3' ends of the primer, written in the 5 '-3' direction, were respectively complementary in part to positions 1562-1537 of the chicken MMP-2 sequence. A second downstream primer, designed to encode a terminus site of the chicken MMP-2 protein at position 552, to encode a cysteine residue at position 551, and to contain an internal EcoRI endonuclease restriction site for insertion into a GST vector, had the nucleotide sequence (5 'TCTGAATTCT-GCCACAGT-TGAAGG3' (SEQ ID NO 41)). The 5 'and 3' ends of the primer, written in the 5 '-3' direction, were respectively complementary in part to positions 1666-1643 of the chicken MMP-2 sequence. A third downstream primer, designed to encode a terminus site of the chicken MMP-2 protein at position 637, and to contain an internal EcoRI endonuclease restriction site for insertion into a GST vector, had the sequence of nucleotides (5 'TTGAA-TTCTTCTACAGTTCA3' (SEQ ID NO 42)). The 5 'and 3' ends of the primer, written in the 5 '-3' direction, were respectively complementary in part to positions 1932-1912 of the chicken MMP-2 sequence. The chicken MMP-2 carboxy terminus regions fixed by the above upstream and downstream primers, which are used in particular combinations to produce the fusion proteins containing at least one cysteine residue designed as described above, were amplified separately for 30 cycles with a tempering temperature of 55C, in accordance with the manufacturer's instructions for the Expand High Fidelity PCR System System (Boehringer Mannheim). The resulting amplification products were purified separately, digested with the restriction enzymes BamH1 and / or EcoRI as necessary, and re-purified before ligation into the appropriate GST fusion protein vector, either pGEX-3X or pGEX-1? T, as indicated above by the reading frame of the upstream oligonucleotide primer. To ligate the amplified MMP-2 products, the vectors were also digested, as well as dephosphorylated prior to the ligation reaction. The BL21 cells of the competent E. coli strain were then transformed separately with the vector constructs containing the resulting MMP-2 by heat shock. The resulting colonies were then screened for incorporation of the appropriate fusion protein coding plasmid by polymerase chain reaction, and the production of the appropriate sized GST fusion protein, prior to dideoxy sequencing of positive clones, for verify the integrity of the introduced coding sequence. The purification of the recombinant GST fusion proteins was then performed, using IPTG-induced phase-order cultures of magnitude, essentially as described above to produce the other GST-MMP-2 fusion proteins. The results of the inhibition of cell binding assays with different chicken MMP-2 proteins, as well as with other peptides, indicate that intact MMP-2, the CTMMP-2 (2-4) fusion protein of residues 445-637 and peptide 66203 (SEQ ID NO 5), but not MMP-2 (1-445) and control peptide 69601, inhibited adhesion of CS-1 cells expressing β3 to vitronectin but not to laminin, and by the same, inhibited the binding of the vitronectin receptor (ovv3) to vitronectin by interfering with the normal c-vß3 binding activity. Other fusion proteins CTMMP-2 7-1 from residues 274-637, 10-1 from residues 292-637 and 4-3 from residues 274-400 had less effect on cell adhesion compared to 2-4. In addition to the chicken MMP-2 GST fusion proteins described above, two human MMP-2 GST fusion proteins were produced, to express amino acid regions 203-631 and 439-631 of the mature human MMP-2 proenzyme polypeptide. The indicated regions correspond respectively to the chicken MMP-2 regions 203-637 and 445-637. Human MMP-2-GST fusion proteins were produced by polymerase chain reaction, as described above for the chicken MMP-2-GST fusion proteins, using a cDNA template which encoded the entire framework of MMP-2 open human reading provided by Dr. WG Stetler-Stevenson at the National Cancer Institute, Bethesda, Maryland, United States. The upstream 5 'primer sequences were designed based on the previously published sequence of human MMP-2 (Collier et al., J. Biol. Chem., 263: 6579-6587 (1988), and to encode a restriction Internal EcoRI introduced, to allow the insertion of the amplified products, within the appropriate expression vector. An upstream primer, designed to encode a start site of the human MMP-2 protein at position 203 after an internal designed EcoRI endonuclease restriction site, for insertion into the GST vector of pGEX-1ΔT, had the nucleotide sequence (5 'GATGAATTCTACTG-CAAGTT3 * (SEQ ID NO 43)). The 5 'and 3' ends of the primer corresponded respectively to positions 685-704 of the human MMP-2 open reading frame sequence. Another upstream primer, designed to encode a start site of the human MMP-2 protein at position 439 after an internal designed EcoRI restriction site, for insertion into the GST vector of pGEX-1ΔT, had the nucleotide sequence (5 'CACTGAATTCATCTGCAAACA3' (SEQ ID NO 44)). The 5 'and 3' ends of the primer corresponded respectively to positions 1392 and 1412 of the human MMP-2 open reading frame sequence. Each of the above primers was used separately with a downstream primer, having the 5 'and 3' ends respectively complementary to the 1998 and 1978 bases of the human MMP-2 sequence, terminating distant to the reading frame of MMP-2, and directs the termination of the protein after amino acid residue 631. The amplified products produced expressed fusion proteins that contained amino acid residues 203-631 (SEQ ID NO 45) and 439-631 (SEQ ID NO 18). ) of human MMP-2. The resulting polymerase chain reaction products were purified, digested with EcoRI and re-purified for ligation into a plasmid pGEX-1ΔT which was similarly digested and dephosphorylated prior to the ligation reaction. The cells were transformed as described above. Other human MMP-2 fusion proteins containing amino acid residues 410-631 (SEQ ID NO 17), 439-512 (SEQ ID NO 19), 439-546 (SEQ ID NO 20), 510- were also prepared. 631 (SEQ ID NO 21) and 543-631 (SEQ ID NO 22), as described above, for use in the methods of this invention. B. Ligand-Receptor Fixation Test The synthetic peptides prepared in Example 1, along with the MMP-2 fusion proteins described above, were further screened by measuring their ability to antagonize the binding activity of the receptor. in purified ligand-receptor binding assays. The method for these fixation studies has been described by Barbas et al., Proc. Nati Acad. Sci., USA, 90: 10003-10007 (1993), Smith et al., J. Biol. Chem., 265: 11008-11013 (1990), and Pfaff et al., J. Biol. Chem., 269: 20233- 20238 (1994), whose descriptions are incorporated into the present by reference. Herein is disclosed a method of identifying antagonists in a ligand-receptor binding assay in which the receptor is immobilized to a solid support and the ligand and antagonists are soluble. Also described is a ligand-receptor binding assay in which the ligand is immobilized to a solid support, and the receptor and antagonists are soluble. Briefly, the selected purified integrins were separately immobilized in Titertek microtiter wells at a coating concentration of 50 nanograms (ng) per well. Purification of the receptors that are used in ligand-receptor binding assays is well known in the art, and can be obtained rapidly with familiar methods-for one of ordinary skill in the art. After incubation for 18 hours at 4C, the non-specific binding sites on the plate were blocked with 10 milligrams / milliliter. (mg / ml) of bovine serum albumin (BSA) in saline regulated with Tris. For the inhibition studies, different concentrations of peptides selected from Table 1 were tested for their ability to block the binding of 125 I-vitronectin or 125 I-fibrinogen to integrin receptors., avß3 and aIIbß3. Although these ligands exhibit optimal binding for a particular integrin, vitronectin for ovv3 and fibrinogen for? FIIb? 3, inhibition of binding studies using peptides to block the binding of fibrinogen to any receptor, allowed the exact determination of the amount in micromoles (uM) of peptide needed to inhibit the mean-maximum binding of the receptor to the ligand. Radiolabelled ligands were used at concentrations of 1 nM, and binding was attacked separately with unlabeled synthetic peptides.

After a three-hour incubation, the free ligand was removed by washing and the fixed ligand was detected by gamma counting. The data from the trials in which the selected cyclic peptides listed in Table 1 were used, to inhibit the binding of the receptors and the radiolabelled fibrinogen, to the immobilized avß3 and o-IIbβ3 receptors separately, were highly reproducible with the error between data points typically below 11 percent. As shown in Table 2, the IC50 data in micromoles (IC50 uM) are expressed as the average of duplicate data points ± the standard deviation. Table 2 Peptide No. ot, .ß, (IC.n uM) a, tJ, (ICn uM 62181 1.96 ± 0.62 14.95 ± 7.84 62184 0.05 + 0.001 0.525 ± 0.10 62185 0.085 ± 0.16 100 ± 0.001 62187 0.05 ± 0.001 0.26 ± 0.056 62186 57.45 ± 7.84 100 ± 0.001 62175 1.05 ± 0.07 0.63 + 0.18 62179 0.395 ± .21 0.055 + 0.007 Thus, the cyclic peptides containing RGD or derivatized by RGD 62181, 62184, 62185 and 62187, each having a residual amino acid D-, exhibited preferential inhibition of fibrinogen binding to the o-vß3 receptor, as measured by the lower concentration of peptide required for medium-maximal inhibition, as compared to that for the o-IIBβ3 receptor. In contrast, the other cyclic peptides containing RGD or derivatized by RGD, 62186, 62175 and 62179, were not as effective in blocking the binding of fibrinogen to avß3 or exhibited preferential inhibition of fibrinogen binding to o-? IBß3j in comparison a o-vß3. These result are consistent with those recently published by Pfaff, et al., J. Biol. Chem., 269: 20233-20238 (1994) in which the cyclic peptide RGDFV (where F indicates an amino acid residue D-) specifically inhibited binding of fibrinogen to the integrin avß3 and not to the integrins? íIIbß3 or o-5ß !. Similar binding inhibition assays were performed with linearized peptides having or lacking an RGD motif, whose sequences were derived from the av receptor subunit, the receptor subunit or the amino acid residues of the vitronectin ligand. In Table 1 the sequences of the linear peptides, 62880 (amino acid residues 35-49 derived from VN), 62411 (amino acid residues 676-687 derived from av) are listed; 62503 (amino acid residues 655-667 derived from av) and 62502 (amino acid residues 296-306 derived from or: IIb). Each of these peptides was used in separate assays to inhibit the binding of either vitronectin (VN) or fibrinogen (FG) to either aβrβ3 or o-vβ3. Table 3 shows the IC50 data in micromoles (IC50 uM) of an individual assay for each experiment.

Table 3 Peptide No. «IIb 3 IC50 (μM) Oívß IC50 (μM) FG VN FG VN 62880 4. twenty . 98 < 0 1 0. 5 62411 > 100 > 100 > 100 > 100 62503 > 100 > 100 > 100 > 100 62502 90 5 > 100 > 100 The results of inhibition of ligand binding assays to selected integrin receptors with linearized peptides show that only peptide 62880 was effective in inhibiting media-maximal fixation of either fibrinogen or vitronectin to c.vß3, as it was measured by the lowest concentration of the peptide required for medium-maximal inhibition compared to the? IIbβ3 receptor. None of the other linearized peptides were effective in blocking the binding of the ligand to avß3, although peptide 62502 was effective in blocking the binding of vitronectin to o-? Ibß3. In other ligand-receptor binding assays performed as described above, with the exception that detection of binding or inhibition thereof was with enzyme-linked immunosorbent assay and goat anti-rabbit IgG conjugated with peroxidase, it was shown that the ligands vitronectin, MMP-2 and fibronectin at a range of 5-50 ng / well, and listed in the order of effectiveness, they were fixed to the immobilized avß3 receptor, while the collagen did not. In addition, the ability of the peptides to inhibit the binding of either MMP-2 or immobilized a? Vß3 vitronectin was evaluated, with peptides 69601 (SEQ ID NO 6) and 66203 (SEQ ID NO 5). Only peptide 66203 was effective in inhibiting the binding of any substrate to the ovv3 receptor, whereas control peptide 69601 failed to have no effect with any ligand. The specificity of MMP-2 binding to integrin receptors was confirmed with a solid-phase receptor binding assay, in which it was shown that iodinated MMP-2 was fixed to ofvß3 and not to αβIIbβ3 that had been immobilized on a solid phase (300 cpm fixation against approximately 10 CPM fixation). The ability of a peptide derived from, or MMP-2 fusion protein to inhibit the specific binding of MMP-2 to o-vß3, was demonstrated in a comparable assay, the results of which are shown in Figure 23. The fusion protein GST-CTMMP-2 (445-637) (also referred to as CTMMP-2 (2-4)) prepared as described above, labeled GST-MAID, inhibited the binding of iodinated MMP-2 to c- vß3, while GST alone had no effect with fixation CPM levels comparable to or wells receiving no inhibitor at all (labeled NT). The MMP-2 fusion protein referred to as CTMMP-2 (274-637), also referred to as CTMMP-2 (10-1), failed to inhibit MMP-binding 2 tagged to avß3. The specificity of the receptor interaction with antagonists derived from MMP-2 was confirmed with the binding and inhibition of solid phase binding assays. The CTMMP-2 (2-4), labeled in Figure 24 as [1251] GST2-4, was set to cfvß3 and not to c-IIbß3, while CTMMP-2 (10-1), labeled in Figure 24 as [1251] GST10-1, it was not fixed to any receptor in the solid phase assay in vitro. In addition, the binding of GST2-4 labeled by unlabelled GST2-4 was challenged. Therefore, the ligand-receptor assay described herein can be used to screen both circular and linearized synthetic peptides exhibiting selective specificity for a particular integrin receptor, specifically o-vß3, as used as a vitronectin receptor antagonist. (o-vß3) in the practice of this invention. 5. Characterization of the Chicken Chorioallantoic Membrane (CAM) Not Treated A. Preparation of the CAM Angiogenesis can be induced in the chick chorioallantoic membrane (CAM) after normal embryonic angiogenesis has resulted in the formation of blood vessels mature. It has been shown that angiogenesis is induced in response to specific cytokines or tumor fragments, as described by Leibovich et al., Nature, 329: 630 (1987) and Ausprunk et al., Am. J. Pathol. , 79: 597 (1975). Chicken chorioallantoic membranes were prepared from chicken embryos for subsequent induction of angiogenesis and inhibition thereof, as described in Examples 6 and 7, respectively. 10-day-old chicken embryos were obtained with Mclntyre Poultry (Lakeside, CA), and incubated at 37C with 60 percent moisture. A small hole was made through the shell, at the end of the egg, directly over the air bag, with the use of a small drill bit (Dremel, Division of Emerson Electric Co. Racine Wl). A second hole was drilled on the wide side of the egg, in a region devoid of embryonic blood vessels, previously determined by candling the egg. Negative pressure was applied to the original hole, which resulted in the CAM (chorioallantoic membrane) moving away from the shell membrane, and creating a false air sac on the chicken chorioallantoic membrane. A square window of 1.0 centimeter (cm) x 1.0 centimeter was cut through the shell, on the chorioallantoic membrane of loose chicken, with the use of a small model emery wheel (Dremel). The small window allowed direct access to the fundamental chicken chorioallantoic membrane. Then the resulting chicken chorioallantoic membrane preparation was used either at 6 days of embryogenesis, a stage marked by active neovascularization, without additional treatment to chick chorioallantoic membrane that reflects the model used to evaluate the effects on embryonic neovascularization, or was used 10 days after embryogenesis, where angiogenesis had decreased. The latter preparation was therefore used in this invention to induce renewed angiogenesis in response to treatment with cytokine or contact with the tumor, as described in Example 6. B. Histology of CAM To analyze the structure Microscopic examination of chick embryo chorioallantoic membranes and / or human tumors that were resected from chicken embryos, as described in Example 8, chick chorioallantoic membranes and tumors were prepared for frozen sectioning, as described in FIG. Example 3A. Sections of six micras (um) thickness of the frozen blocks were cut in a cryostat microtome for immunofluorescence analysis. Figure 4 shows a typical photomicrograph of an area devoid of blood vessels in an untreated 10-day-old chicken chorioallantoic membrane. Since angiogenesis in the chick chorioallantoic membrane system is decreasing for this stage of embryogenesis, the system is useful in this invention to simulate the production of new vasculature from existing vessels of adjacent areas within the areas of the membrane chorioallantoic chicken that is currently lacking in glasses. C. Integrine Profiles in the CAM Detected by Immunofluorescence To view the tissue distribution of the integrin receptors present in the chick chorioallantoic membrane tissues, frozen sections of 6 micras were fixed in acetone of both tumor tissue and chicken embryo chorioallantoic membrane, for 30 seconds, and stained by immunofluorescence with 10 micrograms / milliliter (μg / ml) mAb CSAT, a monoclonal antibody specific for the β-integrin subunit, as described by Buck et al., J Cell Biol. , 107: 2351 (1988) and thus were used for controls, or LM609 as prepared in Example 2. The primary spotting was followed by spotting with a dilution of 1: 250 of goat anti-mouse rhodamine labeled secondary antibody (Tago ), to allow the detection of the primary immunoreaction product. The sections were then analyzed with a Zeiss immunofluorescence compound microscope. The results of the immunofluorescence analysis show that the mature blood vessels present in an untreated 10-day-old chicken embryo expressed the ßx integrin subunit (Figure 5A). In contrast, in a serial section of the tissue shown in Figure 5A, no immunoreactivity was revealed with LM609 (Figure 5B). Therefore, the avß3 integrin detected by the LM609 antibody was not actively expressed by the mature blood vessels present in a 10-day-old untreated chicken embryo. As shown in the chicken chorioallantoic membrane model and in the following Examples, while the blood vessels are experiencing new growth in normal embryogenesis or induced either by cytokines or tumors, the blood vessels are expressing avß3. However, after active neovascularization, once the vessels have stopped developing, avß3 expression decreases to levels not detectable by immunofluorescence analysis. This regulation of the expression of a: vß3 in blood vessels that undergo angiogenesis, in contrast to the lack of expression in mature vessels, allows the unique ability of this invention to control and inhibit angiogenesis, as shown in the following Examples, using the CAM angiogenesis assay system. In other profiles, the metalloproteinase MMP-2 and c.vß3 were co-localized in the endothelial cells undergoing angiogenesis, three days after induction by bFGF in the 10-day-old chicken chorioallantoic membrane model. MMP-2 was expressed only minimally in vessels lacking the o-vß3 receptor. In addition, MMP-2 was colocalized with avß3 in blood vessels associated with angiogenic M21-L tumor in vivo (tumors that resulted from the injection of human M21-L melanoma cells into the dermis of human skin grafts). which grew in SCID mice, as described in Example 11), but not with blood vessels not previously associated with tumor. Similar results were also obtained for the selective association of MMP-2 and avß3 carrying CS-1 melanoma tumors in the chicken chorioallantoic membrane model, but not with CS-1 cells lacking avß3. 6. CAM Angiogenesis Assay A. Angiogenesis Induced by Growth Factors It has been shown that angiogenesis is induced by cytokines or growth factors, as referred to in Example 5A. In the experiments described herein, the angiogenesis in the chick chorioallantoic membrane preparation described in Example 5 was induced by growth factors that were applied locally to the blood vessels of the chick chorioallantoic membrane, as described in FIG. I presented. Angiogenesis was induced by placing a Whatman filter disc (Whatman filter paper No. 1) of 5 millimeters (mm) X 5 millimeters, saturated with Hanks Balanced Salt Solution (HBSS, GIBCO, Grand Island, NY ), or HBSS containing 150 nanograms / milliliter (ng / ml) of basic fibroblast growth factor (bFGF) (Genzyme, Cambridge, MA) in the chicken chorioallantoic membrane of a 10-day-old chicken embryo, in a region devoid of blood vessels, and then the windows were sealed with tape. In other trials, 125 nanograms / milliliter of bFGF were also effective to induce the growth of blood vessels. For assays where the inhibition of angiogenesis was evaluated with intravenous injections of antagonists, angiogenesis was first induced with 1-2 microgram / milliliter of bFGF in fibroblast growth medium. Angiogenesis was monitored by photomicroscopy after 72 hours. The chicken chorioallantoic membranes were frozen instantaneously, and 6 um cryose sections were fixed with acetone, and stained by immunofluorescence, as described in Example 5C, with 10 micrograms / milliliter of either CSAT anti-βx monoclonal antibody or LM609. The immunofluorescence photomicrograph in Figure 5C shows the increased expression of orvß3 during angiogenesis induced by bFGF in chick chorioallantoic membrane, in contrast to the absence of o.vß3 expression in an untreated chicken chorioallantoic membrane, as shows in Figure 5B. La? Vß3 was rapidly detectable in many (75 percent to 80 percent) of the vessels in chicken chorioallantoic membranes treated with bFGF. In addition, the βx integrin expression did not change from that seen in an untreated chicken chorioallantoic membrane, since β2 was also rapidly detectable in stimulated blood vessels. The relative expression of integrins vß3 and β2 was then quantified, during angiogenesis induced by bFGF, by means of co-focal laser image analysis of the cryostat sections of chick chorioallantoic membrane. The stained sections were then analyzed with a Zeiss laser co-focal microscope. Twenty-five vessels stained with LM609 and 15 stained with CSAT (average size ~ 1200 square millimeters, range of 350 to 3,500 square millimeters) were selected from random fields, and average rhodamine fluorescence was measured for each vessel per unit area, in arbitrary units using laser co-focal image analysis. The data are expressed as the mean fluorescence intensity in arbitrary units of vessels ± standard error (SE). The results plotted in Figure 6 show that avß3 spotting was significantly (four times higher) increased in chicken chorioallantoic membranes treated with bFGF, as determined by the Wilcoxon Rank Sum Test (P < 0.0001), while that the staining of ßx was not significantly different with the treatment with bFGF. The chicken chorioallantoic membrane assay was also used to examine the effect of another potent angiogenesis inducer, tumor necrosis factor-alpha (TNFo), on the expression of β2 and β3 integrins, it was found that impregnated filter disks either with bFGF or TNFa, and placed in the chicken chorioallantoic membranes of 10-day embryos, they promote local angiogenesis after 72 hours.The results are shown in the photomicrographs of chick chorioallantoic membranes either untreated (Figure 7A ), treated with bFGF (Figure 7B) or treated with TNFa (Figure 7C). Blood vessels are rapidly differentiated in preparations treated with both bFGF and TNFa, but are not present in the chorioallantoic membrane of untreated chickens. Therefore, local application of a growth factor / cytokine resulted in the induction of angiogenesis of mature vessels in an adjacent area within the area originally devoid of blood vessels. In view of the blood vessels induced by bFGF and the concomitant expression of avß3 as shown in Figure 5C, the treatment of TNFα results in comparable activities. These findings indicate that blood vessels of both human and chicken, involved in angiogenesis, show increased expression of ovv3. Consistent with this, the expression of avß3 in cultured endothelial cells can be induced by different in vitro cytokines, as described by Janat et al., J. Cell Physiol., 151: 588 (1992).; Enenstein et al., Exp. Cell Res., 203: 499 (1992) and Swerlick et al., J. Invest. Derm., 99: 715 (1993). Examples 7A and 7B show the effect on growth factor-induced angiogenesis by the antibody and peptide inhibitors. B. Embryonic Angiogenesis The preparation of chick chorioallantoic membrane to evaluate the effect of angiogenesis inhibitors on the natural formation of embryonic neovasculature was that of embryonic chicken embryo 6 days, as described above. At this stage of development, the blood vessels are undergoing growth again and, in this way, it provides a useful system to determine if avß3 participates in embryonic angiogenesis. The chicken chorioallantoic membrane system was prepared as described above, with the exception that the test was performed on embryonic day 6 rather than on day 10. In Example 7C the effect on embryonic angiogenesis is presented by treatment with the antibodies and peptides of this invention. C. Tumor-Induced Angiogenesis To investigate the role of avß3 in tumor-induced angiogenesis, different fragments of avß3-negative human melanoma and carcinoma were used in the chicken chorioallantoic membrane assay, which were previously grown, and isolated from the 17-day chicken embryo chick chorioallantoic membrane, as described by Brooks et al., J. Cell Biol. , 122: 1351 (1993), and as described herein. The fragments induced extensive neovascularization in the presence of pH regulator alone. Angiogenesis was induced in the chick chorioallantoic membrane assay system by direct apposition of a tumor fragment in the chicken chorioallantoic membrane. The chick embryo chicken chorioallantoic membrane preparation was identical to the procedure described above. In place of a filter paper disk, a fragment of 50 milligrams (mg) to 55 milligrams by weight of one of M21-L human melanoma tumor, human lung carcinoma UCLAP-3 tumor, cell line was placed FG of human pancreatic carcinoma (Cheresh et al., Cell, 58: 945-953, 1989), or HEp3 cell line of human laryngeal carcinoma, all of which were avß3-negative tumors, in the chicken chorioallantoic membrane in one area originally devoid of blood vessels. The human melanoma cell line M21-L, human lung carcinoma cell line UCLAP-3, human laryngeal carcinoma cell line HEp3, all avß3-negative, were used to grow solid human tumors in the cells. Chicken chick chicken chorioallantoic membranes. First, a single 8 x 106 cell suspension of M21-L, UCLAP-3, and FB or 5 x 105 HEp3 cells was applied to chick chorioallantoic membranes in a total volume of 30 microliters of sterile HBSS. The windows were sealed with tape, and the embryos were incubated for 7 days to allow the growth of human tumor lesions. At the end of the 7 days, now a 17-day embryo, the chicken chorioallantoic membrane tumors were resected and trimmed to leave them free of the surrounding chicken chorioallantoic membrane tissue. Tumors were sliced into tumor fragments from 50 milligrams to 55 milligrams, for use in either angiogenesis or tumor growth assays. The tumor fragments were placed in a new 10-day chicken embryo chick chorioallantoic membrane assembly, as described in Example 6A in an area devoid of blood vessels. Tumors that grew in vivo in chicken chick chicken chorioallantoic membranes were stained to see the expression of avß3 with -mAb LM609, as described in Example 3A. No specific staining of tumor cells was observed, indicating a lack of expression of avß3. Subsequently, these chicken chorioallantoic membrane tumor preparations were subsequently treated., as described in Examples 7D and 7E, to measure the effects of antibodies and peptides on tumor-induced angiogenesis. The chick chorioallantoic membrane tumor preparations were also treated as described in Examples 8, 9, and 12, to measure the effects of antibodies and peptides on the regression of tumors and apoptosis of angiogenic blood vessels and vascular cells. 7. Inhibition of Angiogenesis as Measured in the CAM Sampling A. Inhibition of Growth Factor Induced Angiogenesis by Local Application of Inhibitors 1) Treatment with Monoclonal Antibodies To determine if avß3 plays an active role in angiogenesis, disks were placed filter saturated with bFGF or TNFa on chicken chorioallantoic membranes, then monoclonal antibodies (also referred to as mAb), LM609 (specific for avß3), CSAT (specific for ßx), or P3G2 or also P1F6 ( both specific for avß5) to the preparation. Angiogenesis was induced in chicken chorioallantoic membranes of 10-day-old chicken embryos by filter discs saturated with bFGF. the disks were then treated with 50 milliliters of HBSS containing 25 milligrams of mAb in a total volume of 25 microliters of sterile HBSS at 0, 24, and 48 hours. At 72 hours, chicken chorioallantoic membranes were harvested and placed in a 35 millimeter Petri dish, and washed once with 1 milliliter of phosphate buffered saline. The lower side of the filter paper and the tissue of the chicken chorioallantoic membrane were then analyzed under a stereo Olympus microscope, with two observers as a double-blind. The inhibition of angiogenesis was considered significant when chicken chorioallantoic membranes exhibited a reduction of >50 percent in the infiltration of blood vessels of the chicken chorioallantoic membrane directly under the disc. The experiments were repeated four times by antibody, with 6 or 7 embryos per condition. The results of the effects of mAb treatment on angiogenesis induced by bFGF are shown in Figures 8A-8B. An untreated chicken chorioallantoic membrane preparation, devoid of blood vessels, is shown in Figure 8A to provide a comparison with the induction of blood vessels by bFGF shown in Figure 8B and the effects thereon by the mAbs in Figures 8C-8E.

Approximately 75 percent of these chicken chorioallantoic membranes treated with mAb LM609 exhibited an inhibition of > 50 percent of the angiogenesis, as shown in Figure 8E, and many of these appeared devoid of vessel infiltration. In contrast, pH regulator control (Figure 8A) and discs treated with mAbs CSAT (Figure 8C) and P3G2 (Figure 8D) consistently showed extensive vascularization. Identical results were obtained when angiogenesis with TNFa was induced. To examine the effects of these same antibodies on mature blood vessels previously existing from the normal vessel development adjacent to the areas devoid of vessels, filter discs saturated with mAbs were placed in the vascularized regions of the chicken chorioallantoic membranes of 10-day embryos. , who did not receive the local application of cytokine. None of the three mAbs affected the previously existing vessels, as assessed by visualization under a stereo microscope. Therefore, mAb LM609 selectively inhibited only the growth of new blood vessels, and did not affect mature blood vessels present in adjacent areas. This same effect was seen with the application of synthetic peptides applied either locally or intravenously, as described in Examples 7A2) and 7E2), respectively. 2) Treatment with Synthetic Peptides Chiral chorioallantoic membrane tests were also performed with the synthetic peptides of this invention, to determine the effect of cyclic and linearized peptides on growth factor induced angiogenesis. The peptides were prepared as described in Example 1, and 80 ug of peptide was presented in a total volume of 25 microliters of sterile HBSS. The peptide solution was immediately applied to the chicken chorioallantoic membrane preparation, and then again at 24 and 48 hours. After 72 hours the filter paper and surrounding chicken chorioallantoic membrane tissue were dissected, and viewed as described above. The results of this assay revealed to be similar to those shown in Figures 9A-9C, as described in Example 7E2), where synthetic peptides were injected intravenously into tumor-induced blood vessels. Here, with the control peptide, 62186, the blood vessels induced by bFGF remained undisturbed, as shown in Figure 9A. In contrast, when the cyclic RGD peptide, 62814, was applied to the filter, the formation of blood vessels was inhibited leaving the area devoid of new vasculature. This effect was similar in appearance to that shown in Figure 9B, as described in Example 7E2) below. In addition, as also shown in Figure 9C for peptides injected intravenously, in areas in which mature blood vessels were present, still far from the placement of the filter saturated with growth factor, no effect was seen with the local treatment of synthetic peptides in these external vessels. The inhibitory activity of the peptides on angiogenesis is therefore limited to the areas of angiogenesis induced by the growth factors, and does not affect the adjacent mature vessels previously existing, nor did it result in any cytotoxicity harmful to the area surrounding. Similar tests were performed with the other synthetic peptides prepared in Example 1 and listed in Table 1. 3) Treatment with MMP-2 Peptide Fragments To demonstrate the biological effects of the MMP-2 peptide fragments on angiogenesis, chicken chorioallantoic membrane assays were performed as described above, with the exception that angiogenesis was induced with saturated filter disks for 10 minutes with bFGF at a concentration of 1.0 microgram / milliliter in HBS. The discs were then placed in the chicken chorioallantoic membrane in an area that was reduced in the number of previously existing vessels. The fusion protein CTMMP-2 (410-637) with C-terminal was then locally applied, prepared as described above, or the fusion protein (RAP) associated with the control GST receptor (1.5 micrograms in 30 microliters of HBSS), to the filter disk once a day for a total of three days. At the end of the incubation period, the embryos were sacrificed and the filter disc and the chicken chorioallantoic membrane tissue of the fundament were resected, and analyzed for angiogenesis with a stereo microscope. Angiogenesis was quantified by counting the number of branching points of the blood vessels that occur within the confines of the filter discs. It is considered that the branched blood vessels correspond mainly to emerging new angiogenic blood vessels. The quantification was performed as a double blind, by means of at least two independent observers. The results are expressed as the Angiogenic index, where the angiogenic index is the number of branching points (stimulated by bFGF) minus the number of branching points (not stimulated by the control) per filter disc. The experiments routinely had 6-10 embryos per condition. The results of the chicken chorioallantoic membrane angiogenesis assay are shown in Figures 25A-D, 26 and 27. In Figure 25, a series of photographs divided into four figures, Figures 25A-D, illustrates the comparison of inhibited angiogenesis in the presence of the CTMMP-2 fusion protein (CTMMP-2 (410-637)) (Figures 25C). -D), and not inhibited in the presence of the control GST fusion protein (Figures 25A-B). Figures 26 and 27 are bar graphs illustrating the angiogenesis index of chicken chorioallantoic membrane angiogenesis assays with CTMMP-2, the same fusion proteins as before, compared to controls (bFGF fusion protein alone or GST -RAP). In Figure 27, the results of two separate experiments (# 1 and # 2) are shown, using the CTMMP-2 fusion protein (410-637). These results demonstrated in the three figures indicate that a CTMMP-2 fusion protein or polypeptide containing a C-terminal domain of MMP-2, is a composition useful for inhibiting angiogenesis mediated by bFGF, by inhibiting avß3. B. Inhibition of Growth Factor-Induced Ankyogenesis by Intravenous Application of Inhibitors 1) Treatment with Monoclonal Antibodies The effect on growth factor-induced angiogenesis with monoclonal antibodies injected intravenously into the chorioallantoic membrane preparation was also evaluated. of chicken, for use in this invention. The preparation of chick chicken embryo chicken chorioallantoic membranes for intravenous injections was essentially as described in Example 7A with some modifications. During the candling examination procedures, prominent blood vessels were selected and marks were made on the egg shell to indicate their positions. Holes were drilled in the shell and chicken chorioallantoic membranes were removed, and filter papers saturated with bFGF were placed in chick chorioallantoic membranes, as described above. The windows were sealed with sterile tape, and the embryos were placed back in the incubator. Twenty-four hours later, a second small window was carefully cut on the lateral side of the egg shell, directly over the previously selected prominent blood vessels. The outer shell of the egg was carefully removed, leaving the embryonic membranes intact. The shell membrane was made transparent with a small drop of mineral oil (Perkin-Elmer Corp., Norwalk, Connecti-cut, United States), which allowed the blood vessels to be easily visualized. Sterile mAbs, or synthetic peptides were inoculated, the latter of which are subsequently described, directly into the blood vessels once with a 30 gauge needle at a dose of 200 micrograms of IgG per embryo, in a total volume of 100 microliters of sterile PBS. The windows were sealed with tape, and the embryos were allowed to incubate for up to 72 hours. The filter discs and the tissues of the surrounding chicken chorioallantoic membrane were analyzed, as described above. To determine the location of LM609 mAb in chick chorioallantoic membrane tissues or in tumor tissues, as shown here and in the following Examples, which were previously inoculated intravenously with LM609, the fixed sections were blocked with 2.5 100% BSA in HBSS for 1 hour at room temperature, followed by spotting with a 1: 250 dilution of goat anti-mouse Rhodamine labeled secondary antibody (Tago). The sections were then analyzed with a Zeiss immunofluorescence compound microscope. The results of intravenous antibody treatment to the CAM preparation of blood vessels induced by bFGF are shown in Figures 10A-10C. In Figure 10A, induced angiogenesis is shown as a result of treatment with bFGF. As shown in Figure 10B, no change was seen for the presence of bFGF-induced vasculature with intravenous exposure to mAb P3G2, an anti-av5 antibody. In contrast, as shown in Figure 10C, the treatment of the chicken chorioallantoic membrane preparation of angiogenesis induced by bFGF with LM609, an anti-avß3 antibody, resulted in complete inhibition of the growth of new vessels within the area of filter. The inhibitory effect on angiogenesis results, therefore, from the inhibition of avß3 receptor activity by the anti-avß3-specific antibody LM609. Since blockade of avß5 does not inhibit the formation of neovasculature within the filter site of chick chorioallantoic membranes, avß5 is not essential in comparison with avß3 for the growth of new vessels. 2) Treatment with Synthetic Peptides For chicken chorioallantoic membrane preparations in which angiogenesis was induced with 1-2 ug / ml bFGF as described above, synthetic peptides 69601 (control) were injected separately intravenously. and 66203 (SEQ ID NO 5), within the chicken chorioallantoic membrane preparations 18 hours after the bFGF induction of angiogenesis. The preparations were maintained for an additional 36-40 hours, after which the number of branching points was determined as described above. Figure 28 shows the results where peptide 66203 completely inhibited angiogenesis induced by bFGF, in contrast to the absence of inhibition with the control peptide. In other assays, peptide 85189 (SEQ ID NO 15) was evaluated to inhibit angiogenesis induced by bFGF in chick chorioallantoic membrane assay over a dose range of 10 μg / embryo at 300 μg / e brio. The assay was performed as described above. Figure 29 shows the results, where the lowest effective dose was 30 μg, with 100 and 300 μg almost completely inhibiting angiogenesis. In still other assays, peptide 85189 was compared with peptides 69601 and 66203 to see anti-angiogenesis activity. The assay was performed as described above, with the exception that 50 μg of peptide was used. The results, marked in Figure 30, showed that peptides 66203 (labeled 203) and 85189 (labeled 189), were effective inhibitors of bFGF-mediated angiogenesis, compared to controls treated with bFGF (labeled bFGF) and treated with 69601 (tagged 601). The effectiveness of the different salt formulations of peptide 85189 in similar chicken chorioallantoic membrane tests induced by bFGF was also assessed. The peptides were used at 100 μg / embryo. The same peptide sequence in HCl (peptide 85189) and in TFA (peptide 121974), inhibited angiogenesis induced by bFGF with the peptide formulated with HCl being slightly more effective than that in TFA (the respective number of branch points for peptide 85189 against 121974 is 30 against 60). Chicken chorioallantoic membranes that were not treated, labeled as "cytokine-free" had approximately half the branching points as those seen with bFGF treatment, respectively 70 versus 190. Treatment with control peptide 69601 , had no effect on the inhibition of angiogenesis (230 branch points). The other synthetic peptides that were prepared in Example 1 were injected intravenously separately into the blood vessels induced by the growth factor in the chicken chorioallantoic membrane preparation, as described above. The effect of the peptides on the viability of the vessels was similarly assessed. 3) Fragment Treatment of MMP-2 With the protocol described above, the effect of the fusion proteins MMP-2, CTMMP-2 (2-4), also referred to as CTMMP-, was also evaluated. 2 (445-467) and CTMMP-2 (10-1), which are also referenced as CTMMP-2 (274-637). The assay was performed as described above, with the exception that 50 μg of fusion protein was administered to the embryos treated with bFGF. The effect of the treatment of the fusion protein was evaluated at 24 hours, 48 hours and 72 hours. The results for these selected time periods are shown in Figures 31A-L, where angiogenesis was evaluated in a photographic manner under non-treatment assay conditions, bFGF treatment, bFGF treatment followed by CTMMP-2 (2-4). ), labeled as bFGF + MAID (MAID = inhibition domain of angiogenesis MMP-2), and treatment of bFGF followed by CTMMP-2 (10-1), labeled as bFGF + Control. The significant induction of angiogenesis after 48 and 72 hours, followed by the treatment of bFGF was almost completely inhibited only by exposure to CTMMP-2 (2-4). The extent of inhibition with CTMMP-2 (2-4) was greater than that seen with CTMMP-2 (10-1), which exhibited some anti-angiogenesis activity in vivo. The other compositions of MMP-2, complete MMP-2, fragments and fusion proteins, which were prepared as described above, were also injected intravenously separately into the blood vessels induced by the growth factor in the preparation of the Chorioallantoic chicken membrane, as described above. The effect of the peptides on the viability of the vessels was similarly assessed. C. Inhibition of Embryonic Angiogenesis by Topical Application. 1) Treatment with Monoclonal Antibodies To determine whether avß3 participates or not in embryonic angiogenesis, the effect of LM609 on the new growth of blood vessels in chicken chorioallantoic membranes in 6-day-old embryos was examined, a stage marked by active neovascularization, as described in Example 5A. The chicken chorioallantoic membrane assay was prepared as described in Example 6C with the subsequent topical application of disks saturated with mAbs placed on the chicken chorioallantic membranes of 6-day-old embryos in the absence of cytokines. After 3 days, they were resected and photographed. Each experiment included 6 embryos per group and was repeated 2 times.

The LM609 antibody (Figure 11C), but not the CSAT (Figure HA) or P3G2 (Figure 11B), prevented vascular growth under these conditions; this indicates that avß3 plays a substantial role in embryonic neovascularization that was independent of the aggregated growth factors for the induction of angiogenesis. 2) Treatment with Synthetic Peptides The synthetic peptides that were prepared in the Example 1, were added separately to the preparation of the chorioallantoic chicken embryo membrane that was prepared above and as described in Example 5A2) by either topical application to chick chorioallantoic membrane, or intravenous application to chickens. blood vessels. The effect of the peptides on the viability of the vessels was similarly assessed. D. Inhibition of Tumor-Induced Angiogenesis by Topical Application 1) Treatment with Monoclonal Antibodies In addition to the angiogenesis assays described above, wherein the effects of the anti-avß3 antagonists, LM609 and different peptides, on the In embryonic angiogenesis, the role of avß3 in tumor-induced angiogenesis was also investigated. Fragments of human negative M21-L melanoma of avß3 that were cultured and isolated from the chicken chorioallantoic membrane of a 17-day-old chicken embryo were used as an inducer. The fragments were prepared as described in Example 6C. As described above in Example 7A1), mAbs were applied topically separately to the tumor fragments at a concentration of 25 ug in 25 ul of HBSS and then the windows were sealed with adhesive tape. The MAbs were added again in the same way, at 24 hours and at 48 hours. At 72 hours, tumors and surrounding chicken chorioallantoic membrane tissues were analyzed, as described above in Example 7A1). As described in Example 6C, the tumors were initially derived by transplantation of cultured M21-L cells, which did not express the ißtegrin avß3 as described by Felding-Habermann et al., J. Clin. Invest., 89: 2018 (1992), on the chorioallantoic membranes of chicken embryos of 10-day-old chickens. These negative avß3 fragments induced extensive neovascularization in the presence of pH regulator alone, or mAbs CSAT (anti-ßx) or P3G2 (anti-avß5). In contrast, mAb LM609 (anti-avß3) prevented the infiltration of most vessels within the tumor mass and the chorioallantoic membrane of surrounding chicken. In order to quantify the effect of mAbs on tumor-induced angiogenesis, blood vessels entering the tumor were counted within the focal plane of chick chorioallantoic membrane under a stereoscopic microscope by two observers in a double-blind fashion. Each bar of data presented in Figure 12 represents the average number of vessels SE from 12 chicken chorioallantic membranes in each group representing duplicate experiments. This quantitative analysis revealed a three-part reduction in the number of vessels entering tumors that were treated with mAb LM609, compared to tumors that were treated with pH regulator or the other mAbs, P3G2 or CSAT (P < 0.0001), as determined by the Wilco-xon Rank Sum Test. The fact that M21-L tumors do not express avß3 indicates that mAb LM609 inhibits angiogenesis by directly affecting blood vessels, rather than tumor cells. These results correspond to the histological distribution of avß3 in the cancerous tissue biopsies shown in Figure 3A-3D, where the distribution of avß3 was limited to the blood vessels in the tumor, and not to the tumor cells themselves. 2) Treatment with Synthetic Peptides The synthetic peptides that were prepared in Example 1 are applied topically., including the peptides derived from MMP-2 and the fusion proteins, to the angiogenic assay system of the chicken-induced chorioallantoic membrane of the tumor, as described above. The effect of the peptides on the viability of the vessels was similarly assessed. E. Inhibition of Tumor-Induced Angiogenesis by Intravenous Application 1) Treatment with Monoclonal Antibodies Tumor-induced blood vessels that were prepared as described in Example 7E1), were also treated with mAbs applied by intravenous injection. Tumors were placed on the chicken chorioallantic membranes, as described in 7D1), the windows were sealed with adhesive tape and 24 hours later, 200 μg of purified mAbs were inoculated intravenously into the blood of chicken embryos. as previously described. As described in Example 8 below, after this period of time, tumors were resected and analyzed by their weight to determine the effect of antibody exposure on tumor growth or suppression. 2) Treatment with Synthetic Peptides The effects of exposure of the peptide to the tumor-induced vasculature in the chick chorioallantoic membrane assay system were also assessed. The chicken chorioallantoic membrane preparation was used per tumor as described above, with the exception that instead of the intravenous injection of a mAb, synthetic peptides, prepared as described in Example 1, were injected intravenously in a manner separated inside visible blood vessels.

The results of chicken chorioallantoic membrane tests with the cyclic peptide, 66203, containing HCl salt, and control peptide, 62186 are shown in Figures 9A-9C. In Figure 9A the treatment with the control peptide it did not cause the abundant large blood vessels that were induced by the tumor treatment to grow in an area originally devoid of blood vessels from the chicken chorioallantoic membrane. In contrast, when the cyclic RGD peptide, 66203, an antagonist for the avß3 was applied to the filter, the formation of blood vessels was inhibited, leaving the area devoid of new vasculature, as shown in Figure 9B. The inhibitory effect of the peptide that contained RGD was specific and was localized as proven by an absence of any harmful effects to the vessels that are located adjacent to the place. Thus, in Figure 9C, when the inhibitory peptides are intravenously injected into the chick chorioallantoic membrane assay system, no effect was seen on the previously existing mature vessels present in chick chorioallantoic membrane in areas Adjacent, but distant, from the location of the tumor. The previously existing vessels in this location were not affected by the inhibitory peptide that flowed within these vessels, although the generation of new vessels was inhibited from these vessels that previously existed within the tumor mass. Thus, it has been shown that synthetic peptides including 66203 and 62184, of which was previously shown in the ligand-receptor assays in Example 4 that were avß3 antagonists, inhibit angiogenesis that is limited to vessels that they are experiencing development, and not to mature vessels that previously existed. In addition, the intravenous infusion of the peptides does not result in any harmful cytotoxicity in the surrounding area, as demonstrated by the intact vasculature in Figure 9C. Similar tests were performed with the other synthetic peptides that were prepared in Example 1 and listed in Table 1, together with the MMP-2 compositions of this invention. 3) MMP-2 Fragment Treatment A CS-1 (ß3-negative) tumor was prepared on a chorioallantoic chicken membrane, as described above. After 24 hours of tumor growth, a composition of the MMP-2 fragment, designated CTMMP-2 (2-4) and prepared as described in Example 4A, was intravenously administered to a 50 μg fragment in 100 μl of solution Saline regulated by phosphate. After 6 days, the tumor was evaluated to see its mass. Tumors that were treated with CTMMP-2 (2-4) were reduced in the growth rate by approximately 50 percent, when compared to the growth rate of control tumors that were treated with CTMMP-2 (10). -1) or with the control of saline solution regulated by phosphate. In this way, the avß3 antagonist inhibited tumor growth. 8. Inhibition of Tumor Tissue Growth With Antagonists at "ß. As measured in the CAM Assay as described in Example 7E1), in addition to visually assessing the effect of anti-avß3 antagonists on growth factor or tumor-induced angiogenesis, the effect of the antagonists was also assessed. by measuring any changes in the tumor mass after exposure. For this analysis, the tumor induced angiogenesis chicken chorioallantoic membrane assay system was prepared as described in Examples 6C and 7D. At the end of the 7-day incubation period, the resulting tumors were excised from the chicken chorioallantic membranes and cleaned of any chick-cell residual chorioallantoic membrane tissue, washed with 1 milliliter of phosphate-buffered saline, and They determined the wet weights for each tumor. In addition, the preparation of the tumor for microscopic histological analysis, included the fixation of representative examples of tumors in the Fixer of Bulins during 8 hours and the incrustation in paraffin. The sections were cut serially and stained with hematoxylin and eosin (H & amp; amp;; E) for the microscopic analysis. Gladson et al., J. Clin. Invest., 88: 1924 (1991). The sections were photographed with an Olympus 250x composite microscope. A. Topical Application Table 4 lists the results of the typical human melanoma tumor (M21L), the weights resulting from the topical application of the control pH regulator, P3G2 (anti-avß5) or LM609 (anti- avß3). A number of embryos were evaluated for each treatment, calculating the average weight of the tumor in milligrams (mg), together with the SE of the average, as shown at the end of the table.

Table 4 Embryo No. Treatment mAb Tumor (mg) 1 HBSS 108 2 152 3 216 4 270 5 109 6 174 1 P3G2 134 2 144 3 408 4 157 5 198 6 102 7 124 8 99 1 LM609 24 2 135 3 17 4 27 5 35 6 68 7 48 8 59 Treatment mAb Weight Promise: Tumor (mg) control HBSS 172 + 26 P3G2 171 + 36 LM 609 52 ± 13 Exposure of a human melanoma tumor mass avß3-negative in the assay system of chicken chorioallantoic membrane to LM609, caused the decrease in average weight of the untreated tumor from 172 milligrams + 26 to 52 milligrams ± 13. The P3G2 antibody had no effect on the tumor mass . Thus, blockade of the avß3 receptor by topical application of avß3-specific antibody LM609 resulted in a regression of the tumor mass together with an inhibition of angiogenesis, as shown in the previous Examples. The measured diameter of the tumor mass that resulted from exposure to P3G2 was approximately 8 millimeters to 1 centimeter on average. In contrast, tumors treated with LM609 averaged 2 to 3 millimeters in diameter. The frozen sections of these tumors revealed an intact tumor cytoarchitecture for the tumor that was exposed to P3G2, in contrast to a lack of organized cellular structure in the tumor that was exposed to LM609. Therefore, the activity of the avß3 receptor is essential for an avß3-negative tumor to maintain its nourished mass through the development of the neovasculature that expresses avß3. The avß3 block with the avß3 antagonists of this invention results in the inhibition of angiogenesis within the tumor that ultimately results in the decrease of the tumor mass. B. Intravenous Application Table 5 lists the results of the typical carcinoma tumor weights (UCLAP-3) that result from the intravenous application of the control pH regulator (PBS, phosphate-regulated saline) of the CSAT ( anti-ßj.) or of LM609 (anti-avß3). A number of embryos were evaluated for each treatment, calculating the average tumor weight of each one along with the SE of the average, as shown at the end of the table. Table 5 Embryo No. Treatment mAb Pepsis Tumor (mg) 1 PBS 101 2 80 3 67 4 90 1 CSAT 151 2 92 3 168 4 61 5 70 1 LM609 16 2 54 3 30 4 20 5 37 6 39 7 12 Treatment mAb Weight Promised Tumor (mg) control PBS 85 ± 7 CSAT 108 ± 22 LM 609 30 ± 6 The exposure of a tumor mass of avß3-negative human carcinoma in the chicken chorioallantic membrane assay system to LM609, caused the decrease in the average weight of the untreated tumor from 85 milligrams ± 7 to 30 milligrams ± 6. The CSAT antibody it did not significantly affect the weight of the tumor mass. Therefore, blockade of the avß3 receptor by intravenous application of avß3-specific antibody LM609 resulted in a regression of a carcinoma, as it did for the tumor mass of the anterior carcinoma, together with an inhibition of angiogenesis as shown in the Examples previous In addition, human melanoma tumor growth was similarly inhibited by intravenous injection of LM609. . Tumor Tumor Growth Regression With α, β3 Antagonists As Measured in CAM Assay To further assess the effects of avß3 antagonists on tumor growth and survival, fragments of human melanoma and fragments of human carcinomas were placed lung, pancreas, and larynx, on the chicken chorioallantic membranes of 10-day-old embryos, as described in Example 5A. A. Intravenous Application 1) Treatment with Monoclonal Antibodies a) Treatment with LM609 (Anti-a.ß- and CSAT (Anti-B.) Twenty-four hours after implantation of chicken chorioallantoic membrane with human melanoma carcinoma fragments M21-L avß3-negative, pancreatic carcinoma FG, human lung carcinoma UCLAP-3, or human laryngeal carcinoma HEp3, embryos were injected intravenously with phosphate-buffered saline alone or with a single dose (300 ug / 100 ul) of either mAb LM609 (anti-avß3) or CSAT (anti-ßi). The tumors were allowed to spread for an additional six days. At the end of the incubation period, the tumors were carefully resected and cleaned of tissue from the surrounding chicken chorioallantoic membrane. Two independent researchers performed the dissections, removing only the solid tumor mass that could be easily defined. The tumors had well-defined margins, so the semitransparent thin membrane (CAM), which can be easily distinguished from the solid tumor mass, was removed without disturbing the tumor mass itself. We weighed tumors that were resected and examined morphologically and histologically. As shown in Figure 13, the weights of the wet tumor were determined and compared at the end of the 7 days, with the initial weights of the tumor before the treatments. Each bar represents the average ± S.E. of 5-10 tumors per group. MAb LM609 significantly inhibited tumor growth (p <; 0.001), compared to the controls in all the tumors that were tested. Tumors that were treated with phosphate-regulated saline or CSAT, proliferated in all cases. In contrast, mAb LM609 not only prevented the growth of these tumors, but induced extensive regression in most cases. Importantly, these tumor cells do not express the avß3 integrin, demonstrating that the inhibition of growth was due to the anti-angiogenic effects of this neovasculature antibody, rather than to the tumor cells directly. . Treatment with LM609 (Anti-a "ß, v P3G2 (Anti- Oív s) M21-L human melanoma tumor fragments (50 milligrams) were implanted in chicken chorioallantic membranes of 10-day-old embryos, as described in Example 5A Twenty-four hours later, the embryos were injected intravenously with phosphate-buffered saline alone or with a single dose (300 μg / 100 μl) of either mAb LM609 (anti-avß3) or P3G2 (anti-avß5). The tumors were allowed to propagate as described in Example 9A1) above, and were examined morphologically and histologically as described herein. The representative examples of M21-L tumors treated with mAbs P3G2 (anti-av5) or LM609 (anti-av3) were morphologically examined. Tumors treated with P3G2 were large (8 millimeters in diameter) and well vascularized, while those treated with mAb LM609 were much smaller (3 millimeters in diameter) and lacked detectable blood vessels. Tumors were further examined by preparation of histological sections and staining with hematoxylin and eosin, as described in Example 9A1). As shown in Figure 14 (upper panel), tumors that were treated with mAb P3G2 (anti-avß5) showed numerous viable and actively dividing tumor cells, as indicated by the mitotic figures (arrowheads), as well as by the multiple blood vessels (arrows) through the entire tumor stroma. In contrast, few, if any, tumor cells or blood vessels were detected in the tumors that were treated with mAb LM609 (anti-avß3) (Figure 14, lower panel). These results demonstrate that avß3 integrin antagonists inhibit tumor-induced angiogenesis of human tumors in vivo. It is important to note that the embryos that were examined after seven days of tumor growth (embryonic day 17), appeared normal after a general examination, regardless of whether they had been treated with an avß3 antagonist. These findings indicate that antagonists of this integrin are not toxic to developing embryos. 2) Treatment with Synthetic Peptides Melanoma tumor fragments were implanted Human M21-L (50 milligrams) in the chicken chorioallantic membranes of 10-day-old embryos, as described in Example 5A. Twenty-four hours later, the embryos received a single intravenous injection of 300 μg / 100 μl of either the cycle-RADfV (69601) and / or the cycle-RGDfV (66203). After a total of 72 hours, the tumors were removed, examined morphologically, and photographed with a stereoscopic microscope, as described in Example 9A1). The panels shown in Figures 15A to 15E correspond as follows: Figure 15A duplicates the samples that were treated with the cyclo-RADfV peptide (69601); Figure 15B duplicates the samples that were treated with the peptide cyclo-RGDfV (66203); Figure 15C, adjacent to chick chorioallantoic membrane tissue taken from the same embryos that were treated with the cyclo-RGDfV peptide (66203) and Figures 15D and 15E, the elevated (13x) magnification of the peptide-treated tumors. Figure 15D describes the normal blood vessels of the tumor treated with the control peptide (69601). Figure 15E describes the examples of broken blood vessels of tumors treated with the peptide cyclo-RGDfV (66203) (arrows). The results illustrate that only peptide 66203, in contrast to control peptide 69601, inhibited vessel formation, and furthermore that the vessels in chick chorioallantoic membrane tissue adjacent to the tumor were not affected. Additional tumor regression assays were performed with peptide 85189 reactive to avß3 (SEQ ID NO 15), against 69601 as a control. The assays were performed as described above, with the exception that 100 ug of the peptide was injected intravenously into the chicken chorioallantic membrane at 18 hours after implantation. After a further 48 hours, the tumors were resected and the wet weights were obtained. Figures 32, 33 and 34 respectively show the reduction in tumor weight for tumors UCLAP-3, M21-L and fgM, after intravenous exposure to peptide 85189, in contrast to the lack of effect with either the solution Saline regulated by phosphate or peptide 69601. 10. Regression of Tumor Tissue Growth With Antagonists to "ß. How It Was Measured by the In Vivo Rabbit Eye Model Test The effect of anti-avß3 antagonists on angiogenesis induced by growth factor in naturally transparent structures can be observed, as exemplified by the cornea of the eye. New blood vessels grow from the edge of the cornea of the eye, which has an abundant blood supply, to the center of the cornea, which normally does not have a blood supply. The stimulators of angiogenesis, such as bFGF, when applied to the cornea, induce the growth of new blood vessels from the edge of the cornea. Antagonists of angiogenesis, when applied to the cornea, inhibit the growth of new blood vessels from the edge of the cornea. In this way, the cornea undergoes angiogenesis by invading the endothelial cells from the edge of the cornea into firm corneal tissue filled with collagen, which can be easily seen. Therefore, the rabbit ocular model assay provides an in vivo model for direct observation of the stimulus and inhibition of angiogenesis, after implantation of the compounds directly into the cornea of the eye. A. In Vivo Rabbit Eye Model Assay 1) Growth Factor Induced Angiogenesis Angiogenesis was induced in the rabbit ocular model in vivo with the growth factor bFGF, and is described in the following sections. to. Preparation of Hydron Pills Containing Growth Factor and Monoclonal Antibodies Hydron polymer pills containing growth factor and mAbs were prepared, as described by D 'Amato et al. In Proc. Nati Acad. Sci., USA, 91: 4082-4985 (1994). The individual pills contained 650 ng of the fixed bFGF growth factor to a sucralfate (Carafet, Marion Merrell Dow Corporation) to stabilize the bFGF and ensure its slow release into the surrounding tissue. In addition, the hydron pill was prepared, which contained either 40 ug of the mAb LM609 (anti-avß3), or mAb P1F6 (anti-avß5) in phosphate-buffered saline. The pills were emptied into specially prepared Teflon spikes that had a 2.5 millimeter core drilled into their surfaces. Approximately 12 ul of casting material was placed inside each spike and polymerized overnight in a sterile cover. The pills were then sterilized by uliolet irradiation. b. Treatment with Monoclonal Antibodies Each experiment consisted of three rabbits in which one eye received a pill comprising bFGF and LM609, and the other eye received a pill comprising bFGF and a mouse mAb P1F6 (anti-avß5). The use of the paired eye test to compare the LM609 (anti-avß3) with the other mAb and controls phosphate-buffered saline provides a means for rigorous testing to demonse significant differences between the mAbs that were tested. The P1F6 mAb immunoreacts with the avß5 integrin, which is located on the surface of vascular endothelial cells, but which is not supposedly involved in angiogenesis. To determine whether the P1F6 mAb was involved in angiogenesis or not, pill containing only this mAb was prepared, and tested as described below to confirm that the mAb does not induce angiogenesis. All MAbs that were tested were purified from the ascites fluid, using affinity column chromatography of Protein-A Sepharose CL-4B, in accordance with well-known methods. Thereafter, the levigated immunoglobulin was passed through dialysis against phosphate-regulated saline and treated with Detoxi-gel (Pierce Chemicals) to remove the endotoxin. It has been shown that endotoxin is a powerful angiogenic and an inflammatory stimulant. Therefore, the mAbs were tested for the presence of endotoxin with the Ameto-cyto Chromogenic Chimeobate Test (Bio-Whittaker) and only those mAbs without endotoxin detectable in the rabbit eye model assay were used. A hydron pill comprising bFGF and mAb LM609 (anti-avß3) or P1F6 was inserted, inside a horny bag that forms in the eye of rabbits. The hydron pill also contained sucralfate to stabilize the bFGF during the assay. Individual pills were implanted within surgically created "bags" formed in the middle stroma of the cornea of rabbits. The surgical procedure was performed under sterile technique using a Wild Model M691 operating microscope equipped with a beam splitter to which a camera was mounted to photograph the individual corneas in a photographic manner. A 3 millimeter x 5 millimeter "pouch" was created in the corneal stroma by means of a 3-millimeter incision at half the thickness of the cornea with a Beaver 69 knife. The stroma was dissected peripherally using an iris spatula. the pill was implanted with its peripheral margin 2 millimeters from the edge. During the next 4 days, the bFGF and the mAb were diffused from the implanted pill to the surrounding tissue and the angiogenesis was effected by the same from the edge of the cornea. The representative results of each treatment are described in Figures 16A to 16E. The number of vessels present was quantified and described in terms of clock hours, which are defined as follows. The eye was divided into 12 equal sections, in the same way that a clock is divided into hours. "An hour glass clock" refers to that number of glasses that fill an area of the eye equivalent to one hour of a clock. The five rabbits that received only bFGF exhibited embryonic angiogenesis in which new blood vessels had grown from the edge of the cornea to the center of the cornea, which normally has no blood vessels. One of these rabbits had only 1 hour clock of glasses for the pill. Two of the rabbits that received both bFGF and mAb LM609, had absolutely no angiogenesis that could be detected up to 14 days after surgery. One of these rabbits had 3 foci of hemorrhagic vessels and grafted for day 14. Two of the rabbits that received bFGF and mAb P3G2 (anti-avß5) showed extensive vascularization in which new blood vessels had grown from the edge of the cornea towards inside the cornea. One of these rabbits had only 1 to 2 hours of glasses for the pill. As was evident in the rabbit ocular model assay, no angiogenic effect was observed in normal paralimbal vessels in the presence of bFGF growth factor in rabbits receiving mAb LM609 (anti-avß5). The complete inhibition of corneal angiogenesis by mAb LM609 is substantially greater than that of any anti-angiogenic reagent that has been reported previously. c. Treatment with Polypeptides Each experiment consisted of eight rabbits in which one eye received a pill containing 100 nanograms (ng) of bFGF, and the other eye received a pill containing 1 microgram (ug) of VEFG. The pills were inserted into the corneal pocket as described above, and the cytokines subsequently stimulated the growth of new blood vessels within the cornea. Peptides were administered subcutaneously (sq) in 1 milliliter of phosphate-buffered saline at an initial dose of 50 ug per kilogram of rabbit on the day of insertion of the pill, and daily they were given subcutaneous doses at 20 ug / day. kilogram thereafter.

After 7 days, the corneas were evaluated as described above. Rabbits that received the control peptide 69601, showed substantial growth of corneal blood vessels at 7 days, in eyes stimulated by both vFGF and VEGF. Rabbits that received peptide 85189 showed less than 50 percent of the amount of corneal blood vessel growth, compared to controls in eyes stimulated by vFGF, and almost 100 percent inhibition in eyes stimulated by VEGF . 11. In Vivo Regression of Tumor Tissue Growth With Antagonists to "ß. As measured by the Chimeric Mouse Sampling: Human A chimeric mouse: human in vivo model was generated by replacing a portion of the skin of a SCID mouse with a human neonatal foreskin (Figure 17). After the skin graft was established, the human foreskin was inoculated with carcinoma cells. After a measurable tumor was established, either mAb LM609 (anti-avß3) or phosphate-buffered saline was injected into the tail vein of the mouse. After a period of 2 to 3 weeks, the tumor was excised and analyzed by weight and histology. A. Chimeric Mouse Assay: Human In Vivo The chimeric mouse model was prepared: human in. alive, essentially as described by Yan et al in J. Clin, Invest., 91: 986-996 (1993). Briefly, a 2 cm2 skin patch was removed surgically from a SCID mouse area (6-8 weeks of age) and replaced with a human foreskin. The mouse was anesthetized and the hair removed by shaving, from an area of 5 cm2 on each side of the lateral abdominal region. Two 2 cm2 circular graft beds were prepared by removing the full thickness of the skin down towards the fascia. Full-thickness human skin grafts of the same size, derived from human neonatal foreskin, were placed on the wound beds and sutured in place. The graft was covered with a band-aid, which was sutured to the wound. Micropore fabric tape was also applied to cover the wound. Human melanoma cell lines M21-L or breast carcinoma MDA 23.1 (ATCC HTB 26; avß3 negative by immunoreactivity of tissue sections with mAb LM609) were used to form human solid tumors on human skin grafts in the SCID mice. A suspension of a single cell of M21-L of 5 x 106 or MDA 23.1 cells was injected intradermally into the human skin graft. The mice were then observed for 2 to 4 weeks to allow growth of human tumors that could be measured. B. Intravenous Application 1) Treatment With Monoclonal Antibodies After the growth of measurable tumors, SCID mice, which had been injected with M21L tumor cells, were injected intravenously into the tail vein with either 250 μg of either mAb LM609 (anti-avß3) or phosphate-regulated saline, twice a week for 2 to 3 weeks. After this time, the skin tumors were resected and cleared of surrounding tissue. Several mice were evaluated for each treatment, calculating the average tumor weight of each treatment, as shown at the end of Table 6. Table 6 No. Tumor M21L Treatment Weight Tumor (mg) 1 PBS 158 2 192 3 216 4 227 LM609 195 6 42 7 82 8 48 9 37 10 100 11 172 Treatment Weight Average Tumor (mg) PBS 198 LM609 113 Exposure of the human M21L avß3-negative carcinoma tumor mass in the mouse chimeric test system: human to LM609 (anti-avß3), caused the weight reduction of the tumor treated with phosphate-regulated saline from 198 milligrams to 113 milligrams. The representative examples of M21L tumors that were treated with mAb LM609 (anti-aß3) and phosphate-buffered saline were morphologically examined. Tumors that were treated with phosphate-regulated saline were large (8 to 10 millimeters in diameter) and well vascularized, whereas those treated with mAb LM609 were much smaller (3 to 4 millimeters in diameter) and lacked of detectable blood vessels. In other experiments with M21-L melanoma tumor cells in the human mouse chimeric assay system, the response with mAb LM609 was compared to the response obtained with the synthetic peptide 85189 (SEQ ID NO 15), as compared with the synthetic control peptide 69601 (SEQ ID NO 6). The tests were performed as described above. The results, shown in Figure 35, demonstrate that synthetic peptide 85189 reduced tumor volume to below 25 mm3, as compared to the control peptide where the tumor volume was approximately 360 mm3. The LM609 mAb also significantly reduced tumor volume to approximately 60mm3. Tumors that formed in skin grafts that had been injected with MDA 23.1 cells could be detected and measured. Morphological examination of the established tumors revealed that neovascularization had occurred from the human tissue grafted into the tumor cells 23.1. Thus, blocking the vß3 receptor by intravenous application of the avß3-specific LM609 antibody and the peptides, resulted in a regression in a carcinoma in this model system, in the same way as the chorioallantoic membrane systems. of chicken and rabbit eye model, as described in Examples 9 and 10, respectively. 2) Treatment with Synthetic Peptides In a procedure similar to that described above for monoclonal antibodies, avß3 peptide antagonists were intravenously injected into the tail vein of SCID mice that had measurable M21-L tumors. . In a preliminary analysis, a dose response curve was performed for peptides 69601 (control) and 85189 (test) that were injected over a concentration range of 10 to 250 ug / ml. The average volume and weight of tumors that were resected after treatment were determined and the results are shown in Figures 36A and 36B, respectively. Peptide 85189 was effective in inhibiting M21-L tumor growth over the concentration range that was tested, compared to treatment with the control peptide, with the most effective dose being 250 ug / ml. To analyze the effectiveness of treatment with peptide 85189 over a time course, treatment regimens were evaluated in the same SCID tumor model. In one assay, treatment with any of the 85189 or 69601 peptides was started on day 6, with day 0 being the day of subcutaneous injection of the M21-L tumor of 3 x 106 cells into the skin of the mouse, with Intraperitoneal injections of 250 ug / ml of peptide 85189 or control 69601 every third day, until day 29. The other test was performed identically, with the exception that treatment was started on day 20. At the end of the trials, the tumors were resected and the average volume of the tumor in mm3 was determined. The data was marked as this value +/- the standard error of the average. The results of these tests, shown respectively in Figures 37A and 37B, indicate that peptide 85189, but not 69601, inhibited tumor growth on different days after treatment was started, depending on the particular treatment regimen. In this way, peptide 85189 is an effective avß3 antagonist, both angiogenesis and tumor growth. 12. Stimulation of Vascular Cells to Enter the Cycle of the Cell and Undergo Apoptosis in the Presence of Antagonists of the integrin α "β3 as measured in the CAM Assay The angiogenic process clearly depends on the capacity of the cytokines, such as bFGF and VEGF to stimulate vascular cell proliferation. Mignatti et al., J. Cell Biochem. 471: 201 (1991); Takeshita et al., J. Clin. Invest. , 93: 662 (1994); and Koyama et al., J. Cell. Physiol. , 158: 1 (1994). However, it is also apparent that signaling events can regulate the differentiation of these vascular cells within mature blood vessels. Thus, it is conceivable that interference with signals that are related to either the growth or differentiation of vascular cells that are undergoing new growth or angiogenesis, may result in disturbance of angiogenesis. It has been shown that integrin ligation events participate in both cell proliferation, apoptosis or programmed cell death in vitro. Schwartz, Cancer Res., 51: 1503 (1993); Meredith et al., Mol. Biol. Cell. , 4: 953 (1993); Frisch et al., J. Cell Biol., 124: 619 (1994); and Ruoslahti et al., Cell, 77: 477 (1994). Close examination of the effects of avß3 antagonists on angiogenesis reveals the presence of blood vessels associated with tumor, discontinuous and broken. Therefore, it is possible that the loss of blood vessel continuity is due to selective necrosis or apoptosis of vascular cells. To explore this possibility, chicken chorioallantoic membranes were examined after induction of angiogenesis with growth factor bFGF and treatment with the mAb and cyclic peptides of this invention. A. Treatment with Monoclonal Antibodies Apoptosis can be detected by a variety of methods, which include direct examination of DNA isolated from tissue to detect DNA fragmentation and detection of 3 'OH in intact tissue, with an antibody that specifically detects free 3'OH groups of the fragmented DNA. 1) Analysis of DNA Fragmentation Angiogenesis was induced by the placement of filter disks saturated with bFGF in chicken chorioallantoic membranes of 10-day-old embryos, as described in Example 6A. Histological analysis of chicken chorioallantic membranes with LM609 (anti-avß3) revealed peak avß3 expressions in blood vessels 12 to 14 hours after the onset of angiogenesis with bFGF. Thus, 24 hours after the stimulation with bFGF, the embryos were inoculated intravenously with 100 μl of phosphate-buffered saline alone or phosphate-buffered saline containing 300 μg, either mAb CSAT (anti-ßi) or LM609 (anti-avß3). DNA fragmentation was detected by resecting chick chorioallantoic membrane tissue directly below the filter discs saturated with bFGF 24 to 48 hours after intravenous inoculations with mAb LM609 (anti-avß3), CSAT ( anti-ßj, or phosphate-buffered saline The tissues of the dried chicken-chylous membrane were washed three times with sterile phosphate-buffered saline and finally shredded, resuspended in 0.25 percent bacterial collagenase ( Worthing-ton Biochemical; Freehold, NJ) and incubated for 90 minutes at 37 ° C and vortexed occasionally, DNA was extracted from equal numbers of chicken chorioallantoic membrane cells from a single cell suspension, as described above Bissonette et al., Nature, 359: 552 (1992). Briefly, equal numbers of chicken chorioallantoic membrane cells were dissolved in lOmM Tris-HCl, pH 8.0, 10 mM EDTA in 0.5% Triton X-100. (v / v) (Sigma, St. Louis, MO). Lysates were centrifuged at 16,000 x g for 15 minutes at 4 ° C, to separate the soluble fragmented DNA from the intact chromatin pill. The fragmented DNA was washed, precipitated, and analyzed on a 1.2 percent (w / v) agarose gel. Soluble fragmented DNA was isolated from an equal number of chicken chorioallantoic membrane cells from each treatment, electrophoretically separated on an agarose gel, and visualized by staining with ethidium bromide. No difference was detected in the relative amount of DNA fragmentation that resulted from the three different treatments at 24 hours after treatment. However, at 48 hours after treatment with mAb LM609 (anti-avß3), a significant increase in DNA fragmentation was observed, when compared with embryos that were treated with mAb CSAT (anti-ß-L) or with saline regulated by phosphate alone. 2) Stimulation of Vascular Cells to Enter the Cell Cycle To experimentally examine the role of avß3 in these processes, cells derived from chorioallantic chicken membranes treated with or without bFGF were stained with propidium iodide and they were immunoreacted with mAb LM609 (anti-avß3). Chicken chorioallantoic membranes that were isolated from the embryos were dissociated at 24 and 48 hours after treatment with mAb LM609 (anti-avß3), CSAT (anti-ßx), or phosphate-buffered saline in single-cell suspensions. by incubation with bacterial collagenase, as described above. The single cells were then permeabilized and stained with the Apop Tag Insitu Detection Kit, in accordance with the manufacturer's instructions (Oncor, Gaithersburg, MD). Apop Tag is an antibody that specifically detects free 3'OH groups of fragmented DNA. The detection of these free 3'OH groups is an established method for the detection of apoptotic cells. Gavrieli et al., J. Cell Biol. , 119: 493 (1992). The cells stained with Apop Tag in 0.1% Triton X-100 (v / v) in phosphate-buffered saline were then rinsed and re-suspended in FACS regulator containing 0.5 percent BSA (p. / v), sodium azide at 0.02 percent (w / v) and 200 ug / ml NRase A in phosphate-regulated saline. The cells were incubated for 1.5 hours, washed, and analyzed by fluorescence-activated cell sorting. Cell fluorescence was measured using a FACScan flow cytometer and the data analyzed as described below. Cell fluorescence was measured with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The lateral dispersion (SSC) and the forward scatter (FSC) were determined simultaneously and all the data were collected with a Hewlett Packard computer (HP9000), equipped with FAScan search software (Bectdn Dickinson, Mountain View, CA). The data was analyzed with the software of P.C. Lysis version I (Becton Dickinson, Mountain View, CA). The negative control gates were established by the use of cell suspensions are the addition of primary antibodies from the Apop Tag team. The same gateway regulation was applied to the two cell populations resulting in the analysis of approximately 8,000 cells per different cell treatment. Figure 18 shows the percentage of single cells derived from chicken chorioallantic membranes treated with mAb and stained with Apop Tag as determined by FACS analysis. The black bar represents cells of embryos that were treated 24 hours before the analysis. The dotted bar represents embryo cells that were treated 48 hours before the analysis. Each bar represents the average ± S.E. of the three replicas. As shown in Figure 18, chicken chorioallantoic membranes that were treated two days earlier with mAb LM609 (anti-avß3) showed an increase of 3 to 4 parts in the spotted Apop Tag, as compared to chicken chorioallantoic membranes that were treated either with Saline solution regulated by phosphate alone or with CSAT (anti-ß!). B. Treatment With Synthetic Peptides Chorioallantoic chicken membrane tests were also performed with the growth factor-induced angiogenesis, as described in Example 6A, with the synthetic peptides of this invention, to determine the effect of the cyclic peptides on apoptosis Cyclo-RGDfV (66203) and cyclo-RADfV (69601) peptides were prepared as described in Example 1. Peptide or phosphate-buffered saline solutions were injected into the preparation of chick chorioallantoic membrane at a concentration of 300 ug / ml. At 24 and 48 hours, the filter paper and tissue of the surrounding chicken chorioallantoic membrane were dissected and stained with the Apop Tag to detect apoptosis, as described above in Example 12A2). As shown in Figure 18, chicken chorioallantoic membranes that were treated two days earlier with peptide 69203 (cyclo-RGDfV), showed an increase of 3 to 4 parts in the spotting of Apop Tag, as compared to the membranes. chorioallantoic chicken that was treated with either phosphate-regulated saline alone or with the cyclic 69601 control peptide (cyclo-RADfV). C. Effect of Monoclonal Antibody Treatment on Apoptosis and the Cell Cycle Suspensions of a single cell were also examined to see the chromosomal DNA copy number, by staining with propidium iodide, to determine the effect of the treatment with monoclonal antibodies in the cell cycle and apoptosis by staining with the Apop Tag. Single-cell suspensions of chick chorioallantoic membrane that were treated 24 or 48 hours before with mAb LM609 (anti-avß3) or with CSAT (anti-ßi) or with phosphate-buffered saline, were prepared as described in the Example 12A1). To stain the cells with Apop Tag, cell suspensions were washed three times with a pH regulator containing 2.5 percent (w / v) BSA and 0.25 percent (w / v) sodium azide in saline regulated by phosphate. The cells were then fixed in 1 percent paraformaldehyde (w / v) in phosphate-buffered saline for 15 minutes followed by three washes, as described above. To avoid non-specific fixation, cell suspensions were blocked with 5 percent (w / v) BSA in phosphate-buffered saline overnight at 4 ° C. The cells were then washed as above, stained with Apop Tag, and cell fluorescence was measured with a FACScan, as described above in Example 12A. Cells from each experimental condition were stained with propidium iodide (Sigma, St. Louis, MO) at 10 ug / ml in phosphate-buffered saline for 1 hour, washed twice with phosphate-buffered saline, and they were analyzed to see the typical nuclear characteristics of apoptosis, including condensation and chromatin segmentation. The percentage of apoptotic cells was calculated by morphological analysis of the cells from at least 10 to 15 randomly selected microscopic fields. Figure 19 gives the combined results of single-cell suspensions of chicken chorioallantoic membranes from embryos that were treated either with CSAT (anti-ßx) or with LM609 (anti-avß3), stained with Apop Tag and propidium iodide, and analyzed by FACS. The Y axis represents spotting of Apop Tag (apoptosis), the X axis represents the spotting of propidium iodide (DNA content). The horizontal line represents the negative gateway for Apop Tag spotting. The left and right panels indicate chicken chorioallantoic membrane cells from embryos treated with CSAT and LM609, respectively. The cell cycle analysis was performed by analyzing approximately 8,000 events per condition and the data that is represented in a contour diagram. Samples of single cells that were stained with propidium iodide DNA ink, revealed that 25-30 percent of chick chorioallantoic membrane cells that were treated with LM609 (anti-avß3) 48 hours later of the treatment, showed evidence of condensation and / or nuclear segmentation. These processes are characteristic of cells that are undergoing apoptosis. This is in contrast to chicken chorioallantoic membranes that were treated with CSAT (anti-ßx), where 90-95 percent of the cells showed normal nuclear staining. As shown in Figure 19, consistent with the induction of apoptosis by LM609, a significant number of cells were observed in a peak containing less than one copy of DNA (AO). It has been shown earlier that this peak represents fragmented DNA in late-stage apoptotic cells. Telford et al., Cytometry, 13: 137 (1992). On the other hand, these AO cells stain easily with Apop Tag confirming the ability of this reagent to detect apoptotic cells. However, in addition to the staining of the cells in AO, also a significant number of cells containing more than one copy of DNA were stained with the Apop Tag (Figure 19). These results demonstrate that LM609 has the ability to promote apoptosis among vascular cells that have already entered the cell cycle. In contrast, the cells that were derived from chorioallantoic control chicken membranes that had entered the cell cycle showed minimal Apop Tag staining, consistent with the few apoptotic cells that were detected in chicken chorioallantic membranes treated with the control. Among those cells in chicken chorioallantoic membranes stimulated by bFGF that had entered the cell cycle (S phase and G2 / M), 70 percent showed positive staining with LM609 (anti-avß3). This compares with the 10 percent staining of LM609 that was observed between the cycling cells from chicken chorioallantoic membranes that were treated without bFGF. These findings indicate that after the stimulation of bFGF, most of the cells carrying avß3 showed active proliferation. Taken together, these findings indicate that intravenous injection of LM609 mAb or avß3 cyclic peptide antagonist promotes apoptosis within chick chorioallantoic membrane after induction of angiogenesis. Chicken chorioallantoic membranes were also histologically examined for avß3 expression by immunoreactivity with LM609 and by cells that were undergoing apoptosis by immunoreactivity with Apop Tag. The chicken chorioallantoic membrane sections that were resected from embryos that were treated 48 hours earlier with LM609 (anti-avß3), CSAT (anti-ßx), or the phosphate regulated saline solution that was prepared in Example 5A, were washed, embedded in OTC (Baxter) and snap frozen in liquid nitrogen. Six-micron sections were cut from chicken chorioallantoic membrane tissues, fixed in acetone for 30 seconds, and stored at -70 ° C until used. Tissue sections were prepared for spotting by a brief rinse in ethanol (ETOH) at 70 percent (v / v), followed by three washes in phosphate-buffered saline for 2 hours, followed by incubation with 10 ug / ml of mAb LM609 for 2 hours. The sections were then washed and incubated with an attenuated 1:50 goat anti-mouse conjugated rhodamine IgG solution (Fischer Scientific, Pittsburgh, PA) for 2 hours. Finally, the same sections were washed and stained with the Apop Tag as described in Example 12A2). Stained tissue sections were mounted and analyzed by confocal immunofluorescent microscopy. In Figure 20, panels A to C represent the chicken chorioallantoic membrane tissue from embryos that were treated with CSAT (anti-ßx), and panels D to F represent the tissue of the chorioallantic membrane of chicken from embryos that were treated with LM609 (anti-avß3). Panels A and D describe tissues stained with Apop Tag and visualized by fluorescence (FITC) superimposed on a D.I.C. Panels B and E describe the same tissue stained with mAb LM609 (anti-avß3) and visualized by fluorescence (rhodamine). Panels C and F represent the combined images of the same tissues stained both with Apop Tag, and with LM609, in where the yellow spotting represents the joint location. The bar represents 15 and 50 μm in the left and right panels, respectively. As shown in Figure 20 (A-C), after intravenous injection of CSAT or control of phosphate-regulated saline, staining with Apop Tag was minimal and randomized, indicating a minimal level of apoptosis within the tissue. In contrast, chicken chorioallantoic membranes from embryos that were previously treated with LM609 or with cyclic peptide 203, showed a majority of the vessels that were strongly stained with Apop Tag while minimal reactivity was observed among non-vascular cells. surrounding (Figure 20 DF). On the other hand, when both Apop Tag and LM609 were used to stain these tissues (19C and 19F), a significant joint localization was only observed between these markers in chick chorioallantoic membranes that were derived from embryos that were treated with avß3 antagonists (Figure 20F). These findings demonstrate that after the induction of angiogenesis in vivo, inhibitors of the integrin avß3 selectively promoted the apoptosis of the blood vessels carrying avß3. While angiogenesis is a complex process that includes many molecular and cellular biological eventsSeveral lines of evidence suggest that the vascular cell integrin plays a relatively late role in this process. First, immunohistological analysis reveals that the expression of avß3 on vascular cells reached a maximum of 12-24 hours after the induction of angiogenesis with bFGF. Second, avß3 antagonists disturb angiogenesis induced by multiple activators, suggesting that this receptor is included in the downstream common path of perhaps all the major signaling events that lead to angiogenesis. Third, chicken chorioallantoic membranes that were treated with mAb LM609 or cyclic peptide did not show a significant increase in apoptosis, as measured by staggering the DNA up to 48 hours after treatment with these antagonists. Finally, avß3 antagonists promote apoptosis of vascular cells that have already been induced to enter the cell cycle. The results provided herein provide the first direct evidence that integrin ligation events can regulate cell survival in vivo. Therefore, it is hypothesized that, once angiogenesis begins, the individual vascular cells divide and begin to move towards the angiogenic source, after which, the binding of avß3 provides a signal allowing continuous cell survival , which leads to the differentiation and formation of mature blood vessels. However, if the binding of avß3 is avoided, then the cells do not receive this molecular indication and the cells enter into apoptosis by omission. This hypothesis would also predict that after differentiation has occurred, mature blood vessels no longer require avß3 signaling for survival and therefore are refractory to antagonists of this integrin. Finally, the results presented herein provide evidence that avß3 integrin antagonists can provide a powerful therapeutic approach for the treatment of neoplasia and other diseases characterized by angiogenesis. First, avß3 antagonists break newly formed blood vessels without affecting the existing vasculature. Second, these antagonists did not have a significant effect on the viability of the chicken embryo, suggesting that they are not toxic. Third, angiogenesis was significantly blocked regardless of the angiogenic stimulus. Finally, the systemic administration of avß3 antagonists causes a dramatic regression of several histologically distinct human tumors. 13. Preparation of the Antagonists to "ß3 of Organic Molecule The synthesis of Compounds 7 (96112), 9 (99799), 10 (96229), 12 (112854), 14 (96113), 15 (79959) is described below. , 16 (81218), 17 (87292) and 18 (87293) of the organic avß3 antagonist, and is also shown in the annotated figures. Organic antagonists are also referred to by numbers in parentheses. The resulting organic molecules, referred to as organic mimetics of this invention as defined above, are then used in the methods for inhibiting avß3-mediated angiogenesis, as described in Example 11. For each of the synthesis described below, optical rotations were measured on the Perkin-Elmer 241 ultraviolet ray spectrophotometer and the visible spectrum was recorded on a Beckman DU-70 spectrometer. The XH and 13C NMR spectra at 400 and 500 MHz were recorded on a Bruker AMX-400 and AMX-500 spectrometer. The high resolution mass spectra (HRMS) were recorded on a VG ZAB-ZSE mass spectrometer under fast atom bombardment (FAB) conditions. Column chromatography was performed with 70-230 mesh silica gel. The preparatory FTA was carried out in a Merck Art. 5744 (0.5 mm). The melting points were taken on a Thomas Hoover apparatus. A. Compound 1: t-Boc-L-tyrosine benzyl ester as illustrated in Figure 38 O Jl 1 O - BENCILO NH O HO II O COMPOUND 1 Dicyclohexylcarbodiimide (DCC) (1.5 equivalents) was added to a solution of N- (tert-butoxycarbonyl) -L-tyrosine (t-Boc-L-tyrosine) (1.0 equivalents; Aldrich), in methylene chloride of 0.10 M (M) at 25 ° C and allowed to stir for 1 hour. Then, 1.5 equivalents of benzyl alcohol were added and the mixture was stirred for an additional 12 hours at 25 ° C. The reaction mixture was then diluted with ethyl acetate (0.10 M) and washed twice (2X) with water, once (IX) with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography. Compound 1, t-Boc-L-tyrosine benzyl ester, can also be purchased commercially with Sigma. B. Compound 2: (S) -3- (4- (Bromobutyloxy) pheny1-2-N-tert-butyloxycarbonyl-propionic acid benzyl ester COMPOSITE 2 A benzyl ester mixture of t-Boc-L-tyrosine (2 grams, 5.38 mmol, synthesized as described above), 1,4-dibromobutane (1.9 milliliters, 16.2 mmol; Aldrich), potassium carbonate (5 grams) and 18-crown-6 (0.1 grams; Aldrich) at 80 ° C for 12 hours. After cooling, the precipitate was filtered and the reaction mixture was evaporated to dryness in vacuo. After purifying the crude product by crystallization, using 100 percent hexane to yield 2.5 grams (92 percent) of Compound 2. C. Compound 3: (S) -3- (4- (4-Azidobutyloxy)) benzyl ester phenyl-2-N-tert-butyloxycarbonyl-1-propionic as illustrated in Figure 38, step ii O -. . J-L O "BENCILO N- HN- .O ... OR COMPOUND 3 Compound 2 (2.5 grams, 4.9 mmol) was stirred with sodium azide (1.6 grams, 25 mmol) in dimethylformamide (DMF) (20 milliliters) at 25 ° C for 12 hours. The solvent was then evaporated and the residue treated with water (approximately 10 milliliters) and extracted twice with ethyl acetate. The organic layers were combined, dried over magnesium sulfate and evaporated to yield 2.0 grams (90 percent) of Compound 3 as a colorless syrup FAB-MS: 469 (M + H +). D. Compound 4: (S) -3- (4- (4-Azidobutyloxy) phenyl-2-amino-propionic acid benzyl ester as illustrated in Figure 38 step iii O Jl "O - BENCILO N. OR NH- COMPOUND 4 Compound 3 (2.0 grams, 4.4 mmol) was dissolved in trifluoroacetic acid (TFA, 2 milliliters) and stirred for 3 hours at room temperature. Evaporation under vacuum yielded 1.6 grams (quantitative) of Compound 4 as a colorless syrup which was used without further purification for the next step. FAB-MS: 369 (M + H +). E. Compound 5: (S) -3- (4- (4-Azidobutyloxy) phenyl-2-butylsulfonamide-propionic acid benzyl ester as illustrated in Figure 38 step iv ° i i,. | H O-BENCILO N3- ^ k! L HN O O S OR COMPOSITE 5 A mixture of Compound 4 (1.6 grams, 4.3 mmol), butanesulfonic acid chloride (0.84 milliliters, 6.6 mmol) and triethylamine (1.5 equivalents) in methylene chloride was stirred. (20 milliliters) for 12 hours at room temperature.

The reaction mixture was then evaporated and the residue was dissolved in ethyl acetate, washed with dilute HCl, aqueous sodium bicarbonate and water. After evaporating it to dryness, the crude product was purified by flash chromatography (silica gel, toluene / ethyl acetate 15: 1), to yield 1.4 grams (67 percent) of Compound 5 as an amorphous solid. F. Compound 6: (S) -3- (4- (4-Aminobutyloxy) pheny1-2-butylsulfonamidopropionic acid benzyl ester as illustrated in Figure 38 step v | OH H7N HN. -O O COMPOUND 6 Compound 5 (1.3 grams, 2.6 mmol) was dissolved in milliliters of ethyl acetate / methanol / water 5/3/1 and 0.2 milliliters of trifluoroacetic acid (TFA) and hydrogenated under hydrogen (1 atmosphere, Parr Shaker apparatus) at 25 ° C in the presence of 100 milligrams of palladium (10 percent on coal). After 3 hours, the catalyst was filtered and the solvent was evaporated to yield Compound 6 as an oily residue. After lyophilization of the water, 1.0 gram (quantitative) of Compound 6 was obtained as a white powder. FAB-MS: 373 (M + H +). G. Compound 7: (S) -3- (4- (4-Guanidinobutyloxy) pheny1-2-butylsulfonamidopropionic acid benzyl ester as illustrated in Figure 38 step vi COMPOUND 7 Compound 6 (200 milligrams, 0.5 mmol), 3,5-dimethylpyrazole-1-carboxamidine nitrate (DPFN) (170 milligrams, 0.8 mmol, Aldrich Chemical Company) and triethylamine were heated. (0.15 milliliters, 1.0 mmol) in dimethylformamide (DMF, 5 milliliters), at 60 ° C for 12 hours. After cooling, the solvent was evaporated in vacuo, and the residue was purified by high performance liquid chromatography (Lichrocart RP-18, acetonitrile gradient / water + 0.3% trifluoroacetic acid 99: 1 to 1:99) to yield 50 milligrams (25 percent) of Compound 7 as a white, amorphous powder, after lyophilization. FAB-MS: 415 (M + H +), melting point: 70 ° C. H. Compound 8: (S) -3- (4- (4-Aminobutyloxy) phenyl-2-N-tert-butyloxycarbonyl-propionic acid as illustrated in Figure 39 step iii - BENCILO i COMPOUND 8 Compound 3 (0.5 grams, 1.07 mmol) was dissolved in 10 milliliters of ethyl acetate / methanol / water 5/3/1 and 0.1 milliliters of trifluoroacetic acid (TFA) and hydrogenated under hydrogen (1 atmosphere; Parr Shaker) at 25 ° C in the presence of 30 milligrams of palladium (10 percent on charcoal). After 3 hours, the catalyst was filtered and the solvent was evaporated to yield Compound 8 as an oily residue. After lyophilization of the water, 370 milligrams (quantitative) of Compound 8 was obtained as a white powder. FAB-MS: 353 (M + H +). I. Compound 9: (S) -3- (4- (4-Guanidinobutyl-xi) pheny1-2-N-tert-butyloxycarbonyl-propionic acid as illustrated in Figure 39 step iv O JJ.i O BENCILO H2N. NH HN O. O NH COMPOSITE 9 Compound 8 (200 milligrams, 0.5 mmol), 3,5-dimethylpyrazole-1-carboxamidine nitrate (DPFN) (170 milligrams, 0.8 mmol, Aldrich Chemical Company) and triethylamine (0.15 milliliters, 1.0 mmol) were heated in dimethylformamide (DMF, 5 milliliters) at 60 ° C for 12 hours. After cooling, the solvent was evaporated in vacuo, and the residue was purified by high performance liquid chromatography (Lichrocart RP-18, acetonitrile gradient / water + 0.3% trifluoroacetic acid 99: 1 to 1:99). to yield 160 milligrams (90 percent) of Compound 9 as a white, amorphous power, after lyophilization. FAB-MS: 395 (M + H +). J. Compound 10: (R) -3- (4- (4-Guanidinobutyl-xi) phenyl 1-2 -butyl sulfamidopropionic acid as illustrated in Figure 39 steps i-vi COMPOUND 10 The identical reaction sequence was used to synthesize Compound 7 to prepare analogue 10 of D-tyrosine, from which 205 milligrams were obtained as a white, amorphous material FAB-MS: 415 (M + H +) as follows, using intermediate Compounds 100-600 to form Compound 10: 1) Compound 100: t-Boc-D-tyrosine benzyl ester as illustrated in Figure 40 HO i- O - BENCILO NH O.

COMPOUND 100 Dicyclohexylcarbodiimide (DCC) (1.5 equivalents) was added to a solution of N- (tert-butoxycarbonyl) D-tyrosine (t-Boc-L-tyrosine) (1.0 equivalent, Aldrich) in methylene chloride 0.10 M, a 25 ° C and allowed to stir for 1 hour. Then, 1.5 equivalents of benzyl alcohol were added and the mixture was stirred for an additional 12 hours at 25 ° C. The reaction mixture was then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography. 2) Compound 200: (R) -3- (4- (4-Bromobutyloxy) phenyl-2-N-tert-butyloxycarbonyl-propionic acid benzyl ester as illustrated in Figure 40 step i "BENCILO COMPOSITE 200 A benzyl ester mixture of t-Boc-D-tyrosine (2 grams, 5.38 mmol, synthesized as described above), 1,4-dibromobutane (1.9 milliliters, 16.2 mmol, Aldrich), was heated at 80 ° C during 12 hours. After cooling, the precipitate was filtered and the reaction mixture was evaporated to dryness in vacuo. The crude product was then purified by crystallization, using 100 percent hexane to yield 2.5 grams (92 percent) of Compound 200. 3) Compound 300: (R) -3- (4- (4-Azidobutyloxy) phenyl-2-N-tert-butyloxycarbonyl-propionic acid benzyl ester as illustrated in Figure 40 step ii COMPOUND 300 Compound 200 (2.5 grams, 4.9 mmol) was stirred with sodium azide (1.6 grams, 25 mmol) in dimethylformamide (DMF) (20 milliliters) at 25 ° C for 12 hours. The solvent was then evaporated and the residue treated with water (approximately 10 milliliters) and extracted twice with ethyl acetate. The organic layers were combined, dried over magnesium sulfate and evaporated to yield 2.0 grams (90 percent) of Compound 300 as a colorless syrup. FAB-MS: 469 (M + H +). 4) Compound 400: (R) -3- (4- (4-Azidobutyloxy) phenyl-2-aminopropionic acid benzyl ester as illustrated in FIG. 40 step iii O-, I-BENCILO N3 ^. - .. - 0 -1 -.?; ÑH2 COMPOSITE 400 Compound 300 (2.0 grams (4.4 mmol)) was dissolved in trifluoroacetic acid (TFA, 2 milliliters) and stirred for 3 hours at room temperature. Evaporation under vacuum yielded 1.6 grams (quantitative) of Compound 400 as a colorless syrup which was used without further purification for the next step. FAB-MS: 369 (M + H +). 5) Compound 500: (R) -3- (4- (4-Azidobutyloxy) pheny1-2-butylsulfonamido-propionic acid benzyl ester as illustrated in Figure 40 step iv O 11 O - BENCILO N í. i,, C 3 3 ---- HN. BEAR COMPOSITE 500 A mixture of Compound 400 (1.6 grams; 4. 3 mmol), butanesulfonic acid chloride (0.84 milliliters, 6.6 mmol) and triethylamine (1.5 equivalents), in methylene chloride (20 milliliters) for 12 hours at room temperature.

The reaction mixture was then evaporated and the residue was dissolved in ethyl acetate, washed with dilute HCl, aqueous sodium bicarbonate and water. After evaporating to dryness, the crude product was purified by flash chromatography (silica gel, toluene / ethyl acetate 15: 1) to yield 1.4 grams (67 percent) of Compound 500 as an amorphous solid. 6) Compound 600: (R) -3- (4- (4-Amino-butyloxy) phenyl-2-butylsulfonamido-propionic acid as illustrated in Figure 40 step v OR OH H2N. HN COMPOSITE 600 Compound 500 (1.3 grams (2.6 mmol)) was dissolved in 20 milliliters of ethyl acetate / methanol / water 5/3/1 and 0.2 milliliters of trifluoroacetic acid (TFA) and hydrogenated under hydrogen (1 atmosphere; from Parr Shaker) at 25 ° C in the presence of 10 milligrams of palladium (10 percent on charcoal). After 3 hours, the catalyst was filtered and the solvent was evaporated to yield Compound 600 as an oily residue. After lyophilization of the water 1.0 gram (quantitative) of Compound 600 was obtained as a white powder. FAB-MS: 373 (M + H +). 7) Compound 10: (R) -3- (4- (4-Guanidino-butyloxy) phenyl-2-butylsulfonamido-propionic acid as illustrated in Figure 40 step vi Compound 600 was heated (200 milligrams; mmol), 3,5-dimethylpyrazole-l-carboxamidine nitrate (DPFN) (170 milligrams, 0.8 mmol, Aldrich Chemical Company) and triethylamine (0.15 milliliters, 1.0 mmol) in dimethylformamide (DMF, 5 milliliters), at 60 ° C for 12 hours After cooling, the solvent was evaporated in vacuo, and the residue was purified by high performance liquid chromatography (Lichrocart RP-18, acetonitrile gradient / water + trifluoroacetic acid at 0.3 percent 99: 1 a 1:99) to yield 50 milligrams (25 percent) of Compound 10 as a white, amorphous powder, after lyophilization FAB-MS: 415 (M + H +), melting point: 70 ° CK Compound 11: benzyl ester of (S) -3- (4- (4-Azidobutyloxy) pheny1-2-camphorsulfonamido-propionic acid as illustrated in Figure 4 COMPOSITE 11 A mixture of Compound 4 (1.0 gram, 2.7 mmol), 10-camphorsulfonic acid chloride (6.6 mmol; Aldrich) was stirred.

Company) and triethylamine (1.5 equivalents) in methylene chloride (20 milliliters) for 12 hours at room temperature. The reaction mixture was then evaporated and the residue was dissolved in ethyl acetate, washed with dilute HCl, aqueous sodium bicarbonate and water. After evaporating it to dryness, the crude product was purified by flash chromatography (silica gel, toluene / ethyl acetate 15: 1), to yield 1.4 grams (67 percent) of Compound 11 as an amorphous solid. L. Compound 12: (S) -3- (4- (4-Guanidino-butyloxy) phenyl-2-10-camphor-sulfonamido-propionic acid as illustrated in Figure 41 steps i-ii COMPOUND 12 Compound 12 was obtained after hydrogenation and guanylation of Compound 11, in accordance with the following conditions: Step i: Compound 11 (1.3 grams (2.6 mmol)) was dissolved in 20 milliliters of ethyl acetate / methanol / water 5/3/1 and 0.2 milliliters of trifluoroacetic acid (TFA) and hydrogenated under hydrogen (1 atmosphere, Parr Shaker apparatus) at 25 ° C, in the presence of 100 milligrams of palladium (10 percent on carbon). After 3 hours, the catalyst was filtered and the solvent evaporated to yield the intermediate amine as an oily residue. After lyophilization of the water, 1.0 gram (quantitative) of the intermediate amine was obtained as a white powder, which was carried out as follows: Step ii: The intermediate amine compound which was previously formed (200 milligrams; 0.5 mmol), 3,5-dimethylpyrazole-l-carboxamidine nitrate (DPFN) (170 milligrams, 0.8 mmol, Aldrich Chemical Company) and triethylamine (0.15 milliliters, 1.0 mmol), in dimethylformamide (DMF, 5 milliliters) at 60 ° C for 12 hours. After cooling, the solvent was evaporated in vacuo, and the residue was purified by HPLC (Lich-rocart RP-18, acetonitrile gradient / water + trifluoroacetic acid 0.3 percent 99: 1 to 1:99), to yield 50 milligrams (25 percent) of Compound 12 as a white, amorphous powder, after lyophilization. FAB-MS: 509.6 (M + H +). M. Compound 13: (S) -3- (4- (5-Bromopenthyloxy) f-2-N-tert-butyloxycarbonyl-propionic acid benzyl ester as illustrated in Figure 41 O O - BENCILO HN -BOC RB O COMPOSITE 13 A benzyl ester mixture of t-Boc-L-tyrosine (4.5 grams, 12.1 mmol, Compound 1 synthesized as described above), 1,5-dibromopentane (5 milliliters, 36.7 mmol, Aldrich), potassium carbonate was heated. (10 grams) and 18-crown-6 (0.25 grams; Aldrich) at 80 ° C for 12 hours. After cooling, the precipitate was filtered and the reaction mixture was evaporated to dryness in vacuo. After purifying the crude product by crystallization, using 100 percent hexane to yield 5.35 grams (85 percent) of Compound 13. N. Compound 14: (S) -3- (4- (4-Guanidino-pentyloxy) pheny1-2-butylsulfonamido-propionic acid as illustrated in Figure 41 steps i-v COMPOSITE 14 The reaction sequence of step 5 of the bromine-azide exchange, dissociation of Boc, sulfonylation with butanesulfonic acid chloride and guanylation with 3,5-dimethylpyrazole-1-carboxamidine nitrate, was performed in an identical manner to the previous procedures , using the intermediate compounds 1-6 to form Compound 7 or the procedures using the Compounds 100-600 to form Compound 10, as described above. Compound 14 was obtained as a white powder FAB-MS: 429 (M + H +). 0. Compound 15: 3- (4-Amino-phenyl) -5- (4 - (2-carboxy-2-amino-ethyl) phenoxy) methyl-2-oxazolidinone dihydrochloride as shown in Figure 42 1) Synthesis of the starting material 2-N-BOC-amino-3- (4-hydroxy-phenyl) propionate for Compound 15 COMPOSITE 15 O The starting material 2-N-BOC-amino-3- (4-hydroxy-phenyl) propionate was obtained by esterification of (D or L), N- (tert-butoxycarbonyl) -L (D) tyrosine (t-Boc-L (D) -tyrosine) (1.0 equivalent; Sigma) in 0.10 M methanol and 1% diluted HCl. The reaction mixture was stirred at 25 ° C for 12 hours and then neutralized by potassium carbonate and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. . The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography to obtain 2-N-BOC-amino-3- (4-hydroxy-phenyl) propionate. 2) Synthesis of the starting material 3-p-N-BOC-amidino-phenyl-5-methansulfonyloxy-methy1-2-oxazolidinone for Compound 15: 3-step procedure as follows: p-Amino-benzonitrile was stirred (1.0 equivalent); Aldrich) in methylene chloride (0.10 M), with 2,3-epoxypropanol (1.0 equivalents, Aldrich) for 12 hours at 25 ° C. The solvent was then removed in vacuo and the crude 4- (2,3-dihydroxypropyl-lamino) benzonitrile was taken to the next step as follows: 4- (2,3-dihydroxypropylamino) benzonitrile was stirred (1.0 equivalent, as described above), in dimethylformamide (0.10 M) at 25 ° C with diethyl carbonate (1.1 equivalents; Aldrich) and potassium terbutylate (1.1 equivalents; Aldrich) at 110 ° C for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazolidine and carried to the next step as follows: stirred 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazo-lidin (1.0 equivalent, as described above), in methylene chloride (0.10 M) at 25 ° C with 1.1 equivalent of hydrogen sulphide, 1.1 equivalents of methylene iodide, and 1.1 equivalents of ammonium acetate. The reaction mixture was stirred for 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain amidine, which was carried to the next step as follows: 1.0 equivalents of amidine were protected, synthesized as described above, with 1.1 equivalents of BOC-ON (2- (BOC-oximino) -2-phenylacetonitrile; Aldrich) in methylene chloride (0.10 M), at 25 ° C and stirred for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was esterified in methylene chloride of 0.10 M and 1.1 equivalents of methanesulfonyl chloride. The reaction mixture was stirred at 0 ° C for 6 hours and then cooled with water (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over sulfate. of magnesium. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography to obtain 3-p-N-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone. 3) Coupling of intermediate 2-N-BOC-amino-3- (4-hydroxy-phenyl) propionate with 3-pN-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone to form the protected form of the Compound 15, 3- (4-BOC-anedinophenyl) -5- (4- (2-methoxy-carbonyl-2-N-BOC-aminoethyl) phenyloxymethyl-2-oxazolidinone A mixture of 1.9 grams of 2- -BOC-amino-3- (4-hydroxy-phenyl) ropionate (as described above), 20 milliliters of dimethylformamide (DMF) and NaH (1.0 equivalents), for 30 minutes at room temperature. they added 1.8 grams of 3-pN-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone (as described above), in 10 milliliters of dimethylformamide (DMF) and stirred again for 15 minutes at room temperature. The reaction mixture was then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate, then the solvent was removed under vacuum. and the crude product was purified by silica gel column chromatography, to obtain the protected form of Compound 15, 3- (4-BOC-ami-dinophenyl) -5- (4- (2-methoxy-carbonyl-2- N-BOC-aminoethyl) phenoxylmethyl-2-oxazolidinone, which was taken to the next step. 4) Deprotection of the protected form of Compound 15 to form Compound 15: 3- (4-amidinophenyl) -5- (4- (2-carboxy-2-amino-ethyl) phenoxy) methyl-2-oxazolidinone dihydrochloride. Figure 42 Treatment of the protected form of Compound 15, 3- (4-BOC-amidinophenyl) -5- (4- (2-methoxy-carbonyl-2-N-BOC-aminoethyl) phenyloxymethyl-2-oxazolidinone (1.0 equivalent; synthesized as described above), with 4 milliliters of 2N NaOH for 4 hours at room temperature. The mixture was then followed with 40 milliliters of 2N HCl solution in dioxane, which was added by dripping from 0 ° C to 25 ° C for 3 hours. The reaction mixture was then cooled with sodium bicarbonate (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography, to obtain Compound 15: 3- (4-amidinophenyl) -5- (4- (2-carboxy-2-) dihydrochloride amino-ethyl) phenoxy) methyl-2-oxazolidinone; melting point 165 ° C (d). P. Compound 16: 3- (4-amidinophenyl) -5- (4- (2-carboxy-2-N-butylsulfonylaminoethyl) phenoxy) methyl-2-oxazolidinone as shown in Figure X (old 141 1) Synthesis of the starting material 2-N-butyl-sulfonylamino -3- (4-hydroxy-phenyl) propionate for Compound 16 .COOH NH \ v \ _ N O NH-SO2 H, N O COMPOSITE 16 The starting material 2-N-butyl-sulfonyl-amino-3- (4-hydroxy-phenyl) propionate was obtained by esterification of (tyrosine (D or L)) (1.0 equivalent, Sigma), in methanol 0.10 M and 1 percent HCl diluted. The reaction mixture was stirred at 25 ° C for 12 hours and then neutralized by potassium carbonate and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. . The solvent was then removed in vacuo and the crude product was taken as follows: A mixture of the above compound (4.3 mmol), butanesulfonic acid chloride (6.6 mmol) and triethylamine (1.5 equivalents) was stirred in methylene chloride (20 milliliters). ) for 12 hours at room temperature. The reaction mixture was then evaporated and the residue was dissolved in ethyl acetate, washed with dilute HCl, aqueous sodium bicarbonate and water. After evaporation to dryness, the crude product was purified by flash chromatography (silica gel, toluene / ethyl acetate 15: 1) to yield the title compound. 2) Synthesis of the starting material 3-p-BOC-amidino-pheny1-5-methanesulfonyloxy-methyl-2-oxazolidinone for Compound 16: 3-step procedure as follows: The p-amino-benzonitrile was stirred (1.0 equivalents; Aldrich) in methylene chloride (0.10 M), with 2, 3-epoxypropanol (1.0 equivalents, Aldrich) for 12 hours at 25 ° C. The solvent was then removed in vacuo and the crude 4- (2,3-dihydroxypropyl-lamino) benzonitrile was taken to the next step as follows: 4- (2,3-dihydroxypropylamino) benzonitrile was stirred (1.0 equivalent, as described above), in dimethylformamide (0.10 M) at 25 ° C with diethyl carbonate (1.1 equivalents; Aldrich) and potassium terbutylate (1.1 equivalents; Aldrich) at 110 ° C for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazolidine and carried to the next step as follows: stirred 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazo-lidin (1.0 equivalent, as described above), in methylene chloride (0.10 M) at 25 ° C with 1.1 equivalent of hydrogen sulphide, 1.1 equivalents of methylene iodide, and 1.1 equivalents of ammonium acetate. The reaction mixture was stirred for 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain amidine, which was carried to the next step as follows: 1.0 equivalents of amidine were protected, synthesized as described above, with 1.1 equivalents of BOC-ON (2- (BOC-oximino) -2-phenylacetonitrile; Aldrich) in methylene chloride (0.10 M), at 25 ° C and stirred for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was esterified in methylene chloride of 0.10 M and 1.1 equivalents of methanesulfonyl chloride. The reaction mixture was stirred at 0 ° C for 6 hours and then cooled with water (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over sulfate. of magnesium. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography to obtain 3-p-N-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone. 3) Coupling of the intermediate 2-N-butylsulfonylamino-3- (4-hydroxy-phenyl) propionate with the 3-pN-BOC-amidino-phenyl-5-methanesulfoniloxy-methyl-2-oxazolidinone to form the protected form of the Compound 16, 3- (4-BOC-amidinophenyl) -5- (4- (2-methoxycarbonyl-2-N-butylsulfonylaminoethyl) phenyloxymethyl-2-oxazolidinone A mixture of 1.9 grams of 2-N-butyl-sulfonylamino- 3- (4-hydroxy-phenyl) propionate (as described above), 20 milliliters of dimethylformamide (DMF) and NaH (1.0 equivalents), for 30 minutes at room temperature.

After stirring, 1.8 grams of 3-p-N-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone (as described above) was added in 10 milliliters of dimethylformamide (DMF) and stirred again for 15 minutes at room temperature. The reaction mixture was then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography, to obtain the protected form of Compound 16, 3- (4-BOC-amidino-phenyl) -5- (4- (2 - methoxy-carbonyl-2-N-BOC-aminoethyl) phenoxyl-methyl-2-oxazolidinone, which was taken to the next step 4) Deprotection of the protected form of Compound 16 to form Compound 16: 3- (4- amidinophenyl) -5- (4- (2-carboxy-2-N-butylsulphonylamino oethyl) phenoxy) methyl-2-oxazolidinone. Figure 42 Treatment of the protected form of Compound 16, 3- (4-BOC-amidinophenyl) -5- (4- (2-methoxy-carbonyl-2-N-butyl-sulfonylaminoethyl) phenyloxymethyl-2-oxazolidinone (1.0 equivalent; synthesized as described above), with 4 milliliters of 2N NaOH for 4 hours at room temperature, then the mixture was followed with 40 milliliters of 2N HCl solution in dioxane, which was added by dripping from 0 ° C to 25 ° C. C for 3 hours, then the reaction mixture was reacted with sodium bicarbonate (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. Then the solvent was removed in vacuo and the crude product was purified by silica gel column chromatography, to obtain Compound 16: 3- (4-amidinophenyl) -5- (4- (2-carboxy-2-N -butylsulfonyl-aminoethyl) phenoxy) methyl-2-oxazolidinone; mp 236-237 ° CQ Compound 17: 3- (4-amidinof nyl) -5- (4- (2-carboxy-2-N-propyl-sulfonylamino-ethyl) phenoxy) methyl-2-oxazolidinone as shown in Figure 42 1) Synthesis of the starting material 2-N-propyl- sulfonylamino-3- (4-hydroxy-f-nyl) propionate for Compound 17: COMPOUND 17 The starting material 2-N-propyl-sulfonyl-amino-3- (4-hydroxy-phenyl) propionate was obtained by esterification of (tyrosine (D or L)) (1.0 equivalent, Sigma), in methanol 0.10 M and 1 percent HCl diluted. The reaction mixture was stirred at 25 ° C for 12 hours and then neutralized by potassium carbonate and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. . The solvent was then removed under vacuum and the crude product was taken as follows: A mixture of the above compound (4.3 mmol), butanesulfonic acid chloride (6.6 mmol: Aldrich) and triethylamine (1.5 equivalents) was stirred in methylene chloride (20 milliliters) for 12 hours at room temperature. The reaction mixture was then evaporated and the residue was dissolved in ethyl acetate, washed with dilute HCl, aqueous sodium bicarbonate and water. After evaporation to dryness, the crude product was purified by flash chromatography (silica gel, toluene / ethyl acetate 15: 1), to yield the title compound. 2) Synthesis of the starting material 3-p-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone for Compound 17: 3-step procedure as follows: The p-amino-benzonitrile was stirred (1.0 equivalent; Aldrich) in methylene chloride (0.10 M), with 2,3-epoxypropanol (1.0 equivalents, Aldrich) for 12 hours at 25 ° C. The solvent was then removed in vacuo and the crude 4- (2,3-dihydroxypropyl-ene) benzonitrile was taken to the next step as follows: 4- (2,3-dihydroxypropylamino) benzonitrile was stirred. (1.0 equivalent, as described above), in dimethylformamide (0.10 M) at 25 ° C with diethyl carbonate (1.1 equivalents, Aldrich) and potassium terbutylate (1.1 equivalents, Aldrich) at 110 ° C for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazolidine and carried to the next step as follows: stirred 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazo-lidine (1.0 equivalent); as described above), in methylene chloride (0.10 M) at 25 ° C with 1.1 equivalents of hydrogen sulfide, 1.1 equivalents of methylene iodide, and 1.1 equivalents of ammonium acetate. The reaction mixture was stirred for 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain amidine, which was carried to the next step as follows: 1.0 equivalents of amidine were protected, synthesized as described above, with 1.1 equivalents of BOC-ON (2- (BOC-oximino) -2-phenylacetonitrile; Aldrich) in methylene chloride (0.10 M), at 25 ° C and stirred for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was esterified in methylene chloride of 0.10 M and 1.1 equivalents of methanesulfonyl chloride. The reaction mixture was stirred at 0 ° C for 6 hours and then cooled with water (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over sulfate. of magnesium. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography to obtain 3-p-N-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone. 3) Coupling of the intermediate 2-N-propylsufonylamino-3- (4-hydroxy-phenyl) propionate with the 3-pN-BOC-amidino-pheny1-5-methansulfonyl-2-oxazolidinone to form the protected form of the Compound 17, 3- (4-BOC-amidinophenyl) -5- (4- (2-methoxycarbonyl-2-N-propyl-sulfonylaminoethyl) phenyloxyl-methyl-2-oxazolidinone A mixture of 1.9 grams of 2-N- was stirred. propyl-sulfonylamino-3- (4-hydroxy-phenyl) propionate (as described above), 20 milliliters of dimethylformamide (DMF) and NaH (1.0 equivalents), for 30 minutes at room temperature. 1.8 grams of 3-pN-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone (as described above), in 10 milliliters of dimethylformamide (DMF) and stirred again for 15 minutes at room temperature. the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was removed in vacuo and the crude product was purified by silica gel column chromatography, to obtain the protected form of Compound 17, 3- (4-BOC-amidino-phenyl) -5- (4- (2-methoxy) -carbonyl-2-N-propylsulfonylaminoethyl) -phenoxylmethyl-2-oxazolidinone, which was taken to the next step. 4) Deprotection of the protected form of Compound 17 to form Compound 17: 3- (4-amidinophenyl) -5- (4- (2-carboxy-2-N-propylsulfonylaminoethyl) phenoxy) methyl-2-oxazolidinone. Figure 42 Treatment of the protected form of Compound 17, 3- (4-BOC-amidinophenyl) -5- (4- (2-methoxy-carbonyl-2-N-propyl-sulfonylaminoethyl) phenyloxylmethyl-2-oxazolidinone (1.0 equiv. (synthesized as described above), with 4 milliliters of 2N NaOH for 4 hours at room temperature, then the mixture was followed with 40 milliliters of 2N HCl solution in dioxane, which was added dropwise from 0 ° C to 25 ° C for 3 hours, then the reaction mixture was cooled with sodium bicarbonate (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over Magnesium sulfate The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography to obtain Compound 17: 3- (4-amidinophenyl) -5- (4 - (2-carboxy- 2-N-propylsulfonylaminoethyl) phenoxy) methyl-2-oxazolidinone; melting point 200 ° C (d). R. Compound 18: 3- (4-amidinophenyl) -5- (4- (2-carboxy-2-N-ethyl-sulfonylamino-ethyl) phenoxy) methyl-2-oxazolidinone as shown in Figure 42 1) Synthesis of the starting material 2-N-ethyl-sulfonylamino-3- (4-hydroxyphenyl) propionate for Compound 18: COMPOSITE 18 The starting material 2-N-ethyl-sulfonyl-amino-3- (4-hydroxy-phenyl) propionate was obtained by esterification of (tyrosine (D or L)) (1.0 equivalent, Sigma), in methanol 0.10 M and 1 percent HCl diluted. The reaction mixture was stirred at 25 ° C for 12 hours and then neutralized by potassium carbonate and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. . The solvent was then removed in vacuo and the crude product was taken as follows: A mixture of the above compound (4.3 mmol), butanesulfonic acid chloride (6.6 mmol, Aldrich) and triethylamine (1.5 equivalents) was stirred in methylene chloride ( 20 milliliters) for 12 hours at room temperature. The reaction mixture was then evaporated and the residue was dissolved in ethyl acetate, washed with dilute HCl, aqueous sodium bicarbonate and water. After evaporation to dryness, the crude product was purified by flash chromatography (silica gel, toluene / ethyl acetate 15: 1), to yield the title compound. 2) Synthesis of the starting material 3-pN-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone for Compound 18: 3-step procedure as follows: The p-amino-benzonitrile was stirred (1.0 equivalent; Aldrich) in methylene chloride (0.10 M), with 2,3-epoxypropanol (1.0 equivalents, Aldrich) for 12 hours at 25 ° C. The solvent was then removed in vacuo and the crude 4- (2,3-dihydroxypropyl-lamino) benzonitrile was taken to the next step as follows: 4- (2,3-dihydroxypropylamino) benzonitrile was stirred (1.0 equivalent, as described above), in dimethylformamide (0.10 M) at 25 ° C with diethyl carbonate (1.1 equivalents; Aldrich) and potassium terbutylate (1.1 equivalents; Aldrich) at 110 ° C for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazolidine and carried to the next step as follows: stirred 3- (4-cyanophenyl) -5-hydroxymethyl-2-oxazo-lidin (1.0 equivalent, as described above), in methylene chloride (0.10 M) at 25 ° C with 1.1 equivalent of hydrogen sulphide, 1.1 equivalents of methylene iodide, and 1.1 equivalents of ammonium acetate. The reaction mixture was stirred for 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was purified by silica gel column chromatography, to obtain amidine, which was carried to the next step as follows: 1.0 equivalents of amidine were protected, synthesized as described above, with 1.1 equivalents of BOC-ON (2- (BOC-oximino) -2-phenylacetonitrile; Aldrich) in methylene chloride (0.10 M), at 25 ° C and stirred for 6 hours. Then, the reaction mixture was diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and then the crude product was esterified in methylene chloride of 0.10 M and 1.1 equivalents of methanesulfonyl chloride. The reaction mixture was stirred at 0 ° C for 6 hours and then cooled with water (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over sulfate. of magnesium. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography to obtain 3-p-N-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone. 3) Coupling of intermediate 2-N-ethylsulfonylamino-3- (4-hydroxy-phenyl) propionate with 3-pN-BOC-amidino-phenyl-5-methanesulfonyl-2-oxazolidinone to form the protected form of the Compound 18, 3- (4- BOC-amidinophenyl) -5- (4- (2-methoxycarbonyl-2-N-ethyl-sulfonylaminoethyl) phenyloxyl-methyl-2-oxazolidinone A mixture of 1.9 grams of 2-N- was stirred. ethyl-sulfonylamino-3- (4-hydroxy-phenyl) propionate (as described above), 20 milliliters of dimethylformamide (DMF) and NaH (1.0 equivalents), for 30 minutes at room temperature.

After stirring, 1.8 grams of 3-p-N-BOC-amidino-phenyl-5-methansulfonyloxy-methyl-2-oxazolidinone (as described above) was added in 10 milliliters of dimethylformamide (DMF) and stirred again for 15 minutes at room temperature. The reaction mixture was then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography, to obtain the protected form of Compound 18, 3- (4-BOC-amidino-phenyl) -5- (4- (2 - methoxy-carbonyl-2-N-ethylsulfonylaminoethyl) -phenoxylmethyl-2-oxazolidinone, which was taken to the next step 4) Deprotection of the protected form of Compound 18 to form Compound 18: 3- (4-amidinophenyl) - 5- (4- (2-carboxy-2-N-ethylsulphonylaminoethyl) phenoxy) methyl-2-oxazolidinone. Figure 42 Treatment of the protected form of Compound 18, 3- (4-BOC-amidinophenyl) -5- (4- (2-methoxy-carbonyl-2-N-ethylsulfonylaminoethyl) phenyioxy) methyl-2-oxazolidinone (1.0 equivalent; synthesized as described above), with 4 milliliters of 2N NaOH for 4 hours at room temperature. The mixture was then followed with 40 milliliters of 2N HCl solution in dioxane, which was added by dripping from 0 ° C to 25 ° C for 3 hours. The reaction mixture was then cooled with sodium bicarbonate (5 equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2X with water, IX with brine and dried over magnesium sulfate. The solvent was then removed in vacuo and the crude product was purified by silica gel column chromatography to obtain Compound 18: 3- (4-amidinophenyl) -5- (4 - (2-carboxy-2-N- ethylsulphonylaminoethyl) phenoxy) methyl-2-oxazolidinone; melting point 212 ° C (d). 14. Inhibition of Growth Factor Induced Angiogenesis as Measured in CAM Assay by Intravenous Application of Organic Mimetics of Ligand c B- ^ The effect of growth factor-induced angiogenesis with mimetics was also evaluated. of an ocvß3 ligand that was injected intravenously into the chick chorioallantoic membrane preparation for use in this invention. The 10-day-old chicken chorioallantoic membrane preparation was used, as described above in Example 5A. Twenty-four hours after the initiation of bFGF-induced angiogenesis, the organic mimetics referred to as compounds 16 (81218), 17 (87292) and 18 (87293) were injected separately intravenously into the preparation of the chicken chorioallantoic membrane in a volume of 100 ul at a concentration of 1 milligram / milliliter (100 ug / embryo), in tetraglycol-saline regulated by 20 percent phosphate with a pH of 7.0. In parallel trials, compounds 7 (96112), 9 (99799), 10 (96229), 12 (112854) and 14 (96113) were similarly evaluated. The effects of the organic mimetics were analyzed 48 hours later, when the quantification was carried out by counting the number of branching points of the blood vessels in the area of the filter disc in a dual blind approach. The results are shown respectively in Figures 43 and 44. In Figure 43, compounds 14 (96113), 10 (96229), 9 (99799) and 12 (112854), in decreasing order of inhibition, were effective in reducing the number of branching points of new blood vessels, compared to with the induction of the control bFGF and in comparison with the compound 7 (96112). In Figure 44, compounds 17 (87292) and 18 (87293) exhibited anti-angiogenic properties, as compared to the untreated bFGF control and treatment with compound 16 (81218). In a third test, organic compounds 7 (96112), 10 (96229) and 14 (96113) were evaluated as inhibitors of angiogenesis induced by bFGF together with peptides 69601 and 66203. For this assay, 250 ug / ml of organic compounds were administered 18 hours after treatment with bFGF, as described in Example 7B. The results are shown in the Figure 28, wherein as before, compounds 14 (96113) and 10 (96229) almost completely inhibited the formation of new blood vessels, induced by bFGF. Thus, the examples mentioned above demonstrate that integrin ovv3 plays a key role in angiogenesis induced by a variety of stimuli and as such, avß3 is a valuable therapeutic target with antagonists 0 £ vß3 of this invention, for diseases characterized by neovascularization. It is considered that the above written specification is sufficient to allow one skilled in the art to practice the invention. The present invention should not be limited in scope by the deposited cell line, since the deposited modality is proposed as a single illustration of one aspect of the invention, and any cell line that is functionally equivalent is within the scope of this invention. . The deposit of material does not constitute an admission that the written description contained herein is inadequate to allow the practice of any aspect of the invention, including the best mode thereof, nor should it be construed as limiting the scope of the claims to the specific illustration that it represents. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description, and fall within the scope of the appended claims.

LIST OF SEQUENCES (iii) NUMBER OF SEQUENCES: 45 (2) INFORMATION FOR THE SEQUENCE NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) OTHER INFORMATION: / label = BOC- GRGDFV-OMe / note = "BOC means the N-terminal protective butyloxycarbonyl group, OMe means a C-terminal methyl ester, arginine is in the second position." (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCALIZATION: 1..6 (D) OTHER INFORMATION: / label = OMe / note = "OMe means the C-terminal protective methyl ester group." (ix) ) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) OTHER INFORMATION: / label = D-Arg / note = "a prefix A" D "in D-Arg means that arginine in position 2 it is a D-amino acid. " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Gly Arg Gly Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) ) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) ) OTHER INFORMATION: / label = BOC / note = "BOC stands for the N-terminal tertbutyloxycarbonyl blocking group." (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) OTHER INFORMATION: / label = OH / note = "OH means a free C-terminal carboxylic acid." (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) OTHER INFORMATION: / label = D-Arg / note = "a prefix" D "in D-Arg means that the Arginine in position 2 is a D-amino acid. " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Gly Arg Gly Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) ) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) OTHER INFORMATION: / label- H / note = "H means a free N-terminal amine." (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) OTHER INFORMATION: / label- OH / note = "OH means a C-terminal carboxylic acid - free." (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 1..6 (D) OTHER INFORMATION: / label- D-Arg / note- "a prefix" D "in D-Arg in the position 2 means that arginine is a D-amino acid. " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Gly Arg Gly Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) ) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 2 (D) OTHER INFORMATION : / note- "Arg is a D-amino acid." (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Gly Arg Gly Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) ) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 4 (D) OTHER INFORMATION : / note- "Arg is a D-amino acid." (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Arg Gly Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 4 (D) OTHER INFORMATION: / note- "Phe is a D-amino acid." (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Arg Ala Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 5 (D) OTHER INFORMATION: / note- "Val is a D-amino acid. ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Arg Gly Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Tyr Thr Wing Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val Phe 1 5 10 15 (2) INFORMATION FOR SEQUENCE NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 2 (D) OTHER INFORMATION: / note- "Ala is a D-amino acid." (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Arg Ala Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 5 (D) OTHER INFORMATION: / note- "Phe is a D-amino acid. (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: Wing Arg Gly Asp Phe Leu 1 5 (2) INFORMATION FOR SEQUENCE NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION: 5 (D) OTHER INFORMATION: / note- "Phe is a D-amino acid. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: Gly Arg Gly Asp Phe Leu 1 5 (2) INFORMATION FOR SEQUENCE NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) ) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: Thr Arg Gln Val Val Cys Asp Leu Gly Asn Pro Met 1 5 10 (2) INFORMATION FOR SEQUENCE NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: Gly Val Val Arg Asn Asn Glu Ala Leu Ala Arg Leu Ser 1 5 10 (2) INFORMATION FOR SEQUENCE NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide ( v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14: Thr Asp Val Asn Gly Asp Gly Arg His Asp Leu 1 5 10 (2) INFORMATION FOR SEQUENCE NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY : peptide (B) LOCATION: 5 (D) OTHER INFORMATION: / note- "Methylation is in the amino terminus (NH2) of the valine residue. "(Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: Arg Gly Asp Phe Val 1 5 (2) INFORMATION FOR SEQUENCE NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) ) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: peptide (B) LOCATION : 5 (D) OTHER INFORMATION: / note- "Methylation is at the amino (NH2) terminus of the valine residue." (Xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16: Arg Gly Glu Phe Val 1 5 (2) ) INFORMATION FOR SEQUENCE NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 222 amino acids (B) TYPE: amino acid (D) TOPOLOGY: "linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: Lys Gly He Gln Glu Leu Tyr Gly Ala Ser Pro Asp He Asp Leu Gly 1 5 10 15 Thr Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu He Cys Lys 20 25 '30 Gln Asp He Val Phe Asp Gly He Wing Gln He Arg Gly Glu He Phe 35 40 45 Phe Phe Lys Asp Arg Phe He Trp Arg Thr Val Thr Pro Arg Asp Lys 50"55 60 Pro Met Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Glu Leu Pro Glu 65 70 75 8Q Lys He Asp Wing Val Tyr Glu Wing Pro Gln Glu Glu Lys Wing Val Phe 85 90 95 Phe Wing Gly Asn Glu Tyr Trp He Tyr Ser Wing Being Thr Leu Glu Arg 100 105 110 Gly Tyr Pro Lys Pro Leu Thr Ser Leu Gly Leu Pro Pro Asp Val Gln 115 120 125 Arg Val Asp Wing Wing Phe Asn Trp Ser Lys Asn Lys Lys Thr Tyr He 130 135 140 Phe Wing Gly Asp Lys Phe Trp Arg Tyr Asn Glu Val Lys Lys Lys Met 145 150 155 160 Asp Pro Gly Phe Pro Lys' Leu He Wing Asp Wing Trp Asn Wing He Pro 165 170 175 Asp Asn Leu Asp Wing Val Val Asp Leu Gln Gly Gly Gly His Ser Tyr 180 185 190 Phe Phe Lys Gly Wing Tyr Tyr Leu Lys Leu Glu Asn Gln Ser Leu Lys 195"200 205" Ser Val Lys Phe Gly Ser He Lys Ser Asp Trp Leu Gly Cys 210 215 220 (2) INFORMATION FOR SEQUENCE NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 193 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: He Cys Lys Gln Asp He Val Phe Asp Gly He Wing Gln He Arg Gly 1 5 10 15 Glu He Phe Phe Phe Lys Asp Arg Phe He Trp Arg Thr Val Thr Pro 25 30 Arg Asp Lys Pro Met Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Glu 40 45 Leu Pro Glu Lys He Asp Ala Val Tyr Glu Ala Pro Gln Glu Glu Lys 50 55 60 Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp He Tyr Ser Wing Ser Thr 65 70 75 80 Leu Glu Arg Gly Tyr Pro Lys Pro Leu Thr Ser Leu Gly Leu Pro Pro 85 90 95 Asp Val Gln Arg Val Asp Ala Ala Phe Asn Trp Ser Lys Asn Lys Lys 100 105 110 Thr Tyr He Phe Wing Gly Asp Lys Phe Trp Arg Tyr Asn Glu Val Lys 115 120 125 Lys Lys Met Asp Pro Gly Phe Pro Lys Leu He Wing Asp Wing Trp Asn 130 135 140 Ala He Pro Asp Asn Leu Asp Ala Val Val Asp Leu Gln Gly Gly Gly 145 150 155 160 His Ser Tyr Phe Phe Lys Gly Wing Tyr Tyr Leu Lys Leu Glu Asn Gln 165 170 ~ 175 Ser Leu Lys Ser Val Lys Phe Gly Ser He Lys Ser Asp Trp Leu Gly 180 185 190 Cys (2) INFORMATION FOR SEQUENCE NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: He Cys Lys Gln Asp He Val Phe Asp Gly He Wing Gln He Arg Gly 1 5 10 15 Glu He Phe Phe Phe Lys Asp Arg Phe He Trp Arg Thr Val Thr Pro 20 25 30 Arg Asp Lys Pro Met Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Glu 35 40 45 Leu Pro Glu Lys He Asp Wing Val Tyr Glu Wing Pro Gln Glu Glu Lys 50 55 60 Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp 65 70 (2) INFORMATION FOR SEQUENCE NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 108 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: He Cys Lys Gln Asp He Val Phe Asp Gly He Ala Gln He Arg Gly 1 - 5 10 15 Glu He Phe Phe Phe Lys Asp Arg Phe He Trp Arg Thr Val Thr Pro 20 25 30 Arg Asp Lys Pro Met Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Glu 40 45 Leu Pro Glu Lys He Asp Wing Val Tyr Glu Wing Pro Gln Glu Glu Lys 50 55 60 Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp He Tyr Ser Wing Ser Thr 65 - 70 75 80 Leu Glu Arg Gly Tyr Pro Lys Pro Leu Thr Ser Leu Gly Leu Pro Pro 85 90 95 Asp Val Gln Arg Val Asp Ala Ala Phe Asn Trp Ser 100 105 (2) INFORMATION FOR SEQUENCE NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 122 amino acids (B) TYPE: amino acid (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 21: Glu Tyr Trp He Tyr Ser Ala Be Thr Leu Glu Arg Gly Tyr Pro Lys 1 5 10 15 Pro Leu Thr Ser Leu Gly Leu Pro Pro Asp Val Gln Arg Val Asp Wing 20 25 30 Wing Phe Asn Trp Ser Lys Asn Lys Lys Thr Tyr He Phe Wing Gly Asp 35-40 45- Lys Phe Trp Arg Tyr Asn Glu Val Lys Lys Lys Met Asp Pro Gly Phe 50 55 60 Pro Lys Leu He Wing Asp Wing Trp Asn Wing Pro Pro Asp Asn Leu Asp 65"70 75 80 Wing Val Val Asp Leu Gln Gly Gly Gly His Ser Tyr Phe Phe Lys Gly 85 90 95 Ala Tyr Tyr Leu Lys Leu Glu Asn Gln Ser Leu Lys Ser Val Lys Phe 100, 105 110 Gly Ser He Lys Ser Asp Trp Leu Gly Cys 115 120 (2) INFORMATION FOR SEQUENCE NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: Phe Asn Trp Ser Lys Asn Lys Lys Thr Tyr He Phe Wing Gly Asp Lys 1 5 10 15 Phe Trp Arg Tyr Asn Glu Val Lys Lys Lys Met Asp Pro Gly Phe Pro 20 25 30 Lys Leu He Wing Asp Wing Trp Asn Wing He Pro Asp Asn Leu Asp Wing 35 40 45 Val Val Asp Leu Gln Gly Gly His Ser Tyr Phe Phe Lys Gly Wing 50 55 60 Tyr Tyr Leu Lys Leu Glu Asn Gln Ser Leu Lys Ser Val Lys Phe Gly 65 70 75 80 Ser He Lys Ser Asp Trp Leu Gly Cys 85 (2) INFORMATION FOR SEQUENCE NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 228 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: Lys Gly He Gln Glu Leu Tyr Glu Val Pro Pro Asp Val Glu Pro Gly 1 5 10 15 Pro Gly Pro Gly Pro Gly Pro Gly Pro Arg Pro Thr Leu Gly Pro Val 20 25 30 Thr Pro "Glu Leu Cys Lys His Asp He Val Phe Asp Gly Val Wing Gln 35 40 45 He Arg Gly Glu He Phe Phe Phe Lys Asp Arg Phe Met Trp Arg Thr 50 55 60 Val Asn Pro Arg Gly Lys Pro Thr Gly Pro Leu Leu Val Wing Thr Phe 65 70 75 80 Trp Pro Asp Leu Pro Glu Lys He Asp Wing Val Tyr Glu Ser Pro Gln 85 90 95 Asp Glu Lys Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp Val Tyr Thr 100 105 110 Wing Ser Asn Leu Asp Arg Gly Tyr Pro Lys Lys Leu Thr Ser Leu Gly 115 120 125 Leu Pro Pro Asp Val Gln Arg He Asp Wing Ala Phe Asn Trp Gly Arg 130 135 140 Asn Lys Lys Thr Tyr He Phe Ser Gly Asp Arg Tyr Trp Lys Tyr Asn 145 150 155 160 Glu Glu Lys Lys Lys Met Glu Leu Wing Thr Pro Lys Phe He Wing Asp 165 170 175 Ser Trp Asn Gly Val Pro Asp Asn Leu Asp Wing Val Leu Gly Leu Thr 180 185 190 Asp Ser Gly Tyr Thr Tyr Phe Phe Lys Asp Gln Tyr Tyr Leu Gln Met 195 200 205 Glu Asp Lys Ser Leu Lys He Val Lys He Gly Lys I Have To Be Asp 210 215 220 Trp Leu Gly Cys 225 (2) INFORMATION FOR SEQUENCE NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 193 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24: Leu Cys Lys His Asp He Val Phe Asp Gly Val Ala Gln He Arg Gly 1 5 10 15 Glu He Phe Phe Phe Lys Asp Arg Phe Met Trp Arg Thr Val Asn Pro 25 30 Arg Gly Lys Pro Thr Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Asp 40 45 Leu Pro Glu Lys He Asp Wing Val Tyr Glu Ser Pro Gln Asp Glu Lys 50 55 60 Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp Val Tyr Thr Wing Ser Asn 65 _ 70 75 80 Leu Asp Arg Gly Tyr Pro Lys Lys Leu Thr Ser Leu Gly Leu Pro Pro 85 90 95 Asp Val Gln Arg He Asp Wing Wing Phe Asn Trp Gly Arg Asn Lys Lys 100 105 110 Thr Tyr He Phe Ser Gly Asp Arg Tyr Trp Lys Tyr Asn Glu Glu Lys 115 120 125 Lys Lys Met Glu Leu Wing Thr Pro Lys Phe He Wing Asp Ser Trp Asn 130 135 140 Gly Val Pro Asp Asn Leu Asp Wing Val Leu Gly Leu Thr Asp Ser Gly 145 150 155 160 Tyr Thr Tyr Phe Phe Lys Asp Gln Tyr Tyr Leu Gln Met Glu Asp Lys 165 170 175 Ser Leu Lys He Val Lys He Gly Lys He Ser Ser Asp Trp Leu Gly 180 185 - 190 Cys (2) INFORMATION FOR SEQUENCE NO: 25 : (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: Leu Cys Lys His Asp He Val Phe Asp Gly Val Ala Gln He Arg Gly 1 5 10 15 Glu He Phe Phe Phe Lys Asp Arg Phe Met Trp Arg "Thr Val Asn Pro 20 25 30 Arg Gly Lys Pro Thr Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Asp 40 45 Leu Pro Glu Lys He Asp Wing Val Tyr Glu Ser Pro Gln Asp Glu Lys 50 55 60 Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp 65 70 (2) INFORMATION FOR SEQUENCE NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 108 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 26: Leu Cys Lys His Asp He Val Phe Asp Gly Val Ala Gln He Arg Gly 1 5 10 15 Glu He Phe Phe Phe Lys Asp Arg Phe Met Trp Arg Thr Val Asn Pro 20 25 30 Arg Gly Lys Pro Thr Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Asp 35 40 '45 Leu Pro Glu Lys He Asp Wing Val Tyr Glu Ser Pro Gln Asp Glu Lys 50"55 60 Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp Val Tyr Thr Wing Ser Asn 65 70 75 80 Leu Asp Arg Gly Tyr Pro Lys Lys Leu Thr Ser Leu Gly Leu Pro Pro 85 90 95 Asp Val Gln Arg He Asp Wing Wing Phe Asn Trp Gly 100 105 (2) INFORMATION FOR SEQUENCE NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 122 amino acids (B) TYPE: amino acid (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27: Glu Tyr Trp Val Tyr Thr Ala Ser Asn Leu. Asp Arg Gly Tyr Pro Lys 1 5 10 15 Lys Leu Thr Ser Leu Gly Leu Pro Pro Asp Val Gln Arg He Asp Wing 20 25 30 Wing Phe Asn Trp Gly Arg Asn Lys Lys Thr Tyr He Phe Ser Gly Asp 40 45 Arg Tyr Trp Lys Tyr Asn Glu Glu Lys Lys Lys Met Glu Leu Wing Thr 50 55 60 Pro Lys Phe He Wing Asp Ser Trp Asn Gly Val Pro Asp Asn Leu Asp 65 70 75 80 Wing Val Leu Gly Leti Thr Asp Ser Gly Tyr Thr Tyr Phe Phe Lys Asp 85 90"95 Gln Tyr Tyr Leu Gln Met Glu Asp Lys Ser Leu Lys He Val Lys He 100 105 110 Gly Lys He Ser Ser Asp Trp Leu Gly Cys 115 120 (2) INFORMATION FOR SEQUENCE NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 28 : Phe Asn Trp Gly Arg Asn Lys Lys Thr Tyr He Phe Ser Gly Asp Arg 1 5 10 15 Tyr Trp Lys Tyr Asn Glu Glu Lys Lys Lys Met Glu Leu Wing Thr Pro 20 25 30 Lys Phe He Wing Asp Ser Trp Asn Gly Val Pro Asp Asn Leu Asp Wing 35 40 45 Val Leu Gly Leu Thr Asp Ser Gly Tyr Thr Tyr Phe Phe Lys Asp Gln 50 55 60 Tyr Tyr Leu Gln Met Glu Asp Lys Ser Leu Lys He Val Lys He Gly 65 70 75 80 Lys He Ser Ser Asp Trp Leu Gly Cys 85 (2) INFORMATION FOR SEQUENCE NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2123 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI -SENSE: NO (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 132..2123 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: AATTCCGGCA AAAGAGAAAA CGGTGCAGAG AGTTAAGATG TGCAGATAAG CAACTAGTGC 60 ACTGTGCAGC CAAAGTAACT GACAGTCAGT CAGAGAAATC TTTTAAAGAG GATTGCAAAA 120 ATATAGGCAG A ATG AAG ACT CAC AGT GTT TTT GGC TTC TTT TTT AAA GTA 170 Met Lys Thr His Ser Val Phe Gly Phe Phe Phe Lys Val 1 5 10 CTA TTA ATC CAA GTG TAT CTT TTT AAC AAA ACT TTA GCT GCA CCG TCA 218 Leu Leu He Gln Val Tyr Leu Phe Asn Lys Thr Leu Ala Wing Pro Ser 15 - 20 25 CCA ATC ATT AAG TTC CCT GGA GAC AGC ACT CCA AAA ACA GAC AAA GAG 266 Pro He He Lys Phe Pro Gly Asp Ser Thr Pro Lys Thr Asp Lys Glu 30 35"40 45 CTA GCA GTG CA TAC CTG AAT AAA TAT TAT GAT TGC CCA AAA GAC AAT 314 Leu Wing Val Gln Tyr Leu Asn Lys Tyr Tyr Gly Cys Pro Lys Asp Asn 50 55 60 TGC AAC TTA TTT GTA TTG AAA GAT ACT TTG AAG AAA ATG CAG AAA TTT 362 Cys Asn Leu Phe Val Leu Lys Asp Thr Leu Lys Lys Met Gln Lys Phe 65"70 75 TTT GGG CTG ---- CCT GAA ACA GGA GAT TTG GAT CAA AAC ACA ATT GAG ACA 410 Phe Gly Leu Pro Glu Thr Gly Asp Leu Asp Gln Asn Thr He Glu Thr 80 85 90 ATG AAG AAA CCC CGC TGT GGT AAC CCC GAT GTG GCC AAT TAC AAC TTC 458 Met Lys Pro Arg Cys Gly Asn Pro Asp Val Wing Asn Tyr Asn Phe 95 100 105 TTT CCA AGA AAG CCA AAA TGG GAA AAG AAT CAT ATA ACA TAC AGG ATT 506 Phe Pro Arg Lys Pro Lys Trp Glu Lys Asn His He Thr Tyr Arg He ------ 110 -115 120 125 ATA GGC TAT ACC CCG GAT TTG GAT CCT GAG ACA GTA GAT GAT GCC TTT 554 He Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Wing Phe 130 135 140 GCC CGA GCC TTT AAA GTC TGG AGT GAT GTC ACG CCA CTG AGA "TTT AAC 602 Wing Arg Wing Phe Lys Val Trp Ser Asp Val Thr Pro Leu Arg Phe Asn 145 150 155 CGA ATA AAT GAT GAG GAG GAC GAC ATT ATG ATT AAT TTT GGC CGA TGG 650 Arg He Asn Asp Gly Glu Wing Asp He Met He As As Phe Gly Arg Trp 160 - 165 170 GAA CAT GGT GAT GGC TAT CCA TTT GAT GGC AAA GAT GGT CTC CTG GCT 698 Glu His Gly Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Wing 175 180 185 CAC GCC TTT GCA CCG GGG CCA GGA ATT GGA GGA GCC TCC CAT TTT GAT 746 His Wing Phe Wing Pro Gly Pro Gly He Gly Gly Asp Ser His Phe Asp 190 195 200 205 GAT GAT GAA CTG TGG ACT CTT GGA GAA GGG CAA GTG GTT AGA GTA AAG 794 Asp Asp Glu Leu Trp Thr Leu Gly Glu Gly Gn Val Val Arg Val Lys 210 215 220 TAT GGA AAT GCA GAT GGT GAA TAC TGC AAA TTT CCC TTC TGG TTC AAT 842 Tyr Gly Asn Wing Asp Gly Glu Tyr Cys Lys Phe Pro Phe Trp Phe Asn 225 230 235 GGT AAG GAA TAC AAC AGC TGC ACA GAT GCA GGA CGT AAT GAT GGA TTC 890 Gly Lys Glu Tyr Asn Ser Cys Thr Asp Wing Gly Arg Asn Asp Gly Phe 240 245 250 CTC TGG TGT TCC ACA ACC AAA GAC TTT GAT GCA GAT GGC AAA TAT GGC 938 Leu Trp Cys Ser Thr Thr Lys Asp Phe Asp Wing Asp Gly Lys Tyr Gly 255-260"265 TTT TGT CCC CAT GAG TCA CTT TTT ACA ATG GGT GGC AAT GGT GAT GGA 986 Phe Cys Pro His Glu Ser Leu Phe Thr Met Gly Gly Asn Gly Asp Gly 270"" 275 280 285 CAG CCC TGC AAG TTT CCC TTT AAA TTT CAA GGC CAG TCC TAT GAC CAG 1034 Gln Pro Cys Lys Phe Pro Phe Lys Phe Gln Gly Gln Ser Tyr Asp Gln 290 295 300 TGT ACA ACA GAA GGC AGG ACA GAT GGA TAC AGA TGG TGT GGA ACC ACT 1082 Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr Thr 305 310 315 GAA GAC TAT GAT AGA GAT AAG AAA TAC GGA TTC TGC CCA GAA ACT GCC 1130 Glu Asp Tyr Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro Glu Thr Wing 320 325 330 ATG TCA ACA GTT GGT GGA AAT TCA GAA GGA GCT CCT TGT GTA TTC CCC 1178 Met Ser Thr Val Gly Gly Asn Ser Glu Gly Wing Pro Cys Val Phe Pro 335"340 345 TTC ATC TTC CTT GGG AAT AAA TAC GAC TCC TGT ACA AGT GCA GGT CGC 1226 Phe He Phe Leu Gly Asn Lys Tyr Asp Ser Cys Thr Ser Wing Gly Arg 350 355 360 --- 365 AAT GAT GGC AAG CTG TGG TGT GCT TCT ACC AGC AGC TAT GAT GAT GAC 1274 Asn Asp Gly Lys Leu Trp Cys Wing Ser Thr Ser Ser Tyr Asp Asp Asp 370 375 380 CGC AAG TGG GGC TTT TGT CCA GAT CAA GGA TAC AGT CTC TTC TTG GTT 1322 Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val 385 390 395 GCT GCC CAC GAA TTT GGC CAT GCG ATG GGA TTA GAG CAC TCC GAG GAC 1370 Ala Ala His Glu Phe Gly His Wing Met Gly Leu Glu His Ser Glu Asp 400 405 410 CCA GGA GCT CTC ATG GCC CCG ATC TAC ACC TAC ACC AAG AAC TTC CGC 1418 Pro Gly Ala Leu Met Ala Pro He Tyr Thr Tyr Thr Lys Asn Phe Arg 415"420 425 CTT TCT CAG GAT GAC ATT AAG GGG ATT CAG GAG CTA TAT GAA GTA TCA 1466 Leu Ser Gln Asp Asp He Lys Gly He Gln Glu Leu Tyr Glu Val Ser 430 --435 440 445 CCT GAT GTG GAA CCT GGA CCA GGG CCA GCA CCA GGG CCA GGA CCA CGT 1514 Pro Asp Val Glu Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Arg 450 455 460 CCT ACC CTT GGA CCT GTC ACT CCA GAG CTC TGC AAG CAC GAC ATT GTA 1562 Pro Thr Leu Gly Pro Val Thr Pro Glu Leu Cys Lys His Asp He Val 465 470 475 TTT GAT GGA GTT GCA CAA ATT AGA GGA GAA ATA TTT TTC TTC AAA GAC 1610 Phe Asp Gly Val Wing Gln He Arg Gly Glu He Phe Phe Phe Lys Asp 480 485 490 AGA TTC ATG TGG AGG ACT GTA AAC CCT CGA GGA AAA CCC ACA GGT CCT - 1658 Arg Phe Met Trp Arg Thr Val Asn Pro Arg Gly Lys Pro Thr Gly Pro 495 -500 505 CTT CTC GTT GCT ACA TTC TGG CCT GAT CTG CCA GAG AAA ATC GAT GCT 1706 Leu Leu Val Wing Thr- Phe Trp Pro Asp Leu Pro Glu Lys He Asp Wing 510 515 520"525 GTC TAC GAG TCC CCT CAG GAT GAG AAG GCT GTA TTT TTT GCA GGA AAT 1754 Val Tyr Glu Ser Pro Gln Asp Glu Lys Wing Val Phe Phe Wing Gly Asn 530 535 540 GAG TAC TGG GTT TAT ACA GCC AGC AAC CTG GAT AGG GGC TAT CCA AAG 1802 Glu Tyr Trp Val Tyr Thr Ala Ser Asn Leu Asp Arg Gly Tyr Pro Lys 545 550 555 AAA CTC ACC AGC CTG GGA CTA CCC CCT GAT GTG CAA CGC ATT GAT GCA 1850 Lys Leu Thr Ser Leu Gly Leu Pro Pro Asp Val Gln Arg He Asp Wing 560 565 570 GCC TTC AAC TGG GGC AGA AAC AAG AAG ACA TAT ATT TTC TCT GGA GAC 1898 Wing Phe Asn Trp Gly Arg Asn Lys Lys Thr Tyr He Phe Ser Gly Asp 575 580"" "585 AGA TAC TGG AAG TAC AAT GAA GAA AAG AAA AAA ATG GAG CTT GCA ACC 1946 Arg Tyr Trp Lys Tyr Asn Glu Glu Lys Lys Lys Met Glu Leu Wing Thr 590 595 600 605 CCA AAA TTC ATT GCG GAT TCT TGG AAT GGA GTT CCA GAT AAC CTC GAT 1994 Pro Lys Phe He Wing Asp Ser Trp Asn Gly Val Pro Asp Asn Leu Asp 610 615 620 GCT GTC CTG GGT CTT ACT GAC AGC GGG TAC ACC TAT TTT TTC AAA GAC 2042 Wing Val Leu Gly Leu Thr Asp Ser Gly Tyr Thr Tyr Phe Phe Lys Asp 625 630 635 CAG TAC TAT CTA CAA ATG GAA GAC AAG AGT TTG AAG ATT GTT AAA ATT 2090 Gln Tyr Tyr Leu Gln Met Glu Asp Lys Ser Leu Lys lie Val Lys He 640 645 650 GGC AAG ATA AGT TCT GAC TGG TTG GGT TGC TG 2123 Gly Lys He Ser Ser Asp Trp Leu Gly Cys 655 660 (2) INFORMATION FOR SEQUENCE NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 663 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30: Met Lys Thr His Ser Val Phe Gly Phe Phe Phe Lys Val Leu Leu He 1 5 10 15 Gln Val Tyr Leu Phe Asn Lys Thr Leu Ala Wing Pro Ser Pro He He 20 - 25 30 Lys Phe Pro Gly Asp Ser Thr Pro Lys Thr Asp Lys Glu Leu Ala Val 35 40 45 Gln Tyr Leu Asn Lys Tyr Tyr Gly Cys Pro Lys Asp Asn Cys Asn Leu 50 55 60 Phe Val Leu Lys Asp Thr Leu Lys Lys Met Gln Lys Phe Phe Gly Leu 65 70 75 80 Pro Glu Thr Gly Asp Leu Asp Gln Asn Thr He Glu Thr Met Lys lys 85 90 95 Pro Arg Cys Gly Asn Pro Asp Val Wing Asn Tyr Asn Phe Phe Pro Arg 100 105 110 Lys Pro Lys Trp Glu Lys Asn His He Thr Tyr Arg He He Gly Tyr 115 120 125 Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Ala Phe Ala Arg Ala 130 135 140 Phe Lys -Val Trp Ser Asp Val Thr Pro Leu Arg Phe Asn Arg He Asn 145 150 155 160 Asp Gly Glu Wing Asp He Met He Asn Phe Gly Arg Trp Glu His Gly 165 170 .175 Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Wing His Wing Phe 180 185 190 Wing Pro Gly Pro Gly He Gly Gly Asp Ser His Phe Asp Asp Asp Glu 195 200 205 Leu Trp Thr Leu Gly Glu Gly Gln Val Val Arg Val Lys Tyr Gly Asn 210 -. 210 - 215 220 Wing Asp Gly Glu Tyr Cys Lys Phe Pro Phe Trp Phe Asn Gly Lys Glu 225 230 235 - 240 Tyr Asn Ser Cys Thr Asp Wing Gly Arg Asn Asp Gly Phe Leu Trp Cys 245 250 255 Being Thr Thr Lys Asp Phe Asp Wing Asp Gly Lys Tyr Gly Phe Cys Pro 260 265 270 His Glu Being Leu Phe Thr Met Gly Gly Asn Gly Asp Gly Gln Pro Cys 275 280 285 Lys Phe Pro Phe Lys Phe Gln Gly Gln Ser Tyr Asp Gln Cys Thr Thr 290 -. 290 - 295 300 Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr Thr Glu Asp Tyr 305 310 315 320 Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro Glu Thr Wing Met Ser Thr 325 330 335 Val Gly Gly Asn Ser Glu Gly Wing Pro Cys Val Phe Pro Phe He Phe 340 345 350 Leu Gly Asn Lys Tyr Asp Ser Cys Thr Ser Wing Gly Arg Asn Asp Gly 355 360 365 Lys Leu Trp Cys Wing Ser Thr Ser Ser Tyr Asp Asp Asp Arg Lys Trp 370 375 380 Gly Phe Cys Pro A-sp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala His 385 390 395 ._ 400 Glu Phe Gly His Wing Met Gly Leu Glu His Ser Glu Asp Pro Gly Wing 405 410 415 Leu Met Ala Pro He Tyr Thr Tyr Thr Lys Asn Phe Arg Leu -Ser Gln 420 425 430 Asp Asp He Lys Gly He Gln Glu Leu Tyr Glu Val Ser Pro Asp Val 435 440 445 Glu Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Arg Pro Thr Leu 450 455 460 Gly Pro Val Thr Pro Glu Leu Cys Lys His Asp He Val Phe Asp Gly 465 ~ 470 475 480 Val Ala Gln He Arg Gly Glu He Phe Phe Phe Lys Asp Arg Phe Met 485 490 495 Trp Arg Thr Val Asn Pro Arg Gly Lys Pro Thr Gly Pro Leu Leu Val 500 505 510 Wing Thr Phe Trp Pro Asp Leu Pro Glu Lys He Asp Wing Val Tyr Glu 515 520 525 Ser Pro Gln Asp Glu Lys Wing Val Phe Phe Wing Gly Asn Glu Tyr Trp 530"535 540 Val Tyr Thr Ala Ser Asn Leu Asp Arg Gly Tyr Pro Lys Lys Leu Thr 545 550 555 560 Be Leu Gly Leu Pro Pro Asp Val Gln Arg He Asp Wing Wing Phe Asn 565 570 575 Trp Gly Arg Asn Lys Lys Thr Tyr He Phe Ser Gly Asp Arg Tyr Trp 580 585 590 Lys Tyr Asn Glu Glu Lys Lys Lys Met Glu Leu Wing Thr Pro Lys Phe 595 600 605 He Wing Asp Ser Trp Asn Gly Val Pro Asp Asn Leu Asp Wing Val Leu 610 615 620 Gly Leu Thr Asp Ser Gly Tyr Thr Tyr Phe Phe Lys Asp Gln Tyr Tyr 625 630 635 640 Leu Gln Met Glu Asp Lys Ser Leu Lys He Val Lys He Gly Lys He 645 650 _ 655 Ser Ser Asp Trp Leu Gly Cys 660 - (2) INFORMATION FOR SEQUENCE NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs ( B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31: ATTGAATTCT TCTACAGTTC A 21 (2) INFORMATION FOR SEQUENCE NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iü) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (i) DESCRIPTION OF SEQUENCE: SEQ ID NO: 32: ATGGGATCCA CTGCAAATTT C 21 (2) INFORMATION FOR SEQUENCE NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 33: GCCGGATCCA TGACCAGTGT TO 21 (2) INFORMATION FOR SEQUENCE NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL : NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34: GTGGGATCCC TGAAGACTAT G 21 (2) INFORMATION FOR THE SEQUENCE NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO ( xi) DESCRIPTION OF THE SE CAUTION: SEQ ID NO: 35: AGGGGATCCT TAAGGGGATT C. twenty-one (2) INFORMATION FOR SEQUENCE NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 36: CTCGGATCCT CTGCAAGCAC G 21 (2) INFORMATION FOR SEQUENCE NO: 37: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iü) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 37: CTCGGATCCT CTGCAAGCAC G 21 (2) INFORMATION FOR SEQUENCE NO: 38: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 38: GCAGGATCCG AGTGCTGGGT TTATAC 26 (2) INFORMATION FOR SEQUENCE NO: 39: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL : NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39: GCAGAATTCA ACTGTGGCAG AAACAAG 27 (2) INFORMATION FOR SEQUENCE NO: 40: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 40: GTAGAATTCC AGCACTCATT TCCTGC 26 (2) INFORMATION FOR SEQUENCE NO: 41: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 41: TCTGAATTCT GCCACAGTTG AAGG 24 (2) INFORMATION FOR SEQUENCE NO: 42: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 42: ATTGAATTCT TCTACAGTTC A 21 (2) INFORMATION FOR SEQUENCE NO: 43: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 43: GATGAATTCT ACTGCAAGTT 20 (2) INFORMATION FOR SEQUENCE NO: 44: ( i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 44: CACTGAATTC ATCTGCAAAC A 21 (2) INFORMATION FOR SEQUENCE NO: 45: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 429 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: C-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Tyr Cys Lys Phe Pro Phe Leu Phe Asn Gly Lys Glu Tyr Asn Ser Cys 1 5 10 15 Thr Asp Thr Gly Arg Ser Asp Gly Phe Leu Trp Cys Ser Thr Thr Tyr 20 25 30 Asn Phe Glu Lys Asp Gly Lys Tyr Gly Phe Cys Pro His Glu Ala Leu 35 40 45 Phe Thr Met Gly Gly Asn Wing Glu Gly Gln Pro Cys Lys Phe Pro Phe 50 55 60 Arg Phe Gln Gly Thr Ser Tyr Asp Ser Cys Thr Thr Glu Gly Arg Thr 65 70 75 80 Asp Gly Tyr Arg Trp Cys Gly Thr Thr Glu Asp Tyr Asp Arg Asp Lys 85 90 95 Lys Tyr Gly Phe Cys Pro Glu Thr Wing Met Ser Thr Val Gly Gly Asn 100 105 110 Ser Glu Gly Wing Pro Cys Val Phe Pro Phe Thr Phe Leu Gly Asn Lys 115 120 125 Tyr Glu Ser Cys Thr Ser Wing Gly Arg Ser Asp Gly Lys Met Trp Cys 130 135 140 Wing Thr Thr Wing Asn Tyr Asp Asp Asp Arg Lys Trp Gly Phe Cys Pro 145 150 155 160 Asp Gln Gly Tyr Ser Leu Phe Leu Val Wing Ala His Glu Phe Gly His 165 170 175 Wing Met Gly Leu Glu His Ser Gln Asp Pro Gly Wing Leu Met Wing Pro 180 185 190 He Tyr Thr Tyr Thr Lys Asn Phe Arg Leu Ser Gln Asp Asp He Lys 195 200 205 Gly He Gln Glu Leu Tyr Gly Wing Ser Pro Asp He Asp Leu Gly Thr 210 215 220 Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu He Cys Lys Gln 225 230 235 240 Asp He Val Phe Asp Gly He Wing Gln He Arg Gly Glu He Phe Phe 245 250 255 Phe Lys Asp Arg Phe He Trp Arg Thr Val Thr Pro Arg Asp Lys Pro 260 265 270 Met Gly Pro Leu Leu Val Wing Thr Phe Trp Pro Glu Leu Pro Glu Lys 275 280 285 He Asp Wing Val Tyr Glu Wing Pro Gln Glu Glu Lys Wing Val Phe Phe 290 295 300 Wing Gly Asn Glu Tyr Trp He Tyr Ser Wing Being Thr Leu Glu Arg Gly 305 310 315 320 Tyr Pro Lys Pro Leu Thr Ser Leu Gly Leu Pro Pro Asp Val Gln Arg 325 330 335 Val Asp Wing Wing Phe Asn Trp Ser Lys Asn Lys Lys Thr Tyr He Phe 340 345 350 Wing Gly Asp Lys Phe Trp Arg Tyr Asn Glu Val Lys Lys Lys Met Asp 355 360 365 Pro Gly Phe Pro Lys Leu He Wing Asp Wing Trp Asn Wing He Pro Asp 370 375 380 Asn Leu Asp Ala Val Val Asp Leu Gln Gly Gly His Ser Tyr Phe 385 390 395 400 Phe Lys Gly Wing Tyr Tyr Leu Lys Leu Glu Asn Gln Ser Leu Lys Ser 405 410 415 Val Lys Phe Gly Ser He Lys Ser Asp Trp Leu Gly Cys 420 425

Claims (43)

1. CLAIMS 1. A manufacturing article, comprising packaging material and a pharmaceutical agent contained within said packaging material, wherein said pharmaceutical agent is effective to inhibit angiogenesis in a tissue and wherein said packaging material comprises a label indicating that said pharmaceutical agent can be used to treat conditions by inhibition of angiogenesis, and wherein said pharmaceutical agent comprises an inhibitory amount of angiogenesis of a β-β3 antagonist comprising a polypeptide having an amino acid residue sequence that includes a of the carboxy terminal domain of matrix metalloproteinase, said polypeptide capable of binding to vß3 integrin.
2. 2. The article of manufacture of claim 1, wherein said polypeptide includes an amino acid residue sequence shown in SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.
3. 3. The article of manufacture of claim 1, wherein said tissue is inflamed and said condition is arthritis or rheumatoid arthritis. .
4. 4. The article of manufacture of claim 1, wherein said tissue is a solid tumor or solid tumor metastasis.
5. The article of manufacture of claim 1, wherein said tissue is retinal tissue and said condition is retinopathy, diabetic retinopathy or macular degeneration.
6. 6. A vß3 antagonist, comprising a polypeptide having an amino acid residue sequence that includes a portion of the carboxy terminal domain of matrix metalloproteinase, said polypeptide capable of binding to integrin? Vss3.
7. The antagonist of claim 6, wherein said polypeptide includes an amino acid residue sequence shown in SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.
8. 8. The antagonist of claim 6, wherein said polypeptide is a fusion protein.
9. 9. The antagonist of claim 6, wherein said polypeptide has an amino acid residue sequence shown in SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.
10. 10. A pharmaceutical agent comprising an arvß3 antagonist according to claim 6, in a pharmaceutically acceptable carrier, in an amount sufficient to inhibit angiogenesis in a tissue.
11. A method for inhibiting angiogenesis in a tissue, comprising administering to said tissue a composition comprising an angiogenesis inhibiting amount of a vß3 antagonist.
12. The method of claim 11, wherein said antagonist is a fusion protein, a polypeptide, a poly-peptide derivative, a cyclic polypeptide, a monoclonal antibody or an organic mimetic compound.
13. The method of claim 11, wherein said avß3 integrin preferably inhibits fibrinogen that binds avß3 compared to fibrinogen that binds to orIIbβ3.
14. The method of claim 11, wherein said c-vß3 antagonist comprises a polypeptide having an amino acid residue sequence that includes a portion of the carboxy terminal domain of matrix metalloproteinase, said polypeptide capable of binding to avß3 integrin.
15. 15. The method of claim 11, wherein said polypeptide includes an amino acid residue sequence shown in SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.
16. 16. The method of claim 11, wherein said polypeptide is a fusion protein.
17. 17. The method of claim 11, wherein said polypeptide has an amino acid residue sequence shown in SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.
18. 18. The method of claim 11, wherein said tissue is inflamed and said angiogenesis is angiogenesis of inflamed tissue.
19. 19. The method of claim 18, wherein said tissue is arthritic.
20. 20. The method of claim 19, wherein said arthritic tissue is present in a mammal with rheumatoid arthritis.
21. 21. The method of claim 11, wherein said tissue is the retinal tissue of a patient with diabetic retinopathy and said angiogenesis is retinal angiogenesis.
22. 22. The method of claim 11, wherein said tissue is a solid tumor or solid tumor metastasis and said angiogenesis is tumor angiogenesis.
23. 23. The method of claim 11, wherein said administration comprises intravenous, transdermal, intrasynovial, intramuscular or oral administration.
24. The method of claim 22, wherein said administration is conducted in conjunction with chemotherapy.
25. 25. The method of claim 11, wherein said administration comprises a single dose intravenously.
26. 26. A method of inducing regression of solid tumor tissue in a patient, comprising administering to said patient a composition comprising a therapeutically effective amount of a integrin antagonist? Fv? 3 sufficient to inhibit neovascularization of a solid tumor tissue.
27. 27. The method of claim 26, wherein said antagonist is a fusion protein, a polypeptide, a polypeptide derivative, a cyclic polypeptide, a monoclonal antibody, or an organic mimetic compound.
28. 28. The method of claim 26, wherein said avß3 antagonist is the vß3 antagonist according to claim 6.
29. 29. A method of inhibiting growth of solid tumor tissue undergoing neovascularization in a patient, comprising administering to said patient a composition comprising a therapeutically effective amount of an integrin xvß3 antagonist sufficient to inhibit the growth of solid tumor tissue.
30. 30. The method of claim 29, wherein said antagonist is a fusion protein, a polypeptide, a polypeptide derivative, a cyclic polypeptide, a monoclonal antibody or an organic mimetic compound.
31. 31. The method of claim 29, wherein said avß3 antagonist is the vß3 antagonist according to claim 6.
32. 32. A method for treating a patient with inflamed tissue in which neovascularization is occurring, comprising administering to said patient a composition comprising a therapeutically effective amount of a vß3 integrin antagonist.
33. 33. The method of claim 32, wherein said antagonist is a fusion protein, a polypeptide, a polypeptide derivative, a cyclic polypeptide, a monoclonal antibody or an organic mimetic compound.
34. 34. The method of claim 32, wherein said avß3 antagonist is the o.vß3 antagonist according to claim 6.
35. 35. A method for treating a patient in which neovascularization is occurring in retinal tissue, comprising administering to said patient a composition comprising an inhibitory amount of neovascularization of a vß3 integrin antagonist.
36. 36. The method of claim 35, wherein said antagonist is a fusion protein, a polypeptide, a polypeptide derivative, a cyclic polypeptide, a monoclonal antibody or an organic mimetic compound.
37. 37. The method of claim 35, wherein said vß3 antagonist is the avß3 antagonist according to claim 6.
38. 38. A method for treating a patient with restenosis in a tissue, where migration of smooth muscle cells occurs after of angioplasty, which comprises administering to said patient a composition comprising a therapeutically effective amount of an avß3 integrin antagonist.
39. 39. The method of claim 38, wherein said antagonist is a fusion protein, a polypeptide, a polypeptide derivative, a cyclic polypeptide, a monoclonal antibody or an organic mimetic compound.
40. 40. The method of claim 38, wherein said avß3 antagonist is the ovv3 antagonist according to claim 6.
41. 41. A method of reducing the blood supply to a tissue required to sustain new growth of said tissue in a patient. , which comprises administering to said patient a therapeutically effective composition of a sufficient avß integrin antagonist to reduce said blood supply to said tissue.
42. 42. The method of claim 41, wherein said antagonist is a fusion protein, a polypeptide, a polypeptide derivative, a cyclic polypeptide, a monoclonal antibody or an organic mimetic compound.
43. 43. The method of claim 41, wherein said c-vß3 antagonist is the o-vß3 antagonist according to claim 6.