An Ephrin Mimetic Peptide That Selectively Targets the EphA2 Receptor

Abstract

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologicversus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolarK D values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration. Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologicversus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolarK D values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration. Originally identified as regulators of neural development, the Eph family of receptor tyrosine kinases and their ephrin ligands are also critical for vascular development and pathological forms of angiogenesis (1Flanagan J.G. Vanderhaeghen P. Annu. Rev. Neurosci. 1998; 21: 309-345Google Scholar, 2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar, 3Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Google Scholar). For example, the EphA2 receptor and ephrin-A1, a ligand for EphA2, are coordinately expressed in the vasculature of human tumors and mouse xenograft tumors grown from human cancer cells (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar). The EphA2 receptor plays a critical role in tumor necrosis factor α (TNFα) 1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein -induced neovascularization because TNFα up-regulates ephrin-A1, which causes receptor activation in blood vessels (5Pandey A. Shao H. Marks R.M. Polverini P.J. Dixit V.M. Science. 1995; 268: 567-569Google Scholar). Similarly, the homeobox transcription factor Hox B3 promotes angiogenesis by up-regulating ephrin-A1 (6Myers C. Charboneau A. Boudreau N. J. Cell Biol. 2000; 148: 343-351Google Scholar). Furthermore, EphA2 signaling is required for the formation of endothelial capillary tubes in vitro (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 7Daniel T.O. Stein E. Cerretti D.P. St. John P.L. Robert B. Abrahamson D.R. Kidney Int. Suppl. 1996; 57: S73-S81Google Scholar) and promotes the formation of blood vessel-like structures by melanoma cells (8Hess A.R. Seftor E.A. Gardner L.M. Carles-Kinch K. Schneider G.B. Seftor R.E. Kinch M.S. Hendrix M.J. Cancer Res. 2001; 61: 3250-3255Google Scholar). The expression of EphA2 appears to be restricted to “activated” adult blood vessels as this receptor has not been detected in either embryonic or adult quiescent blood vessels (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 9Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Google Scholar,10Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624Google Scholar). Ephrin-A1 has also not been detected in adult blood vessels, although it is present in the embryonic vasculature (11McBride J.L. Ruiz J.C. Mech. Dev. 1998; 77: 201-204Google Scholar).In addition to being present in tumor endothelial cells, EphA2 and ephrin-A1 are up-regulated in the transformed cells of a wide variety of tumors including breast, prostate, colon, skin, and esophageal cancers (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 12Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306Google Scholar, 13Walker-Daniels J. Coffman K. Azimi M. Rhim J.S. Bostwick D.G. Snyder P. Kerns B.J. Waters D.J. Kinch M.S. Prostate. 1999; 41: 275-280Google Scholar, 14Easty D.J. Herlyn M. Bennett D.C. Int. J. Cancer. 1995; 60: 129-136Google Scholar, 15Nemoto T. Ohashi K. Akashi T. Johnson J.D. Hirokawa K. Pathobiology. 1997; 65: 195-203Google Scholar). Many factors increase EphA2 expression in cancer cells including the H-Ras oncogene, E-cadherin, members of the p53 family of transcriptional regulators, DNA damage, and loss of estrogen receptors and c-Myc (16Andres A.C. Reid H.H. Zurcher G. Blaschke R.J. Albrecht D. Ziemiecki A. Oncogene. 1994; 9: 1461-1467Google Scholar, 17Dohn M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar, 18Zelinski D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar).Because the tumor vasculature is discontinuous and leaky in nature, it is possible to utilize the up-regulation of EphA2 and ephrin-A1 to deliver cancer-eradicating agents to both blood vessels and tumor cells (19Dvorak H.F. Nagy J.A. Dvorak J.T. Dvorak A.M. Am. J. Pathol. 1988; 133: 95-109Google Scholar). Indeed, systemically administered biological agents can easily penetrate into tumors from the blood circulation (20Essler M. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2252-2257Google Scholar). Selectively targeting EphA2 and ephrin-A1 is a challenging task because these proteins belong to large families of closely related proteins (21Eph Nomenclature Committee Cell. 1997; 90: 403-404Google Scholar). One approach that has been successfully used to identify peptides that exhibit selectivity for their targets is to screen random peptide libraries displayed on the surface of filamentous bacteriophage (22Koivunen E. Wang B. Ruoslahti E. J. Cell Biol. 1994; 124: 373-380Google Scholar, 23Koivunen E. Arap W. Valtanen H. Rainisalo A. Medina O.P. Heikkila P. Kantor C. Gahmberg C.G. Salo T. Konttinen Y.T. Sorsa T. Ruoslahti E. Pasqualini R. Nat. Biotechnol. 1999; 17: 768-774Google Scholar, 24Smith G.P. Science. 1985; 228: 1315-1317Google Scholar, 25Devlin J.J. Panganiban L.C. Devlin P.E. Science. 1990; 249: 404-406Google Scholar). Therefore, we used a phage display approach to search for peptides that bind selectively to the extracellular domains of EphA2 and ephrin-A1. Remarkably the two peptides identified bind selectively to EphA2, but not other Eph receptors, and antagonize ephrin binding. Intriguingly, at least one of the peptides has bioactive properties in that it stimulates EphA2 tyrosine phosphorylation and signaling. In addition, this peptide delivers phage particles to EphA2-expressing cells and thus can have therapeutic value in delivering agents to tissues that express EphA2. Thus, Eph receptor-binding peptides with different agonistic and drug targeting activities can have important therapeutic applications.DISCUSSIONWe have used phage display to isolate novel peptides that selectively bind to the EphA2 receptor, a cell-surface protein present in pathologic angiogenic vasculature, including tumor vasculature, and in many types of cancer cells (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Thus, targeting this receptor should allow therapeutic intervention in cancer and other diseases. The YSA peptide could be used to deliver cytotoxic agents to blood vessels of diseased tissues. Indeed, vascular-targeted peptides coupled to chemotherapeutic drugs, toxins, or proapoptotic peptides can decrease tumor growth, suppress clinical arthritis, or destroy prostate tissue (32Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Google Scholar, 33Olson T.A. Mohanraj D. Roy S. Ramakrishnan S. Int. J. Cancer. 1997; 73: 865-870Google Scholar, 34Ellerby H.M. Arap W. L.M. Kain R. R. G.D. S. R. Ruoslahti E. Pasqualini R. Nat. 1999; Scholar, W. W. M. Kain R. D. S. H.M. Pasqualini R. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar, E. H.M. Pasqualini R. Ruoslahti E. Res. 2001; Scholar). The YSA peptide has the in that it stimulates EphA2 which likely of the receptor and the peptide N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar, P. T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; Scholar). Therefore, or could be to selectively cells H.M. Arap W. L.M. Kain R. R. G.D. S. R. Ruoslahti E. Pasqualini R. Nat. 1999; Scholar). Furthermore, activation of EphA2 signaling by the YSA peptide should and of EphA2-expressing cancer cells H. T. Rhim J.S. E. Wang Cell Biol. 2001; Scholar, N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar). is with the that EphA2 activation with of and prostate cancer cells and the of EphA2 D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar, N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar). Intriguingly, EphA2 activation could cells to the YSA peptide is used to deliver cytotoxic agents M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google peptides by on receptors bind ligand-binding E. Ruoslahti E. J. Biol. 268: Scholar, B. H. T. Ruoslahti E. H. 1999; Scholar, R. E. Ruoslahti E. J. Cell Biol. 1995; Scholar, D. Ruoslahti E. J. Biol. 1999; Scholar) and can receptor-binding of ligands R. E. Ruoslahti E. J. Cell Biol. 1995; Scholar, A. D. M. J.L. S. D. E. Proc. Natl. Acad. Sci. U. S. A. 90: Scholar, R.M. H. A. S. P. D. 1995; Scholar). that the YSA and peptides bind to the of EphA2 that the high affinity with M. M. Nature. 2001; Scholar). the peptides with the ligand-binding domain of EphA2 and antagonize ephrin binding. at least one of the peptides causes EphA2 phosphorylation and to the YSA and as bind to a affinity is by the peptide and of the which to the affinity of EphA2 M. M. Nature. 2001; also a the two of Eph YSA and peptides bind selectively to EphA2. these peptides have that and are not by the and the the two related YSA and peptides to receptor-binding of including a is and is a present in both the and the of and high affinity allow the of peptides that bind with high affinity to Eph phage display could also be used to isolate peptides that bind to of the Eph receptors Eph receptors have been in types of cancer and should be as possible therapeutic targets (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Indeed, we have peptides that bind not receptor expressed in blood vessels and up-regulated in tumors (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Eph receptors could also be targets for nerve where ephrin mimetic peptides could to the of up-regulated at D. T. T. C. S. J. R. C. H. U. 1999; Scholar, J.D. J. 1999; Scholar, J. J. Neurosci. 2001; peptides that selectively target Eph receptor also represent for to with the activity of a receptor, a not possible by the In addition, peptides be to the of different Eph receptors in cells expressing receptors. ephrin with have value as to Eph receptor and as to in Originally identified as regulators of neural development, the Eph family of receptor tyrosine kinases and their ephrin ligands are also critical for vascular development and pathological forms of angiogenesis (1Flanagan J.G. Vanderhaeghen P. Annu. Rev. Neurosci. 1998; 21: 309-345Google Scholar, 2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar, 3Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Google Scholar). For example, the EphA2 receptor and ephrin-A1, a ligand for EphA2, are coordinately expressed in the vasculature of human tumors and mouse xenograft tumors grown from human cancer cells (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar). The EphA2 receptor plays a critical role in tumor necrosis factor α (TNFα) 1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein1The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein -induced neovascularization because TNFα up-regulates ephrin-A1, which causes receptor activation in blood vessels (5Pandey A. Shao H. Marks R.M. Polverini P.J. Dixit V.M. Science. 1995; 268: 567-569Google Scholar). Similarly, the homeobox transcription factor Hox B3 promotes angiogenesis by up-regulating ephrin-A1 (6Myers C. Charboneau A. Boudreau N. J. Cell Biol. 2000; 148: 343-351Google Scholar). Furthermore, EphA2 signaling is required for the formation of endothelial capillary tubes in vitro (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 7Daniel T.O. Stein E. Cerretti D.P. St. John P.L. Robert B. Abrahamson D.R. Kidney Int. Suppl. 1996; 57: S73-S81Google Scholar) and promotes the formation of blood vessel-like structures by melanoma cells (8Hess A.R. Seftor E.A. Gardner L.M. Carles-Kinch K. Schneider G.B. Seftor R.E. Kinch M.S. Hendrix M.J. Cancer Res. 2001; 61: 3250-3255Google Scholar). The expression of EphA2 appears to be restricted to “activated” adult blood vessels as this receptor has not been detected in either embryonic or adult quiescent blood vessels (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 9Ruiz J.C. Robertson E.J. Mech. Dev. 1994; 46: 87-100Google Scholar,10Ganju P. Shigemoto K. Brennan J. Entwistle A. Reith A.D. Oncogene. 1994; 9: 1613-1624Google Scholar). Ephrin-A1 has also not been detected in adult blood vessels, although it is present in the embryonic vasculature (11McBride J.L. Ruiz J.C. Mech. Dev. 1998; 77: 201-204Google Scholar). In addition to being present in tumor endothelial cells, EphA2 and ephrin-A1 are up-regulated in the transformed cells of a wide variety of tumors including breast, prostate, colon, skin, and esophageal cancers (4Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Google Scholar, 12Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306Google Scholar, 13Walker-Daniels J. Coffman K. Azimi M. Rhim J.S. Bostwick D.G. Snyder P. Kerns B.J. Waters D.J. Kinch M.S. Prostate. 1999; 41: 275-280Google Scholar, 14Easty D.J. Herlyn M. Bennett D.C. Int. J. Cancer. 1995; 60: 129-136Google Scholar, 15Nemoto T. Ohashi K. Akashi T. Johnson J.D. Hirokawa K. Pathobiology. 1997; 65: 195-203Google Scholar). Many factors increase EphA2 expression in cancer cells including the H-Ras oncogene, E-cadherin, members of the p53 family of transcriptional regulators, DNA damage, and loss of estrogen receptors and c-Myc (16Andres A.C. Reid H.H. Zurcher G. Blaschke R.J. Albrecht D. Ziemiecki A. Oncogene. 1994; 9: 1461-1467Google Scholar, 17Dohn M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar, 18Zelinski D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar). the tumor vasculature is discontinuous and leaky in nature, it is possible to utilize the up-regulation of EphA2 and ephrin-A1 to deliver cancer-eradicating agents to both blood vessels and tumor cells (19Dvorak H.F. Nagy J.A. Dvorak J.T. Dvorak A.M. Am. J. Pathol. 1988; 133: 95-109Google Scholar). Indeed, systemically administered biological agents can easily penetrate into tumors from the blood circulation (20Essler M. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2252-2257Google Scholar). Selectively targeting EphA2 and ephrin-A1 is a challenging task because these proteins belong to large families of closely related proteins (21Eph Nomenclature Committee Cell. 1997; 90: 403-404Google Scholar). One approach that has been successfully used to identify peptides that exhibit selectivity for their targets is to screen random peptide libraries displayed on the surface of filamentous bacteriophage (22Koivunen E. Wang B. Ruoslahti E. J. Cell Biol. 1994; 124: 373-380Google Scholar, 23Koivunen E. Arap W. Valtanen H. Rainisalo A. Medina O.P. Heikkila P. Kantor C. Gahmberg C.G. Salo T. Konttinen Y.T. Sorsa T. Ruoslahti E. Pasqualini R. Nat. Biotechnol. 1999; 17: 768-774Google Scholar, 24Smith G.P. Science. 1985; 228: 1315-1317Google Scholar, 25Devlin J.J. Panganiban L.C. Devlin P.E. Science. 1990; 249: 404-406Google Scholar). Therefore, we used a phage display approach to search for peptides that bind selectively to the extracellular domains of EphA2 and ephrin-A1. Remarkably the two peptides identified bind selectively to EphA2, but not other Eph receptors, and antagonize ephrin binding. Intriguingly, at least one of the peptides has bioactive properties in that it stimulates EphA2 tyrosine phosphorylation and signaling. In addition, this peptide delivers phage particles to EphA2-expressing cells and thus can have therapeutic value in delivering agents to tissues that express EphA2. Thus, Eph receptor-binding peptides with different agonistic and drug targeting activities can have important therapeutic have used phage display to isolate novel peptides that selectively bind to the EphA2 receptor, a cell-surface protein present in pathologic angiogenic vasculature, including tumor vasculature, and in many types of cancer cells (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Thus, targeting this receptor should allow therapeutic intervention in cancer and other diseases. The YSA peptide could be used to deliver cytotoxic agents to blood vessels of diseased tissues. Indeed, vascular-targeted peptides coupled to chemotherapeutic drugs, toxins, or proapoptotic peptides can decrease tumor growth, suppress clinical arthritis, or destroy prostate tissue (32Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Google Scholar, 33Olson T.A. Mohanraj D. Roy S. Ramakrishnan S. Int. J. Cancer. 1997; 73: 865-870Google Scholar, 34Ellerby H.M. Arap W. L.M. Kain R. R. G.D. S. R. Ruoslahti E. Pasqualini R. Nat. 1999; Scholar, W. W. M. Kain R. D. S. H.M. Pasqualini R. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar, E. H.M. Pasqualini R. Ruoslahti E. Res. 2001; Scholar). The YSA peptide has the in that it stimulates EphA2 which likely of the receptor and the peptide N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar, P. T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; Scholar). Therefore, or could be to selectively cells H.M. Arap W. L.M. Kain R. R. G.D. S. R. Ruoslahti E. Pasqualini R. Nat. 1999; Scholar). Furthermore, activation of EphA2 signaling by the YSA peptide should and of EphA2-expressing cancer cells H. T. Rhim J.S. E. Wang Cell Biol. 2001; Scholar, N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar). is with the that EphA2 activation with of and prostate cancer cells and the of EphA2 D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar, N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar). Intriguingly, EphA2 activation could cells to the YSA peptide is used to deliver cytotoxic agents M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google peptides by on receptors bind ligand-binding E. Ruoslahti E. J. Biol. 268: Scholar, B. H. T. Ruoslahti E. H. 1999; Scholar, R. E. Ruoslahti E. J. Cell Biol. 1995; Scholar, D. Ruoslahti E. J. Biol. 1999; Scholar) and can receptor-binding of ligands R. E. Ruoslahti E. J. Cell Biol. 1995; Scholar, A. D. M. J.L. S. D. E. Proc. Natl. Acad. Sci. U. S. A. 90: Scholar, R.M. H. A. S. P. D. 1995; Scholar). that the YSA and peptides bind to the of EphA2 that the high affinity with M. M. Nature. 2001; Scholar). the peptides with the ligand-binding domain of EphA2 and antagonize ephrin binding. at least one of the peptides causes EphA2 phosphorylation and to the YSA and as bind to a affinity is by the peptide and of the which to the affinity of EphA2 M. M. Nature. 2001; also a the two of Eph YSA and peptides bind selectively to EphA2. these peptides have that and are not by the and the the two related YSA and peptides to receptor-binding of including a is and is a present in both the and the of and high affinity allow the of peptides that bind with high affinity to Eph phage display could also be used to isolate peptides that bind to of the Eph receptors Eph receptors have been in types of cancer and should be as possible therapeutic targets (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Indeed, we have peptides that bind not receptor expressed in blood vessels and up-regulated in tumors (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Eph receptors could also be targets for nerve where ephrin mimetic peptides could to the of up-regulated at D. T. T. C. S. J. R. C. H. U. 1999; Scholar, J.D. J. 1999; Scholar, J. J. Neurosci. 2001; peptides that selectively target Eph receptor also represent for to with the activity of a receptor, a not possible by the In addition, peptides be to the of different Eph receptors in cells expressing receptors. ephrin with have value as to Eph receptor and as to in have used phage display to isolate novel peptides that selectively bind to the EphA2 receptor, a cell-surface protein present in pathologic angiogenic vasculature, including tumor vasculature, and in many types of cancer cells (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Thus, targeting this receptor should allow therapeutic intervention in cancer and other diseases. The YSA peptide could be used to deliver cytotoxic agents to blood vessels of diseased tissues. Indeed, vascular-targeted peptides coupled to chemotherapeutic drugs, toxins, or proapoptotic peptides can decrease tumor growth, suppress clinical arthritis, or destroy prostate tissue (32Arap W. Pasqualini R. Ruoslahti E. Science. 1998; 279: 377-380Google Scholar, 33Olson T.A. Mohanraj D. Roy S. Ramakrishnan S. Int. J. Cancer. 1997; 73: 865-870Google Scholar, 34Ellerby H.M. Arap W. L.M. Kain R. R. G.D. S. R. Ruoslahti E. Pasqualini R. Nat. 1999; Scholar, W. W. M. Kain R. D. S. H.M. Pasqualini R. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar, E. H.M. Pasqualini R. Ruoslahti E. Res. 2001; Scholar). The YSA peptide has the in that it stimulates EphA2 which likely of the receptor and the peptide N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar, P. T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; Scholar). Therefore, or could be to selectively cells H.M. Arap W. L.M. Kain R. R. G.D. S. R. Ruoslahti E. Pasqualini R. Nat. 1999; Scholar). Furthermore, activation of EphA2 signaling by the YSA peptide should and of EphA2-expressing cancer cells H. T. Rhim J.S. E. Wang Cell Biol. 2001; Scholar, N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar). is with the that EphA2 activation with of and prostate cancer cells and the of EphA2 D.P. Zantek N.D. Walker-Daniels J. Peters M.A. Taparowsky E.J. Kinch M.S. J. Cell. Biochem. 2002; 85: 714-720Google Scholar, N.D. Azimi M. M. Wang R. Kinch M.S. Cell 1999; Scholar, K. Stewart J.C. Kinch M.S. Cancer Res. 2002; Scholar). Intriguingly, EphA2 activation could cells to the YSA peptide is used to deliver cytotoxic agents M. Jiang J.Y. Chen X.B. Oncogene. 2001; 20: 6503-6515Google Scholar). peptides by on receptors bind ligand-binding E. Ruoslahti E. J. Biol. 268: Scholar, B. H. T. Ruoslahti E. H. 1999; Scholar, R. E. Ruoslahti E. J. Cell Biol. 1995; Scholar, D. Ruoslahti E. J. Biol. 1999; Scholar) and can receptor-binding of ligands R. E. Ruoslahti E. J. Cell Biol. 1995; Scholar, A. D. M. J.L. S. D. E. Proc. Natl. Acad. Sci. U. S. A. 90: Scholar, R.M. H. A. S. P. D. 1995; Scholar). that the YSA and peptides bind to the of EphA2 that the high affinity with M. M. Nature. 2001; Scholar). the peptides with the ligand-binding domain of EphA2 and antagonize ephrin binding. at least one of the peptides causes EphA2 phosphorylation and to the YSA and as bind to a affinity is by the peptide and of the which to the affinity of EphA2 M. M. Nature. 2001; also a the two of Eph The YSA and peptides bind selectively to EphA2. these peptides have that and are not by the and the the two related YSA and peptides to receptor-binding of including a is and is a present in both the and the of and high affinity allow the of peptides that bind with high affinity to Eph receptors. phage display could also be used to isolate peptides that bind to of the Eph receptors Eph receptors have been in types of cancer and should be as possible therapeutic targets (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Indeed, we have peptides that bind not receptor expressed in blood vessels and up-regulated in tumors (2Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). Eph receptors could also be targets for nerve where ephrin mimetic peptides could to the of up-regulated at D. T. T. C. S. J. R. C. H. U. 1999; Scholar, J.D. J. 1999; Scholar, J. J. Neurosci. 2001; Scholar). Bioactive peptides that selectively target Eph receptor also represent for to with the activity of a receptor, a not possible by the In addition, peptides be to the of different Eph receptors in cells expressing receptors. ephrin with have value as to Eph receptor and as to in and for and John for the and for on the