This project was originally submitted in response to PAR-98-096, THERAPEUTIC MODULATION OF ANGIOGENESIS IN DISEASE. In this revised project, we seek to develop antibodies and peptides that modulate angiogenesis through the endothelial cell (EC) angiostatin receptor, recently shown by us to be cell surface ATP synthase. ATP synthase on the surface of tumor EC may play a role both in supplemental energy production under low oxygen conditions or in endothelial signaling events involved in tumor angiogenesis. Polyclonal antibodies against subunits of ATP synthase compete with angiostatin for cell surface EC binding and block the ability of angiostatin to inhibit EC proliferation and migration. Polyclonal antisera against the b-subunit also exhibit a direct EC inhibitory effect exceeding that of angiostatin. Furthermore, we recently discovered a second ATP producing enzyme on the EC surface, nucleoside diphosphate kinase h1 (NDPK h1), that is dramatically inhibited by angiostatin (manuscript submitted for publication, see Appendix). These and other findings support the hypothesis that angiostatin exerts its anti-proliferative effect on EC through disruption of surface ATP synthesis. It is likely that the recognized ability of angiostatin to inhibit growth and metastasis of many tumors in vivo is also mediated by inhibition of EC surface ATP synthesis. However, angiostatin itself has poor potential as a therapeutic agent in humans because of limitations in production, stability, and affinity. Our discovery of two EC targets of angiostatin offers the possibility of developing more robust compounds that can block angiogenesis in human cancer and other proliferative diseases. The goal of this project is to develop antibodies and peptides that can substitute for angiostatin to inhibit angiogenesis in breast cancer through direct interactions with the ATP synthesizing apparatus on the surface of EC. The promise of these compounds as therapeutic agents justifies an intensive effort to develop appropriate humanized monoclonal antibodies and targeted peptide phage display libraries. Candidate antibodies and peptides will be screened for EC inhibitory activity using established assays that measure proliferation, migration, and tube formation. In addition, we have developed a novel assay for EC surface ATP synthesis that will be used to screen compounds for inhibitory activity. Compounds that exhibit inhibitory activity in any of these assays will be screened for their ability to inhibit tumor growth in vivo using a human breast cancer xenograft model. Because of the exposed intravascular localization of the ATP synthesizing enzymes, this project will yield a collection of anti-angiogenic compounds with strong potential for rapid translation into clinical studies.
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