Lysine binding fragments of angiostatin

Details Lysine binding fragments of angiostatin Lysine binding fragments of angiostatin

1999-07-07

2003-03-25

Fredman, Jeffrey (Department: 1637)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C514S002600

Reexamination Certificate

active

06538103

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns peptide fragments of angiostatin containing lysine-binding sites of angiostatin, including small molecules that mimick their functions, which can be used as anti-angiogenic agents for the treatment of cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, atherosclerotic plaque formation, and any disease process that involves angiogenesis.
BACKGROUND OF THE INVENTION
Different than de novo vascularization, angiogenesis is the process of neovascularization from pre-existing blood vessels. It has pronounced effects in a wide array of physiological conditions, such as placenta development and embryogenesis. An imbalance of the angiogenic process has been shown to contribute to pathological disorders such as diabetic retinopathy, rheumatoid arthritis, and psoriasis (Folkman, J. (1995)
Nat. Med.
1, 27-31; Folkman, J. (1995)
New Engl. J. Med.
333, 1757-1763). Particularly, both primary and metastatic tumors need to recruit neovessels for their growth expansion (Folkman, J. (1971)
New Engl. J. Med.
285, 1182-1186; Folkman, J., and Shing, Y. (1992)
J. Biol. Chem.
267, 10931-10934).
There is a mounting body of evidence supporting the concept that angiogenesis is a prerequisite for tumor growth. Such a correlation has been established by blocking positive regulators of angiogenesis or utilizing negative regulators to suppress angiogenesis that resulted in a delay or regression of experimental tumors. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are potent mitogens and strong chemoattractants for endothelial cells (Shing Y., Folkman J., Sullivan, R., Butterfield, C., Murray, J., Klagsbrun, M. (1984)
Science
223, 1296-1298; Maciag, T., Mehlnan, T., Friesel, R., and Schreiber, A. (1984)
Science
225, 932-935; Ferrara, N. and Henzel, W. J. (1989)
Biochem. Biophys. Res. Commun.
161, 851-855). Their roles in inducing tumor angiogenesis have been demonstrated in a variety of human tumors (Nguyen, M., Watanabe, H., Budson, A. E., Richie, J. P., Hayes, D. F., and Folkman, J. (1994)
J. Natl. Cancer Inst.
86, 356-361; Dvorak, H. F., Sioussat, T. M., Brown, L. F., Berse, B., Nagy, J. A., Sotrel, A., Manseau, E. J., Van de Water, L., and Senger, D. R. (1991)
J. Exp. Med.
174, 1275-1278). Antibodies neutralizing VEGF or FGF caused a marked decrease of tumor growth via angiogenic inhibition (Gross, J. L., Herblin, W. F., Dusak, B. A., Czerniak, P., Diamond, M. D., Sun, T., Eidsvoog, K., Dexter, D. L., and Yayon, A. (1993)
J. Natl. Cancer Inst.
85(2), 121-131; Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and Ferrara, N. (1993)
Nature
362(6243), 841-844). Such anti-angiogenic and anti-tumor effects were also observed by antagonizing the corresponding receptors for these endothelial cell mitogens. A soluble form of Tie-2 receptor, a receptor tyrosine kinase preferentially expressed on vascular endothelium, exhibited effective inhibition of tumor angiogenesis (Lin, P., Polverini, P., Dewhirst, M., Shan, S., Rao, P. S., and Peters, K. (1997)
J. Clin. Invest.
100(8), 2072-2078). Similarly, a dominant negative mutant of the VEGF receptor, Flk-1, prevented VEGF-induced angiogenesis by dimerizing and deactivating the endogenous Flk-1 (Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., and Ullrich, A. (1994)
Nature
367(6463), 576-579). In addition, negative regulators of angiogenesis, such as angiostatin, endostatin, and antagonists for integrin av&bgr;3, displayed profound anti-tumor activities in vivo (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994)
Cell
79, 315-328; O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997)
Cell
88(2), 277-285; Brooks, P. C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F. H., and Cheresh, D. A. 1995)
J. Clin Invest.
96(4), 1815-1822). TNP-470 and Interferon alfa-2a also manifested clinical evidence that tumor growth could be therapeutically intervened using an anti-angiogenic approach (Ingber, D., Fujita, T., Kishimoto, S., Sudo, K., Kanamaru, T., Brem, H., and Folkman, J. (1990)
Nature
348, 555-557; Ezekowitz, R. A., Mulliken, J. B., and Folkman, J. (1992)
N. Engl. J. Med.
326, 1456-1463).
Angiostatin was initially isolated from urine and sera of mice bearing Lewis Lung carcinoma (O'Reilly, M. S., et al., (1994)
Cell
; U.S. Pat. No. 5,639,725). It is an approximately 38-45kD internal fragment of plasminogen, which consists of 4 triple-disulfide bridged kringle structures. Both kringle 1 and 4 have lysine-binding sites, which are responsible for anchoring the plasminogen molecule on fibrin-rich blood clots (Wiman, B. and Collen, D. (1978)
Nature
272, 549-545). Angiostatin was originally generated from plasminogen by proteolytic cleavage with porcine pancreatic elastase (O'Reilly, M. S., et al., (1994)
Cell
). Subsequent studies have shown that angiostatin can be generated from plasminogen by a variety of physiological and pathological proteases, including macrophage-derived metalloelastases (Dong, Z., Kumar, R., Yang, X., Fidler, I. J. (1997)
Cell
88(6), 801-810), members of matrix metalloproteinase (MMP) family, such as matrilysin (MMP-7) or gelatinase B/type IV collagenase (MMP-9) (Patterson, B. C. and Sang, Q. A. (1997)
J. Biol. Chem.
272(46), 28823-28825), and Urokinase (Gately, S., Twardowski, P., Stack, M. S., Cundiff, D. L., Grella, D., Castellino, F. J., Enghild, J., Kwaan, H. C., Lee, F., Kramer, R. A., Volpert, O., Bouck, N., and Soff, G. A. (1997)
Proc. Natl Acad. Sci. USA
94(20), 10868-10872). Angiostatin was found to inhibit endothelial cell proliferation in vitro and block growth factor, such as basic FGF, elicited angiogenesis in vivo. Elastase-cleaved angiostatin was shown to induce dormancy of several metastatic and primary tumors, including carcinomas of breast, prostate, colon, and lung (O'Reilly, M. S., et al., (1994)
Cell
; O'Reilly, M. S., Holmgren, L., Chen, C., and Folkman, J. (1996)
Nat. Med.
2(6), 689-692). Angiostatin generated by urokinase cleavage significantly reduced the growth of a murine hemangioendothelioma in vivo (Lannutti, B. J., Gately, S. T., Quevedo, M. E., Soff, G. A., and Paller, A. S. (1997)
Cancer Res.
57, 5277-5280). Recombinant angiostatin also produced anti-tumor effects in vivo via the blocking of tumor angiogenesis (Wu, Z., O'Reilly, M. S., Folkman, J., and Shing, Y. (1997)
Biochem. Biophys. Res. Commun.
236, 651-654; Sim, B. K., O'Reilly, M. S., Liang, H., Fortier, A. H., He, W., Madsen, J. W., Lapcevich, R., and Nacy, C. A. (1997)
Cancer Res
57(7), 1329-1334). These anti-tumor effects were accompanied by a marked reduction of microvessel density within the tumor mass, indicating that suppression of angiogenesis led to the inhibition of tumor growth.
Angiogenesis is a complex process that entails an orchestration of endothelial cell proliferation, migration, basement membrane degradation, and neovessel assembly. The kringle domains of angiostatin were shown to exhibit divergent inhibitory activities for endothelial cell proliferation (Cao, Y., Ji, R. W., Davidson, D., Schaller, J., Marti, D., Sohndel, S., McCance, S. G., O'Reilly, M. S., Llinas, M., and Folkman, J. (1996)
J. Biol. Chem.
271, 29461-29467). Kringle 1-3 manifested a potent inhibition of endothelial cell proliferation whereas kringle 4 had only marginal effect. Kringle conformation was also shown to be essential for the potent anti-proliferative activity of angiostatin.
SUMMARY OF THE INVENTION
Angiostatin (approximately kringle regions 1-4 (“K1-4”) of human plasminogen) has been described as a potent angiogenesis inhibitor. We now demonstrate that the blocking of the lysine-binding sites of angiostatin (K1-4) completely abolishs its inhibitory activities of endothelial cell migration, a process crucial for angiogenesis. The present invention is directed to a frag

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