Antiangiogenic peptides and compositions thereof

Details Antiangiogenic peptides and compositions thereof Antiangiogenic peptides and compositions thereof

2001-09-18

2004-05-18

Winkler, Ulrike (Department: 1648)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C514S012200, C514S013800, C514S014800, C514S015800, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000

Reexamination Certificate

active

06737507

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides for novel antiangiogenic polypeptides and method of use thereof for treatment of diseases or disorders involving abnormal angiogenesis and tissue remodeling-associated conditions.
BACKGROUND
Blood vessels are the means by which oxygen and nutrients are supplied to living tissues and waste products are removed from living tissue. Angiogenesis refers to the process by which new blood vessels are formed. See, for example, the review by Folkman and Shing,
J. Biol. Chem.
267, 10931-10934 (1992). Thus, where appropriate, angiogenesis is a critical biological process. It is essential in reproduction, development and wound repair. However, inappropriate angiogenesis can have severe negative consequences. For example, it is only after many solid tumors are vascularized as a result of angiogenesis that the tumors have a sufficient supply of oxygen and nutrients that permit it to grow rapidly and metastasize. Because maintaining the rate of angiogenesis in its proper equilibrium is so critical to a range of functions, it must be carefully regulated in order to maintain health. The angiogenesis process is believed to begin with the degradation of the basement membrane by proteolytic enzymes, e.g., metalloproteinases (MMPs) and plasminogen activator (PA), secreted from endothelial cells (EC) activated by mitogens such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
Proteolytic activity is also required for the migration of EC into the perivascular stroma. These events are followed by sprout extension and subsequent lumen formation (Ausprunk, D. H., et al.,
Microvascular Res.
14:53-65 (1977)). As is EC “escape” from the parent venule, capillary sprout elongation, lumen formation, and EC migration are all events which are dependent on a shift in the proteolytic balance in favor of enzymatic activity (Ausprunk, D. H., et al.,
Microvascular Res.
14:53-65 (1977), Kalebic, T., et al.
Science,
221:281-283 (1983), and (Moses, M. A., et al.,
Science
248:1408-1410 (1990)). Vascular morphogenesis and invasion are also regulated by shifts in the finely tuned balance between proteases and their inhibitors (Liotta, L. A., et al.,
Cell
64:327-336, (1991); Moses, M. A., et al.,
J. Cell Biochem.
47:1-8 (1991); Herron, G. S., et al.,
J. Biol. Chem.
261:2814-2828 (1986), and Montesano, R., et. al.,
Cell
62:435-445, (1990)).
An accumulating body of evidence suggests that the remodeling of ECM that occurs during normal growth, wound repair and angiogenesis as well as during the development and progression of pathologic conditions including malignant diseases, is accomplished largely through the action of MMPs (Birkedal-Hansen, H.
Cell. Bio.
7:728-735 (1985), Matrisian, L.
Trends Genet.
6:121-125, (1990), and Woessner, J. F.
Acad. Sci.
732:11-21 (1994), and Woessner, J. F.
Ann. N.Y. Acad. Sci.
732:11-21, 1994.
The MMPs are members of a multigene family of metal-dependent enzymes. These proteases have been classified into four broad categories originally based on substrate specificity. These specific enzymes are the collagenases (MMP-1/EC3.4.24.7; MMP-8/EC3.4.24.34;MMP-13), the gelatinases A(MMP-2/EC3.4.24.24) and B(MMP-9/EC3.4.24.35), the stromelysins (MMP-3/EC3.4.24.17:MMP-10/EC3.4.24.22; MMP-1/EC3.4.24.7) including a metalloelastase (MMP-12), the membrane MMPs (MMP-14) (Birkedai-Hansen, H.
Current Opinions in Cell Biol.
7:728-735, 1995. Matrisian, L.
Trends Genet.
6:121-125, 1990. Woessner, J. F.
Ann. N.Y. Acad. Sci.
732:11-21, 1994) and the family of membrane type MMPs (MT-MMP 1-4).
The regulation of MMP activity occurs at several levels including gene transcriptional control, proenzyme activation and inhibition of activated MMPs by endogenous inhibitors. Like many other enzyme families, the MMPs are a key component of a system of “balanced proteolysis” wherein a finely tuned equilibrium exists between the amount of active enzyme and its proteinase inhibitor(s) (Liotta, L. A., et al.,
Cell
64:327-336, (1991)). These native metalloproteinase inhibitors comprise a family of proteins generally referred to as the TIMPS (Tissue Inhibitor of MetalloProteinase) (Docherty, A. J. P., et al.,
Nature
318:66-69, (1985), Carmichael, D. F., et al.
Proc. Natl. Acad. Sci. USA
83:2407-2411, (1986); Moses, M. A., et al.,
J. Cell. Biochem.
47:230-235, (1991); Murray, J. B., et al.,
J. Biol. Chem.
261:4154-4159 (1986); Stetler-Stevenson, W. G., et al.,
J. Biol. Chem.
29:17374-17378, (1989); Pavloff, N., et al.,
J. Biol. Chem.
267:17321-17326, (1992), and DeClerck, T. A., et al.,
J. Biol. Chem.
264:17445-17453 (1989)). They bind to activate MMPs with 1:1 molar stoichiometry. There are also a number of less well-characterized lower molecular weight metalloproteinase inhibitors which await complete purification and identification.
The TIMPs consist of six disulfide-bonded loops. Deletion mutagenesis studies have demonstrated that two structurally distinct domains can be defined, the N-terminal domain consisting of loops 1-3 and the C-terminal domain consisting of loops 4-6 (Murphy, G. Houbrechts., et al.
Biochemistry
30(33):8097-8101, (1991); Willenbrock, F., et al.,
Biochem.
32:4330-4337, (1993), and Nguyen, Q., et al.,
Biochem.
33:2089-2095, (1994)).
Much research attention has been focused on studies aimed at defining the domains of TIMPs that are important to their ability to inhibit MMP activity. Construction of truncated forms of these molecules has provided some insight. Residues 1-126 of TIMP-1 and 1-127 of TIMP-2 which contain three of the six disulfide bonds in the full-length molecules have been expressed in mammalian cells in the absence of the C-terminal region and are secreted in a soluble form (Murphy, G., et al.
Biochemistry
30(33):8097-8101, (1991)). These truncated forms inhibit matrilysin and the catalytic domains of stromelysin and gelatinase A, demonstrating that there is a direct interaction between the N-terminal domain of the TIMPs and the catalytic domains of the MMPs (Murphy, G., et al.
Biochemistry
30(33):8097-8101, (1991); Willenbrock, F., et al.,
Biochem.
32:4330-4337, (1993), and Nguyen, Q., et al.,
Biochem.
33:2089-2095, (1994). The structure of the N-terminal domain of either TIMP-1 or TIMP-2 is not affected by the C-terminal domain (Nguyen. Q., et al.,
Biochemistry
33:2089-2095, (1994)).
A significant number of mutational analyses also support the concept that the NH
2
-terminal domain of TIMP-1 (Cys
1
-Glu
126
) is sufficient for inhibition of MMPs (Wilheim, S. M., et al.,
J. Biol. Chem.
264:17213-17221, (1989), Murphy, G., et al.,
Biochemistry
30(33):8097-8101, (1991); Murphy, G., et al.,
Bio. Chem. Biophys. Acta.
839:214-218, (1985); Stricklin, G. P.,
Collagen Relat. Res.
6:219-228, (1986); Tolley, S. P., et al.,
Protein: Struc., Fuct., Genet.
17:435-437, (1993), and Howard, E. W., et al.,
J. Biol. Chem.
266:13064-13069, (1991)). Furthermore, single-residue mutations in the region bounded by Cys3 and Cys13 caused an increase of 8-fold in K/when compared with wild type TIMP-1 (O'Shea, M., et al.,
Biochemistry
31(42):10146-10151, (1992)). A series of experiments including competition studies with synthetic peptides and localization of epitopes of blocking antibodies revealed that the region marking the transition between the NH2-terminal and COOH-terminal domains of the TIMP-1 molecule may be particularly important for its ability to inhibit collagenase (Bodden, M. K., et al.,
J. Biol. Chem.
269:18943-18952, (1994)).
It is now widely accepted that the N-terminal domain of the TIMPs represent a stable, minimized form of the inhibitor that includes the major site or sites necessary for MMP inhibition (Williamson, R. A., et al.,
Biochem.
33:11745-11759, (1994)). Site-directed mutagenesis studies on TIMP-1 have demonstrated that no single residue is likely to be responsible for MMP inhibition (O'Shea, M., et al.,
Biochem.
10146-10151, (1992)).
The C-terminal domain of TIMPs also makes some binding contribution to th

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