Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 6 to 7 amino acid residues in defined sequence
Reexamination Certificate
1997-08-21
2004-02-03
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
6 to 7 amino acid residues in defined sequence
C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C514S012200, C514S013800, C514S014800, C514S015800, C514S016700, C514S017400
Reexamination Certificate
active
06686444
ABSTRACT:
BACKGROUND OF THE INVENTION
The formation of metastases from a primary tumor is a complex temporal process that involves angiogenesis, invasion of the circulatory system by tumor cells, intravasation of the endothelium, arrest in the venous or capillary bed of the target organ, extravasation, entry into the target parenchyme and proliferation of the secondary tumor in tissue different from its tissue of origin. Throughout this process metastatic tumor cells are constantly interacting with their host tissue modulating their adhesiveness to cells and extracellular matrices, degrading matrices and migrating into interstitial stroma. These events are not unique to tumor metastasis and occur in other processes such as angiogenesis, tissue remodeling, bone remodeling and embryogenesis. Experimental studies have demonstrated that invasion involves the active locomotion of tumor cells through and into tissue barriers. The molecular mechanisms regulating such invasive tumor cell migration and subsequent implantation are poorly understood but it appears that there is organ specificity of colonization.
Several substances that stimulate tumor cell locomotion in vitro have been described. These include factors derived from resorbing bone, liver and smooth muscle. Locomotion of tumor cells can also be induced by endogenous substances in an autocrine fashion. These factors can induce two types of directed cell locomotion: (1) chemotaxis, i.e., directed locomotion of cells up a soluble gradient; or (2) haptotaxis, i.e., migration of cells up a gradient of attached molecules.
Bone metastases are frequently one of the first signs of disseminated disease in certain carcinomas of lung, breast, prostate, kidney or thyroid. Osteoblasts, the bone forming cells, have been shown to secrete substances that induce the chemotaxis and invasion of melanoma and breast cancer cells. One product of osteoblasts that induces the migration of several cell types and is secreted by several tumors is osteopontin.
Osteopontin (Oldberg et al. (1986)
Proc. Natl. Acad. Sci.
USA 83:8819; Oldberg et al. (1986)
J. Biol. Chem.
263:19433-19436) also known as OPN (Wrana et al. (1989)
Nucl. Acid Res.
17:3306), 2ar (Smith, J. H. and Denhardt, D. T. (1987).
J. Cell Biochem.
34: 10-22), transformation-associated secreted phosphoprotein (Senger et al. (1989)
Anticancer Res.
48:1291), or Early T-lymphocyte activation-1 (Patarca et al. (1991)
Proc. Natl. Acad, Sci. USA
88:2736), is a secreted glycosylated phosphoprotein expressed by bone (Oldberg et al. (1986)
J. Biol. Chem.
263: 19433-19436), activated T-lymphocytes (Patarca et al. (1989)
J. Exp. Med.
170:145-161; Patarca et al. (1991)
Proc. Natl. Acad. Sci. USA
88:2736), macrophages (Singh et al. (1990).
J. Exp. Med
171:1931-1942), smooth muscle cells of the vascular system (Giachelli et al. (1991)
Biochem. Biophys. Res. Commun.
177: 867-873), and carcinomas and sarcomas (Senger et al. (1989)
Anticancer Res.
48:1291).
The marked induction of osteopontin during arterial wound healing, immune response, and bone development and remodeling, suggests a role for this protein in these processes. Osteopontin expression by smooth muscle cells is induced upon arterial injury were it is chemotactic to smooth muscle cells and supports the adhesion of endothelial cells. Osteopontin is also abundant in athrosclerotic plaques. Secretion of osteopontin in the early response after T-cell activation is associated with enhanced secretion of IgM and IgG by B-cells (Lampe et al. (1991)
J. Immunol.
147:2902) and is chemotactic to macrophages (Singh et al. (1989)
Anticancer Res.
48:1291). It is constitutively expressed in CD
4−
CD
8−
T lymphocytes from the spontaneously autoimmune MRL/1pr mouse strain (Patarca et al. (1990)
J. Exp. Med.
172:1177-1183). Its circulating levels are elevated in individuals with autoimmune diseases. Osteopontin is also involved in bone development and remodeling. Osteopontin supports the migration and adhesion of osteoclasts and osteoblasts and appears to be chemotactic to osteoprogenitor cells.
Osteopontin is also elevated in sera from patients with advanced metastatic cancer and cellular transformation may lead to enhanced osteopontin expression and increased metastatic activity. Expression of antisense RNA in metastatic Ras transformed fibroblasts resulted in the reduction of the metastatic potential of these cells. The presence of a Gly-Arg-Gly-Asp-Ser (GRGDS, SEQ ID NO:8) cell-surface receptor binding motif within the sequence of osteopontin suggested that osteopontin may be involved in cell attachment and spreading (Oldberg et al. (1986)
Proc. Natl. Acad. Sci.
USA 83:88 19; Oldberg et al. (1986)
J. Biol. Chem.
263:19433-19436). Osteopontin binds to cells via integrin and non-integrin receptors, and is a ligand for &agr;
v
&bgr;
3
, &agr;
v
&bgr;
1
, and &agr;
v
&bgr;
5
integrins. Multiple phosphorylated and nonphosphorylated forms of osteopontin are secreted by cells and are differentially stimulated by tumor promoters (Kubota et al. (1989) Biochem. Biophys. Res. Commun. 162: 1453-1459). In addition, differential attachment of osteoclasts to surfaces coated with osteopontin isolated from various tissues and to phosphorylated and nonphosphorylated osteopontin has been demonstrated. Furthermore, cleavage of osteopontin with thrombin enhances its cell attachment properties. These results suggest that depending on the cell surface receptor repertoire, cells may recognize distinct forms of osteopontin and may respond differently to the form of osteopontin they encounter.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of or identification of the chemotactic regions of the osteopontin polypeptide. This discovery led to the development of chemotactic peptides derived from osteopontin. The peptides (or antagonists of the same) can be used to treat conditions or diseases associated with chemotaxis. These peptides can further be used to treat conditions or diseases which can be treated using osteopontin, e.g., based upon osteopontin's chemotactic properties. For example, the peptides of the present invention can be used to treat or inhibit tumor metastasis, inflammation, osteoporosis and autoimmune disease.
The present invention pertains to osteopontin derived peptides. The peptides are capable of inducing the chemotaxis of several cell types. Examples of cell types include, but are not limited to, endothelial cells, periosteal cells, tumor cells, macrophages and osteoprogenitor cells. The osteopontin derived chemotactic peptides do not appear to mediate cell attachment but rather alter the cytoskeletal organization of the cell and induce migration.
The invention also pertains to an isolated nucleic acid encoding an osteopontin derived peptide(s) of the present invention. The nucleic acid can be used to produce the peptide and also as a therapeutic agent.
Other aspects of the invention include antibodies, e.g., monoclonal antibodies, which are specifically reactive with the above-described peptides. These antibodies can be administered to a subject in the form of a therapeutic composition to modulate the chemotactic effect of the peptides of the invention. The preferred antibody of the invention has the amino acid sequence KFHSHKDKLVLDPKSK (SEQ ID NO:2). The antibodies neutralize the migration of various cell types in response to osteopontin both in vitro and in vivo.
In another aspect, the invention features a therapeutic composition which includes an osteopontin derived chemotactic peptide and a pharmaceutically-acceptable carrier or diluent. The therapeutic composition can be used in the methods described herein.
In another aspect, the invention features a method for modulating tumor invasion in a subject. The method includes administering to a subject a therapeutically effective amount of an antagonist of an osteopontin derived chemotactic peptide such that tumor invasion is modulated.
In another aspect, the invention features a method for promoting wound healing in a subject. T
Children's Medical Center Corporation
Holland & Knight LLP
Low Christopher S. F.
Lukton David
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