Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds antigen or epitope whose amino acid sequence is...
Reexamination Certificate
1998-10-29
2002-06-04
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds antigen or epitope whose amino acid sequence is...
C530S387100, C530S388220, C530S387900, C530S391300
Reexamination Certificate
active
06399064
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is generally in the area of cloning, expression, and regulation of an endothelial cell protein C/activated protein C receptor.
Protein C plays a major role in the regulation of blood coagulation. Patients deficient in protein C usually exhibit life threatening thrombotic-complications in infancy (Seligsohn et al., (1984)
N. Engl. J. Med.
310, 559-562; Esmon, (1992)
Trends Cardiovasc. Med.
2, 214-220) that are corrected by protein C administration (Dreyfus et al., (1991)
N. Engl. J. Med.
325, 1565-1568). In addition, activated protein C (APC) can prevent the lethal effects of
E. coli
in baboon models of gram negative sepsis (Taylor et al., (1987)
J. Clin. Invest.
79; U.S. Pat. No. 5,009,889 to Taylor and Esmon) and preliminary clinical results suggest that protein C is effective in treating certain forms of human septic shock (Gerson et al., (1993)
Pediatrics
91, 418-422). These results suggest that protein C may both control coagulation and influence inflammation. Indeed, inhibition of protein S, an important component of the protein C pathway, exacerbates the response of primates to sublethal levels of
E. coli
and augments the appearance of TNF in the circulation (Taylor et al., (1991)
Blood
78, 357-363). The mechanisms involved in controlling the inflammatory response remain unknown.
Protein C is activated when thrombin, the terminal enzyme of the coagulation system, binds to an endothelial cell surface protein, thrombomodulin (Esmon, (1989)
J. Biol. Chem.
264, 4743-4746; Dittman and Majerus, (1990)
Blood
75, 329-336; Dittman, (1991)
Trends Cardiovasc. Med.
1, 331-336). In cell culture, thrombomodulin transcription is blocked by exposure of endothelial cells to tumor necrosis factor (TNF) (Conway and Rosenberg, (1988)
Mol. Cell. Biol.
8, 5588-5592) and thrombomodulin activity and antigen are subsequently internalized and degraded (Lentz et al., (1991)
Blood
77, 543-550, Moore,K. L., et.al., (1989)
Blood
73, 159-165). In addition, C4bBP, a.regulatory protein of the complement system, binds protein S to form a complex that is functionally inactive in supporting APC anticoagulant activity in vitro (Dahlbäck, (1986)
J. Biol. Chem.
261, 12022-12027) and in vivo (Taylor,et al., 1991). Furthermore, C4bBP behaves as an acute phase reactant (Dahlbäck, (1991)
Thromb. Haemostas.
66, 49-61). Thus, proteins of this pathway not only appear to regulate inflammation, but they also interact with components that regulate inflammation, and they themselves are subject to down regulation by inflammatory mediators.
Given the central role of the protein C pathway in regulating the host response to inflammation and the critical role of the pathway in controlling blood coagulation, it is important to identify and characterize all of the components that interact with the system. This is especially true since the molecular basis of the anti-inflammatory effects of the protein C pathway components have yet to be elucidated at the molecular level.
It is therefore an object of the present invention to provide a cellular receptor for protein C and activated protein C.
It is a further object of the present invention to provide nucleotide sequences encoding the cellular receptor and amino acid characterization of the receptor which allows expression of recombinant native and modified forms of the receptor.
It is another object of the present invention to provide methods of modulating the inflammatory response involving protein C and activated protein C.
SUMMARY OF THE INVENTION
An endothelial cell protein C binding protein (referred to herein as “EPCR”) has been cloned and characterized. The protein is predicted to consist of 238 amino acids, which includes a 15 amino acid signal sequence at the N-terminus, and a 23 amino acid transmembrane region which characterizes the receptor as a type 1 transmembrane protein. The protein binds with high affinity to both protein C and activated protein C (Kd=30 nM) and is calcium dependent. The message and binding function of the receptor are both down regulated by cytokines such as TNF.
These results identify a new member of a complex pathway that, like other members of the pathway, is subject to regulation by inflammatory cytokines, and can therefore be used to modulate inflammatory reactions in which protein C or activated protein C is involved. Inhibition of the inflammatory response can be obtained by infusing soluble EPCR. Alternatively, localizing EPCR to surfaces in contact with blood will render the surfaces anticoagulant by virtue of the ability of EPCR to bind and concentrate the anticoagulant activated protein C at the surface. Alternatively, the function of EPCR can be enhanced by overexpressing the EPCR in endothelium that could be used to coat vascular grafts in patients with vascular disease or on stents in cardiac patients.
REFERENCES:
patent: 3625214 (1971-12-01), Higuchi
patent: 4244946 (1981-01-01), Rivier
patent: 4305872 (1981-12-01), Johnston et al.
patent: 4316891 (1982-02-01), Guilleman et al.
patent: 4629784 (1986-12-01), Stammer
patent: 4782137 (1988-11-01), Hopp et al.
patent: 4789734 (1988-12-01), Pierschbacher
patent: 4792525 (1988-12-01), Ruoslahti et al.
patent: 4906474 (1990-03-01), Langer et al.
patent: 4925673 (1990-05-01), Steiner et al.
patent: 4980286 (1990-12-01), Morgan et al.
patent: 5009889 (1991-04-01), Taylor et al.
patent: 5298599 (1994-03-01), Rezaie et al.
patent: 5695993 (1997-12-01), Fukudome et al.
patent: 5698189 (1997-12-01), Rowe et al.
patent: 5749968 (1998-05-01), Melanson et al.
patent: 5779673 (1998-07-01), Roth et al.
patent: 5804392 (1998-09-01), Esmon et al.
patent: WO 96/05303 (1996-02-01), None
patent: WO 96/20732 (1996-07-01), None
patent: WO 96/21470 (1996-07-01), None
patent: WO98/20041 (1998-05-01), None
Bird et al.Science, vol. 242, pp. 423-426, 1988.*
Bailey et al.J. Pharm. Biomed. Anal., vol. 5, pp. 649-658, 1987.*
Blumberg et al.J. Immunology, vol. 147, pp. 2518-2524, 1991.*
*Abe, et al., “Granulocyte protease and hydrogen peroxide synergistically inactive thrombomodulin of endothelial cells in vitro,”J. Lab. Clin. Med.123(6):874-881 (1994).
*ACCP/SCCM Consensus Conference, “Definitions for Spesis and Organ Failure and Guidelines for the Use of Innovative Therapies in Sepsis,”Chest101(6):1644-1655 (1992).
Agrawal, et al., “Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus,”Proc. Natl. Acad. Sci. USA85(19):7079-7083 (1988).
*Arend, et al., “Building of IL-1&agr;, IL-1&bgr;, and IL-1 Receptor Antagonist by Soluble IL-1 Receptors and Levels of Soluble IL-1 Receptors in Synovial Fluids,”J. Immunol.153:4766-4774 (1994).
*Asakura, et al., “Plasma Levels of Soluble Thrombomodulin Increase in Cases of Disseminated Intravascular Coagulation With Organ Failure,”Am. J. Hematol.38:281-287 (1991).
Askew, B., et al., “Molecular Recognition with Convergent Functional Groups, 6, Synthetic and Structural Studies with a Model Receptor for Nucleic Acid Components”,J. Am. Chem. Soc., 111:1082-1090 (1989).
Bangalore, et al., “High affinity binding sites for activated protein C and protein C on cultured human umbilical vein endothelial cells. Independent of protein S and distinct from known ligands,”Thromb Haemos72(3):465-74 (1994).
*Berg, et al., “Aberrant RNA splicing of the protein C and protein S genes in health individuals,”Blood Coag Fibrinol.7:625-631 (1996).
Blume, et al., “Triple helix formation by purine-rich oligonucleotides targeted to the human dihydrofolate reductase promoter,”Nucleic Acids Res.20(7):1777-84 (1992).
Bock, “Active Site Selective Labeling of Serine Protease with Spectroscopic Probes Using Thioester Peptide Chloromethyl Ketones: Demonstration of Thrombin Labeling Using N&agr;-[(Acetylthio)acetyl]-D-Phe-Pro-Arg-CH2C1,”Biochemistry27:6633-6639 (1988).
*Boehme, et al., “Release of thrombomodulin from endothelial cells by concentrated action of TNF-&agr; and neutrophils: in vivo and in vitro studies,”Immunology87:134-140 (1996).
*Bourin & Lindah
Esmon Charles T.
Fukudome Kenji
Gucker Stephen
Holland & Knight LLP
Kunz Gary L.
Oklahoma Medical Research Foundation
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