Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-02-25
2001-03-27
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S252300, C435S320100, C536S023500, C800S013000
Reexamination Certificate
active
06207419
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for inhibiting thrombin. In particular, the present invention provides heparin cofactor II mutants and methods of administering the same that may be employed to inhibit thrombin action both in vitro and in vivo.
BACKGROUND OF THE INVENTION
The classic model of blood coagulation proposes a series of cascading reactions that result in clot formation. This model has been modified to incorporate initiation, amplification, and propagation phases. Furie and Furie, (1992)
N. Engl. J. Med.
326:800; Roberts and Lozier, (1992)
Hosp. Pract.
27:97. Adding an additional layer of complexity is an array of mechanisms that turn off and/or modulate the coagulation cascade. Thrombin is a key regulatory point in pathways that lead to blood coagulation, as well as those that reverse this process. Circulating prothrombin is cleaved to form thrombin following interaction with other blood clotting factors. Thrombin is a trypsin-like serine protease, which is responsible for the proteolytic conversion of fibrinogen to fibrin, with the aggregation of fibrin molecules resulting in the formation of a “soft” clot.
The proteolytic actions of thrombin are inhibited by interaction with plasma serine proteinase inhibitors (“serpins”), such as heparin cofactor II (HCII), antithrombin III (ATIII), protein C inhibitor (PCI), and &agr;-proteinase inhibitor. Thrombin recognizes a conserved amino acid sequence among serpins, termed the “reactive site loop”, as a potential substrate. Interaction of thrombin with the reactive site loop results in the formation of a thrombin-serpin complex that is essentially irreversible. Complex formation inactivates both the thrombin protease and serpin inhibitory activities. Pratt and Church, (1993)
Blood Coag. and Fibrinol.
4:479.
The inhibitory actions of ATIII, PCI and HCII are accelerated in the presence of heparin (1000-10,000 fold), and the anti-coagulation effects of heparin are believed to be mediated through these plasma serpins. HCII is the most dependent of these inhibitors on glycosaminoglycan; it has only about 10% the inhibitory activity of ATIII or PCI in the absence of heparin. Pratt et al., (1989a)
Thromb. Res.
53:595; Pratt et al., (1989)
Ann. N.Y. Acad. Sci.
556:104; Pratt et al., (1992)
J. Biol. Chem.
267:8795; Pratt and Church, (1992)
J. Biol. Chem.
267:8789. Thrombin inhibition by HCII, but not ATIII, is also accelerated in the presence of dermatan sulfate and dermatan sulfate proteoglycans. Pratt and Church, (1993)
Blood Coag. Fibrinol.
4:479. Moreover, ATIII inhibits most of the serine proteases involved in coagulation, whereas HCII appears to be uniquely specific for thrombin. Church and Hoffman, (1994)
TCM
4:140. The only other known substrates for HCII are chymotrypsin, cathepsin G, and
Streptomyces gresius
Protease B. Church et al., (1985)
Proc. Natl. Acad. Sci. USA
82:6431; Parker and Tollefsen, (1985)
J. Biol. Chem.
260:3501; Pratt et al., (1989b)
Ann. N.Y. Acad. Sci.
556:104.
As described above, heparin is a known anti-coagulant, and it is widely-administered to human and animal subjects for this purpose. Individual response to heparin is quite variable, however, and heparin administration can result in hemorrhaging, thrombocytopenia, and osteoporosis. Stone, (1995)
TCM
5:134; U.S. Pat. Nos. 5,118,793 and 5,102,995 to Tollefsen et al. Accordingly, there exists a need in the art for alternative anti-coagulant agents and therapies.
SUMMARY OF THE INVENTION
Heparin cofactor II (HCII) is a serine protease inhibitor (“serpin”) that acts to inhibit the biological actions of thrombin. The present invention is based on the discovery of novel HCII mutants with hyperactive thrombin inhibitory activity.
One aspect of the present invention is a mutant heparin cofactor II comprising, in combination, a heparin cofactor II and a carboxyl terminal amino acid extension, where the carboxyl terminal extension comprises at least one histidine residue and confers enhanced anti-thrombin activity to the mutant heparin cofactor II. Also provided are isolated DNA encoding the mutant heparin cofactor II, and vectors and host cells containing the same.
A further aspect of the present invention are pharmaceutical compositions comprising the inventive mutant heparin cofactor II molecules.
A further aspect of the present invention is a method of inhibiting blood coagulation in a subject in need thereof, comprising administering a mutant heparin cofactor II comprising a carboxyl terminal extension to the subject in an amount effective to inhibit blood coagulation, where the carboxyl terminal extension confers enhanced anti-coagulant activity to the heparin cofactor II.
Still a further aspect of the present invention is a method of inhibiting blood coagulation in a biological sample comprising adding a mutant heparin cofactor II comprising a carboxyl terminal extension to the biological sample in an amount effective to inhibit blood coagulation, where the carboxyl terminal extension confers enhanced anti-coagulant activity to the heparin cofactor II.
Yet a further aspect of the present invention is a method of regulating wound healing in a subject in need thereof, comprising administering a mutant heparin cofactor II comprising a carboxyl terminal extension to the subject in an amount effective to regulate wound healing, where the carboxyl terminal extension confers enhanced anti-thrombin activity to the heparin cofactor II.
As a further aspect, the present invention provides a method of regulating tissue repair in a subject in need thereof, comprising administering a mutant heparin cofactor II comprising a carboxyl terminal extension to the subject in an amount effective to regulate tissue repair, where the carboxyl terminal extension confers enhanced anti-thrombin activity to the heparin cofactor II.
As a further aspect, the present invention provides a method of inhibiting inflammation in a subject in need thereof, comprising administering a mutant heparin cofactor II comprising a carboxyl terminal extension to the subject in an amount effective to inhibit inflammation, where the carboxyl terminal extension confers enhanced anti-inflammatory activity to the heparin cofactor II.
These an other aspects of the invention are set forth in more detail in the description of the invention below.
REFERENCES:
patent: 5102995 (1992-04-01), Tollefsen et al.
patent: 5118793 (1992-06-01), Tollefsen et al.
patent: 0424351 A2 (1991-04-01), None
Ngu et al (V) The protein folding Problem and Tertiary Structure Prediction (1994) Merz et al. (ed) Birkhauser, Boston, MA p. 433 and 492-95, 1994.*
Merriam Webster's Collegiate Dictionary, 10th Edition, Merriam-Webster Inc., USA pp. 906 and 1192, 1994.*
Ciaccia et al.; Heparin Promotes Proteolytic Inactivation by Thrombin of a Reactive Site Mutant (L444R) of Recombinant Heparin Cofactor II*, The Journal of Biological Chemistry 272:2 888-893 (1997).
Herzog et al.; Complete Nucleotide Sequence of the Gene for Human Heparin Cofactor II and Mapping to Chromosomal Band 22q11†,‡, Biochemistry 30:5 1350-1357 (1991).
Hochuli et al.; Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent, Biotechnology 6:11 1321-1325 (Nov. 1988).
Hochuli; Purification or Recombinant Proteins with Metal Chelate Adsorbent, Genetic Engineering 12:87-98 (1990).
International Search Report, PCT/US99/04137, Date of Mailing Aug. 4, 1999.
Bauman et al.; “Serpindipity”: Enhanced Antithrombotic Activity of a Novel Recombinant Heparin Cofactor II, Blood, 92: 10 Part 2 of 2, (1988).
Bauman et al; Enchanced Antithrombotic Porperties of Heparin Cofactor II Through Site-Directed Mutagenesis, Experimental Biology '98 Meeting, Apr. 1998, SanFrancisco, CA, FASEB J. 12:5 Part II, A954 (Abstr. #5528) (1998).
Berthold et al.; Purification of Recombinant Antigenic Epitopes of the Human 68-kDa (U1) Ribonucleoprotein Antigen Using the Expression System pH6EX3 Followed by Metal Chelating Affinity Chromatography; Protein Expr. Purif
Bauman Susannah J.
Church Frank C.
DeCloux Amy
Myers Bigel Sibley & Sajovec P.A.
Saunders David
The University of North Carolina at Chapel Hill
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