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
2000-03-10
2001-05-15
Nashed, Nashaat T. (Department: 1052)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252330, C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06232098
ABSTRACT:
BACKGROUND OF THE INVENTION
In animals, proteinases are important in wound healing, extracellular matrix destruction, tissue reorganization, and in cascades leading to blood coagulation, fibrinolysis, and complement activation. Proteinases are released by inflammatory cells for destruction of pathogens or foreign materials, and by normal and cancerous cells as they move through their surroundings.
The activity of proteinases is regulated by inhibitors; 10% of the proteins in blood serum are proteinase inhibitors (Roberts et al.,
Critical Reviews in Eukaryotic Gene Expression
5:385-436, 1995). One family of proteinase inhibitors, the Kunitz inhibitors, includes inhibitors of trypsin, chymotrypsin, elastase, kallikrein, plasmin, coagulation factors XIa and IXa, and cathepsin G. These inhibitors thus regulate a variety of physiological processes, including blood coagulation, fibrinolysis, and inflammation.
Proteinase inhibitors regulate the proteolytic activity of target proteinases by occupying the active site and thereby preventing occupation by normal substrates. Although proteinase inhibitors fall into several unrelated structural classes, they all possess an exposed loop (variously termed an “inhibitor loop”, a “reactive core”, a “reactive site”, or a “binding loop”) which is stabilized by intermolecular interactions between residues flanking the binding loop and the protein core (Bode and Huber,
Eur. J. Biochem.
204:433-451, 1992). Interaction between inhibitor and enzyme produces a stable complex which disassociates very slowly, releasing either virgin (uncleaved) inhibitor, or a modified inhibitor that is cleaved at the scissile bond of the binding loop.
One class of proteinase inhibitors, the Kunitz inhibitors, are generally basic, low molecular weight proteins comprising one or more inhibitory domains (“Kunitz domains”). The Kunitz domain is a folding domain of approximately 50-60 residues which forms a central anti-parallel beta sheet and a short C-terminal helix. This characteristic domain comprises six cysteine residues that form three disulfide bonds, resulting in a double-loop structure. Between the N-terminal region and the first beta strand resides the active inhibitory binding loop. This binding loop is disulfide bonded through the P2 Cys residue to the hairpin loop formed between the last two beta strands. Isolated Kunitz domains from a variety of proteinase inhibitors have been shown to have inhibitory activity (e.g., Petersen et al.,
Eur. J. Biochem.
125:310-316, 1996; Wagner et al.,
Biochem. Biophys. Res. Comm.
186:1138-1145, 1992; Dennis et al.,
J. Biol. Chem.
270:25411-25417, 1995).
Proteinase inhibitors comprising one or more Kunitz domains include tissue factor pathway inhibitor (TFPI), tissue factor pathway inhibitor 2 (TFPI-2), amyloid &bgr;-protein precursor (A&bgr;PP), aprotinin, and placental bikunin. TFPI, an extrinsic pathway inhibitor and a natural anticoagulant, contains three tandemly linked Kunitz inhibitor domains. The amino-terminal Kunitz domain inhibits factor VIIa, plasmin, and cathepsin G; the second domain inhibits factor Xa, trypsin, and chymotrypsin; and the third domain has no known activity (Petersen et al., ibid.). TFPI-2 has been shown to be an inhibitor of the amidolytic and proteolytic activities of human factor VIIa-tissue factor complex, factor XIa, plasma kallikrein, and plasmin (Sprecher et al.,
Proc. Natl. Acad. Sci. USA
91:3353-3357, 1994; Petersen et al.,
Biochem.
35:266-272, 1996). The ability of TFPI-2 to inhibit the factor VIIa-tissue factor complex and its relatively high levels of transcription in umbilical vein endothelial cells, placenta and liver suggests a specialized role for this protein in hemostasis (Sprecher et al., ibid.). Aprotinin (bovine pancreatic trypsin inhibitor) is a broad spectrum Kunitz-type serine proteinase inhibitor that has been shown to prevent activation of the clotting cascade. Aprotinin is a moderate inhibitor of plasma kallikrein and plamin, and blockage of fibrinolysis and extracorporeal coagulation have been detected in patients given aprotinin during open heart surgery (Davis and Whittington,
Drugs
49:954-983, 1995; Dietrich et al.,
Thorac. Cardiovasc. Surg.
37:92-98, 1989). Aprotinin has also been used in the treatment of septic shock, adult respiratory distress syndrome, acute pancreatitis, hemorrhagic shock, and other conditions (Westaby,
Ann. Thorac. Surg.
55:1033-1041, 1993; Wachtfogel et al.,
J. Thorac. Cardiovasc. Surg.
106:1-10, 1993). The clinical utility of aprotinin is believed to arise from its inhibitory activity towards plasma kallikrein or plasmin (Dennis et al., ibid.) . Placental bikunin is a serine proteinase inhibitor containing two Kunitz domains (Delaria et al.,
J. Biol. Chem.
272:12209-12214, 1997). Individual Kunitz domains of bikunin have been expressed and shown to be potent inhibitors of trypsin, chymotrypsin, plasmin, factor XIa, and tissue and plasma kallikrein (Delaria et al., ibid.).
Known Kunitz-type inhibitors lack specificity and may have low potency. Lack of specificity can result in undesirable side effects, such as nephrotoxicity that occurs after repeated injections of high doses of aprotinin. These limitations may be overcome by preparing isolated Kunitz domains, which may have fewer side effects than traditional anticoagulants. Hence, there is a need in the art for additional Kunitz-type proteinase inhibitors.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide novel Kunitz inhibitor proteins and compositions comprising the proteins. It is another object of the invention to provide materials and methods for making the Kunitz inhibitor proteins. It is a further object of the invention to provide antibodies that specifically bind to the Kunitz inhibitor proteins.
Within one aspect, the invention provides an isolated protein comprising a sequence of amino acid residues as shown in SEQ ID NO:5, wherein the sequence is at least 90% identical to residues 9 through 59 of SEQ ID NO:2 and wherein the protein has proteinase inhibiting activity. within one embodiment, the protein is from 51 to 81 amino acid residues in length. Within other embodiments, the protein is from 51 to 67 residues in length, preferably from 55 to 62 residues in length. Within another embodiment, the sequence is selected from the group consisting of residues 9 through 59 of SEQ ID NO:2 and residues 9 through 59 of SEQ ID NO:4. Within a further embodiment, the protein consists of from 51-557 contiguous amino acid residues of SEQ ID NO:10. Within an additional embodiment, the protein further comprises an affinity tag. Suitable affinity tags include maltose binding protein, polyhistidine, and Glu-Tyr-Met-Pro-Met-Glu (SEQ ID NO:18).
Within a second aspect, the invention provides an expression vector comprising the following operably linked elements: (a) a transcription promoter; (b) a DNA segment encoding a protein as disclosed above; and (c) a transcription terminator. Within one embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment.
Within a third aspect, the invention provides a cultured cell containing an expression vector as disclosed above, wherein the cell expresses the DNA segment.
Within a fourth aspect of the invention there is provided a method of making a protein having proteinase inhibiting activity comprising culturing a cell as disclosed above under conditions whereby the DNA segment is expressed, and recovering the protein encoded by the DNA segment.
Within a fifth aspect of the invention there is provided an antibody that specifically binds to a protein as shown in SEQ ID NO:2 or SEQ ID NO:4.
These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawing.
REFERENCES:
patent: 5780265 (1998-07-01), Dennis et al.
patent: 5795865 (1998-08-01), Markland et al.
patent: 5795954 (1998-08-01), Lazarus et al.
patent: 5834244 (1998-11-01), Dennis et al.
patent: 5880256 (1999-03-01), Dennis et al.
Conklin Darrell C.
Foster Donald C.
Gao Zeren
Fronda Christian L.
Nashed Nashaat T.
Parker Gary E.
ZymoGenetics Inc.
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