Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1996-02-20
2004-04-13
Grun, James (Department: 1641)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S139100, C424S151100, C424S185100, C424S194100, C424S265100, C435S070210, C435S452000, C435S331000, C435S345000, C436S543000, C436S547000, C436S548000, C530S389800, C530S855000
Reexamination Certificate
active
06719975
ABSTRACT:
The invention relates to a method of producing an antibody specific for a hirudin or a hirudin-like protein.
Hirudin is a polypeptide which may be isolated in small quantities from the salivary glands of the leech
Hirudo medicinalis
(Markwardt F: Untersuchungern uber Hirudin. Naturwissenschaften 41: 537-538, 1955).
Hirudin is a potent and specific inhibitor of thrombin (Chang J Y.: The functional domain of hirudin, a thrombin-specific inhibitor. FEBS Lett. 164: 307-313, 1983). Hirudin binds to thrombin, inhibiting the transformation of soluble fibrinogen into insoluble fibrin and preventing the thrombin mediated activation of factors V, VIII and XIII (Markwardt F.: Pharmacology of hirudin: One hundred years after the first report of the anticoagulant agent in the medicinal leeches. Biomed. Biochem. Acta 44: 1007-1013, 1985).
Thrombin has a greater affinity for hirudin than for the platelet thrombin receptors (Fenton II J W, Landis B. H., Walz D. A., Bing D. H., Feinman R. D., Zabinski M. P., Sonder S. A., Berliner L. J., and Finlayson J. S.: Human thrombin: Preparative evaluation, structural properties and enzymic specificity. In: DH Bind. Ed.: The Chemistry and Physiology of Human Plasma, Pergamon Press, New York, pp. 151-182, 1979) and hence hirudin is able to induce the dissociation of the thrombin-platelet receptor complex (Tam S. W., Fenton J. W. and Detwiler T. C.; Dissociation of thrombin from platelets by hirudin: Evidence for receptor processing. J. Biol. Chem., 254: 8723-8725, 1979), thereby inhibiting the release reaction and platelet aggregation (Hoffmann A., and Markwardt F.: Inhibition of the thrombin-platelet reaction by hirudin. Hemostasis, 14: 164-169, 1984, Reinhold D. S. and Gershman H. Hirudin insensitives thrombin-stimulated platelet release. Thromb. Res., 37: 513-527, 1985).
With a similar mechanism hirudin interferes with the binding of thrombin to the receptors present on endothelial cells and on fibroblasts (Fenton II J. W., Landis B. H., Walz D. A., Bing D. H., Feinman R. D., Zabinski M. P., Sonder S. A., Berliner L. J. and Finlayson J. S.: Human thrombin: Preparative evaluation, structural properties and enzymic specificity. In: DH Bind. Ed.: The Chemistry and Physiology of Human Plasma, Pergamon Press, New-York, pp. 151-182, 1979).
Hirudin also inhibits interaction between thrombin and thrombomodulin, which binds to a common site on thrombin, distinct from the catalytic site (Holfsteenge J., Taguechi H. and Stone S. R.: Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors. Biochem. J. 237:243-251, 1986).
Hirudin therefore reduces the capacity of thrombin to activate protein C, a serine-protease able to inactivate factors V and VIII, necessary for the generation of thrombin itself.
Hirudin is also able to inhibit clot-bound thrombin which is protected from inhibition by the heparin-antithrombin III complex (Weitz II., Hudoba M., Massel D., Maraganore J. and Hirsh J.: Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III independent inhibitors. J. Clin. Invest., 86: 385-391, 1990).
In experimental studies in animals, hirudin proved to be effective in preventing venous and arterial thrombosis (Markwardt F.: Development of hirudin as an antithrombotic Agent. Seminars in Thrombosis and Haemostasis, 15 (3): 269-282, 1989; Heras M., Chesebro J. H., Webster M., Mruk J. S., Grill D. E., Penny W. J., Bowied E. J. W., Badimon L. and Fuster V.: Hirudin, Heparin and Placebo during arterial injury in the Pig. The in vivo role of Thrombin in Platelet. Mediated Thrombosis. Circulation 82: 1476-1484, 1990), vascular shunt occlusions (Kelly A. R., Marzek U. M., Krupski L., Bass A., Cadroy Y., Hauson S. R., and Harker L. A.: Hirudin Interruption of Heparin-Resistant Arterial Thrombus Formation in Baboons. Blood, 77 (5): 1006-1012, 1991) and thrombin-induced disseminated intravascular coagulation (Markwardt F., Howak H. and Hoffman J.: The influence of drugs on disseminated intravascular coagulation DIC. Effects of naturally occurring and synthetic thrombin inhibitors. Thromb. Res. 11: 275-283, 1977).
Although the clinical application of hirudin as an antithrombotic drug was proposed several years ago, this has been severely limited because natural hirudin is not easily available. Nowadays, with genetic engineering methodology and methods of polypeptide purification available, it is possible to produce sufficient amounts of hirudin for preclinical and clinical studies. This fact has renewed interest in natural thrombin inhibitors.
The purification and characterization of different hirudins from the leech
Hirudo medicinalis
was studied in detail and the primary structures of these compounds were determined (Tripier D.: Hirudin, A family of iso-proteins, isolation and sequence determination of new hirudins. Folia Hematol. (Leipz) 115:30-35, 1988). In particular, a form of hirudin designated HV1 was extracted from the whole body of the leech (Boskova I. P., Cherkesova D. V. and Mosolow V. V.: Hirudin from leech heads and whole leechs and pseudo-hirudin from leech bodies. Thromb. Res. 30:459-467, 1983; Dodt J. P., Muller H. P., Seemuller V. and Chang J. Y.: The complete amino acid sequence of hirudin, a thrombin-specific inhibitor. FEBS Lett. 165:180-183, 1984) whereas from the head another form was extracted with a slightly different amino acid sequence, designated HV2 (Harvey R. P., Degryse E., Stefani L., Schamber F., Cazenave J. P., Courtney M., Ialstoshev P. and Lecocq J. P.: Cloning and expression of cDNA coding for the anticoagulant hirudin from the blood sucking leech,
Hirudo Medicinalis
, Proc. Natl. Acad. Sci USA 83:1084-1088, 1986). A third variant designated HV3 has been described (Harvey et al, Proc Natl. Acad. Sci. USA (1986)
1084-1088).
The amino acid sequences of HV1, HV2 and HV3 are SEQ ID Nos. 1, 2 and 3. HV1 and HV2 consist of a single polypeptide chain of 65 amino acids in which the NH
2
-terminal apolar core and the strongly acid c-terminal tail bind to the apolar binding site and to the anion binding exosite of thrombin, thus preventing it from interaction with the substrate (fibrinogen) (Rydel T. J., Ravichandran K. G., Tulinsky A., Bode W., Huber R., Roitsch C. and Fenton II J. W.: The Structure of a Complex of Recombinant Hirudin and Human &agr;-Thrombin. Science, 249:277-280, 1990). In addition, in position 63 a sulfated tyrosine is present; this post-translational modification does not appear to be essential for antithrombin activity (Stone S. R. and Hofsteenge J.: Kinetics of the inhibition of thrombin by hirudin. Biochem., 25: 4622-4628, 1986).
HV3 is identical to HV2 from positions 1 to 32 and then differs from HV1 in the following respects: Gln at position 33 instead of Asp, Lys at position 35 instead of Glu, Asp at position 36 instead of Lys, Gln at position 53 instead of Asp, Pro at position 58 instead of Glu, Asp at position 62 instead of Glu, Ala at position 63 instead of Tyr (SO
3
H), Asp at position 64 instead of Leu and Glu at position 65 instead of Gln.
The N-terminal domain (residues 1 to 39) of hirudin is characterized by three disulfide bonds which stabilize the entire conformation (Chang J. Y.: Production, Properties and Thrombin Inhibitory Mechanism of Hirudin Amino-terminal Core Fragments. J. Biol. Chem., 265 (36): 22159-22166, 1990).
Recently, hirudins have also been detected in other species of leeches. In particular, two polypeptides with antithrombin properties similar to those of hirudin HV1 and having the amino acid sequences depicted in SEQ ID NOs 4 and 5 (referred to hereinafter as P1 and P2) were isolated from the leech
Hirudinaria manillensis
, characterized and produced by genetic engineering (European Application No. 92301721.4). P1 shows 60% homology with hirudin HV1 and does not possess sulfated amino acids.
The isolation and purification of hirudin from natural sources or from recombinant biological material is generally carried out by ion exchange chromatography followed by a se
Gerna Marco
Giorgetti Carla
Lansen Jacqueline
Molinari Antonio
Roncucci Romeo
Arent Fox Kintner Plotkin & Kahn
Farmitalia Carlo Erba S.r.l.
Grun James
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