Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1990-12-11
1993-08-03
Hill, Jr., Robert J.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435212, 435219, 424 9463, C12N 948, C12N 1500
Patent
active
052328473
DESCRIPTION:
BRIEF SUMMARY
This invention relates to proteinaceous molecules having improved t-PA-like activity and to nucleic acid (DNA and RNA) coding for all or part of them.
Tissue type plasminogen activator (t-PA) is a key component of the fibrinolytic system that is the natural counterpart to the clotting cascade in the blood. The maintenance of haemostasis is critically dependent on maintaining a correct balance between these two opposing tendencies In certain disorders such as acute myccardial infarction (MI), where the haemostatic balance has broken down locally resulting in the formation of a thrombus, the administration of fibrinolytic agents has been shown to be a beneficial therapy for the promotion of clot dissolution.
Such thrombolytic therapy has become relatively widespread with the availability of a number of such fibrinolytic agents such as streptokinase, urokinase, APSAC and t-PA itself. Human t-PA has theoretical advantages over other fibrinolytic agents in that it has fibrin affinity and preferentially activates plasminogen bound to the surface of a clot. This results in less systemic activation of plasminogen with correspondingly less depletion of fibrinogen and important clotting factors. In addition, t-PA is a natural human plasma protein and so is less likely to result in undesirable immune responses that may preclude against subsequent treatments with the same agent. The isolation and sequence analysis of both cDNA and genomic clones for t-PA has been described (Pennica et al 1983, Nature 301, 214; Ny et al. 1984, P.N.A.S. 81, 5355-5359) and so the amino acid sequence is known.
One of the major problems with t-PA for the treatment of MI or other thrombotic disorders comes from the extremely short plasma half-life of the molecule which in man is in the order of 5 min (Bounameaux et al in: Contemporary Issues in Haemostasis and Thrombosis Vol I p 85-91, 1985 (Collen et al eds, Churchill Livingstone). This results in the need to administer t-PA by infusion in large doses. The treatment is therefore expensive and requires that the patient be hospitalised before treatment can commence.
An improved thrombolytic agent with the beneficial properties of t-PA but with an extended plasma half-life is therefore sought.
Analysis of the t-PA molecule and its gene have revealed that it is a mosaic protein composed of 5 domains. The large serine protease domain can be recognised by its homology with other serine proteases and is the catalytically active domain involved in plasminogen activation. The 4 N terminal domains consist of a finger domain, so called because of its similarity to the type I homologies of fibronectin that are known to mediate the fibrin binding properties of that molecule, a growth factor domain, identified by its homology to a class of domain commonly observed in plasma proteins of which epidermal growth factor is the prototype sequence and two kringle domains which are homologous to domains in plasminogen, prothrombin and some other plasma proteins. A number of t-PA derivatives bearing deletions of one or more of the N-terminal domains have been described (Matsuo et al. 1985, FEBS Letters, 189, 145-149; van Zonneveld et al, 1986, P.N.A.S. 83, 4670-4674; Larsen et al, 1988, J. Biol. Chem. 263, 1023-1029; Browne et al, 1988, J. Biol. Chem. 263, 1599-1602; Kalyan et al 1988, J. Biol. Chem. 263, 3971-3978) and it is generally accepted through these and other studies that the finger domain and the second kringle domain are important mediators of fibrin binding and the fibrin stimulation of plasminogen activation by t-PA. The first kringle domain is of unknown function. It has been suggested that the growth factor domain may be involved in binding of t-PA to a receptor in the liver that mediates the clearance of t-PA from the circulation.
Various suggestions have been made therefore for deletion derivatives of t-PA that lack various N-terminal domains including the GF domain. In particular, evidence has been presented that such molecules may have an extended half life in vivo. Larsen et al describe varia
REFERENCES:
patent: 4963357 (1990-10-01), Bell et al.
patent: 5108901 (1992-04-01), Anderson et al.
Craig Stewart
Dawson Keith
Edwards Richard M.
Fallon Anthony
Allen Marianne Porta
British Bio-technology Limited
Hill Jr. Robert J.
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