Fibrinolytic enzymes

Details Fibrinolytic enzymes Fibrinolytic enzymes

1996-05-24

2004-01-27

Nashed, Nashaat T. (Department: 1652)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

C435S219000, C435S226000

Reexamination Certificate

active

06682733

ABSTRACT:

FIELD OF INVENTION
The present invention relates to fibrinolytically active plasminogen activators of the tissue type, DNA-sequences coding for same, pharmaceutical compositions containing same and processes for their production.
In particular, this invention relates to tissue-type plasminogen activators (t-PA) which have been modified in such a way that a; the uptake of the enzyme by the liver is reduced and b; the enzyme is essentially resistant to inactivation by plasma inhibitors. As a result the modified t-PA:s covered by this invention are characterized by a longer biological half-life than the t-PA preparations (native or recombinant) previously used.
Another aspect of this invention relates to the expression of t-PA, native or modified, in eucaryotic cells. More particularly, the invention relates to specific DNA sequences containing mRNA processing signals and which induce high production of recombinant proteins in heterologous cells.
BACKGROUND ART
Vascular disorders such as myocardial infarction, pulmonary embolism, stroke, deep vein thrombosis, periferal arterial thrombosis and other vascular thromboses are caused by partial or total occlusion of a blood vessel by blood clots. The clot which consists of a fibrin network can be dissolved by firinolytic enzymes. Plasmin is one such fibrinolytic enzyme which is present in the blood as an inactive proenzyme, plasminogen. Plasminogen activators convert plasminogen to plasmin, which in turn degrades the fibrin to soluble fragments. Thus, plasminogen activators can be use to induce thrombolysis.
The tissue plasminogen activator is regarded to be highly suitable for thromblytic treatments since it is a physiological compound with affinity for fibrin, and which activates plasminogen efficiently only in the presence of fibrin (Camiolo et al,
Proc. Soc. Exp. Biol. Med
., 138, pp. 277-280, 1971 and Ranby, M.,
Biochem. Biophys. Acta
, 704, pp. 461-469, 1982). Thus, it is a clot selective fibrinolytic agent suitable for intravenous administration. Other plasminogen activators such as streptokinase, a bacterial protein, or urokinase, isolated from urine, activates plasminogen but are not clot selective. As a result circulating plasmin is generated which may cause a haemorrhagic potential because the circulating plasmin degrades clotting factors such as fibrinogen, factor VIII and factor V.
Clinical studies have demonstrated the thrombolytic effectiveness of t-PA for treatment of acute myocardial infarction. (The TIMI Study Group,
N. Enql. J. Med
., 312, p. 932, 1985 and Verstraete et al,
Lancet
, 1, pp. 842-847, 1985). However, due to the rapid clearance of t-PA by the liver (Korninger et al,
Thromb. Haemostas
., 46, pp. 658-661, 1981) high doses 50-90 mg had to be given as a continuous infusion in order to induce efficient thrombolysis. The biological half-life of t-PA in man is only a few minutes Tiefenbrunn et al,
Circulation
, 71, pp. 110-116, 1985), and only a small fraction of the activator will actually reach the clot. Another factor which further reduces the amount of t-PA available for clot lysis is the reaction with plasma inhibitors. It has been shown that t-PA forms complexes with a number of plasma protease inhibitors including the recently discovered plasminogen activator inhibitors of endothelial and placental type. (Rijken et al,
J. Lab. Clin. Med
., 101, pp. 285-294, 1983; Wiman et al,
J. Biol. Chem
., 259, pp. 3644-3647, 1984; and Lecander et al,
British Journal of Haemathology
, 57, pp. 407-412, 1984).
The modifications of t-PA according to the present invention solves both the problem of the short biological half-life due to the liver clearance and the sensitivity to inactivation by plasma inhibitors.
The DNA sequences containing the information for t-PA and derivatives thereof can be introduced into appropriate vectors for expression in eucaryotic cells. The fibrinolytic activity produced by the transiently transfected or stably transformed host cells may be measured by using standard assays for plasminogen activators. The eucaryotic expression vectors described herein may be constructed by techniques well known by those skilled in the art, using components such as replicons, enhancers, promoters etc from natural sources or chemically synthesized by conventional procedures.
Established cell lines, as well as normal diploid cells, are suitable as hosts. A large number of different cell lines are usable for expression of t-PA or derivatives thereof. For example, different hamster cell lines such as CHOd
−
, CHOK1 and BHK, monkey cell lines such as CV-1 and COS, mouse cell lines such as C127 and 3T3, as well as human cell lines may be used. Other hosts such as insect cells as well as transgenic organisms may also be used for the production of t-PA or t-PA derivatives.
After introduction into a suitable host cell, the t-PA coding DNA sequences may be contained and propagated either as stably integrated into the host cell genome or in extrachromosomal form.
The sequences comprising the t-PA gene is preferentially present in the cells in multiple copies. Different strategies for amplification of t-PA gene copy number may be exploited. For stable integration of the vector DNA into the host cells chromosomal DNA, and for the subsequent amplification of the integrated vector DNA, an amplifiable selectable gene is included in the t-PA expression vector. Chinese hamster ovary cells (CHO) are presently preferred together with the dihydrofolate reductase (DHFR) gene as an amplifiable selectable marker gene. (Kaufman et al,
Mol. Cell. Biol
., 7, pp.
1750-1759, 1985).
Another amplification system is based on the use of papilloma virus DNA, especially bovine papilloma virus 1 (BPV). All or part of the virus genome is used to obtain stable transformation of mouse cells such as C127 or 3T3. The viral genome contains information for the maintenance of the vector DNA as a stable extrachromosomal element at a high copy number Sambrook et al,
Embo J
., 1, pp. 91-103, 1985).
In the eucaryotic host-vector systems discussed above, the expression of t-PA molecules or variants of the t-PA molecule is influenced by different upstream and downstream regulating DNA elements.
We have isolated and characterized a DNA fragment (KGH 11) from a human genomic library , which contains downstream processing signals such as polyadenylation signal etc from the human t-PA gene. The appropriate DNA fragment contains a part of the sequence in the last exon of the human t-PA gene. Since this segment also is represented in the cDNA it provides the possibility to use a unique restriction enzyme site in the overlapping region as a fusion site for ligation of the two elements (
FIG. 3
) For the eucaryotic expression systems analyzed, the production levels from this homologous construction were significantly higher than from expression vector constructions which are identical except for the processing signals downstream of the t-PA gene.
Recombinant DNA and other biotechnological techniques have been employed in order to obtain efficient production of t-PA for treatment of vascular diseases. Promising clinical studies were first performed with t-PA isolated from human melanoma cells. (Coken D,
Circulation
, 72, pp. 18-20, 1985).
Amino acid sequence analysis of the human melanoma t-PA (Wallén et al,
Eur. J. Biochem
., 132, pp. 681-686, 1983) provided the necessary information for the synthesis of DNA probes which were used for the isolation of, first a partial cDNA (Edlund et al,
Proc. Natl. Acad. Sci. USA
, 80, pp. 349-352, 1983), and subsequently a complete cDNA coding for the entire protein (FIG.
1
).
Other examples where cDNA:s coding for t-PA have been isolated and where attempts have been made to produce t-PA in heterologous cells are referred to in European patent applications 93619 and 178105. See also reference (Pennica et al,
Nature
, 301, pp. 214-221, 1983).
Amino acid sequence determinations of various preparations of human t-PA have revealed differences in the N-terminal starting position. Due to differences in processin

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