WO1998005762A1

WO1998005762A1 - Plasminogen activator capable of being activated by thrombin

Info

Publication number: WO1998005762A1
Application number: PCT/EP1997/003925
Authority: WO
Inventors: Ulrich Kohnert; Stephan Fischer
Original assignee: Roche Diagnostics Gmbh
Priority date: 1996-08-02
Filing date: 1997-07-21
Publication date: 1998-02-12
Also published as: AU3769797A; BR9711015A; KR20000029755A; ZA976876B; CN1226926A; JP2000504941A; EP0917567A1; TR199900193T2; CA2262751A1; TW450996B

Abstract

A plasminogen activator (t-PA) is (a) modified in such a way that it can be split by thrombin and thus turned into its two-chain form; (b) modified in such a way that its zymogenity is at least 1.2 higher than that of t-PA; and (c) its fibrin binding power is so reduced that more than 50 % of the plasminogen activator can enter a blood clot. This plasminogen activator has an improved fibrin-specificity and less side effects.

Description

Thrombin activated plasminogen activator

The invention relates to a thrombin-activatable plasminogen activator, medicaments for the treatment of thromboembolic disorders, pharmaceutical compositions which contain such a plasminogen activator, and their use

Tissue plasminogen activator (t-PA) is a multi-domain serine protease that catalyzes the conversion of plasminogen to plasmin and is used for fibrinolytic therapy

A large number of t-PA variants and mutations are known; see, for example, the overview articles T R Harris (1987) and J Krause (1988)

Among other things, it is known about the mode of action of t-PA that fibinolysis is partially regulated by the interaction between t-PA and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor from the Se family). 1 on t-PA occurs essentially via amino acids 296-302. A mutation of this region reduces the inhibitory influence of PAI-1 on t-PA (EL Madison et al (1990)) on the mechanism of the interaction between the amino acid region 296-302 of Comprehensive investigations were carried out in t-PA with PAI-1 (see also EL Madison, Nature 339 (1989) 721-723; RV Schohet, Thrombosis and Hae ostasis 71 (1994) 124-128, CJ Refino, Thrombosis and Haemostasis 70 (1993) 313-319, NF Paoni, Protein Engineering 6 (1993) 529-534 and Thrombosis and Haemostasis 70 (1993) 307-312, WF Bennett, J Biol Chem 266 (1991) 5191-3201, D Eastman, Biochemistry 31 (1992) 419-422).

Unmodified human t-PA (hereinafter referred to as t-PA) consists of 527 amino acids in its form in the plasma and can be split into two chains by plasmin, which are then held together by a disulfide bridge. The A chain (also called heavy chain) consists of four structural domains. The finger domains (amino acids 1-49) show certain similarities with the finger structures in fibronectin. The growth factor domain (amino acids 50-86) is to a certain extent homologous to murine and human epidermal growth factors The Kringledomanes (amino acids 87 - 261) are largely homologous to the fourth and fifth Kringledomain of plasminogen. The finger and Kringle-2 domains of t-PA are in the fibrin formation and particularly involved in the stimulation of proteolytic activity by fibrin. The B chain of t-PA (amino acids 276 - 527, protease domain) is a serine protease and largely homologous to the B chains of urokinase and plasmin (TJ R Harris (1987) and J Krause (1988)

The enzymatic activity of t-PA (catalytic activation of plasminogen to plasmin) is low in the absence of fibrin or fibrin cleavage products, but can be increased significantly in the presence of these stimulators (by more than a factor of 10). The mechanism of action of t-PA is in vivo For example, in Korninger and Collen, Thromb Haemostasis 46 (1981) 561-565, t-PA activates plasminogen to plasmin. Plasmin cleaves fibers to soluble fibrin cleavage products. t-PA is formed between amino acid 275 (arginine) and 276 (isoleucm ) split and thereby activated. The two partial chains remain connected by a cysteine bridge

The stimulability of the activity by fibrin, fibrin cleavage products is an essential feature of t-PA, which distinguishes t-PA from the other known plasminogen activators, such as urokinase or streptokinase. The stimulability can be further improved by modifying the amino acid sequence of t-PA A measure of the stimulability is the ratio of the catalytic efficiency (K _cat / K _m ) in the presence and in the absence of fibrin K _ca t is the rate constant of the catalytic reaction and K _m is the Michaelis constant The stimulability of t-PA can be modified by modifying the Amino acids 292 and / or 305 increase 19 to 81 times (EL Madison et al, Science 262 (1993) 419-421

US Pat. No. 5,501,853 discloses t-PA derivatives which are modified in the region of the amino acids 272-280, in particular in the region 274-277 and additionally in the region of the glycosylation sites (117-119 and 184-186). Such t PA derivatives have an improved proteolytic and plasminogenolytic activity, a reduced sensitivity to inhibition, an improved affinity for fibrin and / or an improved fibrin dependency of the plasminogenolytic activity

Wen-Pin Yang et al describes in Biochemistry 33 (1994) 2306-2312 thrombin-activatable plasminogen activators. This chimeric plasminogen activator (59 D8-scu PA-t) was produced from the Fab fragment of an anti-fibin antibody (59 D8) and of the C-terminal part of a thrombin-activatable low-molecular single-chain urokinase plasminogen activator (scu PA-t), which was obtained by deletion of the amino acid Phe-157 and Lys-158 from the low-molecular single-chain urokinase plasminogen activator (scu PA) by site-directed mutagenesis

By K.N. Dawson et al are described in J. Biol. Chem 269 (1984) 15989-15992 plasminogen mutants which can be activated by thrombin. These plasminogen derivatives were obtained by substituting the P3, P2 and PT amino acids of the cleavage site with a thrombin-cleavable sequence

N.F. Paoni. et al. in Protein Engineering 5 (1992) 259-266 describe the modification of t-PA in the amino acid region 296-229. This results in an improved fibrin specificity.

US Pat. No. 5,200,340 describes thrombin-activatable plasminogen activators which are modified in such a way that they contain a thrombin cleavage site for activation. It is further stated that although the growth factor domain (EGF domain) can be deleted in such t-PA derivatives, the fibrin binding domain (finger domain) and the Kringles structures must be retained

From WO 91/09118 and WO 94/10318 it is known that plasminogen can be activated by introducing a thrombin cleavage site from thrombin to plasmin. Since thrombin is contained in the blood clot, this activation should mainly take place on the blood clot. However, the disadvantage of such a method is that modified plasminogen would have to be administered to the patient in large quantities

The object of the invention is to provide improved plasminogen activators which, with high specificity and effectiveness, are able to dissolve blood clots in vivo.

The object is achieved by a plasminogen activator, which starts from human tissue plasminogen activator

a) is modified so that the plasminogen activator can be cleaved by thrombin and is converted into the two-chain form by such cleavage,

b) is modified so that the zymogenicity compared to human t-PA is at least 1.2 times, preferably 2 times greater, and c) whose fibrin binding is reduced to such an extent that the plasminogen activator can penetrate more than 50% into a blood clot.

Such a plasminogen activator acts specifically on the blood clot and therefore shows significantly fewer side effects than the known plasminogen activators.

The starting point is the sequence of the human tissue plasminogen activator. According to the invention, "starting from human tissue plasminogen activator" means that the sequence of the plasminogen activator according to the invention is derived from the sequence of the human plasminogen activator. On the one hand, this means that the structural features (domains) characteristic of tPA are at least partially structurally preserved. It has been shown, for example, that plasminogen activators in which the structure of the curling 2 and / or protease domain are still preserved and which additionally carry the modifications according to the invention can be used for the purposes of the invention. It is also possible for further domains to be obtained and for the features of the invention to arise from deletions, mutations and / or additions of amino acids. The changes in the amino acid sequence can be carried out by methods known to the person skilled in the art, such as site directed mutagenesis or PCR.

In a preferred embodiment, the plasminogen activator according to the invention is modified compared to human tissue plasminogen activator so that the cleavage of the plasminogen activator according to the invention by plasmin between the amino acids Pl (275) and PT (276) is reduced. In the plasminogen activators according to the invention, the cleavage by plasmin is preferably reduced by 10% or more, particularly preferably by 20% or more and very particularly preferably by 50% or more. It is not necessary for the plasmin cleavage to be completely eliminated. In particular, thrombin activation can be improved by plasmin cleavage of the plasminogen activator according to the invention.

However, it is also preferred to reduce the cleavability to such an extent that no physiologically relevant cleavage takes place in vivo. This can drastically reduce side effects. It is achieved that the degradation of coagulation parameters is drastically reduced in vivo and not, as in the case of known plasminogen activators, the bleeding time is significantly prolonged. The cleavage by plasmin can be determined in an in vitro test. The plasminogen activator is incubated with increasing amounts of plasmin for five minutes at 25 ° C and then SDS electrophoresis is carried out in an acrylamide gel with 12.5 - 15% acrylamide, depending on the size of the plasminogen activator (U. Kohnert et al., Protein Engineering 5 (1992) 93-100).

The modification of the plasminogen activator in such a way that between Pl and PT (nomenclature according to Schechter, J. and Berger, A., Biochem Biophys. Res. Commun. 27 (1967) 157-162) no more cleavage or a reduced cleavage by plasmin can take place in a manner familiar to any person skilled in the art. Plasmin splits the sequence R 275-1 276 (amino acid names in the one-letter code). For example, by modifying one or both amino acids, the cleavage by plasmin can be eliminated or at least drastically reduced

Plasmin can also be reduced by modifying positions P4 - P3 * (amino acids 272 - 278). P2 is preferably converted into a small, preferably hydrophobic and / or non-aromatic amino acid, such as P. As a result, in addition to reducing the plasmin cleavage, a thrombin cleavage can surprisingly be achieved.

One or more of the following general conditions are preferably observed:

(P) P4: Any amino acid (but not P if P2 is P, preferably L, I, V) (Q) P3: Any amino acid

(F) P2: hydrophobic amino acid (F, H, G, V, L, I, T, A or P, particularly preferably P) (R) Pl: R or K, preferably R.

(I) Pl '. V or I, preferably I.

(K) P2 ': V, L, I or K, preferably V

(G) P3 ': G

P4 is particularly preferably converted into V, P2 into P, P2 'into V. This results in the particularly preferred cleavage point (272-278) VQPRIVG (SEQ ID NO: 1). A thrombin cleavage site can also be introduced according to the prior art, but a corresponding mutation is preferably introduced in the region of amino acids 264-288.

A modification of t-PA to introduce a - finity to thrombin can also be done by modifying loops 459-471, autolysis loop 417-425 and / or amino acids Q 475, K 505 and / or E 506

The specificity of an enzyme for its substrate essentially depends on the sequence of the cleavage site (primary sequence). For serine proteases, such as thrombm and plasmin, the PI residue is the essential specific determinant. The specificity of folded protein substrates also depends on the characteristic contact between the enzyme (Thrombin or plasmin) and substrate (plasminogen activator) as well as on the conformation and flexibility of the cleavage site Since the primary specificities of plasmin and thrombin are very similar (both cleave after arginine at the Pl position), it is not readily possible to use plasminogen activators to obtain, in which the cleavage by plasmin (plasma inactivability) is reduced and the cleavage by thrombin (thrombin inactivability) is present or is substantially greater than the plasma inactivability (at least factor 2-10). Surprisingly, however, it was found that by modification at the secondary binding sites between E nzyme and substrate and modifications of the structural and dynamic properties of the activation loop such properties can be obtained

The introduction of a thrombin-specific cleavage site is preferably carried out with a simultaneous reduction in the plasmin cleavage by changing the primary specificity by mutations in the activation loop between amino acids 264 and 288. The mutations mentioned above in the range 272-277 (P4-P2 ') are particularly preferred.

Thrombin cleavage can be improved by modifying the flexibility and / or accessibility of the binding loop. G 265 (mutation leads to less flexibility) or R 267 (mutation leads to a change or cleavage of the salt bridge between G 265 and E 410) is particularly preferred for this purpose Insertions in the range between 264 and 267 are also preferred. R 267 is particularly preferably modified in S (D. Lamba et al, J Mol. Biol 258 (1996) 117-135) A mutation with which flexibility can be increased is the change in R 267 in S, preferably in connection with mutations in P4 in F, P3 in G, P2 in P and P2 'in V.

Thrombin cleavage and specificity can be further improved by changing the secondary specific binding sites. For this purpose, the loop 459 - 471 can be deleted completely or partially. This loop consists of the amino acids

GDTRSGGPQANLH. (SEQ ID NO: 2)

In this loop, the region PQANLH (SEQ CD NO: 3) is preferably completely or at least partially deleted (preferably H being retained) or its amino acid sequence is changed.

The following sequences in the range from amino acids 272 to 277 are preferably used:

GIPRIV (SEQ ID NO: 4)

AQPRK (SEQ ID NO: 5)

It is also advantageous to have the sequence in the amino acid range between 265 and 277 (original t-PA sequence GLRQYSQPQFRIK (SEQ ID NO: 6))

GLSQASQGIPRIV SEQ ID NO: 7). The sequence GLRQYSQAQGIPRIV (SEQ ID NO: 8) is also preferred for this area.

In this sequence the amino acids G and I are inserted between Q and P (original sequence: Q and F) for extension. Such an insertion is preferably carried out at any point in the range between 264 and 276.

Also particularly preferred are compounds according to the invention in which the cleavage site (SEQ ID NO: 1) with one or more, preferably all of the mutations P4 in F, P3 in G, P2 in P and P2 'in V and the mutation R 267 in S is combined.

In a preferred embodiment of the invention, the plasminogen activator additionally contains a mutation which indicates the activity of the single-chain form but not the activity the two-chain form is drastically reduced and the zymogenicity is improved by a factor of 1.2, preferably by a factor of 2 or more.

Zymogenicity is the quotient of the activity of the two-chain form and the activity of the single-chain form. The activity is determined amidolytically. Such a plasminogen activator achieves high selectivity and effectiveness of thrombus dissolution in vivo with drastically reduced side effects. Suitable and preferred mutations of the single-chain form are described, for example, in E.L. Madison et al., Science 262 (1993) 419-421.

To increase the zymogenicity (ratio of the amidolytic activity of the two-chain form to the activity of the single-chain form of the plasminogen activator), it is particularly preferred (also for all plasminogen activators derived from human tissue plasminogen activator) to K 429 in Q and / or H 417 in T. modify and / or prevent or interfere with the interaction between K 429 and H 417.

Particularly preferred compounds according to the invention contain the cleavage sites (272-278) VQPRIVG (SEQ ED NO: 1) with the additional mutation K 429 in Q and / or H 417 in T.

The reduction in fibrin binding can be done by deleting or mutating the domain of t-PA which is specific for fibrin binding (fibrin binding domain, finger domain) in such a way that fibrin binding via the finger domain cannot or only to a small extent (none functional finger domain). By reducing the fibrin binding it is achieved that the plasminogen activator can penetrate the clot (preferably more than 50%) and is distributed evenly. There it is cleaved by thrombin and unfolds its activity in the active two-chain form. A plasminogen activator, the fibrin binding of which is reduced in this way, no longer shows any high-affinity fibrin binding typical of tP A. This specific mode of action increases the potency of the plasminogen activator and, in particular, drastically reduces side effects. The penetration of the plasminogen activator according to the invention into a clot can be determined in an in vitro model. The extent of clot penetration and distribution in the clot can be determined visually. The plasminogen activator described in US Pat. No. 5,223,256 is used as the standard for assessment, which penetrates into the clot, distributes itself homogeneously and thus by definition represents the 100% value (determined at a concentration of 3 μg / ml). As a further standard, recombinant human tissue plasminogen activator according to EP-B 0 093 619 is used, which by definition is not included in the clot penetrates and essentially binds to the surface. The investigation of the clot penetration is carried out as described in Example 3c. A comparison of these standards shows that "non-penetration into the clot" means that the vast majority (80% or more) of the plasminogen activator is in the first quarter of the clot, whereas with a "homogeneous distribution" at least 50% of the plasminogen activator penetrate further into the clot and are thus located in the remaining three quarters.

The specificity and effectiveness of the clot dissolution is further increased if a plasminogen activator according to the invention is used which additionally shows no or only very low and non-specific fibrin binding. Such molecules penetrate the inside of the clot and thus ensure an efficient activation of plasminogen to plasmin in the clot. Such plasminogen activators are based, for example, on the protease domain of t-PA (WO 96/17928) or on a substance which essentially contains the Kringle 2 domain and the protease domain, but not the finger domain, as t-PA domains (WO 90/09437, U.S. Patent 5,223,256, EP-B 0 297 066, EP-B 0 196 920).

In a preferred embodiment, the plasminogen activator according to the invention is additionally modified so that it cannot be inhibited by PA1-1. Such a modification is preferably carried out by mutating amino acids 296-302 (Madison, EL et. Al., Proc. Natl. Acad. Sci. USA 87 (1990) 3530-3533) and particularly preferably by replacing amino acids 296-299 (KHRR ) by AAAA (WO 96/01312)

The compounds according to the invention are thrombolytically active proteins which, in contrast to t-PA (Alteplase), are preferably administered as iv. Bolus injection are suitable. They are effective in a lower dose and show practically the same thrombolytic effect as a clinically customary infusion of Alteplase

The increase in specificity and the associated reduction in the side effects of bleeding make the compounds according to the invention an extraordinarily valuable thrombolytic agent for the treatment of all thromboembolic disorders. In contrast to the thrombolytics which have hitherto only been approved in the case of extremely life-threatening diseases such as heart attack and massive pulmonary embolism, the use of such variants opens up the possibility of using thrombolysis by the compounds according to the invention even in the case of less acute life-threatening diseases, such as deep leg vein thrombosis. In addition, the use of thrombolytics on the basis of the compounds according to the invention can now take place much more widely than hitherto, since this is a main obstacle to - lü ¬

widespread use was the risk of bleeding complications. Irrespective of this, the compounds according to the invention can advantageously also be used in acute diseases, such as heart attack or pulmonary embolism.

The plasminogen activators used according to the invention can be produced in eukaryotic or prokaryotic cells according to the methods familiar to the person skilled in the art. The compounds according to the invention are preferably produced by genetic engineering. Such a process is described, for example, in WO 90/09437, EP-A 0 297 066, EP-A 0 302 456, EP-A 0 245 100 and EP-A 0 400 545, which are the subject of the disclosure for such production processes . Mutations can be introduced into the cDNA of t-PA or a derivative thereof by "oligonucleotide-directed site-specific mutagenesis". The "site-specific mutagenesis" is, for example, by Zoller and Smith (1984), modified from T.A. Kunkel (1985) and Morinaga et al. (1984). The method of PCR mutagenesis, which is described, for example, in Ausubel et al (1991), is also suitable

The nucleic acid obtained in this way serves to express the plasminogen activator used according to the invention if it is present on an expression vector suitable for the host cell used.

The nucleic acid sequence of the protein according to the invention can additionally be modified. Such modifications are, for example

Altering the nucleic acid sequence to introduce different restriction enzyme recognition sequences to facilitate the steps of ligation, cloning and mutagenesis,

Changing the nucleic acid sequence for incorporation of preferred codons for the host cell,

Supplementation of the nucleic acid sequence with additional regulatory and transcription elements in order to optimize expression in the host cell.

All further process steps for the production of suitable expression vectors and for expression are known in the prior art and are known to the person skilled in the art. Such are described Methods, for example in Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory anual (1989) Cold Spring Harbor Laboratory Press, New York, USA.

The glycosylated plasminogen activators used according to the invention are produced in eukaryotic host cells. The non-glycosylated plasminogen activators used according to the invention are either produced in eukaryotic host cells, the glycosylated product initially obtained thereby having to be deglycosylated by methods familiar to the person skilled in the art, or preferably by expression in non-glycosylating host cells, particularly preferably in prokaryotic host cells.

E. coli, Streptomyces spec., For example, are prokaryotic host organisms. or Bacillus subtilis. To produce the protein according to the invention, the prokaryotic cells are fermented in a conventional manner and, after the bacteria have been digested, the protein is isolated in a conventional manner. If the protein is obtained in an inactive form (inclusion bodies), it is solubilized and naturalized according to the methods familiar to the person skilled in the art. It is also possible according to the methods familiar to the person skilled in the art to secrete the protein from the microorganisms as the active protein. An expression vector suitable for this preferably contains a signal sequence which is suitable for the secretion of proteins in the host cells used, and the nucleic acid sequence which codes for the protein. The protein expressed with this vector is secreted either into the medium (for gram-positive bacteria) or into the periplasmatic space (for gram-negative bacteria). Between the signal sequence and the sequence coding for the t-PA derivative according to the invention, there is expediently a sequence which codes for a cleavage site which allows the protein to be split off either during processing or by treatment with a protease.

The selection of the base vector into which the DNA sequence coding for the plasminogen activator according to the invention is introduced depends on the host cells used later for expression. Suitable plasmids and the minimum requirements placed on such a plasmid (e.g. origin of replication, restriction sites) are known to the person skilled in the art. In the context of the invention, a cosmid, the replicative double-stranded form of phage (λ, Ml 3) or other vectors known to the person skilled in the art can also be used instead of a plasmid.

In the production of the plasminogen activators according to the invention in prokaryotes without secretion, it is preferred to separate the inclusion bodies that form from the soluble cell particles separate, solubilize the inclusion bodies containing plasminogen activator by treatment with denaturing agents under reducing conditions, then derivatize with GSSG and renaturate the plasminogen activator by adding GSH and denaturing agents in non-denaturing concentration or L-arginine. Such methods for activating t-PA and derivatives from inclusion bodies are described, for example, in EP-A 0 219 874 and EP-A 0 241 022. However, other methods of obtaining the active protein from the inclusion bodies can also be used.

The plasminogen activators according to the invention are preferably purified in the presence of L-arginine, in particular at an arginine concentration of 10-1000 mmol / l.

Foreign proteins are preferably separated off by affinity chromatography and particularly preferably via an adsorber column on which ETI (Erythrina Trypsin Inhibitor) is immobilized. Sepharose®, for example, is used as the carrier material. Cleaning via an ETI adsorber column has the advantage that the ETI adsorber column material can be loaded directly from the concentrated renaturation batch even in the presence of such high arginine concentrations as 0.8 mol / 1. The plasminogen activators according to the invention are preferably purified via an ETI adsorber column in the presence of 0.6-0.8 mol / 1 arginine. The solution used here preferably has a pH of more than 7, particularly preferably between 7.5 and 8.6.

The plasminogen activators according to the invention are eluted from the ETI column by lowering the pH both in the presence and in the absence of arginine. The pH is preferably in the acidic range, particularly preferably between pH 4.0 and 5.5.

Another object of the invention is a pharmaceutical composition containing a thrombolytically active protein according to the invention, the protein preferably containing the protease domain and optionally the Kringel 2 domain of the human tissue plasminogen activator as the only structure causing the thrombolytic activity.

The plasminogen activators used according to the invention can be formulated for the production of therapeutic agents in a manner familiar to the person skilled in the art, the compounds according to the invention usually being combined with a pharmaceutically acceptable carrier. Such compositions typically contain an effective one Amount of 0.1-7 mgkg, preferably 0.7-5 mg / kg and particularly preferably 1-3 mg / kg body weight as a dose. The therapeutic compositions are usually in the form of sterile, aqueous solutions or sterile, soluble dry formulations such as lyophilisates. The compositions usually contain a suitable amount of a pharmaceutically acceptable salt with which an isotonic solution is prepared. Buffers such as arginine buffer, phosphate buffer for stabilizing a suitable pH Value (preferably 5.5-7.5) can be used by any person skilled in the art to determine the dosage of the compounds according to the invention. It depends, for example, on the type of application (infusion or bolus) and the duration of the therapy. Because of their prolonged half-life (with respect to degradation in vivo), the compounds according to the invention are particularly suitable for a bolus application (single bolus, multiple bolus) .A suitable form for a bolus application is, for example, an ampoule which contains 25-1000 mg of the compound according to the invention, a substance which is soluble of the plasminogen activator improved (such as arginine) and buffer contained. The application is preferably intravenous, but also subcutaneously, intramuscularly or intraarterially. Plasminogen activators according to the invention can also be infused or applied locally

The compounds according to the invention can be used as a multiple bolus (preferably as a double bolus). Suitable time intervals are between 20 and 180 minutes, an interval between 30 and 90 minutes is particularly preferred and an interval between 30 and 60 minutes is particularly preferred administration as an infusion over a period of 1 h - 2 days is possible

The compounds according to the invention are particularly suitable for the treatment of all thromboembolic diseases, such as, for example, acute heart attack, cerebral infarction, pulmonary embolism, deep leg vein thrombosis, acute arterial occlusion, etc. The compounds according to the invention are particularly preferred for the treatment of subchronic thromboembolic diseases in which prolonged thrombolysis is carried out must be applied

It is preferred to use the compounds according to the invention in combination with an inhibitor of coagulation (anticoagulant), such as, for example, hepan and / or an inhibitor of platelet aggregation, which increases the vascular effect with few side effects. The administration of anticoagulants can take place at the same time or after the administration of the compound according to the invention. Likewise preferred is the addition of substances which promote blood circulation or substances which improve the microcirculation The following examples, publications, the sequence listing and the illustrations further explain the invention, the scope of which is evident from the patent claims. The described methods are to be understood as examples which describe the subject matter of the invention even after modifications.

"R-PA" is further understood to mean a recombinant plasminogen activator which consists of the domains K2 and P of human t-PA. The production of such plasminogen activators is described, for example, in US Pat. No. 5,223,256

Under r-PA (F274P, K277V) it is to be understood that in the plasminogen activator consisting of domains K2 and P, amino acid 274 (F) was replaced by amino acid P and amino acid 277 (K) by amino acid V (amino acid name analogous to TJ Harris (1987) )

FIG. 1 is a schematic representation of the plasma clot penetration and lysis model. In order to avoid the coagulation of the plasma caused by shear stress, the pressure was generated through a buffer chamber (hatched area). The mixing of the buffer with the plasma over the clot (dotted area) was carried out avoided by installing a bubble trap

1: buffer tank, 2nd peristaltic pump, 3 bubble trap, 4 injection syringe for the fibrinolytic, 5: pipette tip with clot (cross-hatched area), 6 hose clamp, 7: pressure element

FIG. 2 shows the cleavage of r-PA (F274P, K277V) (A) or r-PA (B) by thromboma (for more see Example 8)

FIG. 3 shows the cleavage of r-PA (F274P, K277V) (A) or r-PA (B) by plasmin (for more details see Example 9)

FIG. 4 shows the cleavage of r-PA (P272V, F274P, K277V) by thrombin (for more see Example 10) example 1

Recombinant preparation of the compounds according to the invention

a) Construction of the expression plasmid

The starting plasmid pA27fd, described in EP 0 382 174, contains the following components: tac promoter, lac operator region with an ATG start codon, the coding region for the t-PA mutein, consisting of the Kringle 2 domain and the Protease domain and the fd transcription terminator. The starting vector represents the plasmid pkk 223-3.

The method of Morinaga et al., Biotechnology (1984) 636 was essentially used to introduce the mutations. For the formation of heteroduplex, two suitable fragments are isolated from pA27fd (eg fragment A: the large BamHI fragment, fragment B: the vector linearized with Pvu I).

Table 1 lists the oligonucleotides used and the resulting mutations.

The heteroduplex batch was transformed together with the plasmid pUBS520 into E. coli (Brinkmann et al., Gene 85 (1989) 109). The transformants were selected by adding ampicillin and kanamycin (50 µg / ml each) to the nutrient medium.

The plasmids resulting from the batches are also shown in Table 1.

b) Expression in E. coli

To check the expression performance, the E. coli with the respective plasmid (see Table 1) and pUBS520 in LB medium (Sambrook et al., 1989, Molecular Cloning, Cold Spring Harbor) in the presence of ampicillin and kanamycin (50 μg / ml) grown up to an OD at 550 nm of 0.4. Expression was initiated by adding 5 mmol / 1 IPTG. The culture was incubated for an additional 4 hours. The E. coli cells were then collected by centrifugation and resuspended in buffer (50 mmol / 1 Tris-HC1 pH8, 50 mmol / 1 EDTA); the cells were lysed by sonication. The insoluble protein fractions were collected by centrifugation again and resuspended in the above-mentioned buffer by sonication. The suspension was coated with V * volume of application buffer (250 mmol / 1 Tris-HCl pH 6.8, 10 mmol / 1 EDTA, 5% SDS, 5% Mercaptoethanol, 50% glycerin and 0.005% bromophenol blue) were added and analyzed using a 12.5% SDS polyacrylamide gel. As a control, the same preparation was carried out with a culture of E. coli with the respective plasmids, which had not been induced with IPTG, and separated in the gel. In the preparation of the IPTG-induced culture, after staining the gel with Coomassie Blue R250 (dissolved in 30% methanol and 10% acetic acid), a clear band with a molecular weight of about 40 kD can be seen; this band is not present in the control preparation.

Further steps for the preparation of the active compound correspond to Examples 2 and 3 of EP-A 0 382 174.

Table 1

1) SEQIDNO 9

2) SEQIDNO 10

3) SEQIDNO II

4) SEQIDNO 12

Example 2

In vivo characterization

To test the thrombolytic potency and efficiency of the proteins according to the invention, the rabbit model of neck vein thrombolysis established by D. Collen (J. Clin Invest 71 (1983) 368-376) was used. A radiolabelled thrombus was generated in the animals in the neck vein. The animals were subcutaneously anticoagulated with 100 IU / kg heparin. Alteplase (recombinant wild-type tissue plasminogen activator, "t-PA", commercially available as Actilyse® from Thomae, Biberach, Germany), the protein described in Example 1, streptokinase (commercially available as Streptase® from Behring, Marburg, Germany ) or solvents (0.2 M arginine phosphate buffer) were administered intravenously to the rabbits

The placebo group received an intravenous single bolus injection of 1 mg / kg solvent. The Alteplase group received a total dose of 1.45 mg / kg intravenously, of which 0.2 mg / kg as an initial bolus injection and 0.75 mg / kg as 30 minute infusion, followed directly by 0.5 mg / kg as a 60 minute continuous infusion (total infusion 90 minutes). The streptokinase group received a 60-minute intravenous infusion of 64,000 IU / kg. The group with the protein according to the invention received an intravenous single bolus injection. These are recognized standard rules for Alteplase and streptokinase

Two hours after the start of therapy, the residual thrombus was removed and the extent of the thrombus dissolution (thrombolysis) was determined by means of the decrease in radioactivity in the thrombus. Blood samples for obtaining plasma were taken before therapy and two hours after the start of therapy. The activated thromboplastin time was measured using standard methods. Blood loss due to thrombolytic therapy was also quantified. Before the thrombolytics were administered, a defined skin incision of 4 cm long and 0.3 cm deep was made to the animals on the thigh with the aid of a template and a scalpel.The resulting bleeding came to a standstill as a result of natural coagulation A sponge was placed on the wound, which soaked up the blood from the bleeding newly started by thrombolysis. By weighing the sponge (after deducting its own weight), the amount of blood leaked was measured, thus describing the extent of the bleeding side effect Both Alteplase and the proteins according to the invention from Example 1 are thrombolytically highly active substances and, compared to solvent control, significantly dissolved the thrombi.

Example 3

Comparison of clot lysis activity

a) Carrying out the clot lysis assay

The activity of t-PA and the recombinant proteins of Example 1 was determined in the clot lysis assay.

The sample is adjusted to the respectively required protein concentration by adding buffer (0.06 M Na2HPÜ4, pH 7.4, 5 mg / ml BSA (bovine serum albumin), 0.01% Tween® 80). 0.1 ml of the sample was mixed with 1 ml of human fibrinogen solution (IMCO) (2 mg / ml 0.006 M Na2HP04, pH 7.4, 0.5 mg / ml BSA, 0.01% Tween® 80) and 5 min. incubated at 37 ° C. Then 100 μl each of a plasminogen solution (10 IU / ml 0.06 M Na ₂ HPO ₄ / Η ₃ PO 4, pH 7.4, 0.5 mg / ml BSA, 0.01% Tween® 80) and a thrombin solution (30 U / ml 0.06 M Na ₂ HPO, pH 7.4, 0.5 mg / ml BSA, 0.01% Tween® 80) was added and the test mixture was incubated again at 37 ° C. After two minutes, a Teflon® ball is placed on the fibrin clot and the time until the ball has reached the bottom of the test tube.

b) Determination of the activity in the dynamic plasma model

In this dynamic plasma model, the substances according to the invention are investigated under conditions which are quite similar to the in vivo conditions. The substances are placed in the plasma over the clot under the influence of a peristaltic pressure similar to the pressure caused by the heartbeat.

200 μl of citrate plasma were mixed with 20 μl of a 0.25 mol / 1 CaCl2 solution and incubated at 37 ° C. 0.16 U thrombin was added and the mixture was placed in a 1 ml pipette tip (Eppendorff, Hamburg, FRG). The pipette tip was 2 min. stored vertically at 23 ° C, 60 min. incubated in 0.01 mol / 1 Tris / HCl, pH 7.4, 0.15 mol / 1 NaCl2, 0.025 mol / 1 CaCl2, 0.01% Tween® 80 and added to the clot-lysis apparatus. The clot lysis activity was determined in a switching system assembled from elastic tubes (FIG. 1). The flow is generated by a peristaltic pump and split into two parallel branches. Splits. Branch A contains the 1 ml pipette tip filled with the plasma clot, which closes this branch. Branch B is a blind line running parallel to branch A. The pressure was set to 10 mbar using a hose clamp in branch B. Plasma (1 ml) was added to the clot. The pump was turned on and the stability of each clot checked for 15 minutes. The fibrinolytic (final plasma concentration between 0.5 and 10 and 20 μg / ml for the proteins of Example 1 or CHO-t-PA) was injected using a 1 ml tuberculin syringe with a hypodermic needle for intramuscular injection (Braun, Melsungen, FRG) carefully injected into the plasma. The clot lysis time was calculated as the time difference between the addition of the fibrinolytic enzyme and the drop in pressure to 50% of the value before the addition of the fibrinolytic. The pressure was determined using a water-calibrated piezoelectric pressure detection system and documented using a computer-aided documentation program.

c) Clot penetration in the static model

800 μl human citrate plasma (healthy donor) are mixed with 75 μl Ca buffer (50 mmol / l Tris / HCl, pH 7.2, 0.25 mol / 1 CaCl ₂ ), 20 μl gelatin solution (10% w / v) in 0.9% NaCl) and 100 ml thrombin solution (8 U / ml, 0.05 mol / 1 sodium citrate / HCl, pH 6.5, 0.15 mol / 1 NaCl). 800 μl of this mixture are carefully transferred into a 2 ml column (Pierce, Rockfort, EL, USA). A plasma clot is formed by incubation for three hours at 37 ° C. 2 ml buffer (0.008 mol / 1 Na ₂ HPO ₄ , 0.001 mol / 1 KH ₂ PO ₄ , 0.003 mol / 1 KC1, 0, 137 mol / 1 NaCl, 0.1% bovine serum albumin, 0.01% Tween® 80) are adjusted to the desired concentrations (0, 0.5, 1, 2 and 3 μg / ml) with the plasminogen activator, which was previously inhibited with Glu-Gly-Arg-chloromethyl ketone, and 1 ml of this solution is applied to the surface of the clot . The remaining buffer is discarded. The surface of the clot is washed with 2 ml PBS buffer (0.008 mol / 1 Na HPO, 0.001 mol 1 KH ₂ PO ₄ , 0.003 mol / 1 KC1 and 0.137 mol / 1 NaCl) and the protein is fixed by adding 2 ml glutardialdehyde Solution in PBS. The clot surface is then washed with 2 ml of 50 mmol / 1 Tris / HCl, pH 8.0 and incubated with 1 ml of peroxidase-labeled polyclonal antibodies against t-PA (250 mU / ml). After washing the clot with 1 ml of PBS, the antibody-bound protein is determined by incubation with 3-amino-9-ethylcarbazole, which is converted into an insoluble red color by peroxidase.

Plasminogen activators according to the invention are not concentrated on the surface of the clot, but penetrate into the clot and are distributed evenly. The intensity of the immunologically colored part of the clot increases with increasing concentration of the plasminogen activators according to the invention in the plasma

Example 4

Comparison of fibrin binding

In this example, the thrombolytically active proteins from Example 1 are tested for their ability to bind to fibrin and compared with this property also with Alteplase

Samples of Alteplase and a protein according to the invention were prepared as solutions of 1.5 μg protein ml. Samples (100 μl) of the thrombolytically active protein were then each prepared with 770 μl buffer (0.05 M Tris / HCl, pH 7.4, 0, 15 NaCl, 0.01% Tween® 80), 10 μl bovine serum albumin solution (100 mg / ml), 10 μl aprotinin (3.75 mg / ml), 10 μl bovine thrombin (concentration 100 U / ml) and increasing amounts of Fibrinogen (10 μg / ml to 300 μg / ml) mixed. All solutions were water. It is known that thrombin converts fibrinogen into an insoluble fibrin clot

The components were mixed and incubated at 37 ° C for one hour. The supernatant was then separated from the fibrin clot by centrifugation (15 minutes, 13,000 rpm, at 4 ° C) and the amount of plasminogen activator protein present in the supernatant was determined by a standard ELISA certainly

Example 5

Comparison of plasminogenolytic activity and stimulability

A known procedure for determining the stimulability of plasminogenolytic activity is described by Verheijen et al in Thromb Haemost 48 (1982) 266-269

The fibrinogen fragments acting as a stimulator were produced by treating human fibrinogen with cyanogen bromide (lg human fibrinogen, 1.3 g CNBr in 100 ml water) in 70% v / v formic acid over a period of 17 hours at room temperature, followed by dialysis against distilled water

When carrying out the assay, 5 ng / ml t-PA or an equivalent concentration of the proteins from Example 1 were incubated in 1 ml 0.1 mol / 1 Tris / HCl (pH 7.5), containing 0.1% v / v Tween® 80, 0.13 μmol / 1 Glu-Plasminogen, 0.3 mmol substrate S2251 (chromogenic substrate HD-Val-Leu-Lys-p-nitroanilide HCl) and 120 μg / ml fibrinogen fragments. The mixtures were incubated for two hours at 25 ° C. and the absorption rate at 405 nm was measured without interrupting the reaction against control blank values. The cleavage of the chromogenic substrate S2251 was measured as a measure of the plasminogenolytic activity of the enzyme. The stimulability is calculated as the activity with fibrinogen fragments divided by the activity without fibrinogen fragments.

In each case 25 μl of the sample, correspondingly prediluted with 0.1 mol / 1 Tris, pH 7.5, 0.15% Tween® 80, are pipetted into a “well” of a microtiter plate. Then 200 μl of reagent mixture are added and the absorbance above 405 nm a period of 2 h was determined against the blank value (25 μl, 0.1 mol / 1 Tris, pH 7.5, 0.15% Tween® 80 with 200 μl reagent mixture).

Eprobe ⁼ ( ^E Probe t ^{~ E} BV t) - ( ^E Probe 0 - ^E BV o)

I sample _t = sample value after 2 h BV t ⁼ reagent blank after 2 h

I-sample 0 ⁼ sample value at time t = 0

^E BV 0 ~ reagent blank at time t = 0

Reagent mixture:

5 ml test buffer (0.1 mol / 1 Tris, pH 7.5, 15% Tween® 80)

1 ml t-PA stimulator (1 mg / ml cyanogen bromide fragments from human fibrinogen)

1 ml substrate solution (3 mmol / 1 S2251, H-D-Val-Leu-Lys-pNA, Chromogenix, Moelndal,

SE)

1 ml plasminogen solution (7 U / ml plasminogen, Boehringer Mannheim GmbH)

Calculation of the stimulation factor:

To calculate the stimulation factor, the activity in the presence of the t-PA stimulator is divided by the activity in the absence of the t-PA stimulator. The dilution should be such that an almost identical absorbance is achieved in both preparations

1 ml H ₂ O is added to the reaction mixture without t-PA stimulator instead of 1 ml t-PA stimulator

The activity is measured in the same way both in the absence and in the presence of the stimulator. The stimulation factor F is calculated as follows ^E sample with stimulant ^{x V} he ^d ünnung _Pro be -with stimulator

F = ^E ^x Stimulator sample without dilution _Pro b _e without stimulator

The specific activity is the quotient of plasminogenolytic activity (KU / ml) and protein concentration (mg / ml).

Example 6

Bolus injection of the recombinant proteins of Example 1 of the tissue plasminogen activator induces an effective and safe thrombolysis in a dog model of coronary thrombosis

The thrombolysis by the proteins of Example 1 produced in E. coli can be evaluated in a dog model of thrombosis of the left coronary artery induced by electrical stimulation.

Example 7

Determination of amidolytic activity

To determine the amidolytic activity, 200 ml of buffer (0.1 mol 1 Tris / HCl, pH 7.5, 0.15% Tween® 80) and 200 μl of the plasminogen activator solution (diluted with buffer to a concentration of 1-12 μg / ml) incubated for 5 minutes at 37 ° C. The determination is started for the addition of 200 μl S2288 (6 mmol / l, H-D-Ile-Pro-Arg-P-nitroaniline dihydrochloride, Kabi Vitrum, Sweden). The S2288 substrate was pre-equilibrated at 37 ° C. The amidolytic activity is calculated from the increase in extinction at 405 nm within the first 2.5 minutes, with an extinction coefficient for p-nitroaniline of 9750 1 / mol / cm.

Example 8

Cleavage of r-PA (F274P, K277V) by thrombin

1. Implementation

In each case 44 μg r-PA (F274P, K277V) and r-PA (reference) were preincubated for 15 min at 37 ° C and with the units specified below on human thrombin (Sigma), which was also 15 min at 37 ° C was pre-incubated, mixed and incubated at 37 ° C for 30 min. The samples were then 1: 1 (v / v) with SDS sample buffer (0.125 mol 1 Tris / HCl, pH 8.8, 4.6% (w / v) SDS, 4 mol / 1 urea, 0.1% bromophenol blue, 0.3 mol / 1 dithioerythritol) mixed, incubated for 3 min at 95 ° C and analyzed by SDS-polyacrylamide gel electrophoresis.

2. Result

The cleavage of r-PA (F274P, K277V) by thrombin is shown in FIG. 2. The data show that r-PA (F274P, K277V) is completely converted into the two-chain form by increasing amounts of thrombin. The protease and kringle 2 domains of thrombin-cleaved r-PA (F274P, K277V) run at the same level as the corresponding domains of the two-chain form of r-PA (r-PA (tc)) produced by plasmin digestion.

In contrast to r-PA (F274P, K277V) (A), r-PA (B), which is carried as a reference, is not cleaved by thrombin under the conditions described.

A:

Lane 1 molecular weight standard Lane 2 r-PA

** lane 3 r-PA (tc) lane 4 r-PA (F274P, K277V) lane 5 r-PA (F274P, K277V) + thrombin buffer lane 6 r-PA (F274P, K277V) + 0.055 NIH units thrombin lane 7 r-PA (F274P, K277V) + 0.55 NIH units thrombin lane 8 r-PA (F274P, K277V) + 2.74 NIH units thrombin lane 9 thrombin (5 NTH units)

*

Lane 10: molecular weight standard

B:

Lane 1 molecular weight standard Lane 2 r-PA Lane 3 r-PA (tc) ** Lane 4 r-PA + thrombin buffer Lane 5 r-PA + 0.055 NTH units thrombin Lane 6 r-PA + 0.55 NIH units thrombin Lane 7 r -PA + 2.74 NIH units thrombin lane 8 thrombin (5 NTH units) lane 9 molecular weight standard *) Molecular weight standard: lysozyme (14,307 Da), soybean trypsin inhibitor (20,100 Da), triose phosphate isomerase (26,626 Da), aldolase (39,212 Da), glutamate dehydrogenase (55,562 Da), fructose-6-phosphate kinase (85,204 Da), ß- Galactosidase (116,353 Da), α-2 macroglobulin (170,000 Da).

**) r-PA (tc): two-chain form of r-PA, which was obtained by incubating r-PA with plasmin.

Example 9

Cleavage of r-PA (F274P, K277V) by plasmin

1. Implementation

25 μg each of r-PA (F274P, K277V) and r-PA (reference) were preincubated for 15 min at 37 ° C and with the units specified below on plasmin (human), which was also preincubated for 15 min at 37 ° C , mixed and incubated at 37 ° C for 10 min. The samples were then mixed 1: 1 (v / v) with SDS sample buffer (0 125 mol / 1 Tris / HCl, pH 8.8, 4.6% (w / v) SDS, 4 mol / 1 urea, 0.1% bromophenol blue, 0 3 mol 1 dithioerythritol) mixed, incubated for 4 min at 95 ° C. and analyzed by SDS-polyacrylamide gel electrophoresis.

2. Result

The cleavage of r-PA (F274P, K277V) (A) and the r-PA (B) carried as a reference by plasmin is shown in FIG. 3

The data show that r-PA (B) is converted to the two-chain form by incubation with increasing amounts of plasmin. Under the conditions used, the amount of r-PA used is completely converted into the two-chain form by 25 U plasmin.

On the other hand, r-PA (F274P, K277V) (A) is cleaved much more poorly by plasmin. No significant cleavage of r-PA (F274P, K277V) by plasmin was observed in the incubation with 0.025 U and 0.1 U plasmin. Even with the incubation of 25 μg r-PA (F274P, K277V) with 25 mU plasmin, there was no complete cleavage The protease and kringle 2 domains of thrombin-cleaved r-PA (F274P, K277V) run at the same level as the corresponding domains of the two-chain form of r-PA (r-PA (tc)) produced by digestion with plasmin-Sepharose. ), so that it can be assumed that the cleavage of the variant takes place between amino acids 275 and 276 as in r-PA (counting method according to Harris, Prot. Engineering 1, 449-458 (1987)).

A:

Lane molecular weight standard lane 2 r-PA lane 3 r-PA (tc) ** lane 4 r-PA (F274P, K277V) lane 5 r-PA (F274P, K277V) + 0.25 mU plasmin lane 6 r-PA (F274P, K277V) + 2.5 mU Plasmin track 7 r-PA (F274P, K277V) + 12.5 mU Plasmin track 8 r-PA (F274P, K277V) + 25 mU Plasmin

B:

Lane 1 molecular weight standard Lane 2 r-PA Lane 3 r-PA (tc) ** Lane 4 r-PA + 0.25 mU plasmin Lane 5 r-PA + 2.5 mU plasmin Lane 6 r-PA + 12.5 mU plasmin Lane 7 r-PA + 25 mU plasmin

*) Molecular weight standard: lysozyme (14,307 Da), soybean trypsin inhibitor (20,100 Da), triose phosphate isomerase (26,626 Da), aldolase (39,212 Da), glutamate dehydrogenase (55,562 Da), fructose-6-phosphate kinase (85,204 Da), ß- Galactosidase (116,353 Da), α- _2nd Macroglobulin (170,000 Da).

**) r-PA (tc): two-chain form of r-PA, which was obtained by incubating r-PA with plasmin-Sepharose. Example 10

Cleavage of r-PA (P272V, F274P, K277V) by thrombin

1. Implementation

40 μg of r-PA (P272V, F274P, K277V) were preincubated for 15 min at 37 ° C and mixed with the units below of bovine thrombin (Sigma), which was also preincubated for 15 min at 37 ° C, and mixed for 30 min Incubated at 37 ° C. The samples were then mixed 1: 1 (v / v) with SDS sample buffer (0.125 mol / 1 Tris / HCl, pH 8.8, 4.6% (w / v) SDS, 4 mol 1 urea, 0.1% bromophenol blue, 0.3 mol / 1 dithioerythritol) mixed, incubated for 3 min at 95 ° C and analyzed by SDS-polyacrylamide gel electrophoresis.

2. Result

The cleavage of r-PA (P272V, F274P, K277V) by thrombin is shown in FIG. 4. The data show that r-PA (P272V, F274P, K277V) is completely converted into the two-chain form by increasing amounts of thrombin. The protease and kringle 2 domains of thrombin-cleaved r-PA (P272V, F274P, K277V) run at the same level as the corresponding domains of the two-chain form of r-PA (r-PA (tc)) produced by plasmin digestion. ).

Lane 1: molecular weight standard

Lane 2: r-PA

Lane 3: r-PA (tc) **

Lane 4: r-PA (P272V, F274P, K277V)

Lane 5: r-PA (P272 V, F274P, K277 V) + thrombin buffer

Lane 6: r-PA (P272V, F274P, K277V) + 0.055 NIH units of thrombin

Lane 7: r-PA (P272V, F274P, K277V) + 0.55 NIH units of thrombin

Lane 8: r-PA (P272V, F274P, K277V) + 2.74 NTH units of thrombin

Lane 9: molecular weight standard

*) Molecular weight standard: lysozyme (14,307 Da), soybean trypsin inhibitor (20,100 Da), triose phosphate isomerase (26,626 Da), aldolase (39,212 Da), glutamate dehydrogenase (55,562 Da), fructose-6-phosphate kinase (85- 204 Da), Galactosidase (1 16,353 Da), α-2 macroglobulin (170,000 Da). **) r-PA (tc): two-chain form of r-PA, which was obtained by incubating r-PA with plasmin.

Reference list

Ausubel et al., Current Protocols In Molecular Biology, Vol. 2, Chapter 15 (Greene Publ.

Associates & Wiley Interscience 1991)

Bennett, W.F., J. Biol. Chem. 266 (1991) 5191-5201

Brinkmann et al., Gene 85 (1989) 109

Dawson, K.N. et al., J. Biol. Chem. 269 (1984) 15989-15992

Eastman, D., Biochemistry 31 (1992) 419-422

EP-A 0 219 874

EP-A 0 241 022

EP-A 0 245 100

EP-A 0 302456

EP-A 0 382 174

EP-A 0 400 545

EP-B 0 196 920

EP-B 0 297 066

Harris, T.J.R., Protein Engineering 1 (1987) 449-459

Kohnert, U. et al., Protein Engineering 5 (1992) 93-100

Korninger and Collen, Thromb. Haemostasis 46 (1981) 561-565

Krause, J., Fibrinolysis 2 (1988) 133-142

Kunkel, T.A., Proc. Natl. Acad. Be. USA 82 (1985) 488-492

Lamba, D. et al., J. Mol. Biol. 258 (1996) 117-135

Madison, E.L. et al., Science 262 (1993) 419-421

Madison, E.L. et. al., Proc. Natl. Acad. Be. USA 87 (1990) 3530-3533

Madison, E.L., Nature 339 (1989) 721-723

Morinaga et al., Biotechnology 21 (1984) 634

Paoni, N.F. et al., Protein Engineering 5 (1992) 259-266

Paoni, N.F. Protein Engineering 6 (1993) 529-534

Paoni, N.F., Thrombosis and Haemostasis 70 (1993) 307-312

Refino, C.J., Thrombosis and Haemostasis 70 (1993) 313-319

Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA

Schechter, J. and Berger, A., Biochem. Biophys. Res. Commun. 27 (1967) 157-162 Schohet, RV, Thrombosis and Haemostasis 71 (1994) 124-128

U.S. Patent 5,200,340

U.S. Patent 5,223,256

U.S. Patent 5,501,853

Wen-Pin Yang et al., Biochemistry 33 (1994) 2306-2312

WO 90/09437

WO 91/09118

WO 94/10318

WO 96/01312

WO 96/17928

Zoller and Smith, DNA 3 (1984) 479-488

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: BOEHRINGER MANNHEIM GMBH

(B) STREET: Sandhofer Str. 116

(C) LOCATION: Mannheim

(E) COUNTRY: Germany

(F) POSTAL NUMBER: D-68305

(G) TELEPHONE: 08856 / 60-3446 (H) TELEFAX: 08856 / 60-3451

(ii) DESCRIPTION OF THE INVENTION:

Thrombin activated plasminogen activator

(iii) NUMBER OF SEQUENCES: 12

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30B (EPA)

(vi) DATA OF THE URN REGISTRATION:

(A) APPLICATION NUMBER: EP 96112487.2

(B) REGISTRATION DAY: 02-AUG-1996

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Val Gin Pro Arg Ile Val Gly 1 5

(2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gly Asp Thr Arg Ser Gly Gly Pro Gin Ala Asn Leu His 1 5 10

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Pro Gin Ala Asn Leu His 1 5

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Gly Ile Pro Arg Ile Val 1 5

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ala Gin Pro Arg Ile Lys 1 5 (2) INFORMATION ON SEQ ID NO: 6:

(i) SEQUENCE LABEL:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Gly Leu Arg Gin Tyr Ser Gin Pro Gin Phe Arg Ile Lys 1 5 10

(2) INFORMATION ON SEQ ID NO: 7:

(i) SEQUENCE LABEL:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Gly Leu Ser Gin Ala Ser Gin Gly Ile Pro Arg Ile Val 1 5 10

(2) INFORMATION ON SEQ ID NO: 8:

(i) SEQUENCE LABEL:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Gly Leu Arg Gin Tyr Ser Gin Ala Gin Gly Ile Pro Arg Ile Val 1 5 10 15

(2) INFORMATION ON SEQ ID NO: 9:

(i) SEQUENCE LABEL:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GTACAGCCAG GTTCAGCCTC GCATCGTTGG AGGGCTCT 38

(2) INFORMATION ON SEQ ID NO: 10:

(i) SEQUENCE LABEL:

(A) LENGTH: 53 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CTGCGGCCTG AGCCAGTACA GCCAGTTTGG CCCTCGCATC GTTGGAGGGC TCT 53

(2) INFORMATION ON SEQ ID NO: 11:

(i) SEQUENCE LABEL:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GGAGCGGCTG CAGGAGGCTC ATG 23

(2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE LABEL:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CTACGGCAAG ACCGAGGCCT TGT 23

Claims

claims

1. Plasminogen activator, which is based on human tissue plasminogen activator

b) is modified such that the zymogenicity is at least 1.2 times greater than that of human plasminogen activator and

c) whose fibrin binding is reduced to such an extent that the plasminogen activator can penetrate more than 50% into a blood clot.

2. Plasminogen activator according to claim 1, characterized in that it is modified so that the cleavage by plasmin between the amino acids Pl (275) and Pl '(276) is reduced by more than 10%, preferably by more than 20%.

3. Plasminogen activator according to claims 1 or 2, characterized in that it penetrates a blood clot evenly.

4. Plasminogen activator according to claims 1-3, characterized in that the amino acid region between G 264 and A 288 is modified so that the plasminogen activator is cleavable by thrombin.

5. Plasminogen activator according to claims 1-4, characterized in that the amino acid areas 459 - 471, 417 - 425 and / or the amino acids Q 475, K 505 and / or E 506 are modified.

6. Plasminogen activator according to claims 1-5, characterized in that the amino acids G 265 and / or R 267 are modified and / or at least one amino acid is inserted in the range between 264 and 267.

7. Plasminogen activator according to claims 1-6, characterized in that the finger domain is deleted.

8. Plasminogen activator according to claim 7, characterized in that said plasminogen activator contains only the protease domain or the Kringle 2 domain and the protease domain of human tissue plasminogen activator.

9. Use of a plasminogen activator according to claims 1-8 for the manufacture of a pharmaceutical composition for the treatment of thromboembolic disorders.

10. A process for the recombinant production of a plasminogen activator according to claims 1-8, characterized in that a prokaryotic or eukaryotic host cell is transformed with a vector which is capable of expressing said tissue plasminogen activator, this cell is grown and said cell Plasminogen activator is isolated.

11. Pharmaceutical composition of a plasminogen activator according to claims 1-8 in a therapeutically effective amount and optionally pharmaceutical auxiliaries, fillers or additives.