CA2262751A1 - Plasminogen activator capable of being activated by thrombin - Google Patents

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

CA 022627~1 1999-02-01 Plasminogen activator capable of being activated by thrombin The invention concerns a thrombin-activatable plasminogen activator, pharmaceutical agents for treating thromboembolic diseases, pharmaceutical compositions which contain such a plasminogen activator and their use.

Tissue plasminogen activator (t-PA) is a serine protease composed of several domains which catalyses the conversion of plasminogen into plasmin and is used for fibrinolytic therapy.

Numerous t-PA variants and mutations are known, cf. for example the review articles by T.J.R. Harris (1987) and J. Krause (1988).

With regard to the mechanism of action of t-PA it is known among other things that the fibrinolysis is partially regulated by the interaction between t-PA and plasminogen activator inhibitor 1 (PAI-l, a serine protease inhibitor from the serpine family). PAI-1 binds to t-PA
primarily via the amino acids 296 - 302. A mutation of this region reduces the inhibitory effect of PAI-1 on t-PA (E.L. Madison et al. (1990)). Extensive investigations have been carried out on the mechanism of interaction between the amino acid region 296 - 302 of t-PA
and PAI-1 (cf. also E.L. Madison, Nature 339 (1989) 721 - 723; R.V. Schohet, Thrombosis and Haemostasis 71 (1994) 124 - 128; C.J. Refino, Thrombosis and Haemostasis 70 (1993) 313 - 319; N.F. Paoni, Protein Engineering 6 (1993) 529 - 534 and Thrombosis andHaemostasis 70 (1993) 307 - 312; W.F. Bennett, J. Biol. Chem. 266 (1991) 5191 - 5201, D.
Eastman, Biochemistry 31 (1992) 419 - 422).

Non-modified human t-PA (denoted t-PA in the following) is composed of 527 amino acids in the form which occurs in plasma and can be cleaved by plasmin into two chains which are then still held together by a disulfide bridge. The A chain (also called the heavy chain) is composed of four structural domains. The finger domain (amino acids 1 - 49) has 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 kringle domains (amino acids 87 - 261) are to a large extent homologous to the fourth and fifth kringle domains of plasminogen. The finger and kringle 2 domains of t-PA are particularly involved in the fibrin binding and in the stimulation of the proteolytic activity by fibrin. The B chain of t-PA (amino acids 276 - 527, protease domain) is a serine protease and substantially homologous to the B chains of urokinase and plasmin (T.J.R. Harris (1987) and J. Krause (1988)).

CA 022627~1 1999-02-01 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 substantially increased in the presence of these stimulators (by more than a factor of 10). The mechanism of action of t-PA
in vivo is described for example in Korninger and Collen, Thromb. Haemostasis 46 (1981) 561 - 565. t-PA activates plasminogen to plasmin. Plasmin cleaves fibrin to form soluble fibrin cleavage products. t-PA is cleaved by protease/plasmin present in the blood between amino acid 275 (arginine) and 276 (isoleucine) and is thus activated. In this process the two partial chains remain linked via a cysteine bridge.

The ability to stimulate the activity by fibrin and fibrin cleavage products is an essential feature of t-PA which distinguishes t-PA from the other known plasminogen activators such as e.g. urokinase or streptokinase. The stimulatory potency can be fùrther improved by modifying the amino acid sequence of t-PA. A measure of the stimulatory potency is the ratio of the catalytic efficiency (kCat/Km) in the presence and in the absence of fibrin. kCat is the rate constant of the catalytic reaction and Km is the Michaelis constant. The stimulatory potency of t-PA can be increased by 19-fold to 81-fold by modifying the amino acids 292 and/or 305 (E.L. Madison et al., Science 262 (1993) 419 - 421).

t-PA derivatives are known from the US patent 5,501,853 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.

Thrombin-activatable plasminogen activators are described by Wen-Pin Yang et al., in Biochemistry 33 (1994) 2306 - 2312. This chimeric plasminogen activator (59 D8-scu PA-t) was prepared from the Fab fragment of an anti-fibrin antibody (59 D8) and 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 acids Phe-157 and Lys-158 from the low molecular single chain urokinase plasminogen activator (scu PA) by site directed mutagenesis.

Thrombin-activatable plasminogen mutants are described by K.N.Dawson et al., in J. Biol.
Chem. 269 (1984) 15989 - 15992. These plasminogen derivatives were obtained by substituting the P3, P2 and P1' amino acids of the cleavage site by a thrombin-cleavable sequence.

CA 022627~1 1999-02-01 , N.F.Paoni et al., describe in Protein Engineering 5 (1992) 259 - 266 the modification of t-PA
in the amino acid region 296 - 299. As a result an improved fibrin specificity is obtained.

Thrombin-activatable plasminogen activators are described in the US patent 5,200,340 which are modified such that they contain a thrombin cleavage site for the activation. It is also 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 kringle structures must be preserved.

It is known from WO 91/09118 and WO 94/10318 that plasminogen can be activated by thrombin to form plasmin by introducing a thrombin cleavage site. Since thrombin is contained in the blood clot, it is intended that this activation mainly takes place in the blood clot. However, the disadvantage of such a method is that modified plasminogen has to be ~mini~tered to the patient in large quantities for this.

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

The object is achieved by a plasminogen activator which, based on human tissue-type plasminogen activator, a) is modified in such a way that the plasminogen activator can be cleaved by thrombin and is converted by such a cleavage into the two-chain form, b) is modified in such a way that the zymogenicity is higher by a factor of at least 1.2, preferably by a factor of 2 as compared with human t-PA and c) its fibrin binding is reduced to an extent that the plasminogen activator can penetrate by more than 50 % into a blood clot.

Such a plasminogen activator acts specifically on the blood clot and therefore has substantially less side effects than the known plasminogen activators.

The starting point is the sequence of the human tissue-type plasminogen activator.
Consequently, "based on human tissue-type plasminogen activator" according to the invention means that the sequence of the plasminogen activator of the invention is derived CA 022627~1 1999-02-01 from the sequence of human plasminogen activator. This implies on the one hand that the structural features (domains) characteristic for t-PA are at least partly structurally retained.
For instance, it has been found that plasminogen activators in which the structure of the kringle 2 andlor protease domains is still retained and which in addition bear the modifications according to the invention can be used in the sense of the invention. It is also possible that further domains are retained and the features according to the invention are brought about by deletions, mutations and/or additions of amino acids. The changes of the amino acid sequence can be effected by the methods known to those 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 the human tissue-type plasminogen activator in such a way that the cleavability of the plasminogen activator according to the invention by plasmin between the amino acids P1 (275) and P1' (276) is reduced. Preferably, the cleavability by plasmin in the plasminogen activators according to the invention is reduced by 10% or more, more preferably by 20% or more, and most preferably by 50% or more. In this connection it is not necessary that the cleavability by plasmin is completely abolished. In particular the thrombin activation can be improved by a plasmin cleavability of the plasminogen activator according to the invention.

However, it is also preferred that the cleavability is reduced to the extent that physiologically relevant cleavage no longer takes place in vivo. As a result it is possible to drastically reduce side effects. Consequently the degradation of coagulation parameters is drastically reduced in vivo and the bleeding time is not significantly increased as is the case with known plasminogen activators.

The cleavability by plasmin can be determined in an in vitro test. For this the plasminogen activator is incubated for 5 minutes at 25~C with increasing amounts of plasmin and subsequently an SDS electrophoresis is carried out in acrylamide gel containing 12.5 - lS %
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 it can no longer, or only to a reduced extent, be cleaved by plasmin between P1 and P1' (nomenclature according to Schechter, J. and Berger, A., Biochem. Biophys. Res Commun. 27 (1967) 157 - 162) can be carried out in a manner known to any person skilled in the art. Plasmin cleaves the sequence R 275-I 276 (amino acid names in the single letter code). For example the cleavability by CA 022627~1 1999-02-01 plasmin can be abolished or at least drastically reduced by modifying one or both amino acids.

The cleavability by plasmin can also be reduced by modifying positions P4 - P3' (amino acids 272 - 278). In this case P2 is preferably converted into a small, preferably hydrophobic and/or non-aromatic amino acid such as P. As a result it is surprisingly also possible to achieve a thrombin cleavability in addition to the reduction of the plasmin cleavability.

In this process one or more of the following general conditions are preferably adhered to:

(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) P 1: 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 and P2' into V. This results in the particularly preferred cleavage site (272-278)VQPRIVG. (SEQ ID NO: 1) A thrombin cleavage site can also be introduced according to the state of the art. However, an appropriate mutation is preferably introduced in the region of amino acids 264 - 288.

A modification of t-PA to introduce an affinity for thrombin can also be achieved by modifying the loop 459 - 471, the autolysis loop 417 - 425 and/or the 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). In the case of serine proteases such as thrombin and plasmin the P1 residue is the important 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 P1 position), it is not easily possible to obtain plasminogen activators in which cleavage by plasmin (plasmin activatability) is reduced and cleavage by CA 022627~1 1999-02-01 thrombin (thrombin activatability) is present or is substantially greater than the plasmin activatability (at least a factor of 2-10). However, it was surprisingly found that such properties can be obtained by modifying the secondary binding sites between the enzyme and substrate and by modifying the structural and dynamic properties of the activation loop.

The introduction of a thrombin-specific cleavage site while simultaneously reducing the plasmin cleavability is preferably carried out by changing the primary specificity by mutations in the activation loop between the amino acids 264 and 288. In this case the above-mentioned mutations in the region 272 - 277 (P4-P2') are particularly preferred.
The thrombin cleavability can be improved by modifying the flexibility and/or accessibility of the binding loop. For this it is particularly preferable to modify G 265 (the mutation leads to a lower flexibility) or R 267 (the mutation leads to a change or cleavage of the salt bridge between G 265 and E 410). Insertions in the region between 264 and 267 are also preferred.
R 267 is particularly preferably modified into S (D. Lamba et al., J. Mol. Biol. 258 (1996) 117- 135).

A mutation which can be used to increase the flexibility is to change R 267 into S preferably in combination with mutations of P4 into F, P3 into G, P2 into P and P2' into V.
The thrombin cleavability and specificity can be further improved by changing the secondary specific binding sites. For this the loop 459 - 471 can for example be completely or partially deleted. This loop is composed of the amino acids GDTRSGGPQANLH. (SEQ ID NO:2) The region PQANLH (SEQ ID NO:3) in this loop is preferably completely or at least partially deleted (wherein H is preferably preserved) or its amino acid sequence is modified.

The following sequences are preferably used in the region of amino acids 272 - 277:

GIPRIV (SEQ ID NO:4) AQPRIK (SEQ ID NO:5) CA 022627~1 1999-02-01 It is also advantageous to use the sequence GLSQASQGIPRIV (SEQ ID NO:7) in the amino acid region between 265 and 277 (t-PA original sequence GLRQYSQPQFRIK
(SEQ ID NO:6)). The sequence GLRQYSQAQGIPRIV (SEQ ID NO:8) is also preferred for this region. In this sequence the amino acids G and I are inserted between Q and P (original sequence: Q and F) in order to extend it. Such an insertion is preferably carried out at any site in the region between 264 and 276.

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

In a preferred embodiment of the invention the plasminogen activator additionally contains a mutation which drastically reduces the activity of the one-chain form but not the activity of the two-chain form and thus improves the zymogenicity by a factor of 1.2, preferably by a factor of 2 or higher.

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

In order to increase the zymogenicity (ratio of the amidolytic activity of the two-chain form to the activity of the one-chain form of the plasminogen activator) it is particularly preferred (also for all plasminogen activators derived from human tissue-type plasminogen activator) to modify K 429 into Q and/or H 417 into T and/or suppress 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 ID NO: 1) with the additional mutation K 429 into Q and/or H 417 into T.

.

CA 022627~1 1999-02-01 , .

The fibrin binding can be reduced by deleting the domain of t-PA which is specific for the fibrin binding (fibrin binding domain, finger domain) or by mutating it in such a way that fibrin binding via the finger domain cannot take place or only to a slight extent (no functionally active finger domain). The reduction of fibrin binding enables the plasminogen activator to penetrate into the clot (preferably to an extent of more than 50%) and to disperse uniformly. It is cleaved there by thrombin and exhibits its activity in the active two-chain form. Thus a plasminogen activator whose fibrin binding is reduced in this manner exhibits no longer a high-affinity fibrin binding typical of t-PA. This specific mechanism of action increases the potency of the plasminogen activator and in particular it 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 the distribution in the clot can be determined visually. For the assessment the plasminogen activator described in the US patent 5,223,256 is used as a standard which penetrates into the clot, disperses homogeneously and thus represents the 100 % value according to definition (as determined at a concentration of 3 ,ug/ml). Recombinant human tissue-type plasminogen activator according to EP-B 0 093 619 is used as a further standard which according to definition does not penetrate into the clot and essentially binds to the surface. The procedure described in example 3c is used to examine the clot penetration. A comparison of these standards reveals that "non-penetration into the clot" means that the by far greatest part (80% or more) of the plasminogen activator is present in the first quarter of the clot while in the case of a "homogeneous distribution" at least 50% of the plasminogen activator penetrate further into the clot and thus are present in the other three quarters.

The specificity and effectiveness of clot dissolution is increased further if a plasminogen activator according to the invention is used which in addition has no or only a very slight and unspecific fibrin binding. Such molecules penetrate into the interior of the clot and thus ensure that plasminogen is efficiently activated 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, US 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 in such a way that it cannot be inhibited by PAI-1. Such a modification is preferably achieved by a mutation of the amino acids 296 - 302 (Madison, E.L. et al., Proc.

CA 022627~1 1999-02-01 Natl. Acad. Sci. USA 87 (1990) 3530 - 3533) and particularly preferably by substituting the amino acids 296 - 299 (KHRR) by AAAA (WO 96/01312).

The compounds according to the invention are thrombolytically active proteins which preferably, in contrast to t-PA (Alteplase), are suitable for ~mini~tration as an i.v. bolus injection. They are effective in a lower dose and have practically the same thrombolytic action as a standard clinically infusion of Alteplase.

The increase of the specificity and the associated reduction of the bleeding side effect make the compounds according to the invention an exceptionally valuable thrombolytic agent for the treatment of all thromboembolic diseases. In contrast to the thrombolytic agents that have previously been approved only for extremely life-threatening diseases such as cardiac infarction and massive pulmonary embolism, the use of such variants provides an opportunity of treating thrombolysis by the compounds according to the invention even for less acutely life-threatening diseases such as e.g. deep vein thrombosis. Furthermore thrombolytic agents based on the compounds according to the invention can be used on a much broader basis than hitherto since a major reason preventing their wide-spread use was the risk of bleeding complications. Regardless of this the compounds according to the invention can also be used advantageously in acute diseases such as cardiac infarction or pulmonary embolism.

The plasminogen activators used according to the invention can be produced by methods familiar to a person skilled in the art in eukaryotic or prokaryotic cells. 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 a subject matter of the disclosure of such production processes. Mutations can be introduced by oligonucleotide directed site-specific mutagenesis into the cDNA of t-PA or a derivative thereof. Site-specific mutagenesis is for example described by Zoller and Smith (1984), modified according to T.A. Kunkel (1985) and Morinaga et al. (1984). The PCR mutagenesis process is also suitable which is for example described in Ausubel et al. (1991).

The nucleic acid obtained in this manner is used to express the plasminogen activator according to the invention when it is present on a suitable expression vector for the host cell used.

CA 022627=71 1999-02-01 The nucleic acid sequence of the protein according to the invention can be additionally modified. Such modifications are for example:

Modification of the nucleic acid sequence in order to introduce various recognition sequences for restriction enzymes in order to facilitate the steps of ligation, cloning and mutagenesis.

Modification of the nucleic acid sequence in order to incorporate preferred codons for the host cell.

Extension of the nucleic acid sequence by additional regulation and transcription elements in order to optimize the expression in the host cell.

All further process steps for the production of suitable expression vectors and for the expression are state of the art and familiar to a person skilled in the art. Such methods are described for example in 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.

The production of the glycosylated plasminogen activators used according to the invention is carried out in eukaryotic host cells. The production of the non-glycosylated plasminogen activators used according to the invention is carried out either in eukaryotic host cells in which the glycosylated product that is initially obtained has to be de-glycosylated by methods familiar to a person skilled in the art or preferably by expression in non-glycosylating host cells particularly preferably in prokaryotic host cells.

E. coli, streptomyces spec. or bacillus subtilis are for example suitable as prokaryotic host org~ni.cm~. In order to produce the protein according to the invention the prokaryotic cells are fermented in the usual manner and the protein is isolated in the usual manner after lysing the bacteria. If the protein is produced in an inactive forrn (inclusion bodies) it is solubilized and renatured according to methods familiar to a person skilled in the art. It is also possible to excrete the protein as an active protein from the microorg~ni~m~ according to methods familiar to a person skilled in the art. A suitable expression vector 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 either secreted into the medium (in the case of gram-positive bacteria) or into the CA 022627~1 1999-02-01 periplasmatic space (in the case of gram-negative bacteria) in this process. It is expedient that a sequence is present between the signal sequence and the sequence coding for the t-PA
derivative according to the invention which codes for a cleavage site which enables the protein to be cleaved 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 which are later used for the expression. Suitable plasmids as well as the minimum requirements for such a plasmid (e.g. origin of replication, restriction cleavage sites) are familiar to a person skilled in the art. Instead of a plasmid it is also possible within the scope of the invention to use a cosmid, the replicative double-stranded form of phages (~, M13) or other vectors known to a person skilled in the art.

When plasminogen activators according to the invention are produced in prokaryotes without secretion it is preferable to separate the inclusion bodies that form from the soluble cell particles, to solubilize the inclusion bodies containing the plasminogen activator by treatment with denaturing agents under reducing conditions, subsequently to derivatize with GSSG and to renature the plasminogen activator by adding GSH and denaturing agents in non-denaturing concentration or L-arginine. Such processes 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 processes for isolating 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 by affinity chromatography and particularly preferably by an adsorber column onto which ETI (erythrina trypsin inhibitor) isimmobilized. Sepharose~) is for example used as a support material. The advantage of purifying by means of an ETI adsorber column is that the ETI adsorber column material can be directly loaded with the concentrated renaturation mixture even in the presence of arginine concentrations as high as 0.8 mol/l. The plasminogen activators according to the invention are preferably purified by means of an ETI adsorber column in the presence of 0.6 - 0.8 mol/l arginine. The solution used in this process preferably has a pH of over 7 particularly preferably between 7.5 and 8.6.

CA 022627~1 1999-02-01 The plasminogen activators according to the invention are eluted from the ETI column by lowering the pH in the presence as well as in the absence of arginine. In this process the pH
value is preferably in the acid range particularly preferably between pH 4.0 and 5.5.

A further subject matter of the invention is a pharmaceutical composition cont~ining, a thrombolytically active protein according to the invention, wherein the protein preferably contains the protease domain and optionally the kringle 2 domain of human tissue-type plasminogen activator as the only structure producing the thrombolytic activity.
The plasminogen activators used according to the invention can be formulated in a manner familiar to a person skilled in the art to produce therapeutic agents wherein the compounds according to the invention are usually combined with a pharmaceutically acceptable carrier.
Such compositions typically contain an effective amount of 0.1 - 7 mg/kg preferably 0.7 - 5 mg/kg and especially preferably 1 - 3 mg/kg body weight as the 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 which is used to prepare an isotonic solution. In addition buffers such as arginine buffer, phosphate buffer can be used to stabilize a suitable pH value (preferably 5.5 - 7.5). The level of the dosage of the compounds according to the invention can be determined without difficulty by any person skilled in the art. It is for example dependent on the type of application (infusion or bolus) and the duration of the therapy. Due to their extended half-lives (with respect to the 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 - lO00 mg of the compound according to the invention, a substance enhancing the solubility of the plasminogen activator (such as e.g. arginine) and buffer. The a~mini~tration is preferably intravenously but also subcutaneously, intramuscularly or intraarterially. In addition plasminogen activators according to the invention can be infused or applied locally.

The compounds according to the invention can be ~mini~tered 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 quite especially preferred. In addition it is also possible to a~mini.~ter the compounds according to the invention as an infusion over a period of 1 h - 2 days.

CA 022627~1 1999-02-01 The compounds according to the invention are particularly suitable for the treatment of all thromboembolic diseases such as for example acute cardiac infarction, brain infarction, pulmonary embolism, deep leg vein thrombosis, acute arterial occlusion etc.. The compounds according to the invention are particularly preferably used to keat subchronic thromboembolic diseases in which a longer thrombolysis has to be carried out.

It is preferable to use the compounds according to the invention in combination with an inhibitor of coagulation (anticoagulant) such as e.g. heparin and/or an inhibitor of platelet aggregation which increases the vasodilatory effect with few side effects. The ~(lmini.stration of anticoagulants can take place simultaneously or at a different time to the ~mini.stration of the compound according to the invention. The addition of substances stimulating the blood flow or substances improving the microcirculation is also preferred.

The following examples, publications, the sequence listing and the drawings further elucidate the invention the protective scope of which derives from the patent claims. The described processes are to be understood as examples which still also describe the subject matter of the invention after modifications.

By "r-PA" is understood in the following a recombinant plasminogen activator which consists of the domains K2 and P of human t-PA. The production of such plasminogen activators is described in U.S. patent 5,223,256 for instance.

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

Figure 1 is a schematic representation of the plasma clot penetration and lysis model. In order to avoid the plasma coagulating due to shear strain, the pressure was produced by a buffer chamber (hatched area). Mixing of the buffer with the plasma over the clot (dotted area) was avoided by incorporating a bubble trap.
1: buffer store; 2: peristaltic pump; 3: bubble trap; 4: injection syringe for the fibrinolytic agent; 5: pipette tip with clot (cross-hatched area); 6: tube clamp; 7: pressure element.

Figure 2 shows the cleavage of r-PA (F274P, K277V) (A) and r-PA (B) respectively by thrombin. (For details see example 8).

CA 022627~1 1999-02-01 Figure 3 shows the cleavage of r-PA (F274P, K277V) (A) and r-PA (B) respectively by plasmin. (For details see example 9).

Figure 4 shows the cleavage of r-PA (P272V, F274P, K277V) by thrombin. (For details see example 10).

Example 1 Recombinant production of the compounds according to the invention a) Construction of the expression plasmid The initial plasmid pA27fd described in EP 0 382 174 contains the following components:
tac promoter, lac operator region containing an ATG start codon, the coding region for the t-PA mutein comprising a kringle 2 domain and the protease domain and the fd transcription terminator. The initial vector is the plasmid pkk 223-3.

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

The oligonucleotides that were used and the mutations that resulted from them are listed in table 1.

The heteroduplex preparation was transformed together with the plasmid pUBS520 in E. coli (Brinkm~nn et al., Gene 85 (1989) 109). The transformants were selected by adding ampicillin and kanamycin (50 ,ug/ml in each case) to the nutrient medium.

The plasmids resulting from each of the preparations are also shown in table 1.

b) Expression in E. coli In order to examine the expression yield the E. coli cont~ining the respective plasmid (see table 1) and pUBS520 was cultured in LB medium (Sambrook et al., 1989, MolecularCloning, Cold Spring Harbor) in the presence of ampicillin and kanamycin (50 ~"g/ml of each) up to an OD at 550 nm of 0.4. The expression was initiated by adding 5 mmol/l IPTG.

CA 022627~1 1999-02-01 The culture was incubated for a further 4 hours. Subsequently the E. coli cells were collected by centrifugation and resuspended in buffer (50 mmol/l Tris HCI pH 8, 50 mmol/l EDTA);
the cells were lysed by sonication. The insoluble protein fractions were collected by centrifuging again and resuspended in the above-mentioned buffer by sonication. The suspension was admixed with 1/4 volumes application buffer (250 mmol/l Tris-HCl pH 6.8, 10 mmol/l EDTA, 5 % SDS, 5 % mercaptoethanol, 50 % glycerol and 0.005 % bromophenolblue) and analysed with the aid of a 12.5 % SDS polyacrylamide gel. As a control the same preparation was carried out with a culture of E. coli containing the respective plasmids which was not induced with lPTG and separated in the gel. A distinct band with a molecular weight of about 40 kD can be seen in the preparation of the IPTG-induced culture after staining the gel with coomassie blue R250 (dissolved in 30 % methanol and 10 % acetic acid); this band is not present in the control preparation.

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

Table 1 Mu t a t i o l- s i t e s ~ 01 ~ o n u c l e o t i d e u e d a) 272P--V, 274F ~ P, 277K--V G.-AC.AGC.CA~'.G' ~.CAG CC-.CCC.ATC._~.GGA.GG~ .CTC.TI) ptt ~A-b) 267R--S, 272P ~ F, 273Q ~ G C.l'GC.GGC.CTC .A~'C.CAG.TAC.AGC.CAG.T---.GGC.CC' .CGC.ATC.GTT.GGA.CGG.CTC.T ptt 'A-2 274F ~ P, 277K ~ V ~' c) 429K ~ Q G.GAG.CGG.CTG.CAG.GAG.GCT.CAT.G 3) pttPA-3 d) b) and c) starting from plasmid pttPA-2 and using the oligonucleotide c) pttPA-4 ~' e)417H~ T C.TAC.GGC.AAG.ACC.GAG.GCC.TTG.T4) pttPA-S
f) a) and e) starting from p asmid pttPA-I and using t le oligonucleotide e) pttPA-6 g) b) and e) starting from p asmid pttPA-2 and using t le oligonucleotide e) pttPA-7 1~-I ) SEQ ID NO: 9 2) SEQ ID NO: 10 3) SEQ ID NO: 11 4) SEQ ID NO: 12 CA 022627~1 1999-02-01 Example 2 In vivo characterization The rabbit model of jugular vein thrombolysis established by D. Collen (J. Clin. Invest. 71 (1983) 368-376) was used to examine the thrombolytic potency and efficiency of the proteins according to the invention. In this method a radioactively labelled thrombus was produced in the jugular vein of the animals. The animals were subcutaneously anticoagulated with 100 IU/kg heparin. Alteplase (recombinant wild-type plasminogen activator, "t-PA", commercially available as Actilyse(~' from the Thomae Company, Biberach, Germany), the protein described in example 1, streptokinase (commercially available as Streptase(~ from the Behring Company, Marburg, Germany) or solvent (0.2 M arginine phosphate buffer) were ~mini.stered intravenously to the rabbits.

The placebo group received an intravenous single bolus injection of 1 mg/kg solvent. The Alteplase group were intravenously aArninistered with a total dose of 1.45 mg/kg, 0.2 mg/kg thereof as an initial bolus injection, 0.75 mg/kg as a 30 minute infusion directly followed by 0.5 mg/kg as a 60 minute continuous infusion (total infusion: 90 min.). 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. In the case of Alteplase and Streptokinase these are recognized standard rules.

Two hours after the start of the therapy the residual thrombus was removed and the extent of thrombus dissolution (thrombolysis) was determined by means of the decrease of the radioactivity in the thrombus. Blood samples to obtain plasma were taken before therapy and two hours after the start of therapy. The activated thromboplastin time was measured by means of standard procedures. In addition the blood loss due to the thrombolytic therapy was quantified. For this a defined skin incision of 4 cm length and 0.3 cm depth was made on the thigh of the animals before ~ministering the thrombolytic agents with the aid of a template and a scalpel. The bleeding which this caused came to a standstill due to natural coagulation.
After the therapy had begun a sponge was placed on the wound which absorbed the blood from the bleeding which started again due to the thrombolysis. The amount of blood which escaped was measured by weighing the sponge (after subtracting its net weight) and thus the extent of the bleeding side effect was described.

CA 022627~1 1999-02-01 Alteplase as well as the proteins according to the invention of example I are highly active thrombolytic substances and significantly dissolve the thrombi in comparison to the solvent control.

Example 3 Comparison of the clot Iysis activity a) Procedure of the clot lysis assay In the clot lysis assay the activity of t-PA and the recombinant proteins of example 1 was determined.
The sample was adjusted to the protein concentration required in each case by adding buffer (0.06 M Na2HPO4, pH 7.5, 5 mg/ml BSA (bovine serum albumin), 0.01 %
Tween~) 80). 0.1 ml sample was admixed with 1 ml human fibrinogen solution (IMCO) (2 mg/ml 0.006 M Na2HP04, pH 7.4, 0.5 mg/ml BSA, 0.01 % Tween(~ 80) and incubated for 5 min. at 37~C. Subsequently 100 ~11 each of a plasminogen solution (10 IU/ml 0.06 M
Na2HP04/H3P04, pH 7.4, 0.5 mg/ml BSA, 0.01 % Tween(~) 80) and a thrombin solution (30 U/ml 0.06 M Na2HP04, pH 7.4, 0.5 mg/ml BSA, 0.01 % Tween(~) 80) were added and the test mixture was again incubated at 37~C. After two minutes a Teflon@~ ball was placed on the fibrin clot and the time taken for the ball to reach the bottom of the test tube was stopped.

b) Determination of the activity in a dynamic plasma model In this dynamic plasma model the substances according to the invention are examined under conditions which are quite similar to the in vivo conditions. The substances are added to the plasma via the clot, under the action of a peristaltic pressure which is similar to the pressure caused by the heart beat.

200 ~11 citrate plasma were mixed with 20 !11 of a 0.25 mol/l CaC12 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, GER). The pipette tip was held vertically for 2 min. at 23~C, incubated for 60 min. in 0.01 mol/l Tris/HCl, pH 7.4, 0.15 mol/l NaC12, 0.025 mol/l CaC12, 0.01 % Tween(~ 80 and placed in the clot lysis apparatus. The clot Iysis activity was determined in a switching system (figure 1) composed of elastic tubes. The flow is produced by a peristaltic pump and divided into two parallel branches. Branch A contains the 1 ml pipette tip filled with the plasma clot which closes this branch. Branch B is a blind line which CA 022627~1 1999-02-01 runs parallel to branch A. The pressure in branch B was adjusted to 10 mbar by means of a tube clamp. Plasma (1 ml) was applied to the clot. The pump was switched on and the stability of each individual clot was checked for 15 minutes. The fibrinolytic agent (final plasma concentration between 0.5 and 10 and 20 ~lglml for the proteins of example 1 or CHO-t-PA) was carefully injected into the plasma by means of a 1 ml tuberculin syringe with a hypodermic needle for intramuscular injection (Braun, Melsungen, GER). The clot lysis time was calculated as the time difference between the addition of the fibrinolytic enzyme and the pressure reduction to 50 % of the value before addition of the fibrinolytic agent. The pressure was determined by means of a water-calibrated piezoelectric pressure detection system and documented by means of a computer-aided documentation programme.

c) Clot penetration in a static model 800 ,ul human citrate plasma (healthy donor) is mixed with 75 1ll Ca buffer (50 mmol/l Tris/HCl, pH 7.2, 0.25 moVl CaC12), 20 ~1 gelatin solution (10 % w/v in 0.9 % NaCl) and 100 ml thrombin solution (8 U/ml, 0.05 mol/l sodium citrate/HCl, pH 6.5, 0.15 mol/l NaCl).
800 ,ul of this mixture is carefully transferred to a 2 ml column (Pierce, Rockfort, IL, USA).
A plasma clot is formed by incubating for three hours at 37~C. 2 ml buffer (0.008 mol/l Na2HP04, 0.001 mol/l KH2P04, 0.003 mol/l KCl, 0.137 mol/l NaCl, 0.1 % bovine serum albumin, 0.01 % Tween(~) 80) is adjusted with the plasminogen activator, which was previously inhibited with Glu-Gly-Arg-chloromethyl ketone, to the desired concentrations (0, 0.5, 1, 2 and 3 ~lg/ml) and 1 ml of this solution was 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/l Na2HP04, 0.001 mol/l KH2P04, 0.003 mol/l KCl and 0.137mol/1 NaCl) and the protein is fixed by adding 2 ml glutaraldehyde solution in PBS. Subsequently the clot surface is washed with 2 ml 50 mmolll Tris/HCI, pH 8.0 and incubated with 1 ml peroxidase-labelled polyclonal antibodies against t-PA (250 mU/ml). After washing the clot with 1 ml PBS, the antibody-bound protein is determined by incubating with 3-amino-9-ethylcarbazol which is converted by peroxidase into an insoluble red pigment.

Plasminogen activators according to the invention are not concentrated at the surface of the clot but rather penetrate into the clot and disperse uniformly. The intensity of the immunologically stained part of the clot increases with increasing concentrations of the plasminogen activators according to the invention in the plasma.

CA 022627~1 1999-02-01 Example 4 Comparison of fibrin binding In this example the thrombolytically active proteins of example 1 are examined for their ability to bind to fibrin and also compared with Alteplase with respect to this property.

Samples of Alteplase and a protein according to the invention were prepared as solutions of 1.5 ~lg protein/ml. Subsequently samples (100 ~l) of the thrombolytically active protein were each mixed with 770 ~l buffer (0.05 M Tris/HCl, pH 7.4, 0.15 NaCl, 0.01 % Tween3 80), 10 ',11 bovine serum albumin solution (100 mg/ml), 10 ,ul aprotinin (3.75 mg/ml), 10 !11 bovine thrombin (concentration 100 U/ml) and increasing amounts of fibrinogen (10 ~Lg/ml to 300 ,ug/ml). All solutions were aqueous. It is known that thrombin converts fibrinogen into an insoluble fibrin clot.

The components were mixed and incubated for 1 hour at 37~C. Subsequently the supernatant was separated from the fibrin clot by centrifugation (15 minutes, 13,000 rpm, at 4~C) and the amount of the plasminogen activator protein present in the supernatant was determined by a standard ELISA.

Example 5 Comparison of the plasminogenolytic activity and the stimulatability A known procedure for determining the stimulatability of the plasminogenolytic activity is described by Verheijen et al. in Thromb. Haemost. 48 (1982) 266-269.

The fibrinogen fragments which act as a stimulator were prepared by treating human fibrinogen with cyanogen bromide (1 g 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 with subsequent dialysis against distilled water.

When the assay was carried out 5 ng/nl t-PA or an equivalent concentration of the proteins of example 1 was incubated in 1 ml 0.1 mol/l Tris/HCl (pH 7.5), containing 0.1 % v/v Tween(~3 80, 0.13 ~mol/l Glu-plasminogen, 0.3 mmol substrate S2251 (chromogenic substrate H-D-Val-Leu-Lys-p-nitroanilide HCl) and 120 ~lg/ml fibrinogen fragments. The mixtures were incubated for 2 hours at 25~C and the absorbance rate at 405 nm was measured against control blank values without interrupting the reaction. The cleavage of the chromogenic CA 022627~1 1999-02-01 substrate S2251 was measured as a measure of the plasminogenolytic activity of the enzyme.
The stimulatability is calculated as the activity with fibrinogen fragments divided by the activity without fibrinogen fragments.

In each case 25 !11 of the sample appropriately pre-diluted with 0.1 mol/l Tris, pH 7.5, 0.15 %
Tween@~ 80 is pipetted into a well of a microtitre plate. Subsequently 200 111 reagent mixture is added and the absorbance at 405 nm is determined against the blank value over a period of 2 hours (25 1ll, 0.1 moVl Tris, pH 7.5, 0.15 % Tween(g) 80 with 200 ~11 reagent mixture).

Asample=(Asample t~ABVt)~(Asample O-ABVO) Asample t sample value after 2 h ABV t = reagent blank value after 2 h Asample 0 sample value at time t = 0 ABV 0 = reagent blank value at time t= 0 Reagent mixture:

5 ml test buffer (0.1 moVl Tris, pH 7.5, 0.15 % Tween(~' 80) 1 ml t-PA stimulator (1 mg/ml cyanogen bromide fragments of human fibrinogen) 1 ml substrate solution (3 mmoVl S2251, H-D-Val-Leu-Lys-pNA; Chromogenix, Moelndal, SE) l ml plasminogen solution (7 U/ml plasminogen, Boehringer Mannheim GmbH).

Calculation of the stimulation factor:
In order 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. In each case the dilution should be such that approximately the same absorbance is achieved in both preparations. 1 ml H2O is added instead of 1 ml t-PA stimulator to the reaction mixture without t-PA stimulator.

The activity is measured in the same way in the absence as well as in the presence of the stimulator. The stimulation factor F is calculated as follows:

Asample with stimulator x dilution5ample with stimulator F= ------------------------------- -_ _ ______ __ _ _____ __ Asample without stimulator x dilUti~nsample without stimulator .

CA 022627~1 1999-02-01 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 induce an effective and reliable thrombolysis in a dog model of coronary thrombosis The thrombolysis caused by the proteins of example 1 produced in E. coli can be evaluated in a dog model of a thrombosis of the left coronary artery induced by electrical stimulation.

Example 7 Determination of the amidolytic activity In order to determine the amidolytic activity 200 ml buffer (0.1 moVl Tris/HCl, pH 7.5, 0.15 % Tween(g 80) and 200 ~,11 of the plasminogen activator solution (diluted with buffer to a concentration of 1 - 12 ~lg/ml) are incubated for 5 minutes at 37~C. The determin~tion is started by adding 200 1ll S2288 (6 mmoVl, 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 of the absorbance at 405 nm within the first 2.5 minlltes with an extinction coefficient for p-nitroaniline of 9750 VmoVcm.

Example 8 Cleavage of r-PA (F274P, K277V) by thrombin 1. Procedure In each case 44 llg r-PA (F274P, K277V) and r-PA (standard) were preincubated for 15 min.
at 37~C and mixed with the units of human thrombin (Sigma) stated below which was also preincubated for 15 min. at 37~C, and were incubated for 30 min. at 37~C. Subsequently the samples were mixed at a ratio of 1: 1 (v/v) with SDS sample buffer (0.125 moVl TrislHCl, pH
8.8, 4.6% (w/v) SDS, 4 moVl urea, 0.1% bromophenol blue, 0.3 moVl dithioerythritol), incubated for 3 min. at 95~C and analysed by SDS polyacrylamide gel electrophoresis.

CA 022627~1 1999-02-01 2. Results The cleavage of r-PA (F274P, K277V) by thrombin is shown in Figure 2. The data demonstrates that increasing amounts of thrombin convert r-PA (F274P, K277V) completely into the two-chain form. The protease and kringle 2 domains of r-PA (F274P, K277V) cleaved by thrombin run at the same level as the corresponding domains of r-PA in the two-chain form (r-PA (tc)) prepared by plasmin digestion.

Unlike r-PA (F274P, K277V) (A), under the conditions described r-PA (B) which is used here as a standard is not cleaved by thrombin.

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 NIH 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 NIH units thrombin Lane 6: r-PA + 0.55 NIH units thrombin Lane 7: r-PA + 2.74 NIH units thrombin Lane 8: thrombin (5 NIH units) Lane 9: molecular weight standard*

CA 022627~1 1999-02-01 .

*) Molecular weight standard: lysozyme (14,307 Da), soybean trypsin inhibitor (20,100 Da), triose phosphate isomerase (26,626 Da), aldolase (39,212 Da), gh~t~m~te dehydrogenase (55,562 Da), fructose-6-phosphate-kinase (85,204 Da), 13-galactosidase (116,353 Da), a-2-macroglobulin (170,000 Da).

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

Example 9 Cleavage of r-PA (F274P, K277V) by plasmin l. Procedure In each case 25 ,~g r-PA (F274P, K277V) and r-PA (standard) were preincubated for 15 min.
at 37~C and mixed with the units of plasmin (human) stated below which was also preincubated for 15 min. at 37~C, and were incubated for 10 min. at 37~C. Subsequently the samples were mixed at a ratio of 1: 1 (v/v) with SDS sample buffer (0.125 moVl Tris/HCl, pH
8.8, 4.6% (w/v) SDS, 4 moVI urea, 0.1% bromophenol blue, 0.3 moVl dithioerythritol), incubated for 4 min. at 95~C and analysed by SDS polyacrylamide gel electrophoresis.

2. Results The cleavage of r-PA (F274P, K277V) (A), and of r-PA (B) used here as a standard, by plasmin is shown in Figure 3.

The data demonstrates that r-PA (B) is converted into the two-chain form by incubation with increasing amounts of plasmin. Under the conditions employed the applied amount of r-PA is converted completely into the two-chain form by 25 mU plasmin.

In contrast to this, r-PA (F274P, K277V) (A) exhibits a markedly poorer cleavage by plasmin. When incubating with 0.025 U and 0.1 U plasmin, no significant cleavage of r-PA
(F274P, K277V) by plasmin is observed yet. Even when incubating 25 llg r-PA (F274P, K277V) with 25 mU plasmin, no complete cleavage occurs.

The protease and kringle 2 domains of r-PA (F274P, K277V) cleaved by thrombin run at the same level as the corresponding domains of r-PA in the two-chain form (r-PA (tc)) prepared CA 022627~1 1999-02-01 by digestion with plasmin sepharose so that it has to be assumed that cleavage of the variant takes place between the amino acids 275 and 276 (numbering according to Harris, Prot.
Engineering 1, 449-458 (1987)), as in r-PA.

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) + 0.25 mU plasmin Lane 6: r-PA (F274P, K277V) + 0.25 mU plasmin Lane 7: r-PA (F274P, K277V) + 12.5 mU plasmin Lane 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: lysozyrne (14,307 Da), soybean trypsin inhibitor (20,100 Da), triose phosphate isomerase (26,626 Da), aldolase (39,212 Da), gh~t~ te dehydrogenase (55,562 Da), fructose-6-phosphate-kinase (85,204 Da), 13-galactosidase (116,353 Da), a-2-macroglobulin (170,000 Da).

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

CA 022627~1 1999-02-01 Example 10 Cleavage of r-PA (P272V, F274P, K277V) by thrombin 1. Procedure 40 llg r-PA (P272V, F274P, K277V) were preincubated for 15 min. at 37~C and mixed with the units of bovine thrombin (Sigma) stated below which was also preincubated for 15 min.
at 37~C, and were incubated for 30 min. at 37~C. Subsequently the samples were mixed at a ratio of 1:1 (v/v) with SDS sample buffer (0.125 mol/l Tris/HCl, pH 8.8, 4.6% (w/v) SDS, 4mol/1 urea, 0.1% bromophenol blue, 0.3 mol/l dithioerythritol), incubated for 3 min. at 95~C and analysed by SDS polyacrylamide gel electrophoresis.

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

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 (P272V, F274P, K277V) + thrombin buffer Lane 6: r-PA (P272V, F274P, K277V) + 0.055 NIH units thrombin Lane 7: r-PA (P272V, F274P, K277V) + 0.55 NIH units thrombin Lane 8: r-PA (P272V, F274P, K277V) + 2.74 NIH units 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), ghlt~m~te dehydrogenase (55,562 Da), fructose-6-phosphate-kinase (85,204 Da), 13-galactosidase (116,353 Da), a-2-macroglobulin (170,000 Da).

CA 022627~1 1999-02-01 , .

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

List of References 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 Brinkm~nn 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 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. Sci. 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. Sci. 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. und Berger, A., Biochem. Biophys. Res. Commun. 27 (1967) 157 - 162 CA 022627~1 1999-02-01 Schohet, R.V., Thrombosis and Haemostasis 71 (1994) 124-128 US-Patent 5,200,340 US-Patent 5,223,256 US-Patent 5,501,853 Wen-Pin Yang et al., Biochemistry 33 (1994) 2306 - 2312 Zoller and Smith, DNA 3 (1984) 479 - 488 CA 022627~1 1999-02-01 SEQUENCE LISTING

(1) GENERAL INFORMATION:
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(A) NAME: BOEHRINGER MANNHEIM GMBH
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Gly Asp Thr Arg Ser Gly Gly Pro Gln Ala Asn Leu His . 1 5 10 (2) INFORMATION FOR SEQ ID NO: 3:
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Pro Gln Ala Asn Leu His (2) INFORMATION FOR SEQ ID NO: 4:
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(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonukleotid"

CA 022627~1 1999-02-01 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonukleotid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Claims (11)

What is claimed:

1. One-chain plasminogen activator which, based on human tissue-type plasminogen activator, is modified by deletions, mutations and/or additions of amino acids in such a way that, a) the plasminogen activator can be cleaved by thrombin and is converted by such a cleavage into the two-chain form, b) the zymogenicity is higher by a factor of at least 1.2 as compared with the human plasminogen activator and c) its fibrin binding is reduced to an extent that the plasminogen activator can penetrate by more than 50% into a blood clot.

2. Plasminogen activator as claimed in claim 1, wherein it is modified in such a way that its ability to be cleaved by plasmin between the amino acids P1 (275) and P1' (276) is reduced by more than 10 %, preferably by more than 20 %.

3. Plasminogen activator as claimed in claims 1 or 2, wherein it uniformly penetrates a blood clot.

4. Plasminogen activator as claimed in claims 1 - 3, wherein the amino acid region between G 264 and A 288 is modified in such a way that the plasminogen activator can be cleaved by thrombin.

5. Plasminogen activator as claimed in claims 1 - 4, wherein the amino acid regions 459 - 471, 417 - 425 and/or the amino acids Q 475, K 505 and/or E 506 are modified.

6. Plasminogen activator as claimed in claims 1 - 5, wherein the amino acids G 265 and/or R 267 are modified and/or at least one amino acid is inserted in the region between 264 and 267.

7. Plasminogen activator as claimed in claims 1 - 6, wherein the finger domain is deleted.

8. Plasminogen activator as claimed in claim 7, wherein the said plasminogen activator only contains the protease domain or the kringle 2 domain and the protease domain of human tissue-type plasminogen activator.

9. Use of a plasminogen activator as claimed in claims 1 - 8 for producing a pharmaceutical composition for the treatment of thromboembolic diseases.

10. Process for the recombinant production of a plasminogen activator as claimed in claims 1 - 8, wherein a prokaryotic or eukaryotic host cell is transformed with a vector which is capable of expressing the said tissue plasminogen activator, this cell is cultured and the said plasminogen activator is isolated.

11. Pharmaceutical composition of a plasminogen activator as claimed in claims 1 - 8 in a therapeutically effective amount and optionally pharmaceutical auxiliary substances, fillers or additives.