WO1999009185A1

WO1999009185A1 - Plasminogen activator with improved zymogenity

Info

Publication number: WO1999009185A1
Application number: PCT/EP1998/005055
Authority: WO
Inventors: Richard Engh; Ulrich Kohnert; Martin Renatus; Wolfram Bode; Robert Huber
Original assignee: Roche Diagnostics Gmbh
Priority date: 1997-08-13
Filing date: 1998-08-10
Publication date: 1999-02-25
Also published as: AU9162198A

Abstract

The invention relates to a plasminogen activator of the tissue plasminogen activator type, in which the amino acid area G265-F274 is partially or fully deleted. The inventive plasminogen activator has increased zymogenity with the result that the side-effects of therapeutic application are reduced.

Description

Plasminogen activator with improved zymogenicity

The invention relates to a plasminogen activator with improved zymogenicity, 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, cf. For example, the review articles T.J.R. Harris (1987) and J. Krause (1988).

Among other things, it is known about the mode of action of t-PA that fibrinolysis is partly regulated by the interaction between t-PA and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor from the serpin family). The binding of PAI-1 to t-PA takes place essentially via amino acids 296-302. A mutation in this region reduces the inhibitory influence of PAI-1 on t-PA (EL Madison et al. (1990)). Extensive studies have been carried out on the mechanism of the interaction between the amino acid region 296-302 of t-PA with PAI-1 (cf. also EL Madison, Nature 339 (1989) 721-723; RV Schohet, Thrombosis and Haemostasis 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) 5191-5201, 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 still held together by a disulfide bridge. The A chain (also called heavy chain) consists of four structural domains. The finger domain (amino acids 1-49) shows 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 Kringled domains (amino acids 87-261) are largely homologous to the fourth and fifth Kringled domains of plasminogen. The finger and kringle 2 domains of t-PA are particularly involved in fibrin binding and 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 (TJR Harris (1987) and J. Krause (1988).

The stimulability of the activity by fibrin or by fibrin cleavage products is an essential feature of t-PA, which t-PA from the other known plasminogen activators, such as. B. urokinase or streptokinase differs. 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 _ca {/ K _m ) in the presence and in the absence of fibrin. K ^ is the rate constant of the catalytic reaction and K _m is the Michaelis constant. When amino acids 292 and / or 305 are modified, the stimulability of t-PA can 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 improved proteolytic and plasminogenolytic activity, a reduced sensitivity to inhibition, an improved affinity for fibrin and / or improved fibrin dependence of the plasminogenolytic activity. The mechanism of action of t-PA in vivo is, for example, in Korninger and Collen, Thromb. Haemostasis 46 (1981) 561-565. t-PA activates plasminogen to plasmin. Plasmin splits fibrin into soluble fibrin products. Natural human tPA and also recombinantly produced t-PA are usually essentially in single-chain form. Single-chain t-PA is cleaved in the blood by protease / plasmin between amino acid 275 (arginine) and 276 (isoleucine) in two-chain t-PA, which increases activity. The two partial chains remain connected by a cysteine bridge. The amidolytic activity of single-chain 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.

The enzymatic activity of the single-chain form in therapeutic use is not negligible, which leads to undesirable effects outside the blood clot to be resolved. Dual chain t-PA has greater activity than small chain and plasmin, in the absence of fibrin, than single chain t-PA. However, in the presence of fibrin, the activities of both t-PA forms are approximately the same.

From W.F. Bennett et al., J. Biol. Chem. 266 (1991) 5191-5201 it is known that by modifying the amino acid region around amino acid 275, by modifying regions 339-351 or 410-440, in particular on amino acids 417, 428 and 433, the ratio of the activity of the single-chain and two-chain form (zymogenicity) can be increased by a factor of five to seven, compared to wild-type t-PA.

WO 90/02798 describes tP-A variants with increased zymogenicity, which contain substitutions, deletions or insertions around position 305, preferably 297-305. Furthermore, zymogenic variants are described which contain an amino acid exchange at other positions, including at position 267. The object of the invention is to provide further t-PA mutants which show improved zymogenicity.

The object is achieved by a plasminogen activator of the tissue plasminogen activator type, which is characterized in that the amino acid region G265-F274 is partially or completely deleted.

Surprisingly, it has been shown that the partial or complete deletion of this amino acid region significantly increases the zymogenicity of t-PA and t-PA mutants. This is particularly surprising since it has previously been assumed (Bennett et al., J. Biol. Chem. 266 (1991) 5191-5201) that only the exchange of amino acids leads to an improvement in zymogenicity.

The deletion in the range mentioned enables the zymogenicity to be increased by at least a factor of 2 compared to human t-PA.

According to the invention, a plasminogen activator of the tissue plasminogen activator type is to be understood as a plasminogen activator whose sequence is recognizably derived from the sequence of 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 according to 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.

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 and can be checked in a static clot-lysis model for confirmation. Such a plasminogen activator achieves high selectivity and effectiveness of thrombus dissolution in vivo with drastically reduced side effects.

The amino acid region mentioned according to the invention represents a loop, as can be seen from structural analyzes by the inventors. In the single-chain form of the protein, amino acids 265 - 274 form an "activation loop", which, in confirmation, forms a kind of lid or shield that protects the activation pocket against the ingress of water. In the two-chain form of the activated protein, this activation pocket contains a salt bridge between D 194 and the N-terminal 1 16. This salt bridge is essential for the development of proteolytic activity. In the single-chain form of the protein, the shield formed by the loop stabilizes a salt bridge between D 194 and K 156 by preventing water from entering. This structural stabilization also stabilizes the activity of the single-chain protein. The result of a shortening or complete removal of the loop is that the activation pocket becomes more accessible and an inactive conformation of the single-chain protein is stabilized.

In a preferred embodiment, the fibrin binding of the plasminogen activator according to the invention is additionally 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 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 is essentially a t-PA domain. are the Kringle 2 domain and the protease domain, but not the finger domain (WO 90/09437, US Pat. No. 5,223,256, EP-B 0 297 066, EP-B 0 196 920).

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 (no functional effective 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 split by plasmin 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 high-affinity fibrin binding typical of tPA. The combination of a lack of fibrin binding and zymogenicity increases the potency of the plasminogen activator and 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 does not penetrate the clot 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.

In a further preferred embodiment, the plasminogen activator according to the invention is additionally modified such that it cannot be inhibited by PAI-1. Such 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) 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 i.v. 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 previously only approved for extremely life-threatening diseases such as heart attack and massive pulmonary embolism, the use of such variants opens up the possibility of thrombolysis by the compounds according to the invention even in the case of less acute life-threatening diseases, such as e.g. deep vein thrombosis or arterial occlusion. 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 a main obstacle to widespread use was the risk of bleeding complications. Irrespective of this, the compounds according to the invention can advantageously also be used in the case of 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 by 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". leads. The "site-specific mutagenesis" is described, for example, by Zoller and Smith (1984), modified from TA Kunkel (1985) and Morinaga et al. (1984). The method of PCR mutagenesis, which is described, for example, in Ausubel et al. (1991).

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:

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

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

Supplementation of the nucleic acid sequence to include 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 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 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 periplasmic 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 (eg origin of replication, restriction sites) are familiar 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. When producing the plasminogen activators according to the invention in prokaryotes without secretion, it is preferred to separate the forming inclusion bodies from the soluble cell particles, to solubilize the inclusion bodies containing the plasminogen activator by treatment with denaturing agents under reducing conditions, then to derivatize them with GSSG and to add the plasminogen activator to be renatured from GSH and from denaturing agents in non-denaturing concentration or from 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 amount of 0.1-7 mg / kg, 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 used to prepare an isotonic solution. Buffers such as arginine buffers and phosphate buffers can also be used to stabilize a suitable pH (preferably 5.5-7.5). The amount of the dosage of the compounds according to the invention can be easily determined by any person skilled in the art. It depends, for example, on the type of application (infusion or bolus) and the duration of the therapy. Because of their prolonged plasma half-life, 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 improves the solubility of the plasminogen activator (such as tranexamic acid) and buffer. It is preferably used intravenously, 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 a is particularly preferred Interval between 30 and 60 minutes preferred. In addition, infusion over a longer period of time is also possible.

The compounds according to the invention are particularly suitable for the treatment of all thromboembolic disorders, such as e.g. acute heart attack, cerebral infarction, pulmonary embolism, deep leg vein thrombosis, acute arterial occlusion, etc. In particular with deep leg vein thrombosis or peripheral arterial occlusion, an infusion over several days (e.g. 1-4 days) can be advantageous. The compounds according to the invention are particularly preferably used for the treatment of subchronic thromboembolic disorders in which prolonged thrombolysis has to be carried out.

It is preferred to use the compounds of the invention in combination with an anticoagulant such as. B. heparin and / or an inhibitor of platelet aggregation, whereby the vascular opening effect is increased with minor side effects. The administration of anticoagulants can take place at the same time or with a time delay when the compound according to the invention is administered. Likewise preferred is the addition of substances that promote blood circulation or substances that 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.

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

Under rPA (ΔG265, L266) it is to be understood that amino acids 265 and 266 were deleted in the plasminogen activator consisting of domains K2 and P (amino acid name analogous to TJ Harris (1987)). Figure 1 is a schematic representation of the plasma clot penetration model. In order to avoid coagulation of the plasma induced by shear forces, the pressure was generated by a buffer compartment (hatched). Mixing of buffer and plasma over the clot (dotted) was prevented by using a bubble trap.

1st: buffer reservoir, 2nd: peristaltic pump, 3rd: bubble trap, 4th: syringe for the injection of the fibrinolytic agent, 5th: pipette tip with clot (double hatched), 6th: hose clamp, 7th: pressure element.

example 1

Recombinant preparation of the compounds according to the invention

a) Construction of the expression plasmid (deletion Δ G265, L266)

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.

Essentially, the method of Morinaga et al., Biotechnology (1984) 636 was used to introduce the deletion. 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).

The following was used as the oligonucleotide: CTCCACCTGCAGACAGTACA (SEQ ID NO: 1) 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.

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 lactose. 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-HCl pH8, 50 mmol / 1 EDTA); the cells were lysed by sonication. The insoluble protein fractions were collected by centrifugation and resuspended in the above-mentioned buffer by sonication. V * volume of application buffer (250 mmol of 1 Tris-HCl pH 6.8, 10 mmol / 1 EDTA, 5% SDS, 5% mercaptoethanol, 50% glycerol and 0.005% bromophenol blue) was added to the suspension and using a 12.5% SDS-polyacrylamide gel analyzed. 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. Example 2

In vivo characterization

The rabbit model of neck vein thrombolysis established by D. Collen (J. Clin. Invest. 71 (1983) 368-376) was used to test the thrombolytic potency and efficiency of the proteins according to the invention. A radiolabelled thrombus was created in the animals in the cervical 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), which in Example

1 described protein, streptokinase (commercially available as Streptase® from Behring, Marburg, Germany) or solvent (0.2 M arginine phosphate buffer) were administered intravenously to the rabbits.

The placebo group received a single bolus intravenous injection of 1 mg / kg solvent. The Alteplase group received a total dose of 1.45 mg / kg intravenously, 0.2 mg / kg as an initial bolus injection, 0.75 mg / kg as a 30-minute infusion, immediately followed by 0.5 mg / kg as 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. 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. For this purpose, a defined skin incision of 4 cm in length and 0.3 cm in depth was added to the animals on the thigh before administration of the thrombolytics using a template and a scalpel. The resulting bleeding stopped due to natural coagulation. After the start of therapy, the wound was treated Sponge laid, which sucked up the blood from the bleeding newly started by thrombolysis. The amount of blood leaked was measured by weighing the sponge (after deducting its own weight) and thus the extent of the bleeding side effect was described.

Both Alteplase and the proteins according to the invention from Example 1 are thrombolytically highly active substances and, compared to the 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 prepared by adding buffer (0.06 M Na2HP04, pH 7.4, 5 mg / ml BSA (bovine serum albumin), 0.01% Tween® were mixed 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 5 min. incubated at 37 ° C. Then 100 ul of a plasminogen solution

(10 IU / ml 0.06 M Na ₂ HPθ4 / H ₃ Pθ4, pH 7.4, 0.5 mg / ml BSA, 0.01% Tween® 80) and one

Thrombin solution (30 U / ml 0.06 M Na ₂ HPO4, 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, one

Place the Teflon® ball on the fibrin clot and stop the time until the ball has reached the bottom of the test tube. b) Clot penetration in the static model

800 μl human citrate plasma (healthy donor) are mixed with 75 μl Ca buffer (50 mmol / 1 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 / l NaCl). 800 μl of this mixture are carefully transferred into a 2 ml column (Pierce, Rockfort, IL, USA). A plasma clot is formed by incubation for three hours at 37 ° C. 2 ml buffer (0.008 mol / 1 Na ₂ HPO, 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.) With the plasminogen activator, which was previously inhibited with Glu-Gly-Arg-chloromethyl ketone , 2 and 3 μg / ml) 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 ₂ HPO ₄ , 0.001 mol / 1 KH ₂ PO, 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 of Example 1 are tested for their ability to bind to fibrin and compared with this with Alteplase.

Samples of Alteplase and a protein according to the invention were prepared as solutions of 1.5 μg protein ml. Subsequently, samples (100 μl) of the thrombolytically active protein were each treated 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 of the solutions were aqueous. Thrombin is known to convert fibrinogen to 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 an ELISA using its own standard curve for each variant.

Example 5

Comparison of plasminogenolytic activity and stimulability

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

The fibrinogen fragments acting as 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, followed by Dialysis against distilled water. When carrying out the assay, 5 ng / nl tPA 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 HC1) 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.

25 μl each 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 at 405 nm over a period of 2 h is determined against the blank value (25 μl, 0.1 mol / 1 Tris, pH 7.5, 0.15% Tween® 80 with 200 μl reagent mixture ).

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

^E sample t ⁼ sample value after 2 h

^E BV t ⁼ reagent blank after 2 h

^E Sample 0 ⁼ sample value at time t = 0 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 stimulator ^x dilution sample with stimulator _F =

^E sample without stimulator ^x thinning sample 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® 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 to add 200 μl S2288 (6 mmol / l, HD-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

Determination of the zymogenicity of r-PA (ΔG265, L266)

1. Preparation of r-PA (ΔG265, L266)

5.4 mg of r-PA (ΔG265, L266) are converted into the two-chain form by incubation with 2 ml of plasmin-Sepharose (5 mg of plasmin ml of Sepharose) (45 min at RT) (r-PA (ΔG265, L266). Sepharose is removed by centrifugation.

2. Determination of the zymogenicity of r-PA (ΔG265, L266)

The amidolytic activity of r-PA (ΔG265, L266) and t-PA was determined as described in Example 7. The zymogenicity was calculated as the quotient of the specific activity of the two-chain form and the single-chain form and is significantly increased compared to t-PA.

R e f e r e n z l i s t e

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

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

EP-A 0 382 174

EP-A 0 245 100

EP-A 0 302 456

EP-A 0 400 545

EP-B 0 196 920

EP-B 0 297 066

EP-A 0 219 874

EP-A 0 241 022

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., Nature 339 (1989) 721-723

Morinaga et al., Biotechnology 21 (1984) 634

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

Paoni, NF, Thrombosis and Haemostasis 70 (1993) 307-312 Refmo, CJ, 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, R.V., Thrombosis and Haemostasis 71 (1994) 124-128

U.S. Patent 5,223,256

U.S. Patent 5,501,853

WO 90/02798

WO 90/09437

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: Plasminogen activator with improved zymogenicity

(iii) NUMBER OF SEQUENCES: 1

(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 97113937.3

(B) REGISTRATION DAY: 13-AUG-1997

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LONG: 20 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: 1: CTCCACCTGC AGACAGTACA 20

Claims

Patent claims

1. Plasminogen activator of the tissue plasminogen activator type, characterized in that the amino acid region G265-F274 is partially or completely deleted.

2. Plasminogen activator according to claim 1, characterized in that its fibrin binding is additionally reduced to such an extent that the plasminogen activator can penetrate more than 50% into a blood clot.

3. Plasminogen activator according to claims 1 or 2, characterized in that the finger domain is deleted or functionally ineffective.

4. Plasminogen activator according to claims 1-3, characterized in that said plasminogen activator contains only the protease domain or the Kringel 2 domain and the protease domain of human plasminogen activator.

5. Use of a plasminogen activator according to claims 1-4 for the manufacture of a pharmaceutical composition for the treatment of thromboembolic disorders.

6. A process for the recombinant production of a plasminogen activator according to claims 1-4, characterized in that it transforms a prokaryotic or eukaryotic host cell with a vector which is capable of expressing said tissue plasminogen activator, this cell is grown and said plasminogen activator is isolated.

7. Pharmaceutical composition of a plasminogen activator according to claims 1-4 in a therapeutically effective amount and optionally pharmaceutical auxiliaries, fillers or additives.