GB2247022A - Pro-urokinase derivatives - Google Patents

Abstract

Derivatives of the human fibrinolytic enzyme pro-urokinase and its analogues having decreased affinity for plasmatic inhibitors are described together with the process for preparing them by recombinant DNA technology. These derivatives demonstrate better enzymatic and fibrinolytic properties than natural pro-urokinase, so providing improved thrombolytic treatment. They are characterized by negatively charged residues in those sites of the pro-urokinase potentially responsible for interaction with plasmatic inhibitors, the presence of the negative charge creating a greater resistance to those inhibitors. Those pro-urokinase derivatives can be modifications either of human pro-urokinase itself or of its analogues, such as mutant- by substitution, deletion, insertion or inversion of human pro-urokinase or of the corresponding enzymes obtained from species other than human, or mammals in general. <IMAGE>

Description

q 1 PRO-UROKINASE DERIVATIVES The blood coagulation or hemostatic process

is of vital importance.

In this respect, the formation of a coagulum following tissue damage prevents excessive loss of blood while at the same time reducing the possibility of entry of potentially pathogenic microorganisms.

When the hemorrhage has been arrested and the tissue repaired, the coagulum is spontaneously dissolved by the action of a fibrinolytic system. This latter, like the coagulation system, consists of a series of enzymes and cofactors which on mutual activation result in the final formation of plasmin, which is able to degrade the insoluble constituent fibrin mass of the coagulum into soluble fragments (1).

There are however pathological situations in which a coagulum, formed within a blood vessel not necessarily to arrest a hemorrhagic process but as a response to the pro-coagulating stimulus represented by morphofunctional alterations of the vascular wall, progressively reduces its lumen. In this situation, defined as thrombosis, an insufficiency in the fibrinolytic system can lead to a reduction in blood flow within the vessel, with hypoxic or anoxic consequences for the tissues concerned (2).

Cardiac arrhythmia deriving from thromboembolization, myocardial 1 1 1 1 2 infarction, angina pectoris, pulmonary embolism, ictus, deep venous thrombosis and occlusive peripheral arteriopathy are clinical manifestations of this alteration in the functioning of the menostatic and fibrinolytic system as they derive from the occlusion of one or more blood vessels as the result of the development of a thrombus.

These disorders represent the most important cause of death in industrial countries. In a survey carried out in the United States, Japan, West Germany, United Kingdom, France, Italy and Spain it was found that 46.3% of all deaths in the population were attributable to thrombolytic causes.

To reduce the incidence of death following a thrombotic phenomenon, researchers and pharmaceutical companies throughout the world have continually worked towards the identification and development of medicaments able to promote thrombus lysis.

These medicaments are known as fibrinolytic agents or more commonly thrombolytic agents.

The first commercially available thrombolytic agents were streptokinase (SK) and urokinase (UK) (3, 4).

Their effectiveness in anticoagulative therapy in the treatment of thromboembolic disorders (deep venous thrombosis and pulmonary embolism) has been widely demonstrated by numerous clinical trials conducted during the last decade (3, 5, 6, 7, 8).

Notwithstanding their effectiveness the clinical use of these thrombolytic agents has always been on a small scale and is limited to specialist unit centres, mainly because of the risk of hemorrhagic complications associated with the treatment.

SK and Uk are today known as 1st generation thrombolytic agents.

In contrast, the tissue plasminogen activator (t-PA) (9) and more i:

i 1 1 ii 3 recently human pro-urokinase (pro-UK) (10), both natural proteins, are weak activators of circulating plasminogen but strong activators of the plasminogen linked to fibrin coagula. In other words, these molecules do not cause systemic degradation of the coagulation factors or of alpha 2- antiplasmin, and their clinical use therefore presents less hemorrhagic risk (11, 12, 13).

The present invention relates to pro-urokinase derivatives with better enzymatic and fibrinolytic properties than pro-UK and therefore offering the possibility of alternative thrombolytic treatment.

Pro-UK is a protein with a single-chain structure comprising 411 amino acids, 12 disulphide bridges, glycosylated in position 302 and with a molecular weight of 54 M (14).

It is proteolytically inactive in vitro and can be transformed into the active form by plasmin which specifically hydrolyses the peptide bond between the amino acids lys 158-ile 159 to generate a two-chain structure joined by a disulphide bridge (UK).

Chain A (20,000 daltons) contains 157 amino acids and a domain known as a kringle. This chain also comprises the domain responsible for binding to a cell receptor (15) Chain B (30,000 daltons) consists of 253 amino acids and contains the catalytic domain.

In the pro-UK molecule the plasmin is able to also produce hydrolysis between the amino acids glu 143-1eu 144 to generate a biologically active low molecular weight molecule (16).

This second cutting site has a lower specificity for plasmin than the first.

For a better understanding of the present invention, reference is made hereinafter to the accompanying drawings in which:

4 Figure 1 is a schematic representation of human pro-urokinase and its possible disulphide bridges. The bidimensional structure is obtained by homology with other serin proteases. The arrows indicate the cutting points into two-chain urokinase of high and low molecular weight.

Figure 2 is a schematic representation of the plasmid pFC16 used for expressing the coding gene for human pro-urokinase in E. coli. The human pro-urokinase derivatives were produced with strictly isogenic plasmids.

Figure 3 is a scheme of the mutagenesis approach used for isolating the coding genes for the human prourokinase derivatives, Six primers were synthesized. Of these, 02, 03 and 06 contain the desired modifications, ie 138-Glu, 139-Glu and 303Glu respectively. 02, 03 and 04 also carry a further modification creating a MroI restriction site, which however does not alter the amino acid sequence of the human pro-urokinase.

Figure 4 is a summary scheme of the genetic manipulations involved in isolating the plasmids pFC146, pFC147 and pFC148.

Figures 5(a) and 5(b) show the percentage of residual activity (vertical axis) at different concentrations (ng) of PAI-1 irkdbitor (horizontal axis) for rec pro-UK and for the mutant pro-UK-303-Glu according to the invention.

Three domains can be distinguished in the pro-UK structure (Figure 1), namely: the finger the kringle the proteolytic region.

1 1 1 The finger (aa. cysll-ser47) contains the site of interaction between the molecule and the specific cell receptor for UK.

The kringle (aa. lys48-lysI35) is very similar to those in the plasminogen and t-PA molecules, assigned to fibrin binding. However in contrast to these latter it seems to have no specific affinity for fibrin.

The proteolytic region (aa. lys136-leu411) contains the site of interaction with plasminogen.

Using site-specific mutagenesis techniques we effected various amino acid substitutions within the proteolytic region.

We unexpectedly found that derivatives obtained by introducing negatively charged amino acid residues instead of the serines in position 138 and/or 139 and/or 303, and then converted into the active two-chain form, prove to be more resistant to inactivation by plasmatic inhibitors such as PAI1 (plasminogen activator inhibitor 1) than pro-UK.

These derivatives however retain the enzymatic and fibrinolytic properties of pro-UK, and hence offer the possibility of improved fibrinolytic treatment by virtue of the fact that their respective enzymatic activity is less sensitive to inactivation by plasmatic inhibitors.

The present invention relates to derivatives of the human fibrinolytic enzyme pro-urokinase (pro-UK) or its analogues, and to the process for their preparation by recombinant DNA techniques.

The invention also covers the structural coding genes for said derivatives, the plasmidic vectors containing said genes and suitable bacterial hosts transformed by said vectors.

The invention therefore firstly provides derivatives of human pro- 6 urokinase or its analogues, characterized by a lesser affinity towards plasmatic inhibitors such as PAI-1 (plasminogen activator inhibitor) and the maintaining of the enzymatic and fibrinolytic properties of the natural molecule.

The human pro-urokinase analogues can for example be mutants by deletion, insertion or inversion of human pro-urokinase, or the corresponding natural or synthetic enzymes obtained from species other than human, for example bovine, ovine or mammal in general.

In particular, the invention relates to derivatives of human prourokinase or its analogues in which between one and three amino acid residues present in the protein sequence have been replaced by a like number of negatively charged amino acids. In particular, the serine residues in position 138 and/or in position 139 and/or in position 303 are replaced by aspartic acid or glutamic acid, preferably glutamic acid.

The present invention also provides a method for producing the described derivatives by recombinant DNA techniques.

According to this method said derivatives are prepared by sitespecific mutagenesis of the coding gene for pro-urokinase or its analogues, the mutant genes then being introduced into a suitable expression vector able to direct the synthesis of the new derivatives in a suitable host microorganism.

The invention further provides the coding genes, comprising chemically synthesized polynucleotides, for the aforedefined prourokinase derivatives.

The invention also provides the recombinant expression vectors containing said genes and able to direct the synthesis of said derivatives.

The invention also provides the host micro-organisms transformed by said vectors and hence able to produce said derivatives.

1 1 l 1 i 1 i 1 i 1 1 1 1 1 1 1 i 1 1 1 7 Finally, the invention covers the pharmaceutical compositions containing the aforedefined pro-urokinase derivatives together with one or more pharmacologically acceptable excipients and/or diluents and/or carriers.

Said pharmaceutical compositions can be prepared in a conventional manner using conventional processes and ingredients.

In particular, the pro-urokinase derivatives according to the present invention can be administered parenterally during the course of antithrombotic therapy. The enzyme dose used for the treatment can for example be a total of between 10 and 100 mg in the form of a bolus in a single administration, or in the form of a continuous intravenous infusion for about 12 hours in a total quantity of about 10-100 mg/hour.

As examples of this invention we prepared pro-urokinase derivatives in which the serines are replaced by glutamic acid residues.

The specific examples are: 1) a human pro-urokinase derivative in which the serine in position 138 is replaced by glutamic acid. This derivative, named proUK-138-Glu, was produced in recombinant Escherichia coli cells. 2) a human pro-urokinase derivative in which the serine in position 139 is replaced by glutamic acid. This derivative, named proUK-139-Glu, was also produced in recombinant Escherichia coli cells. 3) a human pro- urokinase derivative in which the serine in position 303 is replaced by glutamic acid. This derivative, named proUK-303-Glu, was also produced in recombinant Escherichia coli cells.

Further human pro-urokinase derivatives with other or greater negative charges in the 138 and 139 or 303 regions or possible combinations of these are also contemplated in the present invention. These modifications can also be made to other 8 derivatives or mutants of human pro-urokinase and not only to the intact molecule. The modifications can also be made to prourokinase other than human (bovine, ovine etc.).

The new human pro-urokinase derivatives were prepared by the genic amplification technique using the enzyme Taq Polymerase (17). This technology is based on the thermostable properties of the enzyme, which by a series of thermal denaturation, renaturation and elongation cycles in vitro, enable specific DNA fragments to be amplified starting from a template DNA and two specific primers.

The various site-specific modifications were produced by introducing the appropriate variations into the sequence of one of the two primers. The template used was the human pro-urokinase vector pFC16 (18) (Figure 2). In this plasmid the human prourokinase gene was placed under the control of regulator sequences able to promote its expression in Escherichia coli.

This plasmid and its characteristics have already been described in a preceding patent application (18).

By the amplification process, DNA fragments are obtained which are homologous with the template except for the mutation as previously introduced at the primer level.

By inserting the new fragments into the original plasmid as replacement for the analogous unmutated fragment, new expression plasmids isogenic with pFC16 are directly constructed, able to promote the production of the new derivatives in Escheichia coli strains. The detailed construction of these new plasmids is indicated in the captions of Figures 3 and 4 and in the following Materials and Methods description, under "Mutagenesis" and "Expansion of the Mutant Genes".

Mutagenesis: The three human pro-urokinase derivatives specifically described in this patent application were constructed i 1 i 1 1 1 1 i 1 1 9 form the plasmid pFC16 containing the human pro-urokinase gene, by in vitro genic amplification using the enzyme Taq polymerase. Figure 3 shows schematically the strategy used in constructing the oligonucleotides used as primers. In the case of the mutants proUK-138-Glu and proUK-139-Glu, because of subsequent subcloning problems it was necessary to effect the amplification of two separate fragments with the introduction of a new MroI restriction site at the junction point (see Figure 3). Although this site causes a variation at the gene nucleotide sequence level, it in no way alters the amino acid sequence of the human pro-urokinase.

The amplification process was effected using Taq polymerase of Perkin Elmer Cetus following the conditions suggested by the supplier. Specifically, 100 pl of a mixture containing 1 ng of DNA template (pFC16), 8 ng/pl of the respective primers, 0.2 mM of dNTP, 50 mM of KC1, 10 mM of Tris pH 8.3, 10 MM Of MgC121 0.01% Of gelatin and 2 units of Taq polymerase (Perkin Elmer Cetus) were subjected to 30 thermal cycles of 1 min 30 sec at 92'C and 2 min 30 sec at 72'C.

On termination of the amplification process the various amplified fragments were digested with suitable restriction enzymes (Figure 4) and subcloned in the vectorial portion BgIII-MroI of the pFC16. In this manner three new expression vectors were obtained and were classified respectively as pFC146, pFC147 and pFC148.

All genetic manipulations were effected as described by Maniatis and collaborators (19).

The various mutants were then checked for direct sequencing of the respective plasmids using the SequenaseII system (United States Biochemical Corporation).

Expression of the Mutant Genes: The three plasmids pFC146, pFC147 and pFc148, like pFC16, have the coding gene for the corresponding human prourokinase derivative under the control of the promoter tryptophan (Ptrp) and of the "Shine DalgarnJ of the bacteriofage 1 MS-2 (MS-2 RBS). The expression of the recombinant mutants was obtained by inserting the various plasmids into a type B E. coli strain (Pasteur Institute Collection No. 54125 - Paris). The transformation, recombinant strain cultivation, expression induction and expression level analysis conditions are essentially equivalent to those already described for pFC16 (18).

The experimental test reported below shows for a representative pro-UK derivative according to the invention, namely proUK-303Glu, a superior resistance to inactivation by the PAI-1 plasmatic inhibitor than the wild type recombinant pro-UK (rec pro-UK).

Equal amounts (100 pl) of recombinant pro-UK and proUK-303-Glu mutant were incubated with 100 pl of 1:1 suspension of plasminogen/plasmin Sepharose for 3 hours at 37C. The percentage conversion from single to two-chain uPA was then assessed by SDSPAGE under reducing conditions and found t be greater than 90%.

The enzymatic activity of 2 ng of the wild type and 2 ng of the 303 mutant were tested by indirect reaction using I pM plasminogen and the synthetic substrate S 2390.

Both convert approximately the same amount of S 2390 at any time point, allowing us to assess for relative PAI-1 sensitivity.

2 ng of the rec pro-UK and the rec proUK-303-Glu were incubated with PAI1 at the concentrations indicated in Figures 5a, 5b for 1 hour at 25C in 100 mM of Herpes buffer, pH 7.5 to which 1 mg/ml of BSA had been added.

After the incubation the remaining enzymatic activity was tested as described before, the background subtracted and the velocity calculated as OD400 13 min - OD4oo 3 min. The residual enzymatic activity of the samples preincubated with the inhibitor is reported in Figures 5a and 5b as percentage of enzymatic activity of the sample with no inhibitor.

Figures 5a and 5b show that at concentrations (ng) of PAIl the 1 ' i 1 li j! li, ii il i 1 i - 1 1 1 1 1 i 1 11 residual activity of the rec proUK-303-Glu is higher than the corresponding residual activity of the wild type rec pro-UK.

The references cited by numbers in parentheses in the introductory part of this description are as follows:

(1) Collen D. and Wnen H.R. The fibrinolytic system in man. CRC Critical Reviews in oncology/hematology, 4, No. 3, 249, 1986; (2) The Molecular Basis of Blood Diseases, Eds. Stamatoyannopoulos G., Nienhuis A.W., Leder P., Majerus P.W. 1987 W.B. Sounders Company; (3) Samama M. and Kehr A., Fibrinolytic and Antifibrinolytic Agents Sem. Hop. Paris, 61, No. 20, 1423, 1985; (4) Zamarron C., Lijnen H.R., Van Hoef B. and Collen D., biological and thrombolytic properties of proenzyme and active forms of human urokinase. I. Fibrinolytic and Fibrinogenolytic properties in human plasma in vitro of urokinase obtained from human urine or by recombinant DNA technology. Thromb. Haemostas. 52, 19, 1984; (5) Maizel A.S. and Bookstein J.J., Streptokinase, urokinase and tissue plasminogen activator: Pharmo-kinetics, relative advantages and methods for maximizing rates and consistency of lysis; Cardiovasc. Intervent. Radiol., 9, 236, 1986; (6) Bell W.R., streptokinase and urokinase in the treatment of pulmonary emboli; Thromb. Haemostas., 35, 57, 1976; (7) Acar J., Vahanian A., Michel P.L., Slama M., Cromier B. and Roger V., Thrombolytic treatment in acute Myocardial Infarction; seminars in Thromb. and Haemost. 13, No. 2, 186, 1987; (8) Gruppo Italiano Per Lo Studio Della Streptochinasi 1 12 Nell'Infarto Miocardio (GISSI) Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction, Lancet, 1, 397, 1986; (9) Hoylaerts M., Ryken D.C., Lijnen H.R. and Collen D., Kinetics of activation of plasminogen by human tissue plasminogen activator: role of fibrin; J. Biol. Chem. 257, No. 6, 2912, 1982; (10) Husain S.S. and Gurewich V., Purification and partial characterization of a single chain, high molecular weight form of urokinase in human urine; Arch. Biochem. Biophys. 220, 31, 1983; (11) Collen, D. and Lijnen H.R., Tissue Plasminogen Activator; Mechanism of action and thrombolytic properties; Haemostasis 16, No. 3, 25, 1986; (12) Pannell R., and Gurewich V., Pro-urokinase. A study of its stability in plasma and of the mechanism of its selective fibrinolytic effect; Blood, 67, 1215, 1986; (13) Gurewich V. and Pannell R., Fibrin binding and zymogenic properties of single chain urokinase (Pro-Urokinase); Seminars in Thromb. and Haemost. 13, No. 2, 146, 1987; (14) Holmes W.E., Pennica D., Blaber M., Rey M.W., Gunzler W.A., Steffens G.J. and Heynecker H.L., cloning and expression of the gene for pro- urokinase in Escherichia coli. Biotechnology 3, 923, 1985; (15) Appella E., Robinson E.A., Ullrich S.J., Stoppelli M.P., Corti A., Cassani G. and Blasi F., The receptor binding sequence of urokinase; H. Biol. Chem., 262, No. 10, 4437, 1987; (16) Stefens G.J., Gunzler W.A., Otting F., Frankens E. and Floh& L., The complete amino acid sequence of low molecular weight urokinase from human urine; Hoppe Seyler's Z. Physiol. chem. 363, 1043, 1982; I! 11 11 i : i i i 1 i i i 1 t i i X 13 (17) PCR Technology; Ed. Erlich H.A., 1989 Stockton Press; (18) European Patent Application No. 365,894; (19) Maniatis T., Fritsch E.F. and Sambrook J., Molecular Cloning, A Laboratory Manual. Cold Spring Harbour 1982.

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Claims (23)

1. Claims:

   Derivatives of human pro-urokinase or its analogues, characterized by a lesser affinity towards plasmatic inhibitors.
2. 2. Derivatives as claimed in claim 1, wherein said analogues consist of mutants by deletion, insertion or inversion of human pro-urokinase.
3. 3. Derivatives as claimed in claim 1, wherein said analogues consist of pro-urokinase obtained from species other than human.
4. 4. Derivatives as claimed in claim 3, wherein the pro- urokinase is obtained from a mammal.
5. 5. Derivatives as claimed in claim 1, wherein between one and three amino acid residues present in the human pro-urokinase or analogue sequence are replaced by a like number of negatively charged amino acids.
6. 6. Derivatives as claimed in claim 5, wherein said residues are the serines in position 138, 139 and 303 and said negatively charged amino acids are glutamic acid or aspartic acid.
7. 7. A process for producing pro-urokinase derivatives as defined in claims 1 to 6, by the use of recombinant DNA technology.
8. 8. A process as claimed in claim 7, wherein said-derivatives are obtained by site-specific mutagenesis of the coding gene for human pro-urokinase or its analogues followed by introduction of the mutant genes into a suitable expression vector able to direct the synthesis of the new derivatives in a suitable host micro- organism.
9. 9. A process as claimed in claim 8, wherein the expression vector is the plasmid pFC146 and the host micro-organism is E.

   i i i 1 1 I 1 j ! i I i:i 1 1 1 1 C, coli.
10. 10. A process as claimed in claim 8, wherein the expression vector is the plasmid pFC147 and the host micro-organism is E. coli.
11. 11. A process as claimed in claim 8, wherein the expression vector is the plasmid pFC148 and the host micro-organism is E. coli.
12. 12. A gene comprising a chemically synthesized polynucleotide and which is coding for the pro-urokinase derivatives defined in claims 1 to 6.
13. 13. A recombinant expression vector containing a gene defined in claim 12 and able to direct the synthesis of the pro-urokinase derivative defined in claim 6.
14. 14. The expression vector pFC146.
15. 15. The expression vector pFC147.
16. 16. The expression vector pFC148.
17. 17. A host micro-organism transformed by an expression vector defined in claim 16.
18. 18. An E. coli type B strain transformed by the expression vector pFC146.
19. 19. An E. coli type B strain transformed by the expression vector pFC147.
20. 20. An E. coli type B strain transformed by the expression vector pFC148.
21. 21. The use of the promoter Ptrp for expression, in an E. coli 4 16 type B strain, of the genes defined in claim 12.
22. 22. The use of the Shine-Dalgarno of the MS-2 fage for expression, in an E. coli type B strain, of the genes defined in claim 12.
23. 23. A pharmaceutical composition containing a pro-urokinase derivative as defined in claims 1 to 6, together with a pharmaceutically acceptable diluent and/or carrier.

    Published 1992 at The Patent Office. Concept Sales Branch. Unit 6. Nine Mile Point. Cw-mfelinfach. Cross Keys. Newport. NP 1:1 i 1 i 1 i i A 1 1 I i i 1 i I 1 1 I i i i 1 i i i i 1 i 1 i i 1 Z House Cardiff Road. Newport. Gwent NP9 1 RH. Further copies may be obtained fron., 7HZ. Printed by Multiplex techniques ltd. St Mary Cray- Kent