AU599372B2 - Tissue-type plasminogen activator (tPA) derivatives and processes for the preparation thereof - Google Patents

Description

ttl'mTF-'^- W ur .H B~ow lilli~l H.i--f~rn 1 i I *ftr, WTlir~g~-- i.H I, «,111. .ilM lU -Who^.* CO M MONWE A'L T H OF A IFS T R A L I A PATENT ACT 1952 COMPLETE SPECIFICAT: (Original) ION 9 FOR OFFICE USE Class Int. Class Application Number: Lodged: -7 it-6 r 7 Complete Specification Lodged: Accepted: Published: Priority: tku1t 48 aand"Jentsqge ~d SOctto 49 te 0 49 tXIIT«t pntor"ng.

Related Art: IT C tt 1.

Name of Applicant: ~ij [1 Address of Applicant: Actual Inventor(s): Address for Service: BOEHRINGER MANNHEIM GmbH Sandhoferstrasse 112-132, D-6800 Mannheim Waldhof Federal Republic of Germany RALF MATTES DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

I r I Complete Specification for the invention entitled: "TISSUE-TYPE PLASMINOGEN ACTIVATOR (tP-A) DERIVATIVES AND PROCESSES FOR THE PREPARATION THEREOF" The following statement is a full description of this invention, including the best method of performing it known to us -1r -2- The present invention is concerned with new derivatives of tissue-type plasminogen activator (tPA).

as well as with a process for the preparation thereof.

The main component of the protein matrix of coagulated blood is polymeric fibrin. This protein matrix is dissolved by plasmin which is formed from plasminogen via activation by so-called plasminogen activators, for example by tissue-type plasminogen activator (tPA). The enzymatic activity of natural tPA ,..o10 or of tPA obtained gene technologically from eukaryonts (catalytic activation of plasminogen to plasmin) is very small in the absence of fibrin or fibrin fission a S" products (FSP) but can be considerably increased by more than a factor of 10 in the presence of these stimulators.

15 The mechanism of action of tPA in vivo is described, for example, by Korniger and Collen (Thromb.

Haemostasis, 46, 561-565/1981). According to these authors, tPA is solit by proteases present in blood at the point in the A- and B-chain marked by a large arrow 0 in Fig. lb of the accompanying drawings and is thus 1* t activated. The two partial chains thereby remain joined via a cysteine bridge. The stimulatability of the activity is a decisive advantage of tPA in comparison with other known plasminogen activators, for example urokinase or streptokinase for example, B.M. Hoylaerts et al., J. Biol. Chem.o 257, 2912-2919/ 19827 W. Nieuwenhuizen et al., Biochemica et Biophysica Acta, 755, 531-533/1983).

I

-3- However, an important disadvantage of tPA is its short half-life. It is known from D. Collen et al., (Circulation, 72, 384-388/1985) that the half-life of tPA is about 2 minutes. This applies not only to the non-split activator but also to the A-chain alone Rijken et al., Haemostasis, 1985, Abstract 0358).

It is assumed that tPA is rapidly taken up in the liver and broken down Fuchs et al., Blood, 65, 539-544/ S 1985). Because of this small half-life, very high doses To 0 of tPA are necessary for therapeutic use, which results in high treatment costs. Furthermore, there is the fear r that tPA in such high doses could act as an immunogen.

Therefore, it is an object of the present invention to provide tPA derivatives which, in comparison with natural tPA, have a considerably lengthened half-life but still display the characteristic biological properties of tPA, namely, the proteolytic activity and the stimulatabilty bf the activity of fibrin.

Thus, according to the present invention, there is provided a tissue-type plasminogen activator (tPA) derivative, wherein it has an amino acid sequence which corresponds to that of natural tPA but in which the amino acids forming the epidermal growth factor-like domain (EGF domain) of the natural tPA molecule are at least partially absent and at least one of the loops I or II, the connecting region, the catalytic -4region and the fibrin finger are present.

Therefore, in the tPA derivative according to the present invention, the amino acid chain of the natural tPA from the NH 2. end including the fibrin finger is attached directly or via spacers with the amino acid chain of the tPA behind the EGF domain, especially of the loop II domain up to the COOH end. Apart from the EGF domain, one of the loops I or II can also be wholly or partly absent.

In the biologically absent tPA, the amino acid chain is folded in characteristic manner and bound intramolecularly by disulphide bridges with the formation of characteristic domains which, commencing at the NH 2 end, are referred to as the fibrin finger domain (amino acids 1 to 49), EGF domain (epidermal growth factor-like domain) (amino acids 50 87), loop domain I (amino acids 88 176), loop domain II (amino acids 177 to 262), connecting region and catalytic region, as is shown in Fig. la of the accompanying drawings. The derivative according to the present invention is characterised in that the so-called EGF domain is at least partially absent and, in addition, the loop domains I and II can also be wholly or partly absent. Preferably, the preponderant or complete remainder of the amino acids of loop domain I up to the first disulphide bridge of loop domain II is absent. The fibrin finger domain, which includes amino acids I to 49 of the tPA molecule, can be shortened in r the places where, in the coding DNA, introns are cut out (arrows with letters), whereby on the NH 2 end there is shovm a leader sequence consisting of 35 amino acids which, in the case of tPA formation in eukaryonts, is present in the first formed tPA precursor.

Exoressed with reference to the numbering of the amino acids in the complete tA sequence, in the case of the tPA derivative according to the present invention, those amino acids are missing which form a section beginning with one of the amino acids 44 to 72 and S ending with one of the amino acids 87 to 179. The Snomenclature proposed by Pennica (Nature, 301, 214-221/ 1983) is hereby used. The section removed preferably ends with one of the amino acids 113 (the first disulphide bridge of loop I lies here) to 179. If loop II is partly or wholly absent, in the derivative accordw20' ing to the present invention, in comparison with tPA, two sections of the amino acid chain are removed, namely the EGF domain and the loop II part, whereas the loop I lying therebetween is present.

Surprisingly, the tPA derivative according to the present invention has a substantially higher half- Life in the organism than natural tPA and, at the same time, possesses the mentioned biological properties, especially its enzymatic activity and stimulatability of the tPA, to an unchanged extent. It is thus possible to achieve the therapeutic effects brought about by tPA with substantially smaller amounts of the tPA derivative according to the present invention. Especially good properties are thereby obtained in the case of tPA derivatives in which the amino acids 45 to 179 or 50 to 87 or 50 to 175 of the complete tPA molecule are absent (molecular weight of about 43000 D).

The new tPA derivatives can be prepared by cutting on the cDNA level with appropriate restriction endonucleases and subsequently again ligating. This modified cDNA is then introduced via an appropriate it vector into prokaryotes or eukaryotes and expressed.

In the case of this embodiment of the process according to the present invention, from the tPA-cDNA there is deleted that section which codes the amino acid sequence which, in comparison with the complete tPA molecule, is absent from the derivative by the use of the appropriate 20 restriction endonucleases, the section coding the desired tPA derivative is bound, the tPA derivative cDNA thus obtained is introduced, via an appropriate vector, into prokaryonts and expressed therein.

As restriction endonucleases, those enzymes are especially preferred which only cut in the region of bp315 to bp 726 of the cDNA sequence (cf. Pennica, Nature, 301, 214-221/1983).

-7- The restriction endonuclease combinations Dra III and Mae III are especially suitable for the removal of amino acids 44 to 179 or Dde I and Mnl I for the removal of amino acids 44 to 171, as well as Dde I for amino acids 44 to 174 and Fnu 4H for the removal of amino acids 52 to 168 and Rsa I for the removal of amino acids 67 to 162.

Insofar as restriction endonucleases have to be used which also cut the cDNA outside of the mentioned S 10 region, a limited digestion can be achieved by means of the time of action. The resultant ends of the DNA fragments have to be modified by appropriate filling or by digestion with Sl nuclease or by introduction of linkers that a phase-correct linking of the remain- S 15 ing amino acid triplets is possible.

After the treatment with restriction endonucleases and subsequent ligation, the cDNA is preferably incorporated into a vector. Appropriate vectors are, for S' example, pBR 322, pKK 223-3, pACYC 177, pRSF 1010, r eukaryontic vectors, such as bovine papilloma virus, ;or shuttle vectors, such as pKCR. For the relinking in the vector, there are used the known methods, preferably with the use of linkers.

Subsequently, these vectors are introduced into prokaryotes and eukaryotes according to known processes and the tPA derivatives expressed. In the case of the use of prokaryotes, a control of the expression is -8preferably provided in known manner, for example by the use of the system lac promotor (or a derivative thereof, such as tac promotor) and lac repressor, or trp promotor and trp repressor or lambda promotor and lambda repressor.

For the preparation of the derivatives, it is also possible to start from a complete genomic DNA fragment which contains introns. For this purpose, the tPA-DNA from the Maniatis gene bank is isolated 10 and introduced into vectors. This takes place via ,aimed deletion of the amino acid coding triplet absent in the tPA derivative according to the present invention in comparison with tPA and of the intron positioned therebetween by means of oligonucleotides. In the case of this embodiment of the process according to the present invention, tPA-DNA is introduced into an appropriate vector, the vector obtained is then hybridised with an oligonucleotide which, with the base sequences which, on both sides, connect on to the base sequences to be removed from the tPA-DNA molecule, has a complementary base sequence, subjected to repair mutagenesis, the so mutated vector then introduced into an appropriate cell and amplified, those amplified vectors are recovered which hybridise with the oligonucleotide and introduced into prokaryotes appropriate therefor and expressed. The oligonucleotide should preferably consist of 18 to 26 nucleotides. Of these -9nucleotides, in each case one half should preferably coincide with the nucleotides to be obtained of the triplets lying in front and behind. This process principle of the "gapped duplex" synthesis is described S in detail by Morinaga et al. (Biotechnology, 2, 634- 639/1984) and by Nagai et al. (Nature, 309, 810/1984).

Furthermore, from the tPA-producing cell lines (see Pennica loc. cit.), there can be isolated the m-RNA and separated by size fractionation. By means :e 10 of natural splice artefacts, the m-RNA derivatives can result which are shortened. By means of cDNA preparation therefrom and investigation of the resultant I clones, those clones can be found which no longer contain the coding region for the amino acids which, in t 15 comparison with tPA, are absent from the tPA derivative, especially amino acids 49 to 175. For this purpose, clones are selected which no longer hybridise with appropriately constructed oligonucleotides appropriate for the removed region. Such shortened cDNA molecules can, in principle, also result by errors in the prepar- 1 ation of cDNA from intact or partly deleted mRNA and be found by hybridisation and sequencing.

tPA derivatisation can also take place on the amino acid level. tPA, for example produced by gene technology and purified, is then cut with proteases which cut in the region between amino acids 43 and 72 and between 87 and 179 and again put together. The fragments to be combined can be isolated from other fragments by gel elec.'rophoresis or other size fractionation.

In the case of the tPA derivatives according to the present invention, it can be advantageous to optimise the distance of fibrin finger-loop II in order to bring about the maximum action of the tPAcharacteristic enzymatic effectiveness of the molecule.

This can take place, for example, by incorporation of short amino acid chains ur of other spacers between the fibrin finger domain and the loop II domain.

A tPA derivative in which amino acids 45 to 179 are absent is especially preferred. This can be produced, for example, on the cDNA plane by cutting with the restriction endonucleases Dra III and Mae III and digestion of the remaining individual strand ends with nuclease Sl and subsequent ligation with ligase. It is then expressed withan appropriate vector system, such as is described, for example in German Patent Application No. P 36 13 401.5, into prokaryonts or eukaryonts.

The tPA derivative resulting in the case of expression into prokaryotes or eukaryotes has a molecular weight of about 43000 D. The derivative resulting in eukaryotes is additionally glycolised and, therefore, migrates somewhat more slowly in the SDS electrophoresis.

Another especially preferred tPA derivative differs from tPA by the absence of amino acids 50 to 175.

-11- For the preparation thereof, there is especially preferred the above-described tPA-DNA method of "gapped duplex" synthesis because it does not require the presence of restriction fission points at the desired positions of the sequence.

The present invention also provides new, pharmacologically active proteins which are derived from tPA but display a better combination of properties in comparison with tPA, as well as a process for the preparation thereof.

The present invention also provides an agent for dissolving a coagulum, containing a tPA derivative according to the present invention. The agent according to the present invention preferably contains a tPA derivative which has the amino acid sequence of tPA but without amino acids 45 to 170.

The following Examples are given for the purpose of illustrating the present invention: Example 1.

Deletion from tPA of the domains determining the half-life time.

1 a) -onstruction of the tPA mutein gene As starting plasmid, there is used the plasmid pBT 95 (DSM 3611 p; see Federal Republic of Germany Patent Specification No. 35 45 126), from which the plasmid pePa 98.1 is produced in the following way: The plasmid is split with the enzyme Bgl II and after- 1 -i -12treated with the nuclease Sl. By further splitting with the enzyme Sca I, there can be obtained preparatively a fragment a which has a size of about 760 base pairs and which contains the nucleotide sequence 192 952 coding tPA (Pennica, loc. cit.). A fragment b is also obtained from pBT 95 by splitting with Sca I and Hind III. There is thereby obtained a fragment of about 1200 base pairs which contains the tPA nucleotide sequence 953 2165. A partner c is produced synthetically as linker and contains the followng sequence: 5'GAATTCTTATGTC3' 3' As partner d in the construction, the plasmid pKK 213-3 (DSM 3694 P) is split with the enzymes Eco RI and Hind III in the polylinker. The partners a d are ligated together. The ligation batch is treated according to the usual technique with T4 ligase and subsequently transformed into Escherichia coli cells (DSM 3689). The transformed cells are cultured on a medium with the addition of 50 ig./ml. ampicillin. From the clones obtained, those are selected which carry the plasmid pePa 98.1 which differs from the starting Splasmids pBT 95 and pKK 223-3 in that, in the poly- Slinker of the plasmid pKK 223-3, it contains, between the Eco RI and Hind III fission point, the tPA nucleotide sequence 192 2165 in the correct sequence. From this plasmid pePa 98.1 is obtained the tPA-coding -13fragment with the tac promotor via Xho II splitting.

This fragment has a size of about 1.85 kb and, on the remaining ends, is completely filled by treatment with Klenow enzyme in the presence of all four desoxynucleoside triphosphates. In a step A, this fragment is split with the enzyme Dra III and the remaining ends digested with the nuclease Sl. The fragment with a size of about 370 base pairs is obtained by gel electrophoresis. In a step B, the same Xho II starting fragment is split with the enzyme Mae III and also then digested with Sl. Subsequently, this batch is additionally treated with the enzyme Sac I. A fragment with a size of about 700 base pairs is isolated therefrom by gel electrophoresis. In a batch C, the vector plasmid pePa fa is cut with the enzyme BamH I and the remaining ends filled with the Klenow enzyme, with the use of all four desoxynucleoside triphosphates, and then split with the enzyme Sac I. The largest fragment is subsequently obtained from this batch by gel electrophoresis.

In a ligation batch, the fragments obtained from batches A, B and C are combined and ligated with the addition of DNA ligase. The ligation mixture is transformed into Escherichia coli cells (DSM 3689), which contain a lac I q plasmid, and the resultant transformants selectioned on nutrient medium with 50 gg./ml. ampicillin.

From the clones thus obtained, those are selected which contain the desired plasmid pePa 129 which differs from -14the starting plasmid pePa 98.1 in that it no longer contains the DNA sequence between the tPA-cDNA sequence positions 301 to 726. Fig. 2 of the accompanying drawings shows the nucleotide seuqence of the mutein so obtained and the amino acid sequence obtained therefrom in which the mino acids 45 to 179 of the tPA are absent.

1 b) Construction of an expression vector for the ai mutein for the expression in Escherichia coli.

From the plasmid pePa 129 prepared according to 1 by digestion with the restriction enzymes Bam H I and Pvu I, thereis obtained the tPA-coding fragment.

The plasmid pACYC 177 (DSM 3693 P) is also split with t'I* Bam H I and limited with Pvu I. Both fragments are I, 15 ligated together and transformed into Escherichia coli cells (DSM 3689) which contain a lac Iq plasmid. By selection on kanamycin and ampicillin with, in each S* case, 25 and 50 gg./ml., respectively, there are obtained transformants from which those are selected which contain the plasmid pePa 137. This differs from the starting vectors in that it contains the mutein gene fragment from pePa 129 and the sequence of the plasmid pACYC 177. A restriction fission point chart of the plasmid pePa 137 is shown in Fig. 3 of the accompanying drawings.

1 c) Construction of an expression vector for the mutein for expression in Pseudomonas putida.

x; i- As vector for Pseudomonas putida, there is used a derivative of the plasmid pRSF 1010 which additionally contains akanamycin-resistant gene from pACYC 177.

This plasmid has the designation pREM 3061 (DSM 3692 P).

This plasmid is linearised by the use of the restriction enzyme Hpa I. From the vector pePa 129 obtained according to 1 by Xho II splitting, there is obtained a fragment with a size of about 1.45 kb which contains the tac promotor and the complete mutein gene sequence.

S 10 By treatment with Klenow fragment in the presence of S, all four desoxynucleoside triphosphates, the Xho II fission points are completely filled. The so treated s t fragment is ligated with the linearised vector pREM 3061 and transformed into Escherichia coli cells (DSM 3689).

I t 15 The transformants are selectioned on medium with 4g./ml. kanamycin. Transformants which contain the I plasmid pePa 143 are selected. This plasmid differs Sfrom the starting vector pREM 3061 in that it contains the complete mutein gene sequence with tac promotor.

20 This plasmid is isolated and introduced by transformation into cells of the strain Pseudomonas putida (DSM 2106). The transformants are selectioned on S' medium with 25 4g./ml. kanamycin and subsequently analysed. They contain the plasmid pePa 143. Additionally, into these transformant cells can also be introduced, by transformation, a plasmid pePa 119 (DSM 3691 which is compatible with pePa 143 and contains -16the lac Iq gene. This plasmid is a derivative of plasmid RP 4. The presence of this plasmid suppresses the production of the mutein in the cells by production of the lac repressor protein. This can repress the transcription from the tac promotor.

Example 2.

Construction of vectors which contain the tPA mutein gene with leader sequence for the secretion and are suitable for expression in CHO cells.

The vector pKCR is used. A plasmid which contains the complete tPA-cDNA is plasmid pBT 95. This plasmid is limitedly split with the restriction enzyme Pst I so that a fragment is obtained which no longer contains the tPA sequence of the cDNA, nucleotide positions 205 1312. From the plasmid pePa 129, produced according to Example 1 which contains the mutein gene, there is obtained, by limited Pst I splitting, the fragment which contain, the nucleotide positions 205 1312 but displays no deletion for the sequence from 322 726. These so obtained fragments are commonly ligated and transformed into Escherichia coli cells (DSM 3689). By the addition of 50 t.g./ml. ampicillin, transformants are obtained and the clonesselected which have had inserted the mutein fragment in the correct orientation. These plasmids are designated with pePa 144. They are introduced into CHO cells, which are DHFR-, according to the method of R. Kaufmann and P. Sharp Mol.

x-~ -17- Biol., 15, 601-621/1982). Under the there-described conditions, tPA mutein is expressed and analysed (Molec. Cell. Biol., 5, 1750-1759/1985).

Example 3.

Expression of the tPA mutein in Escherichia coli cells.

The plasmid pePa 137 pr-oduced according to Example 1 a) and 1 b) is transformed into Escherichia coli cells (DSM 3689) which contain a lac I q plasmid.

The transformants are selectioned on medium with 50 .g./ml.

ampicillin. The cells which carry this plasmid are cultured, with aeration, in nutrient broth up to OD 0.4 and then induced with the addition of .550nm 0.2% lactose or 10 mMole/litre IPTG. The culturina takes place at 37 0 C. After culturing and aerating for 4 hours, the cells are collected and lysed. For this purpose, they are treated for 15 minutes on ice with 0.5 mg./ml. lysozyme (cell concentration 10 OD/ml.).

Tris buffer is used ,(pH 8.6; 0.1 mole/litre Tris-HCl with 10 mMole/litre EDTA and 100 mMole/litre sodium chloride). Thereafter, the cells are fragmented by ultrasonic treatment. The suspension thus obtained is centrifuged for 10 minutes at 20,000 g apd the supernatant is discarded. The sediment contains the tPA mutein in inactive form. This can be dissolved and reactivated by the method described in Federal Republic of Germany Patent Specification No. 35 37 708.

The pellet obtained can also be dissolved by the LI i- C L---~Uprr -18addition of 1% SDS and 100 mMole/litre mercaptoethanol and separated electrophoretically in an SDS polyacrylamide gel. By staining the proteins after electrophoresis has taken place by means of Coomassie blue, the protein bands can be made visible. According to this analysis, the extract contains a protein with a molecular weight of about 43000 Dalton. As expected, the tPA mutein is smaller than the intact tPA obtained *from analogously treated cells with pePa 133 (molecular 0 weight about 57000 Dalton). Not only tPA mutein but also tPA can be shown to be immunologically equal by Sthe Western blot process with anti-tPA-PAK POD conjugate from goats.

Example 4.

Comparison of mutein and tPA with regard to the proteolytic activity and the stimulatability by fibrin.

According to Example 2, from bacteria which contain the plasmid pePa 137 or pePa 133, there is obtained the mutein or tPA. According to the process described in Federal Republic of Germany Patent Specificaton No. 35 37 708, there is obtained corresponding active mutein or tPA. All proteins are present in solution in single-chained form.' Comparable yields are thereby obtained referred to the protein initial concentration (see the following Table). In the test for proteolytic activity, comparable values are obtained for both -19preparations (see the following Table). The proteolytic activity is hereby determined as described in Federal Republic of Germany Patent Specification No.

37 708. The comparison of the activity which is obtained with and without the addition of fibrin fragments shows comparable values for both preparations (see the following Table). According to this, the mutein is not to be differentiated from tPA but rather is identical in its enzymatic properties.

Table tPA mutein mg. protein yield 234.0 75.0 mg. thereof with activity 11.6 5.6 stimulatability factor 11.0 21.6 Example Determination of the half-life time.

The mutein prepared according to Examples 1 to 3 has a comparatively high half-life time in the biological test. Preparations of TPA and mutein which have been obtained according to Example 3 were, for this purpose, compared in the mouse test system. Use was made of the process described by Fuchs et al. (Blut, 65, 539-544/ 25 1985).

Comparable values were found for the plasma clearance for tPA. The mutein, on the other hand, was removed more slowly from the blood, demonstrated by TCA- 125 precipitatable I-mutein. The accumulation in the liver was markedly reduced: only after 20 to minutes were values measured which had already been reached for tPA after only 5 minutes.

Example 6.

Deletion of the domains determining the half-life time from tPA by means of oligonucleotide-induced mutagenesis.

a) Preparation of the tPA vector pePa 133.

I 10 From the plasmid pePa 98.1 prepared according to Example 1 is obtained, by splitting with the enzyme Xho II, an approximately 1.85 kb sized fragment which is preparatively isolated. This fragment contains the tac promotor and lac operator region with an ATG start codon and the tPA nucleotide sequence 192 1809 in Sthe correct sequence. The plasmid pKK 223-3 (DSM 3694 P) is split with the enzyme Bam HI, the larger fragment is recovered preparatively, combined with the Xho II fragment from pePa 98.1 and ligated with the addition of DNA ligase. The ligation mixture is transformed into Escherichia coli cells (DSM 3689) which contain a lac Iq plasmid and the resultant transformants are selectioned on nutrient medium with 50 4g./ml.

ampicillin. From the clones thus obtained are selected those which contain the desired plasmid pePa 126. This differs from the starting plasmid pePa 98.1 in that, with Bam HI and Hind III splitting, there is obtained I- -21an approximately 1.85 kb sized fragment, besides an approximately 4.4 kb sized fragment.

By digestion with the enzymes Bam HI and Pvu I, from this plasmid pePa 126 there is obtained a fragment which has a size of about 2.9 kb and contains the tPA-coding sequence. The plasmid pACYC 177 (DSM 3693 P) is also split with Bam HI and limited with Pvu I. The larger fragment thereby resulting is ligated together with the tPA-coding fragment from pePa 126, with the addition of T4 ligase, and transformed into Escherichia coli cells (DSM 3689) which contain a lac Iq plasmid. By selecting on nutrient medium with kanamycin and ampicillin in amounts of and 50 pg./ml., respectively, there are obtained transformants from which those are selected which contain the plasmid pePa 133. This differs from the starting vectors in that it contains the approximately 1.85 kb sized tPA fragment (after splitting with Bam HI and Hind III) from pePa 126, the sequence of the plasmid pACYC 177.

b) Deletion of the EGF loop I domain from pePa 133.

In principle, there is used the method of Morinaga et al., (Biotechnology, 2, 634-639/1984). Two splitting batches of pePa 133 are preapred. Batch a) is split with Eco RI and the largest fragment is isolated.

Batch b) is split with Xho I and the product thus linearised. The fragment from batch a) is combined -22with batch b) (in each case about 150 fmole) and both DNAs are denatured by brief heating to 95 0 C. This mixture is subsequently incubated at 60 0 C. for about minutes. In addition, an oligonucleotide (75 fmole) is added thereto. Its sequence is as follows: 3' After cooling the mixture to 12 0 there are added thereto 0.25 mM desoxynucleoside triphosphate, 1 mM ATP, 100 mM sodium chloride, 6.5 mM Tris-HCl (pH 8 mM magnesium chloride, 1 mM B-mercaptoethanol, Klenow fragment of the DNS polymerase from Escherichia i coli in an amount of 0.125 U/pl. of batch and T4 ligase o in an amount of 0.0625 U/4l. of batch. The mixture is left for 4 hours at 12 0 C. Subsequently, this batch is 15 transformed in Escherichia coli cells (DSM 3689) which Scontain a lac Iq plasmid. By selection on a nutrient s l medium containing kanamycin and ampicillin in amounts of 25 and 50 4g./ml. of nutrient medium respectively, transformants are obtained. From these are selected those which contain.the plasmid pREM 7685. This differs 4 from the starting vector in that it hybridises specifically with the mentioned oligonucleotide (wash temperature 50 0 and contains a shortened Eco RI fragment.

S. Instead of an approximately 0.61 kb sized fragment in

I

25 pePa 133, there can only be detected a 0.23 kb fragment, besides the other Eco RI fragments.

-23- The so obtained tPA mutein gene can be expressed as described in Example 3 and a protein detected with a size of about 43000 d which has the sequence of a natural tPA in which the amino acids 50 to 175 are absent, i.e. the amino acids 49 and 176 are linked with one another. As described in Example 4, it can also be obtained as active mutein. Its values do not differ from those described in the above Table. As described in Example 5, in the mouse test system there is found an equally slowed down plasma clearance and distinctly reduced liver accumulation.

Example 7.

Deletion of the EGF domain from pePa 133.

From the tPA vector pePa 133 prepared according to Example 6a, there are prepared the splitting batches a) and b) according to Example 6 b) and treated as therein described. However, in addition, to the theredescribed mixture is added 75 4mole of the following oligonucleotide: 5' GCCTGTCAAAACCAGGGCC 3' All further steps are carried out in the manner described in Example 6 Those clones are selected which contained the desired plasmid pREM 7732. This differs from the starting vector by a shortened Eco RI fragment: instead of being about 0.61 kb, this has a size of only about 0.46 kb.

-24- The mutein gene thus obtained is expressed as described in Example 3 and demonstrated as being a protein with a size of about 49000 d with the sequence of a natural tPA molecule in which the amino acids to 87 are absent, i.e. the amino acids 49 and 88 are linked with one another. As described in Example 4, it can also be obtained as active mutein.

II

I It !'s

Claims (27)

1. Tissue-type plasminogen activator (tPA) derivative, wherein it has an amino acid sequence which corresponds to that of natural tPA but in which a sequence section is absent from the complete tPA molecule which begins with one of the amino acids 44 to 72 and ends with one of the amino acids 113 to 179, wherein the amino acids forming the epidermal growth factor-like domain (EGF domain) of the natural tPA molecule are at least partially absent and at least one of the loops I or II, the connecting region, the catalytic region and the fibrin finger are present.

2. tPA derivative according to claim 1, wherein the amino acids following the EGF domain up to at least the first Sdisulphide bridge of the domain of loop I are absent.

3. tPA derivative according to claim 2, wherein the total amino acids of the domain of loop I up to the first j disulphide bridge of the domain of loop II are absent.

4. tPA derivative according to any of the preceeding claims, wherein it has the amino acid sequence of tPA without amino acids 45 to 179. tPA derivative according to claim 5, wherein it has the amino sequence of tPA without amino acids 50 to 175.

6. tPA derivative according to claim 6, wherein it has the amino acid sequence of tPA without amino acids 50 to 87. 26

7. tPA derivatives according to claim 1 which are hereinbefore specifically exemplified.

8. Process for the preparation of tPA derivative according to claim 1, wherein from the tPA-cDNA is cut out that section which codes the amino acid sequence missing from the derivative in comparison with the complete tPA molecule with the use of appropriate restriction endonucleases, the section coding the desired tPA derivative is connected and the tPA I r derivative cDNA thus obtained is introduced via an appropriate vector into prokaryotes or eukaryotes and expressed therein.

9. Process according to claim 9, wherein restriction endonucleases are used which only cut in the region of the base pairs 315 to 726 of the tPA-cDNA sequence. Process according to claim 9 or 10, wherein, as restriction endonucleases, there is used Dde I alone or together with Mnl I or Dra III and Mae III or Pnu 4H alone or Rsa I alone.

11. Process according to any of claims 9 to 11, wherein, as vector, there is used pBR 322, pKK 223-3, pACYC 177, pRSF 1010, bovine papilloma virus or pKCR as hereinbefore S described.

12. Process for the preparation of a tPA derivative according to claim 1, wherein tPA-DNA is introduced into an appropriate vecotr, the vector obtained is hybridised with an oligonucleotide which has a base sequence complementary with ihe base sequences which connect on both sides to the base sequences to be removed from the tPA-DNA molecule, a repair mutagenesis is carried out, the vector so mutated is then 27 introduced into an appropriate cell and amplified and those amplified vectors are recovered which hybridise with the oligonucleotide and introduced into prokaryotes or eukaryotes appropriate therefore and expressed.

13. Process according to claim 13, wherein an oligonucleotide is used with 18 to 26 nucloetides.

14. Process for the preparation of a tPA derivative according to claim 1, wherein the tPA-mRNA is isolated from tPA-producing cells, fractionated according to size, Irt fractions of the size corresponding to the desired tPA derivative are transcribed into cDNA and cloned, those clones are selected which no longer hybridise with an oligonucleotide which hybridises with that region of the tPA-coding cDNA which is absent form the cDNA coding the tPA derivative in comparison with tPA-cDNA, the tPA derivative DNA is recovered therefrom and introduced via an appropriate S vector into prokaryote or eukaryote Process for the preparation of a tPA derivative accorJing to claim 1, wherein tPA is cut with proteases between amino acids 44 and 72 and between 87 and 179 and the end parts formed are again connected.

16. Process according to any of claims 9 to 15 wherein the amplification and/or the expression is carried out in an Escherichia oli strain which contains a lac Iq plasmid as hereinbefore described.

17. Process according to any of claims 10 to 15, wherein the expression is carried out in mouse LTK- cells.

18. Process according to claim 17, wherein Escherichia cli DSM 3698 is used. LS Tr7 28

19. Process according to claim 19, wherein Escherichia coli DSM 3698 which contains plasmid pePa 137, as hereinbefore described, is cultured. Process according to any of claims 9 to 15, wherein the amplification and/or expression is carried out in Pseudomonas putida DSM 2106.

21. Process according to claim 21, wherein Pseudomonas putida DSM 2106 which contains plasmid pePa 143 and optionally pePa 119, as hereinbefore described, is cultured.

22. Process for the preparation of a tPA derivative according to claim 1, substantially as hereinbefore described and exemplified.

23. A tPA derivative according to claim 1, whenever prepared by the process according to any of claims 9 to 23.

24. Plasmid pePa 137, as hereinbefore described. Plasmid pePa 143, as hereinbefore described.

26. Plasmid pePa 144, as hereinbefore described.

27. Plasmid pREM 7685. as hereinbefore described.

28. Plasmid pREM 7732, as hereinbefore described.

29. Agent for dissolving a coagulum, containing a tissue-type plasminogen activator (tPA) derivative according to claim 1 which has an amino acid sequence which corresponds to that of natural tPA but in which the amino acids forming the epidermal growth 29 factor-like domain (EGF domain) and at least the subsequent amino acids up to at least the first disulphide bridge of the domain of loop I of the natural tPA molecule are absent. Agent according to claim 30, wherein it contains a tPA derivative which has the amino acid sequence of tPA without the amino acids 45 to 170.

31. Agent according to claim 30, wherein it contains a tPA derivative which has the amino acid sequence of tPA without the amino acids 50 to 175.

32. Agent according to claim 30, wherein it contains a tPA derivative which has the amino acid sequence of tPA without the amino acids 50 to 87.

33. Agent according to claim 30 for dissolving a coagulum, substantially as hereinbefore described and exemplified. Dated this 30th day of April, 1990. BOEHRINGER MANNHEIM GmbH by its Patent Attorneys DAVIES COLLISON.