NO902405L - Tissue PLASMINOGEN ACTIVATOR DERIVATIVES, DNA SEQUENCE AND EXPRESSION PLASMID, AND PROCEDURE FOR PREPARING THE PLASMID. - Google Patents

Abstract

The tissue plasminogen activator (t-PA) derivative is not glycosylated and has the following amino-acid sequence: <IMAGE>

Description

The invention relates to a new t-PA derivative, a DNA sequence encoding the new t-PA derivative, expression plasmids having a DNA sequence encoding the t-PA derivative, an expression plasmid with a DNA sequence which codes for the t-PA derivative, as well as a method for producing the plasmid, a method for producing the t-PA derivative and an agent for coagulation solution containing the t-PA derivative.

Coagulated blood contains polymeric fibrin as the main component of the protein matrix. Fibrin is dissolved by a fibrinolytic system under physiological conditions in a reaction cascade similar to blood coagulation. The central reaction here is the activation of plasminogen to plasmin, which is effected e.g. using the tissue plasminogen activator t-PA (tissue-type plasminogen activator). Plasmin again dissolves fibrin, which is the main component of the protein matrix of coagulated blood. The enzymatic activity of natural t-PA or t-PA derived genetically from eukaryotes, namely the catalytic activation of plasminogen to plasmin, in the absence of fibrin or fibrinogen cleavage products is very small, but can however be clearly increased in the presence of this protein, namely by a factor of more than 10.

t-PA is cleaved in blood through proteases present in an A and a B chain. Both partial chains are bound via a cysteine bridge. The stimulability of the activity of t-PA is a decisive advantage compared to other known plasminogen activators such as e.g. urokinase or streptokinase (see, e.g., M. Hoylaerts et al., J. Biol. Chem., 257 (1982), pp. 2912-2919; W. Nieuwen-huizen et al., Biochem. Biophys. Acta, 755 (1983), pp. 531-533).

The mechanism of action of t-PA in vivo is described, for example, in Korniger and Coilen, Thromb. Haemostasis, 46 (1981), pp. 561-565. The activity of the enzyme, which is focused on the fibrin surface, makes it appear as a suitable agent for combating pathological vein blockages (e.g. in the case of a heart attack), which for the most part can be confirmed through clinical trials (Coilen et al. , Circulation, 70 (1984),

pp. 1012; Circulation, 73 (1986), p. 511).

However, the rapid reduction in plasma concentration (removal rate) must be considered a disadvantage of t-PA. The consequence of this is that a relatively large amount of t-PA is necessary to achieve an effective lysis of thrombi in vivo. The high treatment doses also have side effects, such as e.g. bleeding, as a result.

In EP 0 196 920, a natural degradation product of t-PA is described which contains only the kringel 2 and protease regions of t-PA and their N-terminal amino acid alanine 160, which is the amino acid sequence described by Pennica et al., Nature, 301 ( 1983), pp. 214-221. However, the removal rate of this t-PA degradation product is not significantly different from the removal rate of natural t-PA. Only through a chemical modification of the catalytic area by placement of a blocking group can an improvement be achieved here.

The purpose of the invention is thus to change t-PA in such a way that the resulting derivative exhibits a greatly reduced removal rate and thus an extended half-life in blood plasma. Thereby, the thrombolytic effect should as well

the stimulability by means of fibrin is achieved.

The object of the invention is thus a tissue plasminogen activator (t-PA derivative, also called K1K2P in the examples), which is characterized by the fact that it is not glycosylated and has the following amino acid sequence:

Surprisingly, it was determined that the deletion of the remaining region present in naturally occurring t-PA has no influence on the thrombolytic activity of the protein, and that the fibrin-dependent stimulability of the mutein is exactly the same as that of naturally occurring t-PA .

A further object of the invention is a DNA sequence which codes for the t-PA derivative according to the invention, and which contains the following sequence:

The DNA sequence according to the invention serves for expression of the t-PA derivative according to the invention when it is present on an expression plasmid. Such an expression plasmid is a further object of the invention, as well as an expression plasmid which has a DNA sequence which deviates, but which nevertheless codes for the t-PA derivative according to the invention. Due to the degeneracy of the genetic code, sequences deviating from the specified DNA sequence are also suitable.

The expression plasmid contains, in addition to an origin of replication ("origin of replication"), preferably next to the sequence coding for the t-PA derivative, in addition a regulatable promoter structure (e.g. tac promoter), an effective terminator (e.g. . fd), a selection marker (eg β-lactamase gene).

A further object of the invention is the plasmid pA33. The production of this plasmid is described in example 1; it contains a DNA sequence that codes for the t-PA derivative according to the invention.

Furthermore, a further object of the invention is a method for producing an expression plasmid according to the invention which is characterized by introducing a DNA sequence which codes for the t-PA protein according to the invention or a derivative thereof, which in addition to kringel I-, the kringel II and protease areas also present additional areas in the t-PA protein in a plasmid and remove the areas that code for amino acids that are not present in the t-PA derivatives according to the invention, by means of directed mutagenesis.

The selection of the plasmid, in which the DNA sequence that codes for the t-PA derivative according to the invention is introduced, depends on the host cells that are later used for expression of the derivative. Suitable plasmids as well as the minimum conditions imposed on such a plasmid (e.g. origin of replication, restriction cleavage site) are known to the person skilled in the art. Within the framework of the invention, however, instead of plasmid, a cosmid, the replicative, double-stranded form of phage (A., M13), and other vectors known to the person skilled in the art can also be used. The method of directed mutagenesis (site-directed mutagenesis) is described by Morinaga et al., Biotechnology, 21

(1984), p. 634, and is carried out mainly as described there.

Furthermore, a further object of the invention is a method for producing a t-PA derivative according to the invention, which is characterized by expressing a plasmid according to the invention in suitable host cells and extracting the product from the culture medium, or after decomposition of the host cells, from the liquid where the decomposition was carried out (e.g. buffer or also the culture medium). Preferably, prokaryotic cells are used as host cells for the production of the t-PA derivative according to the invention. Here, it is also particularly preferred that the resulting so-called "inclusion bodies" (insoluble protein aggregates) are first rinsed from the soluble cell particles, the t-PA-containing inclusion bodies are solubilized by treatment with guanidine hydrochloride, then derivatized with oxygenated glutathione and finally renatures the t-PA derivative by adding L-arginine and guanidine hydrochloride. Exact instructions for activating t-PA from "inclusion bodies" are, for example, revealed in the patent applications EP-A 0 219 874 and EP-A 0 241 022. However, any other methods for extracting the active protein from "inclusion bodies" can is also used according to the invention.

The method according to the invention is preferably carried out in the presence of L-arginine, particularly in a concentration of 25-1000 mmol/1.

The separation of foreign protein according to the invention by means of affinity chromatography is carried out in a preferred embodiment of the invention over an ETI (erythrin trypsin inhibitor) adsorption column. ETI is then fixed on a carrier material (adsorbent), such as e.g. BrCN-"Sepharose". The purification over an ETI adsorption column has the advantage that the ETI adsorption column material can be loaded directly onto the concentrated reoxidation mixture even in the presence of as high arginine concentrations as 0.8 mol/l arginine. An aggregation of p-t-PA which can take place at lower arginine concentrations below 25 mmol/1 is thereby avoided. Particularly preferably, the purification of the p-t-PA preparation takes place over an ETI adsorption column in the presence of 0.2-1.0 M, preferably 0.5-1.0 M, arginine. The KlK2P-/pro-containing solution preferably has a pH of more than 6.5, particularly preferably more than 7.

The elution of the ETI column takes place by means of the pH reduction or by arginine under conditions which enable a good solubility of t-PA, which is expressed in prokaryotes. Preferably, the pH during the elution is in the acidic range, particularly preferably in the range from 4 to 6.

A KlK2P/prop preparation produced according to the invention has a specific t-PA activity of > 0.4-IO<6>IU/mg, preferably S 0.6-IO<6>IU/mg, the stimulability through the fibrinogen cleavage product (activity in the presence of fibrinogen peptides/activity without fibrinogen peptides) is greater than 10 times, preferably greater than 20 times. The purity of the preparation according to the invention is more than 90% and preferably more than 95%, especially more than 98%.

The t-PA derivative according to the invention is thus particularly suitable for use in a medicine for coagulation dissolution, which in turn is a further object of the invention.

The following example illustrates the invention in more detail in connection with the figures.

Figure 1 schematically shows the preparation of the plasmid pA33,

figure 2 shows the time course for t-PA activity-plasma concentration after intravenous bolus infection of commercially obtained t-PA ("Actilyse") compared to the t-PA derivative according to the invention in linear preparation, and

figure 3 shows the concentration course in logarithmic representation.

Example 1

Construction of the plasmid pA33

The plasmid pePa 133, which is described in EP patent application 0 242 836, served as the starting plasmid. All experimental steps for specific mutagenesis, i.e. for deletion of regions F and E from pePa 133 (see figure 1), were carried out mainly using the method of Morinaga et al., Biotechnology,

21 (1984), p. 634. Two cleavage mixtures of pePa 133 were thus prepared. Mixture A was cleaved with EcoRl and the largest fragment isolated. Mixture B was cleaved with Xhol and the product then linearized. For heteroduplex formation, the following oligonucleotide was added:

By colony hybridization with the above-mentioned mutagenesis oligonucleotide, the clones containing the desired plasmid pA33 were found. This plasmid was characterized by restriction analysis and checked for the error in the SspI restriction site located in the portion of the t-PA expression cassette encoding the finger region.

Example 2

Production of active KlK2P/ pro from E. coil

a) Cell lysis and production of inclusion bodies (IBs) 1.6 kg moist cell mass (E. coil, DSM 3689), transformed with the plasmid pA33, was suspended in 10 1 0.1 mol/l tris-HCl, 20 mmol/1 EDTA , pH 6.5, 4°C. To this, 2.5 g of lysozyme was added, and it was incubated for 30 min at 4°C; then complete cell digestion was carried out using high pressure dispersion. To the decomposition solution was added 5 1 0.1 mol/l tris-HCl, 20 mmol/1 EDTA, 6% "Triton X100" and 1.5 mol/l NaCl, pH 6.5, and it was further incubated for 30 min at 4°C. This was followed by the separation of the insoluble components (IBs) by centrifugation.

The pellet was slurried in 10 1 0.1 mol/l tris-HCl,

20 mmol/l EDTA, pH 6.5, it was incubated for 30 min at 4°C and IB preparation isolated by subsequent centrifugation.

b) Liquidation of IBs

8.7 g of IBs (wet weight) were suspended in 100 ml of 0.1 M

tris, 6 M guanidine, 0.1 M DTE, pH 7.5, and it was stirred for 30 min at 25°C.

After setting the pH value to pH 3 with HC1 (25%), a dialysis was carried out against 4 mol/l guanidine-HCl, pH 2.5 (3x3 1, 24 hours, 4°C).

c) Derivatization

The above-mentioned lysate was adjusted with oxidized

glutathione (GSSG) at 50 mmol/1 and with tris at 100 mmol/1, and the pH was titrated to 7.5 with 5 mol/l NaOH. The mixture was incubated at 25°C for 90 min. After setting the pH value to pH 3 with HC1 (25%), a dialysis was carried out against 10 mmol/1 HC1 (3 x 100 1, 48 hours, 4°C).

d) Naturalization

A 10-liter reaction vessel was filled with 0.8 mol/l

Arg/HCl, pH 8.5, 1 mmol/l EDTA, 1 mmol/l GSH (glutathione). The naturalization was carried out at 20°C through the 3-fold addition of 100 ml of the derivative previously dialyzed against 6 mol/l guanidine-HCl, pH 2.5, at intervals of 24 hours.

After naturalization, a material with a specific activity of 50-75 KU/mg was obtained (test according to H. Lill, Z. Gesamte Inn. Med. Ihre Grenzgeb. (1987), 42, pp. 478-486).

e) Concentration of the renaturation mixture

The renaturation mixture can be concentrated as required

over a hemodialyzer.

Example 3

Purification of KlK2P/ pro from E. coli

The purification of KlK2P/pro from E. coil took place by means of affinity chromatography on erythrina-trypsin-inhibitor (ETI)-"Sepharose".

The denaturation mixture was concentrated over a hemodialyzer ("Asahi AM 300") 1:5, dialyzed overnight against 0.8 mol/l Arg/HCl, pH 7.5, and centrifuged. A 0.8 mol/l Arg/HCl, pH 7.5, equilibrated ETI "Sepharose" column (120 ml) was loaded with 1.8 L of dialysate (4 column volumes (SV)/hr) and washed with buffer ( 0.8 mol/l Arg/HCl, pH 7.5, 0.5 mol/l NaCl) until the absorbance of the eluate at 280 nm reached the blank value of the buffer. After a second wash with 5 SV of 0.3 mol/l Arg/HCl, pH 7.0, elution followed with 0.3 mol/l Arg/HCl, pH 4.5.

Example 4

Characterization of purified KlK2P/ pro from E . coli

a) Protein chemical characterization - SDS-PAGE and reverse-phase HPLC

The homogeneity of the material purified by affinity chromatography on ETI-Sepharose was determined by SDS-PAGE and by reverse-phase HPLC (RP-HPLC). After electrophoretic analysis of the purified material, an apparent molecular weight of 50,800 Da ± 2,000 Da was calculated from the relative displacement for K1K2P from E. coli. The densitometric evaluation gave a purity of > 90%.

RP-HPLC is based on the different interaction of proteins with hydrophobic matrices. This property was used as an analytical method for quantifying the degree of purity.

The analysis of the purified KlK2P/pro from B. coli occurred over a "Nucleosil 300" separation column using a trifluoroacetic acid/acetonitrile gradient (buffer A: 1.2 ml trifluoroacetic acid in 1000 ml H 2 O; buffer B: 300 ml H 2 O, 700 ml acetonitrile, 1 ml trifluoroacetic acid, 0-100% The integration of the chromatographic analysis gave a purity of >95%.

- N-terminal sequence

The N-terminal amino acid sequence was determined with an "ABI 470" sequence analyzer with standard program and connected PTH detection. The sequence found, S1-Y2-Q3-V4-I5-D6-T7-R8-A9-T10-C11-Y12-E13-D14, matches the sequence expected from the deduced DNA sequence.

b) Activity determination

The in vitro activity of KlK2P/pro from E. coli was

determined according to H. Lill, Z. Gesamte Inn. With. Ihre Grenzgeb.

(1987), 42, pp. 478-486. The specific activity (SA) was 650,000 IU/mg ± 200,000 IU/mg. The stimulability of KlK2P/pro from E. coli by means of BrCn fragments of fibrinogen (activity in the presence of fibrinogen fragments divided by activity without fibrinogen fragments) was in this test system 25-30.

c) In vitro binding to fibrin

The in vitro binding of KlK2P/pro to fibrin was determined

according to the method described by Higgins and Vehar (Higgins, D.L. and Vehar, G.A. (1987), Biochem., 26, pp. 7786-7791).

It then turned out that KlK2P/pro, in contrast to tPA from CHO cells, did not show any significant fibrin binding.

Example 5

Pharmacokinetics of KlK2P/pro in rabbits

KlK2P/pro was compared for pharmacokinetic properties with "Actilise" in New Zealand white rabbits. Both fibrinolytics were injected intravenously in a dose of 200,000 IU/kg body weight (KG) over 1 min. Plasma samples were taken before and at defined times after the injection. The t-PA activity in plasma was measured with a spectrophotometric test according to J.H. Verheijen et al. (Thromb. Haemostas., 48, p. 266, 1982), modified after H. Lill, Z. Ges. Enter. Med., 42, p. 478, 1987).

A non-linear regression computer program, modified according to H.Y., was used to calculate pharmacokinetic parameters. Huang (Aero-Astronautics Report, 64, Rice University, pp. 1-30, 1969). For curve fitting, a 1- or 2-area model was set up individually differently. Before the calculation, each time the value of the body-specific basic mirror was subtracted from the subsequent values.

KlK2P/pro was eliminated only in two of six animals with a fast a and a slow p phase. In these two animals, the a-phase proportion of the total elimination was 53%. In contrast, all the animals in the "Actilise" group showed a rapid a-phase which accounted for more than 70% of the total elimination. KlK2P/pro was mainly eliminated with only a half-life of 15.1 ±

3.1 min. Actilyse, on the other hand, showed a fast half-life of 1.63 min and a slow half-life of 10.1 min (Table 1). The total plasma clearance of KlK2P/pro was 6.3 ±

1.5 ml/kg/min and is thereby significantly lower than with "Actilyse"

(Cltot= 22.9 ± 9.1 ml/kg/min).

Taken together, KlK2P/pro presented as a t-PA mutant, which compared to the state of the art "Actilise", showed a significantly improved pharmacokinetic profile (Figures 2 and 3).

Example 6

Pharmacodynamics of KlK2P/pro in rabbits

For testing the thrombolytic efficiency, it was used by D. Coilen et al. established rabbit model with jugular vein thrombosis used (J. Clin. Invest., 71, p. 368, 1983). Fibrinolytics or the solvent was injected as a bolus intravenously over 1 min. The thrombolysis rate was then calculated and selected parameters in the coagulation system, as well as the platelet count, were determined (table 2).

At equal doses (200,000 IU/kg KG), KlK2P/pro after intravenous bolus injection gave a statistically significantly higher thrombolysis rate of 50.7 ± 6.5% than "Actilyse" (24.1 + 3.7%). The comparable dose of "Actilise" with regard to thrombolysis efficiency with KlK2P/pro was 800,000 IU/kg KG (table 2).

At equipotent dosing of KlK2P/pro compared to "Actilyse", in relation to the solvent group, there was little effect on the coagulation parameters fibrinogen, plasminogen and alpha2-antiplasmin, which, however, did not differ with regard to the effect of an equipotent dose of "Actilyse".

KlK2P/pro is thus a t-PA mutant which exhibits a thrombolytic effect after bolus injection in the rabbit model with jugular vein thrombosis, and which compared to "Actilise" is significantly increased. KlK2P/pro has thus acquired its fibrin specificity to an extent that also occurs for "Actilyse".

Example 7

Optimized expression in E . coli

To increase the expression yield, the sequence coding for the KlK2P gene was cloned into a plasmid with a higher copy number. For this, the plasmid pePa 126.1 described in German patent application P 39 31 933.4 was used. This plasmid is mainly composed of the vector pKK223-3 and the coding sequence for t-PA, as described in patent application EP-A-0 242 835.

The sequence for an fd terminator was then integrated into this plasmid. Then the plasmid pePa 126.1 was linearized with the restriction enzyme Hind III. The thus cleaved plasmid was gel electrophoretically separated and recovered preparatively. The plasmid pLBUl (Gentz et al. (1981), PNAS 78(8):4963) was restricted with Hind III and a ca. 360 bp Hind III fragment containing the fd terminator, prepared preparatively by gel electrophoresis and gel elution. The linearized plasmid pePa 126.1 and the 360 base pair Hind II fragment from pLBU1 were ligated. The ligation mixture was cotransformed with the plasmid pUBS 500 described in German patent application P 39 31 933.4 in E. coli, DSM 2102. Among the clones, those containing the desired plasmid pePa 126 fd, which differed from the starting plasmid pePa 126.1 in that it contained a second Hind III cleavage site.

From the plasmid pePa 126 fd, two fragments were recovered: a 3.4 kb BamHI/PvuI fragment and an approx. 1.3 kb PvuI/XmaI fragment. Both of the aforementioned fragments were ligated with an approx. 1.6 kb BamHI/XmaI fragment from the plasmid pA33 and transformed with the plasmid pUBS 500 in E. coli. The resulting plasmid was named pA33 fd and can be distinguished from pePa 126 fd by the fact that a restriction mixture with EcoRI contains the second smallest EcoRI fragment from pePa 126 fd with approx. 610 bp 1 length, shortened in pA33 fd by approx. 250 bp.

Claims (15)

1. Tissue plasminogen activator (t-PA) derivative, characterized in that it is not glycosylated and has the following amino acid sequence: 2. DNA sequence, characterized in that it codes for a t-PA derivative according to claim 1 and contains the following sequence: 3. Expression plasmid, characterized in that it has the DNA sequence according to claim 2, or a different DNA sequence coding for a protein according to claim 1 within the framework of the degeneration of the genetic code from there. 4. Plasmid pA33. 5. Method for producing an expression plasmid according to one of claims 3 and 4, characterized by introducing a DNA sequence that codes for the entire t-PA protein or for a derivative thereof, which, in addition to the kringel I, kringel II and protease regions, exhibits additional regions in the t-PA protein, and removes those regions which codes for amino acids which are not present in the t-PA derivative according to claim 1, by means of directed mutagenesis. 6. Method according to claim 5, characterized in that the corresponding c-DNA is used as the DNA sequence for the t-PA protein or a derivative thereof. 7. Method for producing a t-PA derivative according to claim 1, characterized by introducing a plasmid according to one of claims 3 and 4 into suitable host cells and extracting expression products from the culture medium or after digestion of the host cells. 8. Method according to claim 7, characterized in that prokaryotic cells, especially E. coli, are used as host cells. 9. Method according to claim 8, characterized by increasing the yield of active protein separating formed "inclusion bodies", dissolving these by treatment with guanidine hydrochloride, then derivatizing with oxidized glutathione and finally renaturating the t-PA derivative by addition of L-arginine and GSH. 10. Method according to one of claims 5-9, characterized in that an ETI adsorption column is used for chromatographic purification. 11. Method according to one of claims 5-10, characterized in that work is carried out in the presence of 25-1000 mmol/1 L-arginine. 12. Method according to one of claims 5-11, characterized in that the chromatographic purification is carried out over an ETI adsorption column with the concentrate of the reactivation mixture containing 25-1000 mmol/1, preferably 200-1000 mmol/1, L-arginine. 13. Method according to claim 12, characterized in that the concentrate used for chromatographic purification is set to a pH value of more than 7.0. 14. Method according to one of claims 5-13, characterized in that the elution is carried out at a pH value of 4-6. 15. Means for dissolving blood clots, characterized in that it contains a t-PA derivative according to claim 1.