WO1993024624A1

WO1993024624A1 - t-PA MODIFICATION

Info

Publication number: WO1993024624A1
Application number: PCT/JP1992/000679
Authority: WO
Inventors: Hideshi Sato; Sene Takahashi; Takaharu Negoro; Yoshiaki Sudo; Hideo Agui
Original assignee: Sumitomo Pharmaceuticals Company, Limited
Priority date: 1992-05-26
Filing date: 1992-05-26
Publication date: 1993-12-09

Abstract

A tissue plasminogen activator (t-PA) modification prepared by inserting a spacer comprising an oligopeptide composed of several to tens of amino acid residues into a single-strand cleavage site of t-PA. When it is in the form of a single strand, it is lowly active and resistant to plasminogen activator inhibitor 1. When converted into the form of a double strand at a thrombotic part of a patient, it has an activity substantially equivalent to that of natural t-PA. Accordingly, it is remarkably excellent as a therapeutic thrombolytic agent, because it has an efficacy substantially equivalent to that of natural t-PA even in a smaller dose and has less side effects.

Description

Description t-PA Variant Technical Field

The present invention relates to novel t-PA variants. More specifically, a single-chain cleavage site of tissue plasminogen activator (hereinafter referred to as t-PA) is composed of oligopeptides consisting of several amino acids to several tens amino acids. By inserting the protein, the activity is low in the single-stranded state, and it is more resistant to plasmino-inhibit and inhibitor 1 (hereinafter, PAI-1). The present invention relates to a novel t-PA variant which has resistance but exhibits an activity equivalent to that of natural t-PA when converted into a double-stranded chain at the local place of thrombus.

Background art

t-PA converts plasminogen, an inactive zymogen that is present in blood, to plasmin, an active enzyme, by limited hydrolysis. Plasmin breaks down blood clots (fibrin) formed in blood vessels for various reasons. This is called fibrinolysis.

It is known that t-PA has several types of inhibitors. Among them, PAI-1 has a high reaction rate with t-PA, Oli ^ M ^ S-li (References 1 and 2), and PAI-1 exists at high concentrations on vascular endothelial cells and platelets ( Literature 3) is known, and the main t-PA in vivo It is considered an inhibitor. It is thought that the physiological function of PAI-1 is to control fibrinolysis in living organisms (Reference 4).

t-PA has a higher affinity for fibrin than other thrombolytic agents, and has been attracting attention as being capable of specifically lysing fibrin and, consequently, thrombus. However, it has become clear that, when used in clinical practice, reopening of the occluded coronary artery requires much higher doses than expected. At present, t-PA is administered as an initial bolus followed by continuous administration, but a large dose of 100 to 150 mg per patient is required for reopening the coronary artery. It is said that it is necessary (Ref. 5). The reason that such a large dose is required is that the administered t-PA is not due to 1) PAI-1 which is present at high concentration in plasma, platelets, and vascular endothelial cells. It has been pointed out that it is activated (References 6, 7, 8) and that 2) it is cleared from circulating blood by hepatocytes (Reference 9).

As a result of this high-dose administration, t-PA, although having a high fibrin affinity, also degrades fibrinogen in the blood, resulting in the side effect of systemic bleeding tendency. What has happened is still a problem that cannot be ignored.Thus, t-PA with the aim of reducing systemic bleeding tendency using protein engineering techniques has recently been used. Attempts have been made to develop variants, for example, there is a t-PA variant having a low effect on fibrinogen (Reference 10). Alternatively, an attempt has been made to develop a t-PA variant exhibiting resistance to PAI-1 (Reference 11).

Disclosure of the invention

There are single-stranded and double-stranded t-PA, and it is known that single-stranded t-PA is converted to double-stranded by plasmin. Since plasmin is present at high concentrations in the local area of the thrombus, t-PA administered as a single strand is converted to a double strand in the local area of the thrombus.

The present invention utilizes the in vivo conversion of t-PA from single-stranded to double-stranded, and thus has low activity in a single-stranded state and is resistant to PAI-1 It is intended to provide a novel t-PA variant that exhibits the same activity as natural t-PA when converted to a double-stranded chain at the site of thrombus .

The t-PA variant described herein has a low activity when administered as a single chain, and thus suppresses the degradation of fibrinogen, which is considered to be a side effect in blood. In addition, since it is hard to be inactivated by PAI-1, it is expected that the protein will reach the local thrombus while maintaining its activity as t-PA. On the other hand, when it is converted to a double strand at the local place of the thrombus, it shows the same activity as natural t-PA. Therefore, the present invention, when administered as a single chain, is effective as a therapeutic thrombolytic agent in a smaller amount than natural t-PA, and has fewer side effects and safety. It provides a high t-PA variant. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of plasmid pTZB—0000 having a cDNA sequence (white box) encoding natural human t-PA.

FIG. 2 shows a conceptual diagram of the construction of a plasmid encoding t-PA that randomly overlaps around the single-site cleavage site. The white box and the shaded box indicate the upstream and downstream of the single-stranded cleavage site (V) of t-PAcDNA, respectively. A and B indicate restriction enzyme cleavage sites present on the DNA sequence corresponding to positions 258 and 353 of the amino acid sequence, respectively. C indicates the position of the cystine residue.

FIGS. 3A to 3D show schematic diagrams of the construction of plasmids expressing t-PA that randomly overlap around the single-strand cleavage site.

FIG. 4 shows a synthetic oligonucleotide DNA sequence containing a mutated or inserted sequence. X and Z in the sequence of P—X represent four (G, A, T, C) and two (G and C) mixed bases, respectively.

FIG. 5 shows the amino acid sequence deduced from the nucleotide sequence of the overlapping region of plasmid pCDM8-DO9 to -D44. The sequence extracted at the top shows the sequence inserted into the natural t-PA as a result of the duplication.

Figure 6 — (1)-(2) shows a schematic diagram of the construction of plasmid PCDM8-DO9-D44.

Figure 7 — Figures A and B show PCDM 8 — D 1001 The construction schematic diagram of D4001 is shown.

FIG. 8 shows a schematic diagram of the construction of plasmid PCDM8—IKGGXQFR.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have found that by inserting a spacer consisting of oligopeptides of several amino acids to several tens amino acids into the single strand cleavage site of t-PA, Although it has low activity in the form of a main chain and has resistance to PAI-11, it shows activity equivalent to that of natural t-PA when converted to a double-stranded form locally at the thrombus. It was found that a novel t-PA variant could be obtained.

That is, the object of the present invention is

1) A modified t-PA characterized in that an oligopeptide is introduced as a spacer into a single-strand cleavage site of t-PA.

2) a recombinant expression vector capable of expressing DNA encoding the t-PA variant according to 1) above in a transformed bacterium, yeast, or mammalian cell;

3) a bacterium, yeast or mammalian cell transformed with the recombinant expression vector according to 2) above;

4) A therapeutic agent for thrombosis, comprising the t-PA variant according to 1) above as an active ingredient.

is there.

Preferred forms of the modified t-PA according to the above 1) include a t-PA and / or urine at the single-strand cleavage site of the t-PA. A plasmid consisting of oligopeptides around the single-strand cleavage site of a native plasminogen activator (hereinafter referred to as u-PA) or a similar oligopeptide is inserted. T-PA variants. For example: (1) a modified t-PA in which a spacer that randomly overlaps the amino acid sequence around the t-PA single-strand cleavage site is inserted into the t-PA single-strand cleavage site; (2) The t-PA single-strand cleavage site has a t-PA, u-PA, or a spacer consisting of an amino acid sequence around the single-strand cleavage site of both t-PAs inserted. — PA variant, ③t One amino acid IKGGXQFR (X is an arbitrary amino acid) (X is an amino acid) A t-PA variant into which a probe has been inserted may be mentioned. Specific examples of ① are, for example, D11 with a spacer consisting of 11 amino acids inserted, or a spacer consisting of 44 amino acids inserted. An example of such a modification is D44 (Fig. 5). As a specific example of (2), there is a modification called D4001, which is a supplier IIGGRFK. As a specific example of (3), there may be mentioned a variant such as D1021, in which the spacer is IKGGEQFR.

As used herein, the single-stranded cleavage site of t-PA refers to positions 275 and 276 which are single-stranded cleavage sites of natural t-PA. In addition, the single-strand cleavage site of u-PA is the cleavage site of pro-u_PA (single-stranded) to u-PA (double-stranded). 1 5 8 Position Z 1 5 9

The spacer in the present invention is an oligosaccharide consisting of several amino acids to several tens amino acids, and is used when t-PA is cleaved into a double strand. And what can be cut and removed.

The preparation of the modified t-PA of the present invention may be performed, for example, by overlapping the amino acid sequence around the single-strand cleavage site of Δt-PA using a gene or cDNA encoding the site, or t-PA and / or u — a gene, cDNA, or synthetic oligonucleotide that encodes an amino acid sequence or similar sequence around the single-strand cleavage site of PA By inserting the single-stranded cleavage site into a coding gene or cDNA. The cDNA encoding the modified t-PA into which the amino acid sequence of interest has been inserted is linked to an appropriate plasmid or expression vector and introduced into a host. It is used for expression and production of the target substance.

The details are described below.

For production of the modified t-PA of the present invention, a cDNA encoding a naturally occurring t-PA can be used. CDNA encoding native t-PA has already been obtained, e.g., by cloning from Pause melanoma cells, e.g., as described in reference 12; cDNA sequences and amino acid sequences are also known. In addition, it is necessary to modify natural t-PA, for example, to increase half-life. The cDNA sequence and amino acid sequence of the modified t-PA (References 13 and 14, etc.) are also known, and these can also be used.

In the present invention, as one of the methods for preparing a convenient and suitable restriction enzyme recognition site for an operation of inserting a spacer sequence into a single-strand cleavage site and the like, Zola and Smith et al. Site-directed mutagenesis (Ref. 15) is effectively used.

Thereby, for example, an arbitrary restriction enzyme cleavage site can be introduced into an arbitrary position of the t-PAc DNA. That is, E. coli transformed with the double-stranded M13 DNA obtained by incorporating cDNA encoding the amino acid sequence of natural t-PA into an M13-based phage vector. A single-stranded form of M13 DNA was prepared from the culture broth, and the primers were synthesized with synthetic oligonucleotides for mutagenesis having an optional restriction enzyme cleavage site. Post-complementary strand synthesis may be performed to induce mutation. Such a site-directed mutagenesis system can be carried out, for example, using Takanashi Shuzo's Mutan ^™ -G.

Another method of site-directed mutagenesis is a mutagenesis method utilizing the polymerase 'chain' reaction (PCR) method (Reference 16). That is, the cDNA encoding the amino acid sequence of natural t-PA is defined as type II, and two synthetic oligonucleotides for mutagenesis are used as primers and Taq polymerase is used as primer. PCR using melases Thus, mutant DNA fragments can be amplified in vitro using a DNA amplifier.

When synthetic oligonucleotides are used in the spacer sequence, the synthesis of the oligonucleotides by the phosphoamidite method is performed by Applied Biosystems based on the principle shown in Ref. It can be performed using the company's Model 381 ADNA synthesizer. Purification can also be carried out using the company's OPC cartridges for oligonucleotide purification.

In order to confirm the desired mutation and the duplicated or inserted DNA sequence, the DNA sequence can be determined according to the dideoxy method shown in Reference 18.

In order to overlap the amino acid sequence around the single-strand cleavage site of t-PA or to insert an amino acid sequence derived from t-PA or u-PA into the site, the amino acid sequence must be Cleavage or deletion of DNA by a restriction enzyme as described in the Examples using a gene, cDNA, or synthetic oligonucleotide encoding a sequence. Separation, recovery, or ligation of the resulting DNA fragments by agarose or polyacrylamide gel electrophoresis can be performed using genetic manipulations. Most are publicly known, and reference is mainly made to reference 19.

The t-PAc DNA portion is cut out from the double-stranded M13 DNA containing the t-PAc DNA region having the spacer sequence obtained by the above operation, and plasmid or plasmid is extracted. Expression The connection with the cluster can also be made according to Ref.

In order to transform an appropriate host using the expression plasmid thus obtained, the electric pulse method (Reference 20) or the Chen-okama method (Reference 21) can be used. You.

The culture supernatant of the transformed cells contains enough t-PA to be used for measurement of t-PA activity by appropriate dilution.

Unless otherwise specified, various vector DNAs (or plasmid DNAs) used in each production process and Escherichia coli strains and animal cell strains used as their hosts are already widely and widely available. For example, vector DNA and Escherichia coli strain are easily available from Takara Shuzo or Toyobo, and animal cell strains are easily available from Dainippon Pharmaceutical.

The expression vector CDM8 (ori −) shown in the examples of the present specification is a shuttle vector that can be used in prokaryotic and eukaryotic host cells. Suitable host prokaryotes for use in this vector include those that can be restored with a sublesser (supF) into host cells such as Escherichia coli MC1061 / p3 (Reference 22). Microbial strains having a marker gene containing an amber mutation are effective.

Also useful as eukaryotic host cells are animal cultures such as COS-1, COS-7, COPS, WOP, and Chinese Hamster Ovary (CHO) cell lines. Cell lines are included.

The modified t-PA produced in the culture supernatant can be obtained by using zinc chelate-agagarose, concananol black A-agarose, Sephadex G-150 agarose, etc. the method of

(Ref. 23), etc., the t_PA variant of the present invention obtained in such a manner that it can be easily purified is dispensed in a required amount into a container by an ordinary method. However, it can be obtained very easily as a preparation by freeze-drying. This lyophilized product is dissolved in a pharmaceutically acceptable carrier substance, for example, physiological saline, and administered by intravenous or intraarterial or intracardiac injection. Administration is performed by continuous intravenous injection or by injecting part of the planned dose first intravenously and the remaining intravenous infusion.

Next, examples are shown below as examples of the present invention, but the present invention is not limited thereto.

〔Example〕

1. t — P a c D N A Construction of a brassmid p TZB-00 0 to make the region a cassette

Plasmid pTZB—0000 was obtained by incorporating the cDNA of human t—PA into plasmid pTZB (described below).

Plasmid p TZB (Reference 24) is a plasmid prepared by removing all multi-cloning sites other than BamHI of commercially available Plasmid PTZ 18R (manufactured by Toyobo Co., Ltd.). , Plus p p TZB — 0 0 0 (Reference 24) Was prepared by inserting the BamHI fragment (BamHI cassette) completely containing the t-PA translation region into the BamHI site of pTZB (Fig. 1 (Reference 24). ))).

2. Construction of a random-dummy clone bank consisting of a plasmid that encodes t-PA that randomly overlaps around the single-strand cleavage site (positions 275 and 276)

Here, a conceptual diagram of this construction method is shown in Fig. 2 to facilitate understanding of the contents described below. In the figure, the cDNA region coding around the single-strand cleavage site of t-PA (position 275/276: position indicated by ▽) is shown. A and B represent restriction enzyme cleavage sites introduced by site-directed mutagenesis upstream and downstream of the single-strand cleavage site, respectively. In order to randomly overlap around the single-strand cleavage site (position 275/276), do not cut from the upstream or downstream side of the single-strand cleavage site to the single-strand cleavage site After deletion to various lengths, the fragment is cut with restriction enzymes A or B to obtain two types of deleted fragments A and B. Both fragments obtained were ligated with a plasmid capable of expressing t-PA digested with restriction enzymes A and B and ligated to sites A and B by ligation. Plasmids encoding randomly overlapping t-PAs are obtained in the sandwiched region.The t-PAs produced by such plasmids are cut by plasmin. It can be a double-stranded molecule having the same sequence as in nature. .

A. Plasmids p UCEE (AN) and p UCEE (AN) Construction of r

Since no suitable restriction enzyme cleavage sites A and B are found on natural t-PAc DNA, site A is used as Aat II site and site B is used as site B. An Nsp7524V site (hereinafter simply referred to as NspV site) was introduced into cDNA by site-directed mutagenesis using a synthetic oligonucleotide. Both of these mutations are synonymous base substitutions that do not change the amino acid. A schematic diagram of the construction is shown in Figure 3-A.

As a type III for site-directed mutagenesis, a single-shaped M13tv19EE was obtained according to the method described in Reference 24. This M13tV19EE contains codons for amino acids 205 to 362 of t-PA.

Ml3tV19EE (AN) is a single-stranded form of Ml3tv19EE, and its synthetic oligonucleotide P—Aat II shown in Fig. 4 And P-N spV, and were obtained using the Mutan ^™ -G system described above. These synthetic oligonucleotides were synthesized and purified by the same method as described above. This Ml 3 νν 19 Ε Ε (A Ν) was digested with EcoRI and a commercially available plasmid PUC 118 was digested with EcoRI and the 5 ′ end was dephosphorylated. The plasmid was transformed with E. coli JM109 strain, and plasmid p UCEE (AN) with EcoRI-EcoRI (AN) fragments inserted in different directions from each other. pUCEE (AN) r was obtained. p U CEE (AN) inserts t — PA Xba I and Pstl sites in the multicloning site of the pUC vector downstream of the cDNA sequence, while pUCEE (AN) inserts the cDNA sequence. Both sites are located upstream.

B. Preparation of deletion fragments A and N (Fig. 3-B)

After cutting plasmid p UCEE (AN) or p UCEE (AN) r at Xbal and Pstl sites, they react with Exo-nuclease III and Mung bean nuc 1 ease for various times. Thus, various lengths can be deleted from the downstream side or the upstream side of the single-strand cleavage site, respectively. At this time, the DNA was deleted in such a range as to leave a single-strand cleavage site in the amino acid sequence (see FIG. 2). Thereafter, as shown in the figure, random deletion fragments A and N generated by digestion with AatII or NspV were obtained by standard techniques from polyacrylamide gel. Isolated.

C. Plasmid p E U K C-0 0 0 (AN-TGA)

By replacing both deletion fragments obtained by the above-described method with the corresponding region of the expression plasmid having the full-length t-PAc DNA, the region around the single-strand cleavage site was reduced. Expression plasmids encoding random t-PAs are obtained. At this time, by introducing a translation stop codon by site-directed mutagenesis into the transfer region of the expression plasmid used for the transfer, the plasmid derived from the expression plasmid is transferred. If the fragment has returned, it will not be able to express its activity, so if only those that have the activity when expressed after transfection are selected, only those with the deleted fragment will be screened. You can lean.

Fig. 3-C shows a schematic diagram of the construction of the expression plasmid pEUKC-0000 (AN-TGA) into which the translation termination codon (TGA) was introduced. First, in order to introduce a translation termination codon (TGA) by site-directed mutagenesis, the single-chain form of M13tv.l9EE (AN) described above was used as type I.

M 13 t V 19 EE (AN-TGA) is a synthetic oligonucleotide shown in Fig. 4 with the single-stranded M 13 tv 19 EE (AN) as the type III. P-TGA was obtained and obtained using the Mutan ^™ -G system described above. This M13tV19EE (AN-TGA) digested with EcoRI and pTZB-0000AEE (described later) digested with EcoRI and dephosphorylated. Were ligated to obtain pTZB-0000 (AN'TGA). p TZB—

0 0 0 Δ Ε Ε Ε 消セルフ Ε Ε Ε Ε Ε Ε Ε Ε Ε Ε Ε Ε Ε Ε Ε セルフ Ε Ε Ε Eco R with codons for

1—EcoRI region deleted.

The commercially available plasmid p EUK-C1 (Toyobo Co., Ltd.) was used as the expression vector. This vector is suitable for high expression of eukaryotic genes in COS cells. However, this vector originally had one Aatll site that would be inconvenient for subsequent transfer, so it was first digested with AatII and then digested with DNA polymerase I (Klenow Plasmid), the ends were blunt-ended, self-ligated, and transformed into Escherichia coli JM109 strain. The plasmid p EUK—C1 (AatII site disappeared) A-) was obtained. This plasmid was digested with BamHI and then dephosphorylated, and the above-mentioned pTZB-0000 (AN'TGA) digested with BamHI was ligated to E. coli JM The l09 strain was transformed to obtain pEUKC-0000 (AN • TGA).

D. Construction of a random overlapping clone bank (Fig. 3-D)

The random duplicate clone bank is composed of the two types of missing fragments A and N and pEUKC—0 0 (AN

• TGA) was digested with A at II and N spV and dephosphorylated and ligated in triplicate to form E. coli JM1

It was obtained by transforming 09 strains. About 1800 transformants were obtained. The master plate was composed of eight pieces (64 pieces / plate) of LB-agar plate, one for each length and width. 3. Screening of active positive clones The

First, the screening of double-stranded active clones was performed. Of the above random clones, 704 clones were arbitrarily selected and used for screening. Screen two In order to perform the screening efficiently, first, eight clones were grouped together as primary screening, and it was examined whether or not the eight clones contained active clones. Preliminary studies have confirmed that even if eight of them are grouped together and at least one of them contains an active clone, it can be detected by the following attest.

First, bacterial cell cultures were grouped together for 8 clones, and the mixed DNA prepared therefrom was used for transformation of COS-7 cells. In other words, a group of culture media of eight vertical or eight horizontal clones of each master plate is grouped into one group (accordingly, a total of 16 vertical / horizontal plates per plate). Eight clones of mixed DNA were prepared by the usual alkaline method.

Using these mixed DNAs, COS-7 cells were transformed by the above-described Chenyang force method. At this time, the transformation was performed on a small scale using a 24-well multi-tie evening plate.

The activity was examined by a method applying the Flinn overlay overlay assay described in Reference 25.

That is, a mixture of DMEM medium supplemented with 10% serum (FCS), agar, plasminogen, fibrinogen, and thrombin on the transformed cells in the cell. and b Nkyube preparative at over liquid off Lee Buri down the solid phase to form a C 0 ₂ Lee Nkyu base Isseki within one. The ability of t-PA, which is not secreted by cells due to the presence of serum, to some extent double-stranded I know it. As a result of performing this assay on 704 clones (therefore, a total of 176 groups), activity was detected in 35 groups.

Next, as secondary screening, either a clone in which activity was detected in either the vertical or horizontal group on the master plate, or a clone in which only one of the vertical and horizontal activities was detected For a total of 66 clones, DNA was prepared from each clone using the same method as above, and COS-7 cells were transformed and assayed. As a result, 22 active positive clones were selected. The extent of fibrin dissolution varied between clones.

4. Determination of base sequence of overlapping region of overlapping clones with activity

The nucleotide sequence of the overlapping region was determined by the previously described dideoxy method for 22 clones in which the fibrinolytic activity was detected. It contained clones with seven different overlapping sequences. Fig. 5 shows the nucleotide sequences of these seven overlapping regions and the amino acid sequences deduced therefrom. The clones are named p EUKC —D 09, —D ll, one D (1 + 1 1), -D 14, —D 28, -D 30, and —D 44 did.

5. Construction of plasmids p CDM 8_D09, one Dll, -D (1 + 11), one D14, -D28, -D30, and one D44 6 figure)

The t-PA variant was produced in the amount required for the subsequent activity measurement. For this purpose, the cDNA region containing the overlapping region of the above seven clones was changed from pEUK-C1 to CDM8 (ori-), which is a higher expression vector.

Construct A. P C D M 8 — 0 0 0

Upon transfer, a plasmid PCDM8-0.00 containing cDNA encoding natural t-PA was constructed. The construction method is described in reference 24.

B. Construction of p C D M 8 — D 0 9, -D 11, — D (1 + 1 1), — D 14,-D 28, one D 30, and one D 44

The plasmid pEUKC-DO9-D44 described above was purified using CsCl density gradient centrifugation. These purified plasmids are digested with ScaI and EcoRI, and the resulting DNA fragment containing the overlapping region (duplicate fragment) is obtained by standard techniques. Each was isolated from polyacrylamide gel. Similarly, the above-described plasmid p TZ 0—1000 is digested with BglII and Seal, and the resulting 765 base pair fragment Bgl 〖I-Seea. l was isolated. After digestion of the plasmid p CDM8-1000 described above with ScaI and EcoRI, the 5 'end is dephosphorylated, and this is combined with the above seven overlapping fragments and fragments. The ligation was performed with each of the three members, BglII-Serial, and E.coli MC1061 / p3 strains were transformed respectively, and the desired t-PA modification was performed. Plasmid PCDM for high expression of the body 8 — D 09, — D ll, — D (1 + 11), one D14, -D28, one D30, and one D44 were obtained.

6. A t-PA, 裂-（Α, or t-PAZu-PA hybrid amino acid sequence at the single-strand cleavage site (position 275/276) t Brass code to code one PA p CDM 8-D1 0 1,-D 2 0 0 1, — Build D 3 0 0 1, 1 D 4 0 0 1

Four types of 7-amino acids derived from the amino acid sequence around the single-stranded cleavage site of either t-Ρ or υ-PΑ at the single-stranded cleavage site of natural t-PA The sequences, IKGGQFR, IKGGRFK, IIGGQFR and IIGGRFK were inserted.

A. Construction of plasmid pCDM8-0000EP for insertion of amino acid sequence (Fig. 7-A)

EcoRV and Pvul sites were synthesized on PCDM8-0000 cDNA to insert four amino acid sequences into the single-stranded cleavage site of natural t-PA. It was introduced by site-directed mutagenesis using an oligonucleotide.

Ml 3 tv 19 EE (EP) is a synthetic oligonucleotide P—EcoR shown in FIG. V and P—Pvul were anneal and obtained using the Mutan ^™ -G system described above.

The M13tV19EE (EP) digested with EcoRI and the PCDM8-000 digested with EcoRI and dephosphorylated were ligated. E. coli MC 106 By transforming the lZp3 strain, PCDM8-0000EP was obtained.

B. Plasmid p C D M 8 —D 1 0 0 1, -D 2 0 0 1, -D 3 0 0 1, One construction of D 4 0 0 1

Oligonucleotides and amino acids that encode amino acid sequences around the single-strand cleavage site of t-PA and u-PA were synthesized. The sequence is shown in FIG. After annealing these eight synthetic oligonucleotides in four combinations as shown in Fig. 7-B, p CDM 8-0 00 EP was combined with EcoR V and P vu After digestion with I, the 5 'end was dephosphorylated and ligated. These cells are used to transform Escherichia coli MC1061 / 3 strain, and plasmid p coding for t-PA having four inserted amino acid sequences, IKGGQFR, IKGGRFK, IIGGQFR and IIGGRFK, respectively. CDM 8 — D 1 0 1, — D 2 0 0 1, -D 3 0 0 1, 1 D 4 0 1

7. Single-strand cleavage site (positions 275/276) Pako 8 amino acid IKGGXQFR (X is an arbitrary amino acid) t-PA-encoding brassmid Construction of p CDM 8-IKGGXQFR (Fig. 8)

An 8-amino acid sequence IKGGXQFR (X is any amino acid) was inserted into the single-strand cleavage site of natural t-PA.

Oligonucleotides encoding the amino acid sequence IKGGXQFRIKGGLFADI such that X is any amino acid were synthesized using the PCR method described above. That is, 4 Oligonucleotide P—XR and P—X having 32 different base sequences as shown in FIG. 4 were synthesized. After that, the oligonucleotides were subjected to a reaction at 95 ° C for 2 minutes → 36 ° C for 3 minutes → 70 ° C for 1 minute by applying the PCR method described above, annealing and complementing. Chain synthesis was performed.

The above PCR reaction was digested with EcoRV and PvuI, and the above-mentioned pCDM8-0000EP was digested with the same restriction enzyme and the 5 'end was dephosphorylated and digested with the same restriction enzyme. Was ligated. This transformed E. coli MC1061 / p3 strain. Plasmid DNA was prepared from the obtained transformant, and the amino acid of X was estimated by digestion with a restriction enzyme or determination of the nucleotide sequence using the dideoxy method.

As a result, 10 types of clones were obtained in which X was P, E, W, S, L, I, Τ, M, V, or A. These clones are referred to as p CDM 8 — D 1 0 1 1,-D 1 0 2 1,-D 1 0 3 1,-D 1 0 4 1, 1 D 1 0 5 1,-D 1 0 6 1,-D 1 0 7 1,-D 1 0 8 1, 1 D 1 0 9 1, 1 and — Dl 1 0 1.

8. Expression of native t-PA and t-PA variants in COS-1 cells

Four kinds of plasmids p CDM 8-D 11, PCDM 8-D 44, p CDM 8-D 4 0 1, and CDM 8-D 1 0 2 1 are selected from the above t-PA variants Using these and plasmid PCDM8-0000 expressing natural t-PA, COS-1 cells were obtained by the electric pulse method described above. Transformation. After culturing for 24 hours in a DMEM medium containing 10% fetal calf serum and 10 g aprotinin treated with lysine chromatographic medium, the medium is serum-free and aprotinin-free DMEM medium And the culture was continued for another 96 hours. After centrifugation of the culture solution, the supernatant was recovered to obtain t-PA variants Dll, D444, D4001, D1021, and natural t-PA. 90% or more of each t-PA variant and natural t-PA contained in the culture supernatant thus obtained were present in a single-stranded state. This culture supernatant was used as a t-PA sample and used in the subsequent evaluation system. (Evaluation Example)

Evaluation Example 1. Measurement of Amidity Activity

In order to measure the amidolytic activity of the single-stranded and double-stranded t-PA samples described above, the reaction using the chromogenic synthetic substrate S—2288 (Mold) was performed. went.

For the measurement of the double-stranded activity, a single-stranded t-PA sample that had been preliminarily dimerized with plasmin was used. For the double-stranding treatment, a t-PA sample diluted to 0.75 nM was used, and finally, 10 nM plasmin was added to 0.68 nM t-PA. Performed by incubating at 30 ° C for 30 minutes. Then, the plasmin-treated or untreated t-PA sample diluted to 50 ngZml was added to a reaction mixture (S-22828813 3M, aprotinin500 5ηΜ). 0.09% (v / v) Tween 80 and 4.5 mg Zral gelatin containing 0.14 M Tris-HCl pH 7.8 solution)] 2 0 0 1 was mixed, 3 7 ° 1 9 hours under heating in C, and absorbance at 4 0 5 nm using plates leader (0 D _4. ₅₎ were measured, A Mi the absorbance value The stimulus activation value was used.

Table 1 shows the amide activity values of the single-stranded (plasmin OnM) and double-stranded (plasmin 10 nM) t-PA samples, and the number of Z-books. Shows the chain value. Table 1 Plus Min (nM)

s ^, fee 0 1 0 Double strand, / Single strand Natural 0.117 0.560 4.79 (1.00)

D 11 0.060 0.522 8.70 (1.82)

D 44 0.063 0.537 8.52 (1.78)

D 4001 0.060 0.467 7.78 (1.62)

D 1021 0.067 0.505 7.54 (1.57)

Each value represents a 0 D value.

The figures in parentheses indicate the respective magnifications when the natural double-stranded Z-chain is 1.0. Evaluation Example 2. Evaluation of PAI-1 resistance

t — Resistance of PAI to single-stranded or double-stranded PA samples was determined according to the method described in Ref. 5 ng untreated or untreated with plasmin A 20 ml / ml t-PA sample (final concentration 0.5 nM) was mixed with PAI-11 (final concentration 1.67 nM) or water 101 and allowed to react at room temperature for 20 minutes. Thereafter, the S-228288 reaction mixture 200 β1 described in Evaluation Example 1 was added thereto, and the mixture was added to a pre-treader at 47.5 nm after 37.5 hours. The absorbance (OD ₄₀₅ ) was measured and the absorbance value was taken as the activity value.

Table 2 shows that the activity value of P-PA with respect to the activity value of t-PA sample when no PAI-11 was added, and the residual activity when the activity without addition of P1-1-1 was 100%. (%). Table 2

Unit:%

Unit:%

() Shows the OD value when PAI-1 was not added. References

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6)

Industrial applicability

The modified t-PA of the present invention is one in which an oligopeptide is inserted as a spacer at the single-strand cleavage site of t-PA. Compared to natural t-PA, it has almost the same effect in smaller doses, with fewer side effects and higher safety. Therefore, it is extremely useful as a therapeutic thrombolytic agent.

Claims

The scope of the claims

1. A modified t-PA characterized in that an oligopeptide is inserted as a spacer into the single-strand cleavage site of tissue plasminogen activator. .

2. Oligopeptides around the single-strand cleavage site of tissue plasminogen activator and / or urinary plasminogen activator or similar oligos The t-PA variant according to claim 1, which is a goal-guide.

3. A recombinant expression vector capable of expressing a DNA encoding the t-PA variant according to claim 1 or 2 in a transformed bacterium, yeast, or mammalian cell.

4. A bacterium, yeast or mammalian cell transformed with the recombinant expression vector according to claim 3.

5. A therapeutic agent for thrombosis, comprising the modified t-PA according to claim 1 or 2 as an active ingredient.