WO1993020194A1 - Tpa analog - Google Patents

Abstract

A novel tissue plasminogen activator (TPA) analog improved in fibrin specificity is obtained by replacing at least part of the amino acid sequence of TPA, which corresponds to the amino acid sequence between the 276th position (just behind the single strand/double strand cleavage site) and the 306th position of a natural TPA, by the corresponding amino acid sequence of a urinary plasminogen activator. This analog hardly causes the activation of plasminogen and the subsequent decomposition of fibrinogen when it is circulating in the blood, but exhibits the activity of activating plasminogen comparable to that of a natural TPA to thereby cause thrombolysis for the first time when it reaches the locus of thrombus wherein fibrin is present. Thus, it is anticipated that it may hardly bring about a side reaction of generalized bleeding tendency, so that it is prospective as a novel thrombolytic agent.

Description

 Light 4m

 Fine t — PA analogs Technical field

 The present invention relates to novel t-PA analogs. More specifically, in the amino acid sequence of the tissue plasminogen activator (hereinafter referred to as t-PA), an amino acid sequence corresponding to positions 276 to 3006 of natural t-PA. By replacing at least part of the protein with the corresponding amino acid sequence of urinary plasminogen activator (hereafter, UK), the fibrin specificity has been improved. Background Art

t-PA is known to degrade thrombus formed in blood vessels by converting plasminogen, an inactive zymogen, to plasmin, an active enzyme. T-PA has relatively high fibrin specificity, whereas existing thrombolytics, such as UK-strain tokinase, do not have any fibrin specificity. have. For this reason, it has the property of specifically dissolving fibrin and, consequently, thrombus, and has attracted attention as a thrombolytic agent with less side effects such as bleeding. However, it is clear that, in practice, the use of t-PA in clinical practice requires much higher doses than expected for reopening the occluded coronary artery. I got it. For example, it is also said that a large dose of 100 to 15 O mg per patient is necessary for reopening the coronary artery (Reference 1). In practice, it is used in doses of less than about 100 mg per patient, but as a result of this large dose, t-PA is not fibrin-specific, but only slightly in the blood. It is still a non-negligible problem that it also degrades blinogen, resulting in a generalized bleeding tendency. From this point of view, t-PA variants have also been produced for the purpose of imparting higher thrombus specificity, and numerous reports have been made (references 2 to 12). Few have achieved. As a successful example, in Reference 6, thrombus specificity was achieved by replacing all four amino acids from positions 296 to 299 of natural t-PA with alanine. We have produced modified t-PA with improved fibrin specificity 4.4-fold, and plasma clot specificity about 3-fold. Disclosure of the invention

The present invention provides a novel t-PA analog with improved fibrin specificity. In more detail, plasminogen activating activity is very low in the presence of fibrinogen, but close to or higher than natural t-PA in the presence of fibrin. The present invention provides a novel t-PA analog which exhibits a gene activating activity and therefore has improved fibrin specificity. The t-PA analogs of the present invention are less likely to cause activation of plasminogen and subsequent degradation of fipurinogen when circulating in the blood. Since it expresses a plasminogen-activating activity close to that of natural t-PA and dissolves thrombus only when it reaches the local thrombus where Lin is present, it is more than natural t-PA. As compared to known variants with improved fibrin specificity, the fibrin specificity is higher, and therefore the thrombus specificity is higher, and the tendency of systemic blood bleeding is high. It is expected that the side effect is unlikely to occur. Accordingly, the present invention provides a highly safe and novel thrombolytic agent as a therapeutic thrombolytic agent.

 In addition, the t-PA analog of the present invention inhibits the inhibition of t-PA by plasminogen activator inhibitor 1 (hereinafter abbreviated as PAI-1), which is an immediate inhibitor of t-PA. Resistant to it. Therefore, it is expected that the protein will reach the thrombus site while maintaining its activity without being inhibited by PAI-1. Therefore, it is expected that the utility as the therapeutic agent for thrombosis of the present invention is further enhanced in addition to the above-mentioned fibrin specificity. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a schematic diagram showing a restriction enzyme cut map of a plasmid pTZB-000.

FIG. 2A shows a schematic diagram of the construction of plasmid pHSGB and a restriction enzyme recognition site. FIG. 2B is a schematic diagram of a synthetic DNA linker for preparing plasmid pHSGB and its assembly.

 FIG. 3 is a schematic diagram of the assembly of plasmid pHSGB—O00.

 FIG. 4 is a schematic diagram of the assembly of plasmid pHSGB-0000NA.

 FIG. 5 shows the sequence of the synthetic oligonucleotide used for site-directed mutagenesis.

 FIG. 6 shows a sequence of a synthetic oligonucleotide having a partial sequence of UK, a single PA, or both, which was used for partial recombination of the t-PA sequence.

 FIG. 7 is a schematic diagram of the assembly of the plus pH S GB B—C S 1 ο

 FIG. 8 is an assembly schematic diagram of the plasmid pCDM8—CS1. BEST MODE FOR CARRYING OUT THE INVENTION

 The present inventors have found that in the amino acid sequence of t — Ρ ア, the amino acid corresponding to positions 276 to 306 of natural t — PA (the position immediately after the single-stranded Z double-strand cleavage site). Replacing at least part of the amino acid sequence with the corresponding amino acid sequence of the UK will result in a novel t-PA analog with improved fibrin specificity Was found.

 That is, the object of the present invention is

1) 1 ー? Eight amino acid sequences, natural 1:-? A t-PA analog, characterized in that at least part of the amino acid sequence corresponding to positions 6 to 36 is substituted with a corresponding amino acid sequence of UK.

 2) a recombinant expression vector capable of expressing a DNA encoding the t-PA analog according to 1) 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 analog according to 1) above as an active ingredient.

It is.

 Preferred examples of the t-PA analog according to 1) above include, for example,

 1) Gly—Gly—Ser—Va1—Thr—Tyr—The corresponding sequence in the UK from position 300 to position 36 of the amino acid sequence of t-PA. A t-PA analog substituted with V a 1,

 2) The amino acid sequence of t-PA from position 294 to position 306 is the UK equivalent sequence Tyr-Arg-Arg-His-Arg-Gly-G ly-Ser-Val-Thr-Tyr-Va1 substituted t-PA analog,

3) From position 285 to position 30 of the amino acid sequence of t-PA, the corresponding sequence in the UK is G1u—Asn—G1n-Pro-Trp-Phe. -A la-A 1 a — I 1 e- Tyr-Arg-Arg-His—Arg—Gly—Gly-Ser-Val-Thr-Tyr-Va1

 4) t — From position 276 to position 30 of the amino acid sequence of PA, the corresponding sequence of U K is I 1 e-I 1 e-G 1 y-

G ly-G lu-P he-T hr-T hr-I 1 e-G lu-A sn-G ln-Pro-T rp-P he-A la-A l-I le-T yr-A rg—A rg—His-A rg-Gly-Gly-Gly-Ser-Val-Thr-Tyr—t—PA analog substituted with Val,

5) From position 276 to position 298 of the amino acid sequence of t-PA, the corresponding sequence in the UK is I1e-I1e-G1y-Gly-Glu-P. he-T hr one T hr-I 1 e — G lu-A sn-G ln-Pro-T rp — P he-A la — A la — lie — T yr one A rg — A rg — H is A substituted t-PA analog,

Which is highly fibrin specific ο

The position 276 of the natural t — PA referred to here is the position immediately after the single-stranded Z double-strand cleavage site, and corresponds to positions 276 to 306 of the natural t — PA. The amino acid sequence region of K corresponds to the amino acid sequence from 159 to 188, which is the position immediately after the cleavage site of pro-UK (—strand) to UK (double-strand). That is what you do. In the present invention, the corresponding amino acid sequence of UK is the amino acid sequence region of natural t-PA. The amino acid sequence of the natural t-PA is less than that of the natural amino acid corresponding to the natural t-PA, and the corresponding amino acid at position 295 of the natural t-PA in the UK Amino acids are treated as if they do not exist.

 The preparation of the t-PA analogs of the present invention can be performed, for example, using at least some amino acid residues of the amino acid sequence corresponding to positions 276 to 306 of natural t-PA. By replacing the peptide with a peptide consisting of the corresponding amino acid sequence in the UK. Specifically, for example, a cDNA encoding the amino acid sequence of the corresponding region of t-PA is replaced with a cDNA encoding the corresponding amino acid sequence in the UK or a synthetic oligonucleotide. And linking it with an appropriate expression vector, introducing it into the host, and expressing and producing the t-PA analog protein as the final target substance of the present invention. It can be. The details are described below.

For the production of the t-PA analogs of the present invention, cDNA that normally encodes natural t-PA can be used. CDNA encoding native t-PA has already been obtained, for example, as described in Reference 13 by cloning from paused melanoma cells. Therefore, its cDNA sequence and amino acid sequence are also known. In addition, the cDNA sequence and amino acid sequence of a modified t-PA that increases the half-life (for example, References 14 and 15), which are modifications of natural t-PA, are also known. These can also be used. The operation of replacing a specific sequence of cDNA, which is cloned in plasmid and whose sequence is also separated, has already been freely performed (Reference 16). In the present invention, a part of the amino acid sequence corresponding to positions 276 to 306 of natural t-PA can be easily replaced with the corresponding sequence of UK by the same operation. Can be. When the cDNA of natural t-PA is used, there is no suitable restriction enzyme recognition site suitable for substitution around the substitution site in the present invention. Nsp V (TTCG AA), A at the position of amino acid at position 6 (TTCGCA) and at position 309 to 311 (GCATAC) without changing this amino acid. It is convenient to introduce a restriction enzyme recognition site called cc I (GTATAC). As a technique therefor, site-directed mutagenesis by Zora et al. And Smith et al. (Reference 17) is effectively used. The DNA fragment containing the UK sequence to be replaced is prepared by double-stranded oligonucleotides synthesized by the phosphoamidite method (Reference 18). It is convenient to prepare it in the form. In addition, if necessary, a method in which a plurality of short synthetic oligonucleotides are prepared and these are joined together can be employed. The method for replacing the t-PA sequence with the UK sequence is to cut the t-PA sequence by cleaving the introduced NspV and AccI restriction enzyme recognition sites with the restriction enzyme, as described above. Instead, a DNA fragment containing the UK sequence is ligated to this site (Ligea —Shion) is convenient.

 The cDNA encoding the t-PA analog thus produced is ligated to a well-known expression vector such as CDM8 (ori-), and then introduced into an appropriate host. As a result, it is possible to express and produce the t-PA analog protein which is the final target substance of the present invention. As the host, any of prokaryotic organisms such as bacteria and eukaryotic organisms such as mammalian cells and yeast can be used. Examples of animal cell hosts include COS-1 and CH0 cells. Can be An efficient method for introducing an expression vector into a host is the electric pulse method (Reference 19).

The t-PA analogs produced in the culture supernatants include zinc clear agarose, concananol black A agarose, Sephadex G-150 agarose, etc. The t-PA analog thus obtained, which can be easily purified by a known method (Reference 20) or the like, has high fibrin specificity Therefore, when administered to a living body as a thrombolytic agent, it can be used as an active ingredient of a pharmaceutical composition having reduced side effects of systemic blood bleeding as compared with natural t-PA. Further, since the t-PA analog of the present invention has PAI-11 resistance, it can be used as an active ingredient of a pharmaceutical composition which is effective at a lower dose than natural t-PA. In addition, a prescribed amount can be dispensed into a container by a conventional method and freeze-dried, whereby the preparation can be extremely easily made. This lyophilized product is a pharmaceutically acceptable carrier. It is dissolved in a single substance, for example, physiological saline, and administered by intravenous or intracoronary injection. The dose may be about the same as the dose of natural t-PA currently used in clinical practice, but the t-PA analog can be used more than the dose because it has few side effects. Administration is performed by continuous intravenous injection or by injecting part of the planned dose first intravenously and continuous intravenous infusion of the remainder.

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

 EXAMPLES Preparation of t—PA Analogue CS—1

 1. Construction of a plasmid pHSGGB-0000 for cassette-forming the -t-PAC DNA region · The cDNA of t-PA was obtained from Bowes melanoma cells. By incorporating the cDNA into plasmid pTZB (described later), plasmid pTZB—0000 was obtained. Next, the t—P Ac DNA portion was transferred from pTZB—0000 to the plasmid pHSGB to obtain pHSGB—00.

Plasmid p TZB (Reference 21) was prepared by removing all the multi-cloning sites other than BamHI of commercially available Plasmid PTZ 18R (Toyobo Co., Ltd. (T0Y0B0)). The plasmid pTZB-0000 (Reference 21) is a BamHI fragment (Ba) that completely contains the t-PA translation region in the BamHI site of pTZB. mHI cassette) was inserted (Reference 21). Fig. 1 shows a schematic diagram of the restriction enzyme of pTZB-1000. Show.

 The plasmid pHSGB is a multiplex of commercially available plasmid pHSG398 (manufactured by Takara Shuzo Co., Ltd.) (Fig. 2-A), which has the chromium penicol resistance gene as a marker. The entire DNA site was prepared by replacing the synthetic DNA linker (Fig. 2-B). pHSGB-O00 was constructed by inserting the obtained BamHI cassette into the BamHI site of pHSGB (Fig. 3).

 The synthetic DNA linker was prepared as follows. First, the oligonucleotide of sequence a, which is self-complementary as shown in Fig. 2-B, was synthesized. The synthesis was carried out using a type 381A DNA synthesizer manufactured by Applied Biosystems, and the purification was performed using the company's 0PC cartridge. This synthetic oligonucleotide is converted into a double strand (FIG. 2—B, sequence b), digested with Fokl and HindE, and synthesized DNA linker is synthesized. (Figure 2-B, sequence c) was isolated and purified from a polyacrylamide gel by standard techniques.

 This synthetic DNA linker and pHSG398 were digested with HindIII and EcoRI, and the 5 'end was dephosphorylated, and ligated. The strain was transformed to obtain the plasmid pHSGB (Figure 2-A).

This pHSGB is digested with BamHI and the 5 'end is removed. The oxidized product is ligated with a fragment (BamHI cassette) obtained by quenching pTZB-0H with BamHI and isolating it from polyacrylamide gel. Then, Escherichia coli HB101 strain (manufactured by Takara Shuzo Co., Ltd.) was transformed to obtain plasmid pHSGB-0000 (FIG. 3).

 2. Assembling the plasmid p H S GB B—0000 NA for insertion of the U K sequence—

 t In order to replace a part of the PA sequence with the UK sequence, p HSGB-0.0NA containing the NspV and AccI recognition sequences in the t-PA translation region of pHSGB-000 Made. For the preparation, a site-directed mutagenesis technique using a synthetic oligonucleotide was used. Fig. 4 shows a schematic diagram of the assembly.

 The single-stranded form M13tvl9EE (Reference 21) was obtained as type I for site-directed mutagenesis. This M13tV19EE contains the cDNA for the native t-PA amino acids 205-32.

 Ml3mp19E ENA is a single-stranded form of Ml3tv19EE, and the synthetic oligonucleotides P—NspV and P—Acc shown in FIG. I and Anil,

It was obtained using Mutan ^™ -G system (Takara Shuzo Co., Ltd.). These synthetic oligonucleotides were synthesized and purified by the same method as described above.

The 47 2 base pair fragment (Fragment EEN) generated by digestion of this M13m19 ENA with EcoRI. A) was isolated from polyacrylamide gel by a standard method. Escherichia coli HB101 strain was transformed by digesting this fragment EENA and pHSGB-1000 with EcoRI and ligating the one with the 5 'end dephosphorylated. As a result, pHSGB—1000 NA was obtained (FIG. 4).

3. Plasmid pH with UK high-bridge t-PA

Construction of S G B — C S 1 (CS 1 is called C S T U: Renamed ±-)

 Four types of oligonucleotides (CS1M, CS1R, CS2M and CS2R) containing the partial sequence of UK were synthesized. The sequence is shown in FIG. These four synthetic oligonucleotides were annealed, and this was combined with the digestion of pHSG-0000NA with NspV and AccI. Thus, E. coli HB101 strain was transformed to obtain a plasmid pHSGB-CS1 (FIG. 7) having UK hybrid type t-PA.

4. Construction of plasmid pCDM8—CS1 that expresses UK hybrid t-PA in mammalian cells

 Plasmid pCDM8-0000 provided the sequence of the prime motor required to express CS1 in mammalian cells.

 PCDM8-0000 is the expression vector CDM8

 (ori —) with the natural t-Pac DNA portion incorporated therein (Reference 21).

Digest PCDM 8 — 0 0 0 with B gl E and B al I The relevant portion of native t-PAc DNA was removed and the 5 'end was dephosphorylated and the plasmid pHSGB-CS1 described above was digested with BgII and Bal1. Things were ligated. This transforms E. coli MC1061 / 3 (Funakoshi Pharmaceutical Co., Ltd.), and a plasmid p CDM8—CS for expressing the desired UK hybrid t-PA. 1 was obtained (Figure 8).

5. Expression of PCDM8-CS1 in COS-1 cells

 The above-mentioned expression plasmid encoding UK hybrid t-PA, pCDM8-CS1, was used to transform COS-1 cells by the electric pulse method described above. After culturing for 24 hours in a DMEM medium containing 10% fetal calf serum and 10 / Ig Zml of lysine chromatographically treated, the medium was serum-free and aprotinin-free DMEM. The medium was replaced and the culture was continued for another 96 hours. After centrifugation of the culture solution, the supernatant was recovered to obtain a t-PA analog CS-1. 90% or more of CS-1 contained in the thus obtained supernatant was present in a single-stranded state. This culture supernatant was used for the subsequent evaluation system.

Example 2. Preparation of t-PA analog C S-2

In place of CS 1 M, CS 1 R, CS 2 M and CS 2 R in Example 1, CS 3 M, CS 3 R, CS 2 M and CS 2 R shown in FIG. 6 were used. A t-PA analog CS-2 was produced in the same manner as in Example 1 except that the construction of the expression plasmid was as described below. Plasmid ρ CDM 8 — CS2, which expresses the UK hybrid t_PA in mammalian cells, is a plasmid p CDM obtained by introducing a Bg1I site into CDM 8 (ori —). 8 (Bg1I) (Reference 21) digested with Bg1I and then dephosphorylated at the 5 'end, and pHSGB in Example 1—a plasmid pHSGB corresponding to CS1 — It was obtained by ligating CS2 digested with BamHI and transforming E. coli MC1061 / P3.

Example 3.Production of t-PA analog Cs13

 Instead of CS 3 M, CS 3 R, CS 2 M and CS 2 R in Example 2, CS 4 M, CS 4 R, CS 2 M and CS 2 R shown in FIG. 6 were used. A t-PA analog CS-3 was produced in the same manner as in Example 2 except for the above.

Example 4 Preparation of t-PA Analogue CS-4

 Instead of CS 3 M, CS 3 R, CS 2 M and CS 2 R in Example 2, CS 4 M, CS 4 R, CS 5 M and CS 5 R shown in FIG. 6 were used. A t-PA analog CS-4 was produced in the same manner as in Example 2 except for the above.

Example 5 Preparation of t-PA Analogue CS-15

 In place of CS 3 M, CS 3 R, CS 2 M and CS 2 R in Example 2, CS 1 M, CS 1 R, CS 6 M and CS 6 R shown in FIG. 6 were used. Otherwise in the same manner as in Example 2, a t-PA analog CS-5 was produced.

Reference example COS-1 cells were transformed using the expression plasmid PCDM 8-0000 described above encoding native t-PA, and the culture supernatant was recovered to obtain native t-PA. The method was in accordance with 5. of Example 1.

 5 (Evaluation Examples)

 Subsequent evaluations were performed for CS-1, CS-2, CS-3, CS-4, CS-5 and natural t-PA. Evaluation Example 1. Evaluation of fibrin specificity

 Various fibrin cofactors were added to a system for measuring the activation of t-PA using the chromogenic synthetic substrate S-2251, and fibrin was added. The specificity was evaluated according to the method described in Ref.

 1) Fiber added system

 First, 60 international units Zml in a 96-well plate

15 thrombin (PBS containing 0.01% Tween 80) 10

 1 was added. Next, each t-PA sample diluted to 200 ng / ml (but 150 ng / ml for CS-3) in PBS containing 0.01% Tween 80 (protein concentration required for preparation) Is known using polyclonal antibodies against natural t-PA

The measurement was performed by 20 ELISA methods. ) 20 〃 1 was added, and the reaction mixture (plasminogen 135 nM, fibrinogen 0.8; Μ, S-225 378 / zM (0. 0 9% (V / V) Tween 80 and 4.4 mg / ml containing gelatin 0.13 M Tris-HCl pH 7.8 solution)] 200

25 1 was added. 3 After starting the incubation at 7 ° C, Absorption at 405 nm using a rate reader

(◦ D _{4. 5)} was measured over time. At the same time, the absorbance at ₄₉ O nm (0D490) was measured to correct the effect of turbidity due to fibrin, and the 0D49 was measured at each time. The value 〇 D _4. Subtracted from ₅ .

Measured at PA the same manner, the absorbance after a certain time for the concentration - as a static Nda de various native t diluted to a concentration _{(OD 405 - 0 D 4 9} 0) value plot a calibration curve Was prepared, and the relative activity of each t-PA analog to native t-PA was determined based on this calibration curve.

2) Addition of fibrinogen

 The test was performed in the same manner as in the fibrin addition system. However, PBS containing 0.01% Tween 80 was used instead of trombin.

Table 1 shows the relative activity of each t-PA sample to native t-PA. Off Lee Bed Li emissions in addition system reaction 3 0 minutes after full Lee Bed Li Bruno 2 hours after absorbance in one Gen addition system (OD _{4 5} -.. 〇 D ₄₉₎ corresponding to native t - concentration of PA Was determined from the above calibration curve. The fibrin specificity was expressed as a ratio (the activity in the fibrin-added system, the activity in the fibrinogen-added system). Table 1 Fibrin specificity Fibrin coff completion Sample name F g 1) F n 1) F n / 'F g natural 1.0 0 1 .0 0 1 .0 0 cs-1 0 .1 3 1 .0 4 8 .0 0 cs-2 0 .0 7 0 .8 2 1 1 .7

 C S-30 .2 2 1 .2 8 5 .8 2

(cs-40 .0 80 .9 11 1 1 .4) ²⁾ cs-5 0 .0 10 .3 0 3 0 .0

° Fg: fibrinogen, Fn: fibrin

Both the Fg-added system and the Fn-added system were determined from a calibration curve plotted by log-logarithm. (However, CS-4 is a constant plot.) ^{2 3} CS-4 is poor in reliability because the concentration at the time of addition was obtained using 50 ngZml. 'Table 1 shows that the t-PA analog of the present invention has a very low plasminogen activating activity in the fibrinogen-added system compared to the natural t-PA, It was clarified that the purified system had activity close to or higher than that of natural t-PA.

Evaluation Example 2. Evaluation of plasma clot specificity

Using human plasma, each t in plasma and plasma clot — The plasminogen activating activity of PA was measured in a reaction system using the chromogenic synthetic substrate S—2251. The method was performed according to the method shown in Ref.

 1) Plasminogen activating activity in plasma clot

Into a 96-well plate, 60 international units Zml of trombin (PBS containing 0.01% Tween 80) 101 was added. Next, add 40 a1 of each t-PA sample diluted to 200 ng / ml (but 150 ng / ml for CS-3) with PBS containing 0.01% Tween 80. Further, a reaction mixture [plasma 90.1, 9.1 mS—2251 (PBS containing 0.01% Tween 80) 10〃1] .100 01 was added. After starting the 3 7 ° C Day Nkyube DOO, absorbance at 4 0 5 nm using a pre-over preparative rie da one (OD _{4. 5)} was measured over time. At the same time full Lee Bed absorbance at 4 9 O nm in order to correct the influence of turbidity caused by re down (OD _{4 9.)} As 〇 at each time to measure the D _{4 S} Q value OD _4. Subtracted from ₅ .

Measured at PA the same manner, the absorbance after a certain time for the concentration - scan evening as a Nda de various native diluted to a concentration t (OD _{4 5} -.. 〇 D _{4 9)} the value pro Tsu A calibration curve was prepared, and the relative activity of each t-PA analog with respect to native t-PA was determined based on the calibration curve.

 2) Plasma plasminogen activating activity

The measurement was performed in the same manner as the measurement of the plasminogen activation activity in the plasma clot. However, PBS containing 0.01% Tween 80 was used instead of thrombin. Table 2 shows the relative activity of each t-PA sample to natural t-PA. Reactions after 2 hours in plasma mass system, also in the plasma-based absorbance after 4 hours - was determined concentration of native t one PA corresponding to _{(0 D 4 Q 5 0 D} 4 9 0) from the calibration curve . Clot specificity was expressed as the ratio (active in plasma clot Z, activity in plasma).

 Table 2 Plasma clot specificity

 u The value of the plasma clot system was determined from a calibration curve plotted using both numbers.

²⁾ <0.06 and <0.08 represent below the detection limit.

³⁾ Measurement was not possible due to the low concentration of the sample.

As shown in Table 2, the t-PA analog of the present invention has a very low plasminogen activating activity in plasma as compared to natural t-PA, but it has a lower activity than natural t-PA in plasma clots. Close activity It became clear that it had.

Evaluation Example 3. Evaluation of PAI-1 resistance

t The PAI-1 resistance of single- or double-stranded single PA was measured according to the method described in the aforementioned reference 22). Add 66.7 M aprotinin to 6.6 pi 1 to 1 ml of 45 ng / ml t-PA sample, which has been treated with or not treated with plasmin, and then add 20 〃 1 to 2 nM. Mix with PAI-1 or water 101 and react for 20 minutes at room temperature, then add 60 International Units Zml of Thrombin 101 and further add fibrin specificity In the evaluation of, the reaction mixture used was added, and the incubation was started at 37 ° C. Absorbance at 4 0 5 nm after 2 0 minutes (OD _{4. 5)} was measured. At the same time the absorption at 4 9 0 nm in order to correct the influence of turbidity caused by off Lee Bed Li down (0 D _49.) Measured 〇 D _{4. 5} minus that value.

Each t - PA of PAI -. 1 no addition time of (OD _{4 5} -

The ratio of the (0D4Ό5-0D490) value at the time of addition of PAI-11 when the value of (0D490) was 100% is shown in Table 3 as the residual activity (). Show.

Table 3 PAI-1 resistance (residual activity%)

¹⁵ Measurement not possible.

 According to Table 3, the t-PA analogs of the present invention

It became clear that the resistance to PAI-11 was much higher than that of A. References

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22) E 丄 Madison et al., Ature 3 3 9 7 2 1-7 24 (19989) Industrial applicability The t-PA analogs of the present invention, when circulating in the blood, activate plasminogen followed by fibrinogen. It is unlikely to cause degradation of thrombus, and will express plasminogen activating activity close to natural t-PA and dissolve thrombus only when it reaches the local thrombus where fibrin is present. t Compared to known variants with improved fibrin specificity, as compared to PA, the fibrin specificity is higher, and thus the thrombus specificity is higher. It is expected that the side effect of systemic bleeding tendency is unlikely to occur. Therefore, the t-PA analog of the present invention is extremely significant as a novel thrombolytic agent.

Claims

1. In the amino acid sequence of the tissue plasminogen activator, the amino acid sequence corresponding to positions 276 to 3006 of the natural tissue plasminogen activator A t-PA analog, characterized in that at least part of it is substituted with the corresponding amino acid sequence of urinary plasminogen activator.  2. Analysis of urinary plasminogen activator's corresponding amino acid sequence Columns are amino acid sequences from positions 18 to 18 corresponding to positions from 300 to 310 of natural tissue brass minogen activator; G1y-G 2. The t-PA analog according to claim 1, wherein the t-PA analog is an amino acid sequence containing 1 y-Ser-Val-Thr—ι'yr-Va1.  3. The corresponding amino acid sequence of the urinary plasminogen activator has a corresponding amino acid sequence from position 177 to position 177 corresponding to positions 294 to 306 of natural tissue brass minogen activator. Amino acid sequence up to position 18: Tyr-Arg-Arg-His-Arg-Gly-Gly-Ser-Va1 One Thr-Tyr—Val 3. The t-PA analog according to claim 1 or 2, wherein the amino acid sequence comprises an amino acid sequence. 4. The corresponding amino acid sequence of urinary plasminogen activator corresponds to positions 285 to 306 of natural tissue brass minogen activator. 8 Amino acid sequence up to position 8; G 1 υ-A sn — G in — Pro — T rp-P he-A la-A 1 a — I 1 e-T yr-A rg-A rg-H is — A rg — Gly-G 1 y-Ser-Va 1-T hr-T 4. The t-PA analog according to claim 1, 2 or 3, characterized in that it is an amino acid sequence containing yr-Va1.  5. The amino acid sequence of the urinary plasminogen activator corresponds to the amino acid sequence from position 159 to position 156 corresponding to positions 276 to 306 of natural tissue brass minogen activator. Amino acid sequence up to position 18 8; I 1 e — I 1 e-Gly-Gly-Glu-Phe-Thr-Thr-I1 e-Glu-A sn-G 1 n-Pro-T rp-P he-A la-I la-I 1 e-T yr-A rg-A rg-H is-A rg-G ly-G ly-Ser-Va The t-PA analog according to claim 1, 2, 3 or 4, characterized by an amino acid sequence containing 1-Thr-Tyr-Val.  6. The corresponding amino acid sequence of urinary plasminogen activator corresponds to positions 276 to 298 of natural tissue plasminogen activator. Amino acid sequence up to position 80; II e — I 1 e-G 1-G 1 y-G lu-P he-T hr-T hr-I 1 e-G lu-A sn-G ln Claims characterized in that it is an amino acid sequence containing -Pro-Trp-Phe-Ala-A1a-IIe-Tyr-Arg-Arg-His. 2. The t-PA analog according to 1. 7. In transformed bacteria, yeast or mammalian cells A recombinant expression vector capable of expressing a DNA encoding the t-PA analog according to any one of claims 1 to 6.  8. A bacterium, yeast or mammalian cell transformed with the recombinant expression vector according to claim 7.  9. A therapeutic agent for thrombosis, comprising the t-PA analog according to any one of claims 1 to 6 as an active ingredient.