JPH06327473A - New modified t-pa substance - Google Patents

Abstract

PURPOSE:To obtain a new modified t-PA substance improved in fibrin specificity. CONSTITUTION:The modified t-PA substance has a substituted region substituted with an amino acid sequence of a urinary plasminogen activating factor (hereinafter abbreviated to UK) in an amino acid sequence of the t-PA. This modified substance the substituted region, having at least a part of an amino acid sequence which corresponds to the 276- to the 306-positions of the amino acid sequence in the natural type t-PA and is substituted with the amino acid sequence of the UK having a corresponding relationship expressed in the formula and further extending to at least a part of the amino acid sequence corresponding to the 266- to the 275-positions and/or the 307- to the 321-positions of the natural type t-PA. The modified substance is obtained by carrying out the substitution of a desired sequence of t-PA cDNA cloned in a plasmid according to a conventional method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel tissue plasminogen activator (hereinafter abbreviated as t-PA) variant. More specifically, in the amino acid sequence of t-PA, at least part of the amino acid sequence corresponding to positions 266 to 321 of natural t-PA corresponds to urine plasminogen activator (hereinafter abbreviated as UK). The present invention relates to a novel t-PA variant having improved fibrin specificity by being replaced with an amino acid sequence of

[0002]

2. Description of the Related Art Natural t-PA is converted to plasmin which is an active enzyme by hydrolyzing plasminogen which is an inactive zymogen present in plasma. Plasmin decomposes fibrin clots (thrombus) in blood vessels caused by various causes (Reference a).

While existing thrombolytic agents such as UK and streptokinase have no fibrin specificity, t-PA has a relatively high fibrin specificity. Therefore, it has been attracting attention as a thrombolytic agent that has the property of specifically dissolving fibrin and eventually thrombus, and has few side effects such as bleeding. However, in reality tp
When A was used clinically, it became clear that recanalization of the occluded coronary artery required a much higher dose than expected (Reference b). As a result of large dose administration, t-PA
Although it is fibrin-specific, it also decomposes some fibrinogen in the blood, so that the side effect that systemic bleeding tendency eventually occurs is a problem that cannot be ignored. From such a viewpoint, t-PA variants for the purpose of imparting higher thrombus specificity have been produced, and many reports (references c and d) have been made, but the objectives were still achieved sufficiently. There are few things. Literature e is a successful example
At position 296 to 29 in the native t-PA sequence.
We have prepared a t-PA variant with improved thrombus specificity by substituting all four amino acids up to the 9th position with alanine. The effect is that the fibrin specificity is 4.4 times at most, and plasma clump specific. The sex is about 3 times.

[0004]

DISCLOSURE OF THE INVENTION The present invention is a natural type t
-A novel t with improved fibrin specificity compared to PA
-To provide a PA variant. More specifically, in the presence of fibrinogen, the plasminogen activating activity is very low, but in the presence of fibrin, plasminogen activating activity close to or higher than that of natural t-PA is expressed, and therefore the fibrin specificity is increased. An improved novel t-PA variant is provided.

Since the t-PA variant of the present invention has improved fibrin specificity, it is less likely to cause activation of plasminogen and subsequent degradation of fibrinogen when circulating in blood, and the presence of fibrin is present. Only after reaching the local thrombus, the plasminogen activating activity close to that of natural t-PA is expressed and the thrombus is dissolved. Therefore, even if it is used at a dose equivalent to that of natural t-PA, it is systemic. It is expected that the side effect of bleeding tendency will not occur easily.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have recently found that t-PA in which at least a part of the amino acid sequence from the 276th position to the 306th position of natural t-PA has been substituted with the corresponding amino acid sequence of UK. We have found that PA variants show excellent fibrin specificity. As a result of further research, the substitution mutation as described above was further extended, and the substitution region was extended to the 266th position on the N-terminal side and the 321st position on the C-terminal side in natural t-PA. The novel t-P with improved fibrin specificity
It was found that the A variant was obtained.

That is, the gist of the present invention is (1) t-P
In a t-PA variant having a substitution region substituted with the amino acid sequence of UK in the amino acid sequence of A, the natural t
-At least part of the amino acid sequence corresponding to positions 276 to 306 of the amino acid sequence of PA is substituted with the amino acid sequence of UK having the following correspondence, and the substitution region is a natural t-type. -A t-PA variant having a substitution region extending to at least a part of the amino acid sequence corresponding to the 266th position to the 275th position and / or the 307th position to the 321st position of PA,

[0008]

[Chemical 2]

(2) In transformed prokaryotic or eukaryotic cells, the t-P according to the above (1)
A recombinant expression vector capable of expressing a DNA encoding the A variant, (3) a prokaryotic or eukaryotic cell transformed with the recombinant expression vector according to (2) above, and (4) above. (1) A therapeutic agent for thrombosis, which comprises the t-PA variant described in (1) as an active ingredient.

In the present invention, the amino acid sequence of natural t-PA means a sequence consisting of 527 amino acids already reported by Pennica et al. (Reference f). The amino acid sequence of urinary plasminogen activator (UK) refers to a sequence consisting of 411 amino acids previously reported by Holmes et al. (Reference i).

The t-PA variant of the present invention is a natural t-PA.
At least part of the amino acid sequence corresponding to positions 276 to 306 of the amino acid sequence of PA is substituted with the amino acid sequence of UK having the following correspondence, and the substitution region is 266 of natural t-PA. Position 275 and / or 307 to 321 and at least a part of the amino acid sequence corresponding to at least part of the amino acid sequence, which is characterized in that it is 276 of natural t-PA.
The position is located immediately after the cleavage site from single-stranded to double-stranded, and the corresponding amino acid sequence region of UK is the double-stranded form of pro-UK (single-chain precursor) (ie, U
It refers to amino acid residue 159, which is immediately after the cleavage site to K).

[0012]

[Chemical 3]

The correspondence in the amino acid sequence means that the 266th position of natural t-PA corresponds to the 150th position of UK, and the amino acid sequence of t-PA in the upper row corresponds to the amino acid sequence of UK in the lower row. Refers to the relationship. Further, the amino acid sequence in the substitution region of UK referred to in the present invention has two amino acids less than the amino acid sequence in the substitution region of natural t-PA, and the UK corresponding to positions 270 and 295 of natural t-PA. Amino acid (the portion indicated by-in the above sequence notation) corresponds to the amino acid deleted in the substitution of UK with the amino acid sequence. Therefore, in the present invention, 26 of natural t-PA is used.
The amino acid sequence of UK corresponding to positions 6 to 321 is
Refers to the amino acid sequence of positions 150 to 203 of UK.

Preferred examples of the t-PA variant of the present invention include, for example, 26 amino acid sequences of natural t-PA.
From position 6 to position 306, the corresponding sequence of UK, that is, the amino acid sequence of UK substituted from position 150 to position 188 (SEQ ID NO: 1), from the position 276 of the amino acid sequence of natural t-PA Up to position 321 corresponds to the sequence of UK, that is, from position 159 to 2 of the amino acid sequence of UK.
Those substituted at position 03 (SEQ ID NO: 2), or from the position 266 to 32 of the amino acid sequence of natural t-PA
Examples include a sequence corresponding to the UK up to the 1st position, that is, a sequence in which the amino acid sequence of the UK is substituted from the 150th position to the 203rd position (SEQ ID NO: 3). The t-PA variant of the present invention is substituted with the corresponding amino acid sequence of UK in the substitution region as described above, but the amino acid sequence other than the substitution region is not particularly limited. That is, the natural t-P
It may be the same as the amino acid sequence of A, or may be a variant amino acid as long as it does not affect the properties of the t-PA variant of the present invention.

The t-PA variant of the present invention can be prepared, for example, by replacing at least a part of the amino acid sequence of the amino acid sequence corresponding to positions 266 to 321 of natural t-PA with the amino acid of the corresponding part of UK. This can be done by substituting a peptide consisting of the sequence. Specifically, for example, cDNA encoding the amino acid sequence of the region of t-PA
A is substituted with cDNA or synthetic oligonucleotide encoding the corresponding amino acid sequence of UK, ligated with an appropriate expression vector and introduced into a host, and the t-PA modified protein which is the final object of the present invention is obtained. It can be performed by expressing and producing. The details will be described below.

For the production of the modified t-PA of the present invention, a cDNA encoding naturally occurring t-PA can be used. The cDNA encoding native t-PA has already been obtained, for example, by cloning from Bose melanoma cells, as described in, for example, document f, and thus its cDNA sequence and amino acid sequence are also known. In addition, cDNA sequences and amino acid sequences of t-PA variants (for example, documents g and h) having a long half-life for altering natural t-PA are also known, and these can also be used.

T-PA cloned in a plasmid
The method for substituting a specific sequence of cDNA is already in a freely available state, and in the present invention, t
-The amino acid sequence of PA can be replaced. As a method of substitution, it is convenient to amplify the cDNA sequence having the substitution and replace the obtained cDNA fragment with the corresponding sequence of natural t-PA. Specifically, an oligonucleotide consisting of a cDNA sequence having a substitution,
Another oligonucleotide necessary for amplification is used as a primer, and the natural t-PA cDNA is used as a template to prepare the above-mentioned 2
A convenient method is to amplify the region flanked by seed primers and replace the resulting amplified fragment with the corresponding sequence of native t-PA. As a method for amplifying a region sandwiched by two kinds of primers, for example, polymerase chain
The reaction (PCR) method is effectively used (reference j). That is, two types of synthetic oligonucleotides are used as primers, and cDNA is used as a template to amplify DNA by Taq polymerase. To perform this operation, a commercially available automatic DNA amplification device is effectively used. It should be noted that only one type of amplified fragment is used to produce natural t-PA.
When it is not a restriction enzyme fragment that is convenient for replacement with the corresponding sequence of, the two types of amplification fragments can be prepared and ligated together. To do so, for example, Yon and Fried
The method of gene fusion according to d) (reference k) may be used. That is, when two kinds of amplified fragments having substitutions are prepared by the above method, the two are mixed and the PCR reaction is carried out again, the substitutions are introduced and it is convenient to replace with the corresponding sequence of natural t-PA. A fragment having a good restriction enzyme site can be obtained.

The synthesis of the oligonucleotide by the phosphoamidite method may be carried out using, for example, a 381A type DNA synthesizer manufactured by Applied Biosystems Co., Ltd. according to the principle shown in Reference 1. Further purification may be performed using the OPC cartridge for purifying oligonucleotides manufactured by the same company. In addition, in order to confirm the desired substituted or inserted DNA sequence, the principle of the dideoxy method (Reference m) can be used, for example, a method of cycle sequencing using Taq polymerase. For that purpose, for example, 373A type DNA sequencer manufactured by Applied Biosystems is effectively used. In addition, cleavage and deletion of DNA by restriction enzymes as described in Examples, and DNA generated by them
Genetic manipulations such as separation, collection, and ligation of fragments by polyacrylamide gel electrophoresis are all known, and the literature n is mainly referred to.

The cDNA encoding the modified t-PA obtained as described above is ligated to a well-known expression vector such as CDM8 (ori ⁻ ), and then introduced into an appropriate host to give the cDNA. It is possible to express and produce the t-PA variant protein which is the final target substance of the invention. Escherichia coli strains and animal cell strains as hosts are already widespread and easily available unless otherwise specified, and examples of animal cell hosts include COS-1 and CHO cells.
To transform a suitable host with the expression plasmid,
The electric pulse method (reference o) may be used. The culture supernatant of the transformed cells contains the t-PA variant in such an amount that it can be used as it is for various activity measurements by appropriate dilution.

The thus-obtained t-PA variant of the present invention has a high fibrin specificity, and therefore, when administered in vivo as a thrombolytic agent, it has a higher fibrin specificity than natural t-PA. It can be used as an active ingredient of a pharmaceutical composition having a reduced side effect of systemic bleeding tendency. T-P of the present invention
The modified A can be extremely easily obtained as a preparation by dispensing a required amount in a container by a conventional method and lyophilizing it. This lyophilizate is dissolved in a pharmaceutically acceptable carrier substance, for example saline, and administered by intravenous or intraarterial or intracardiac injection. Regarding the dosage, the natural type of t-PA currently used clinically
However, since the t-PA variant of the present invention has few side effects, it can be used in a larger dose than the above dose. The administration method is continuous infusion, or a part of the planned dose is intravenously first and the rest is continuous intravenous infusion.

[0021]

EXAMPLES Examples will be shown below as examples of the present invention, but the present invention is not limited thereto. In addition, the natural t-P in Examples 1 to 3 below.
The substitution region of A is shown in FIG. Example 1. Construction of pUC-CS6 Using pCDM8-CS1, 26 of native t-PA was constructed.
Construction of pUC-CS6, a subclone having an EcoRI fragment of about 480 base pairs, which encodes a sequence in which the amino acid sequence from 6th position to 306th position is replaced with the sequence from 150th position to 188th position (SEQ ID NO: 1) of UK did. pUC-CS
A schematic diagram of the construction of 6 is shown in FIG. 2, and the details are shown below. Note that pCDM8-CS1 is disclosed in JP-A-4-14468.
This is a known plasmid which is the same as pCDM8-CSTU described in Japanese Patent Publication No. 2) and in which the amino acid sequence from position 276 to position 306 of natural t-PA is replaced with the sequence from position 159 to position 188 of UK. . In the present invention pCD
M8-CSTU was designated as pCDM8-CS1 and used in the construction of the following plasmids.

First, 2 from the 266th position of natural t-PA.
Two complementary oligonucleotides (PCS6M, SEQ ID NO: 4 and PCS6R) in which a portion corresponding to the amino acid sequence at position 75 is replaced with the corresponding amino acid sequence of UK, and the front and rear sides thereof match the base sequence of CS1. , SEQ ID NO: 5) was synthesized. Although PCS6M and PCS6R do not correspond to completely complementary parts of the gene, they are designed so that they can be annealed complementarily at 29 bases on the 5'end side of each. Furthermore, the oligonucleotide primers FR124 (SEQ ID NO: 6) and FR53, which are necessary for amplifying the target portion using the PCR reaction,
(SEQ ID NO: 7) was also synthesized. Applied Biosystems 381 for the synthesis of all oligonucleotides
A type A DNA synthesizer was used, and the purification was performed using the OPC cartridge of the same company. As schematically shown in FIG. 2, the synthesized FR53 and PCS6R were used as primers, and pCDM8-CS1 was used as a template for the first-stage P.
DNA fragment FC having a substitution and having a CR reaction
S6A was amplified. On the other hand, PCS6M and FR124 were used as primers to similarly amplify a DNA fragment FCS6B having a substitution.

The conditions for the PCR reaction in the first step are pCD
1 μg of M8-CS1, 50 pmol of each primer in the above combination, 600 μM of dNTP, P
CR reaction buffer [KCl 150 mM, Tris ・ HCl
(PH 8.5) 10 mM, MgCl ₂ 2.5 mM, gelatin 0.2 mg / ml], Taq polymerase 2 uni
ts (manufactured by Takara Shuzo Co., Ltd.) was mixed so as to have a volume of 100 μl, and 1 reaction at 95 ° C. for 1 minute, 57 ° C. for 1 minute and 70 ° C.
Cycle, followed by 94 ° C for 30 seconds, 57 ° C for 1 minute, 70
The reaction at 1 ° C for 1 minute was performed for 9 cycles. The above-mentioned amplified fragments FCS6A and FCS6B were separated and purified by a standard method using polyacrylamide gel electrophoresis, and then subjected to the second-stage PCR reaction under the following conditions.

The conditions for the PCR reaction in the second step were as follows: Fragments FCS6A and FCS6B were 300 n each.
g, primer FR124 and primer FR53 at 10 pmol each, dNTP at 600 μM, PC as described above.
R reaction buffer, Taq polymerase 2 units 10
Mix to give 0 μl, 95 ° C for 1 minute, 57 ° C for 1 minute,
One cycle of reaction at 70 ° C for 1 minute followed by 94 ° C for 30 minutes
Second reaction, 57 ° C. for 1 minute, 70 ° C. for 1 minute was performed for 9 cycles. As a result, a ligation fragment F of about 1100 base pairs was obtained.
Generated by CS6.

The ligated fragment FCS6 thus obtained was digested with EcoRI, and this and the commercially available plasmid pUC118 (Takara Shuzo Co., Ltd.) digested with EcoRI were ligated, and E. coli JM109 was prepared using this reaction solution. The strain competent cells (manufactured by Toyobo Co., Ltd.) were transformed. A recombinant plasmid was extracted from the obtained transformant by a known method. The nucleotide sequence of the fragment inserted into the recombinant plasmid was confirmed by the nucleotide sequence determination method using the dideoxy method, and these were determined from positions 266 to 3 of natural t-PA.
It was confirmed that the portion corresponding to the amino acid sequence up to position 06 encoded a sequence in which the corresponding amino acid sequence of UK (positions 150 to 188) was substituted. One of these was chosen and named pUC-CS6.

Example 2. Construction of pUC-CS7 In the same manner as in Example 1, using pCDM8-CS1, the amino acid sequence of natural t-PA at positions 276 to 321 was a sequence from UK at positions 159 to 203 (SEQ ID NO: : PUC-C, a subclone carrying an approximately 480 base pair EcoRI fragment encoding the sequence replaced by
S7 was constructed. Figure 3 shows a schematic diagram of the construction of pUC-CS7.
The details are shown below.

To construct pUC-CS7, the desired UK
2 new oligonucleotides with substitutions
Kinds (PCS7M, SEQ ID NO: 8 and PCS7R, SEQ ID NO: 9) were synthesized. PCS7M and FR1 primer
24, PCS7R and primer FR53 were used, and the fragments FCS7A and FC were subjected to the first-stage PCR reaction under the same conditions as in Example 1.
S7B was amplified, and they were separated and purified by a method using polyacrylamide gel electrophoresis. The FCS7A and FCS7B thus obtained were
A second-step PCR reaction was performed under the same conditions as in Example 1 except that 6A and FCS6B were used instead. As a result, the ligated fragment FCS7 was generated.

The ligation fragment FCS7 thus obtained was gently partially digested with EcoRI (this ligation fragment was inserted into the region intended for subcloning, Ec.
Because it has an oRI site). This is pU as in Example 1.
It was inserted into the EcoRI site of C118. The nucleotide sequence of the fragment inserted into the plasmid was confirmed by the nucleotide sequence determination method using the dideoxy method, and one of these was determined from position 276 to 321 of natural t-PA.
It was confirmed that the part corresponding to the amino acid sequence at position encoded the sequence in which the amino acid sequence at positions 159 to 203 of UK was substituted. This was named pUC-CS7.

Example 3. Construction of pUC-CS8 Using pUC-CS6 described in Example 1 and the oligonucleotides PCS7M and PCS7R described in Example 2 in the same manner as in Example 2, 26 of native t-PA was prepared.
Construction of pUC-CS8, a subclone retaining an EcoRI fragment of about 480 base pairs, which encodes a sequence in which the amino acid sequence from 6th position to 321st position is replaced with the sequence from 150th position to 203rd position (SEQ ID NO: 3) of UK did. pUC-CS
A schematic diagram of the construction of 8 is shown in FIG. 4, and details thereof are shown below.

In the PCR reaction in the first step, pUC-CS6 was used as the template DNA, and the primers used were PCS7R and universal primer M4 (SEQ ID NO: 10; manufactured by Takara Shuzo Co., Ltd.), PCS7M and universal primer RV. (Sequence No. 11: manufactured by Takara Shuzo Co., Ltd.)
The fragments FCS8A and FCS8B were amplified by being subjected to a CR reaction, and these were separated and purified by a method using polyacrylamide gel electrophoresis. FCS8A and FCS8B thus obtained
Was used in place of FCS6A and FCS6B, and the second-stage PCR reaction was carried out under the same conditions as in Example 1 except that M4 and RV were used as primers.
As a result, the ligated fragment FCS8 was generated.

The ligated fragment FCS8 thus obtained was digested with EcoRI. This was inserted into the EcoRI site of pUC118 as in Example 1. Confirmation of the nucleotide sequence of the fragment inserted into the plasmid is
The nucleotide sequence determination method using the dideoxy method was used, and the portion corresponding to the amino acid sequence from the 266th position to the 321st position of natural t-PA was from the 150th position to the 2nd position of UK.
It was confirmed that it encodes a sequence substituted with the amino acid sequence at position 03. Choose one of these and select pUC
-Named CS8.

Example 4. T-PA containing UK type substitution region
Expression plasmids pCDM8-CS6, pC
Construction of DM8-CS7 and pCDM8-CS8 Expression plasmids pCDM8-CS6, pCDM8-C
FIG. 5 shows a conceptual diagram of the construction of pCDM8-CS8 as a representative of the concept of the construction of S7 and pCDM8-CS8.
Shown in. First, the plasmid pCDM8-ΔEE (Reference p) lacking the EcoRI fragment in the t-PA gene was digested with EcoRI and the 5'end was dephosphorylated. On the other hand, the plasmid pUC-CS6 or pUC-C
S8 was digested with EcoRI or pUC-C, respectively.
S7 was partially digested with EcoRI, and then an EcoRI fragment of about 480 base pairs containing the gene encoding the substitution region was purified and separated by polyacrylamide gel electrophoresis.
The EcoRI digestion and dephosphorylation of the plasmid pCDM8-ΔEE was ligated with the EcoRI fragment obtained from pUC-CS6, pUC-CS7, or pUC-CS8, and E. coli MC1061 /
The p3 strain was transformed. A recombinant plasmid was extracted from the obtained transformant by a known method. An expression plasmid pCD encoding t-PA containing a UK type substitution region was obtained by examining the orientation of the EcoRI fragment inserted in the recombinant plasmid by restriction enzyme digestion.
M8-CS6, pCDM8-CS7, and pCDM8
-CS8 was selected for each.

These three plasmids are pCDM8-
In CS6, natural t-PA positions 266 to 306
The amino acid at position 276 in pCDM8-CS7
Amino acids at positions 321 to 321 correspond to the amino acids at positions 266 to 321 in pCDM8-CS8, respectively (UK positions 150 to 188, 15
It contains the entire region of the t-PA structural gene except that it is substantially replaced with the 9th to 203rd positions and the 150th to 203rd positions).

Example 5. CO of the t-PA variant of the present invention
Expression and secretion in S-1 cells pCDM8-CS6, pCDM8-CS7, and pCD
COS by the above-mentioned electric pulse method using M8-CS8
-1 cells were transformed. 10% fetal bovine serum and 10 μg aprotinin after lysine sepharose chromatography
After culturing in DMEM medium containing 1 / ml for 24 hours, the medium was replaced with serum-free and aprotinin-free DMEM medium, and the culture was continued for further 96 hours. After centrifuging the culture solution, the supernatant was collected, and 1% bovine plasma albumin (BS
The AMEM-containing DMEM medium was added in an amount of 1/9 and stored frozen. This cryopreserved supernatant was used as a modified sample (CS6, CS7,
CS8) and used for the subsequent evaluation. As a control, pCDM8-00 expressing natural t-PA
The cryopreserved supernatant obtained by the same method as described above using 0 (Reference q) was used for the subsequent evaluation (natural type).

Example 6. Quantification of protein amount The protein amount of the t-PA sample in each culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (manufactured by Biopool).

Example 7. Evaluation of fibrin specificity Various fibrin cofactors were added to the plasminogen activating activity measuring system of t-PA using the chromogenic synthetic substrate S-2251 to evaluate the fibrin specificity. The method was performed according to the method described in Reference e. 1) Fibrin system First, 10 μl of 60 international units / ml thrombin (PBS containing 0.01% Tween 80) was added to the wells of a 96-well plate. Next, 200ng with PBS containing 0.01% Tween 80
Each variant sample (natural type, CS6, CS
7, CS8) 20 μl was added, and the reaction mixture [plasminogen 135 nM, fibrinogen 0.8 μM,
S-2251 378 μM (0.09% (v / v) Tween 80 and 4.4 mg / ml gelatin-containing 0.13 M Tris-HCl pH
7.8 solution)] 200 μl was added. After incubating at 37 ℃, 405nm using a plate reader.
Absorbance (OD ₄₀₅ ) was measured with time. At the same time, to correct the effect of turbidity due to fibrin, 490
Absorbance at nm (OD ₄₉₀ ) was measured and at each time the OD ₄₉₀ value was subtracted from OD ₄₀₅ . As a standard, natural t-PA diluted to various concentrations was measured by the same method, and the absorbance (OD) after a certain period of time with respect to the concentration was measured.
₄₀₅ -OD ₄₉₀₎ value to produce a plotted calibration curve, the t-PA based on the calibration curve for the t-PA native
The relative activity of the variants was determined.

2) Fibrinogen system The same procedure as for the fibrin system was carried out. However, PBS containing 0.01% Tween 80 was used instead of thrombin. 3) Fibrinogen cyanogen bromide-plasmin degradation product addition system The procedure was the same as for the fibrin system. However, fibrinogen and thrombin were not added, and instead 0.8 μM of a fibrinogen cyanogen bromine bromide decomposition product (made according to literature r) was added to the reaction mixture. 4) Desafib-X addition system It carried out like fibrin system. However, fibrinogen and thrombin were not added, and 0.073 μM Desafib-X (manufactured by BioPool) was added to the reaction mixture instead.

Table 1 shows each modified sample (CS6, CS7, C
The relative activity of S8) against natural t-PA is shown.
In addition, fibrin-based and fibrinogen cyanogen bromide-
After 30 minutes the reaction in plasmin degradation product addition system, the reaction after 1 hour at Desafibu -X addition system, absorbance after 2 hours in fibrinogen system (OD ₄₀₅ -
OD ₄₉₀ ) was used for evaluation. The concentration of natural t-PA corresponding to the obtained absorbance was determined from the calibration curve. The fibrin specificity was expressed as a ratio of "activity in fibrin system" / "activity in fibrinogen system". Further, in the stimulator display in the table, Fg is a fibrinogen system, FF is a fibrinogen cyanogen bromide-
Plasmin degradation product addition system, desaF is desafib-X
Addition system, Fn refers to fibrin system. Also, Fn / Fg
Is the ratio of "activity in fibrin system" / "activity in fibrinogen system". In addition, the numerical value in each addition system (or system) is a numerical value when a sample diluted to 100 ng / ml on the left side and 200 ng / ml is used on the right side.

[0039]

[Table 1]

From Table 1, the modified t-PA of the present invention shows that the fibrinogen system, the fibrinogen cyanogen bromide-plasmin degradation product-added system and the desafib-X-added system showed plasminogen as compared with the native t-PA. Although the activating activity is very low, it was revealed that the fibrin system has higher activity than the native t-PA, and thus has higher fibrin specificity than the native t-PA. .

References a) European Patent Application Publication No. 0041766 A2 b) The TIMI study group: N. Engl. J. Med., 320,
618, 1989. c) JP 62-24 d) JP 62-253380 e) International publication No. 9002798 f) Pennica et al .: Nature, 301, 214, 1983. g) JP 63 -501335 h) S. Yoshitake et al .: Thromb. Haemostasis, 6
2, 542, 1982. i) Holmes et al .: BIO / TECHNOLOGY, 3,923,1985 j) HA Erlich et al .: PCR technology, 1990. k) J. Yon et al .: Nuc. Acid Res., 17, 4895, 198
9. l) SL Beaucage et al .: Tetrahedron Letters, 2
2, 1859, 1981. m) F. Sanger et al .: Proc. Natl. Acad. Sci. USA,
74, 5463, 1977. n) T. Maniatis et al .: Molecular Cloning, A Labo
ratory Manual, Cold Spring Harbor Laboratory. 198
2. o) Shinichiro Takayama, Cell Engineering 6, 771, 1987. p) Japanese Patent Application No. 4-125595 q) Japanese Patent Application No. 2-87005 r) Blombach et al .: Nature, 218, 130, 1968.

[0042]

INDUSTRIAL APPLICABILITY The t-PA variant of the present invention is a novel t-PA variant having an increased fibrin specificity, and as a result, thrombosis which reduces the side effect of t-PA such as systemic bleeding tendency. It is expected to be used as a therapeutic agent.

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 39 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr 1 5 10 15 Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His Arg 20 25 30 Gly Gly Ser Val Thr Tyr Val 35

SEQ ID NO: 2 Sequence length: 45 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala 1 5 10 15 Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys Gly 20 25 30 Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr 35 40 45

SEQ ID NO: 3 Sequence length: 54 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr 1 5 10 15 Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His Arg 20 25 30 Gly Gly Ser Val Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Cys 35 40 45 Trp Val Ile Ser Ala Thr 50

SEQ ID NO: 4 Sequence length: 65 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No sequence CAG AAA ACC CTG CGG CCT CGG TTT AAA ATT ATT GGG GGA GAG TTC ACC 48 ACC ATC GAG AAC CAG CC 65

SEQ ID NO: 5 Sequence length: 47 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence ATTTTAAACC GAGGCCGCAG GGTTTTCTGG CCGCAGGTGG AGCAGGA 47

SEQ ID NO: 6 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence TTGTCACGAA TCCAGTCTAG GTAGTT 26

SEQ ID NO: 7 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No sequence GC TAC GTC TTT AAG GCG GGG AAG TAC 26

SEQ ID NO: 8 Sequence length: 50 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No sequence CTC ATC AGC CCT TGC TGG GTG ATT TCT GCC ACC CAC TGC TTC CAG GAG 48 AG 50

SEQ ID NO: 9 Sequence length: 50 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence GCAGAAATCA CCCAGCAAGG GCTGATGAGA GAGCCTCCAC ACACGTAGGT 50

SEQ ID NO: 10 Sequence length: 17 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes sequence GTTTTCCCAG TCACGAC 17

SEQ ID NO: 11 Sequence length: 17 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No sequence CAG GAA ACA GCT ATG AC 17

[Brief description of drawings]

FIG. 1 is a schematic sequence showing a substitution region of natural t-PA in Examples 1 to 3 of the present invention.

FIG. 2 shows a schematic diagram of the assembly of plasmid pUC-CS6.

FIG. 3 shows a schematic diagram of the assembly of plasmid pUC-CS7.

FIG. 4 shows a schematic diagram of the assembly of plasmid pUC-CS8.

FIG. 5 shows a conceptual diagram of the construction of an expression plasmid pCDM8-CS8 encoding t-PA containing a UK type substitution region.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12N 15/58 ZNA // (C12N 9/64 C12R 1:91)

Claims (8)

[Claims] 1. A substitution region obtained by substituting the amino acid sequence of urinary plasminogen activator (hereinafter abbreviated as UK) in the amino acid sequence of tissue plasminogen activator (hereinafter abbreviated as t-PA). In the modified t-PA having 3 to 3 from the 276th position of the amino acid sequence of natural t-PA.
At least a part of the amino acid sequence corresponding to position 06 has been substituted with the amino acid sequence of UK having the following corresponding relationship, and the substitution region is a natural t-P
A from 266th to 275th and / or 307th to 3rd
A modified t-PA having a substitution region extending to at least a part of the amino acid sequence corresponding to position 21. [Chemical 1] (Note that in the amino acid sequence, the correspondence relationship refers to the relationship in which the 266th position of natural t-PA corresponds to the 150th position of UK, and hereinafter the upper sequence corresponds to the lower sequence. The-in the notation corresponds to the state in which the amino acid was deleted in the substitution of UK with the amino acid sequence.) 2. A t-PA in which the substitution region is at least a natural type.
2. The t-P according to claim 1, comprising positions 276 to 306.
A variant. 3. The substitution region is from the 266th position to the 306th position of natural t-PA, and the substitution region after substitution is 15 of UK.
The t according to claim 1, which is a sequence from position 0 to position 188.
-PA variants. 4. The substitution region is from the 276th position to the 321st position of natural t-PA, and the substitution region after replacement is 15 of UK.
The t according to claim 1, which is a sequence from the 9th position to the 203rd position.
-PA variants. 5. The substitution region is at positions 266 to 321 of natural t-PA, and the substitution region after substitution is 15 of UK.
The t according to claim 1, which is a sequence from position 0 to position 203.
-PA variants. 6. Recombinant expression capable of expressing the DNA encoding the t-PA variant according to claim 1 in transformed prokaryotic cells or eukaryotic cells. vector. 7. A prokaryotic or eukaryotic cell transformed with the recombinant expression vector according to claim 6. 8. The t-PA according to claim 1.
A therapeutic agent for thrombosis, which comprises a variant as an active ingredient.