WO1996004371A1

WO1996004371A1 - Tissue plasminogen activator dimer

Info

Publication number: WO1996004371A1
Application number: PCT/JP1995/001541
Authority: WO
Inventors: Kazuya Mori
Original assignee: Nippon Shinyaku Co., Ltd.
Priority date: 1994-08-04
Filing date: 1995-08-03
Publication date: 1996-02-15

Abstract

A novel tissue plasminogen activator dimer having a high fibrinolytic activity and a remarkably lowered antigenicity.

Description

Technical Field The present invention relates to a novel human tissue plasminogen activator dimer having fibrinolytic activity. BACKGROUND ART Myocardial infarction and cerebral thrombosis are serious cardiovascular injuries that are the leading causes of death in Japan. In these diseases, blood clots are first formed, blood flow to the organs downstream of the embolized blood vessels is reduced, and the tissues eventually become necrotic.

Brasminogen activator promotes fibrinolytic action by activating brasminogen. Therefore, myocardial infarction and cerebral thrombosis can be prevented or treated by administering a plasminogen activator as a vegetable and dissolving the thrombus.

Human tissue brassminogen activator is a glycoprotein with a molecular weight of about 70,000 and consists of 527 amino acids (Beni Rika et al .: Nature 301, pp. 214-221 (1983)), and has three N-glycosylation sites. At the asparagine residue at positions 117, 184 and 448. There are 17 disulfide bonds, and the SH group of the 83rd cysteine residue remains free.

Human tissue plasminogen activator is secreted in the form of a single-chain polypeptide, but the bond between the arginine residue at position 275 and the isoleucine residue at position 276 is broken by plasmin. It becomes a main chain. Both single and double strands have a plasminogen activating effect. The molecule from the N-terminus of the truncated polypeptide (called A chain or heavy chain) is composed of several domains. That is, a finger domain (F), an EGF domain (E), a kringle domain (K1 and K2) and the like. Molecules from the C-terminus (called B chains or light chains) have a protease domain (Koren et al .: Drugs 38, pp. 346-388). (1989)).

Two subtypes of human tissue plasminogen activator are known. These are called evening eve I and type II, and type I has a sugar chain at the 184th asparagine in the K2 domain, but type II lacks it (Sperman et al .: J. Biol. Chem. 264, pp. 14100-14111 (1989), Palek et al .: Biochemistry 28, pp. 7670-7679 (1989)). The two types of submersible eves can be separated from each other by lysine sepharose column chromatography. Type I has a lower affinity for lysine sepharose than type II and has a lower activating ability to activate plasminogen in a fibrin-dependent manner (Wittwer et al .: Biochemistry 28, pp. 7662-7669 (1989)).

Human tissue plasminogen activator produced by recombinant DNA technology has been used clinically to treat myocardial infarction. Human tissue plasminogen activator has a higher specificity for fibrin and higher thrombolytic activity than perokinase, which has been commonly used for the treatment of myocardial infarction, so that relatively small doses are required. Was considered a more convenient vegetable.

However, actual treatment required higher doses than expected.

In order to solve this problem, various mutants have been created with the aim of prolonging the presence of human tissue plasminogen activator in blood and increasing its specific activity (Rizunen and Koren: Thronb Haemostas 66). , Ρρ.88-110 (1991), German Patent No. 390,581, German Patent No. 3,836,200). However, none of them have been put to practical use due to problems such as having antigenicity.

As described above, there has not yet been found a substance having a property that can be put to practical use, exceeding the activity of a conventional human tissue plasminogen activator and having extremely low antigenicity. DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel human tissue protein derived from human cells, having high specific activity and low antigenicity, as a vegetative useful for the prevention and treatment of cardiovascular disorders. It is an object of the present invention to provide a homologue of a rasminogen activator.

In view of the above circumstances, the present inventors have conducted intensive studies and have found that, from culture supernatants of Bowes cells, a stronger and differenter than human tissue plasminogen activator known so far. The active substance was obtained.

The novel human tissue plasminogen activator homolog according to the present invention is a dimer of a single-stranded or double-stranded human tissue plasminogen activator, and is typically dimerized through a disulfide bond. Forming the body. Such an i-isomer can be obtained by separating type I and type II human tissue brassminogen activator from human cell culture supernatant. The human cells used are not particularly limited as long as they are human-derived cells, such as cultured cells and non-cultured cells, and include, for example, pause cells.

Human cells are cultured in an appropriate medium, for example, Eagle's basic medium supplemented with sodium bicarbonate, L-glutamine and heat-treated newborn calf serum. By purification, the human tissue plasminogen activator 2 i of the present invention can be easily obtained.

Various methods used in protein chemistry can be used as separation and purification means. For example, affinity chromatography such as lysine sepharose chromatography, electrophoresis under native conditions using various suitable buffers, gel filtration using agarose gel as a carrier, or other suitable separation methods Can separate human tissue plasminogen activator dimer from previously known human plasminogen activator (type I, type II).

If necessary, it can be stored in a stable form by freeze-drying after purification. At that time, lyophilization may be carried out by adding an appropriate stabilizer such as human serum albumin in an appropriate buffer such as purine buffer.

Various properties of the human tissue plasminogen activator dimer according to the present invention are shown below.

When performing Immunob opening using an anti-human tissue plasminogen activator antibody, the human tissue plasminogen activator dimer according to the present invention is a protein having a molecular weight of about 120,000 which reacts with this antibody. (See Fig. 2). The human tissue plasminogen activator dimer according to the present invention has the same molecular weight as the human tissue plasminogen activator single S-form when analyzed by SDS-polyacrylamide electrophoresis after passing through 2-mercaptoethanol. (Figure 3).

The human tissue plasminogen activator dimer, type I or type II human tissue plasminogen activator according to the present invention is converted to an N-glycosidase (hereinafter referred to as PNGase) that cleaves an N-linked 耱 chain. Upon processing, all plasminogen activators become the same molecule i (see Figure 4A).

* When the human, tissue I, or type II human tissue plasminogen activator is treated with exo-saminidase H (hereinafter referred to as EndoH), which cleaves the oligomannose structure, All show a decrease in molecular weight (see Figure 4B).

These findings indicate that the plasminogen activator of the present invention is a human tissue plasminogen activator dimer (hereinafter referred to as a dimer) formed via a disulfide bond.

Table 1 shows the indirect hydrolysis activity of human Glu-plasminogen and the plasmin chromogenic substrate S-2251 under stimulating conditions in the presence of this dimeric fibrinogen fragment. The dimer according to the present invention has a much higher specific activity than the type I or type II plasminogen activator monomer. Normally, when dimerization occurs, the active site is expected to become less accessible to the substrate due to steric harm, and the activity is expected to decrease.However, the specific activity of the type II dimer is rather changed due to the change in steric structure. It shows the unexpected property of rising.

The direct hydrolysis activity of the type II dimer on the synthetic plasminogen substrate, S-2288, is similar to that of the type I and type II plasminogen activator monomers. In other words, it is presumed that the structure of the active site does not change even if it is dimerized. However, indirect activity in the presence of fibrinogen using the poor chromogenic group S-2251 indicates that the type II di-isomer has more than twice the specific activity of the type I and type II plasminogen activator monomers. Indicated. The reason for this is that dimerization changes the conformation of the secondary binding site for brassminogen (Gepert et al .: Arch. Biochem. Bioph. 297, pp. 205-212 (1992)), and Activity rises It seems to have done.

As described above, the type IIi-isomer of the present invention is a novel and useful substance having completely different properties from conventionally known human tissue plasminogen activator.

Another advantage is that the dimer can be easily prepared.

Humans with non-type II tissue plasminogen activator dimers derived from paused cells that form dimers via disulfide bonds, resulting in similar structural changes that result in increased specific activity Plasminogen activator homologs are, of course, included within the scope of the present invention.

In summary, the present invention can provide a substance having high fibrinolytic activity.

When the human tissue plasminogen activator 2; ft body according to the present invention is used for the treatment of thrombosis, the required dose of the dimer is the required amount (weight ratio) of the type I or type II monoi body. Less than one-half, for example one-quarter. Moreover, since the dimer of the present invention is derived from human tissue, the antigenicity is extremely low. Therefore, side effects such as shock and allergy are unlikely to occur. The practicality of the present invention is also high in this regard.

The dimer of the present invention can be administered to a patient in need of treatment for thrombosis in an appropriate dosage form. The dose depends first on the severity of the disease and the patient's condition, but it is obvious that the dose should be used within the range of a dose that is effective and does not show toxicity. Usually, it is better to first intravenously inject a dimer of 200 to 800 IU / kg, preferably 400 IU / kg, and administer the same device 12 hours later. The route of administration is preferably parenteral, especially intravenous. It is practical to dissolve the dimer in sterile saline and administer it intravenously. Other suitable dosage forms and administration methods are also described, for example, in Remington's Pharmaceutical Sciences (Arthur 'Fozor, Ed., 16th edition Mack Publishing Co., Eastern, Pa. (1980)). May be applied.

The i-isomer becomes a monomer when it is transmitted. This indicates that the dimer forms a dimer through disulfide bonds. Human tissue plasminogen activator contains many cysteine residues forming an intramolecular disulfide bond in its amino acid primary structure, but the 83rd cysteine residue remains free This cysteine residue is another type I It forms a disulfide linkage with the cysteine residue at the same position of the I plasminogen activator molecule, indicating that it is a dimer. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a lysine Sepharose affinity column chromatogram.

In Fig. 1A, X indicates the electrical conductivity of arginine (nS / cm) in the concentration gradient, and 〇 indicates the direct hydrolysis activity (mOD / min) of plasminogen activator on the chromogenic substrate S-2288. Represents the antigen i (〃g / nl) of the plasminogen activator.

In Fig. 1B, 〇 indicates the direct hydrolysis activity (fflOD / min) of the plasminogen activator for the chromogenic substrate S-2288, and 色 indicates the chromogenic substrate S with Glu-plasminogen using FCB as a stimulant. indirect activity in the presence of Fipuri down against -2251 to ^{(DiOD / (min 2 / nig} / ml) xlO), represents. FIG. 2 shows the immunoplotting of the plasminogen activator obtained by lysine Sepharose affinity column chromatography.

I and II indicate the positions of the single-chain type I monomer and the single-chain type II monomer, respectively, and the arrows indicate the position of the high-molecular plasminogen activator (type II dimer). FIG. 3 shows immunopro- moting after electrophoresis of a type I single-chain monomer, a type II single-chain monomer, and a type II-single-chain dimer in their original state.

Lane 1 is a mixture of type I monomer, type II monomer and type II dimer, lane 2 is plasminogen activator before fractionation, lane 3 is type I monomer, lane 4 is evening Lane 5 represents the evening II II S monomer, respectively. Fig. 4 shows the swimming state of type I monomer, type II single-chain monomer, and Eve II—two-chain main body treated with sugar chain-cleaving enzymes. It shows the immunoblotting after the movement.

In FIG. 4A, lane 1 represents the type I monomer, lane 2 represents the type I single-stranded I i, and lane 3 represents the type II single-stranded duplex. “+” Indicates a product treated with PNGase, and “1” indicates a product not treated with an enzyme.

In FIG. 4B, lane 1 represents the type I monomer, lane 2 represents the type II monomer, and lane 3 represents the type II monomer. + Indicates EndoH-treated product, and 1 indicates ffl without enzyme treatment. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail with reference to examples below, but these examples do not limit the present invention.

ELISA kits for human tissue plasminogen activator, human blasmin, and plasminogen activator used as preparative products used in this example were purchased from American Diagnostics. Lysine Sepharose and Alkaline Phosphatase Synthetic Anti-Egret IgG were purchased from Sigma Chemical Co., and Chromogenic Substrates S-2288, S-2251, Human Fibrinogen and Human Glu-Blasminogen were purchased from ViviVitorum. Molecular weight markers were purchased from Amersham, EndoH from Behringer Mannheim, and PNGase from Oxford Glico 'Systems. A commercially available anti-human tissue plasminogen activator polyclonal antibody can be obtained from the market, but a commercial product from Dr. Hiroshi Sakata of Jichi Medical University was used. Pause cells obtained from ATCC were used as human-derived cells. Example 1

Pause cells were cultured according to the method of Rigikin et al. (Rigikin et al .: J. Biol. Chem. 256, pp. 7035-7041 (1981)).

The fraction containing the single-chain brassminogen activator dissolved in 1 M ammonium bicarbonate, prepared from the culture solution according to the method of Rigikin et al., Was added to buffer E (10 mM sodium phosphate buffer (pH 8.0), 0.15 M KSCN> 0.001¾ teen 80, 25 KIU / ml abrotinin) to 800 / g / ml „

PCT / JP95 / 01541

After dilution to a minimum, the solution was applied to a lysine sepharose column (0.931 cm) pre-equilibrated with buffer E. The following operations are all 4. Went on C.

After washing the column with 24 ml of buffer E, a linear concentration gradient of 0-0.25 M L-arginine was prepared in buffer E to elute the plasminogen activator. Elution was performed with buffer E containing 0.25M L-arginine, followed by buffer Ε containing 0.5 · (Fig. 1). All fractions were collected, and their respective hydrolytic activities against the chromogenic substrate S-2288 were measured. Active fractions 52-63, 70-83 and 110-123 were grouped together and designated as Type I monomer, Eve II monomer and Type II dimer fraction in that order.

300 1 samples were taken from the fifth fraction from the beginning of the fractionation and dialyzed sufficiently against 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl and 0.05¾ tween 80 each. Thereafter, the antigen content of the plasminogen activator of each product and the indirect hydrolysis activity in the presence of a fiprinogen fragment (FCB) were measured.

All activity measurements were performed in 96-well microtiter plates (Coaster). Went on C. Enzyme activity was measured with a Molecular Devices force kinetic microplate reader.

That is, each well in a 96-well microplate was coated with a phosphate buffer (pH 8.0) containing 1 calf serum albumin by treating at 25 ° C for 16 hours, followed by washing with water and room temperature. And dried. The amount of the antigen of the brassminogen activator was measured using an immunoassay kit of American Diagnostica using a polyclonal antibody.

The direct hydrolysis activity was measured using the chromogenic substrate S-2288. The reaction was carried out in Tris-HCl (pH 7.5) containing ImM S-2288, 0.05% tween 80, 0.1% BSA and 0.15M NaCl. The enzyme activity was calculated by measuring the change in absorbance at 405 nm. The results are shown in FIG. 1 as the change in absorbance per minute (mOD / min) per l zg / ml of antigen per 100 ml of reaction solution.

The measurement of indirect activity in the presence of fibrin was performed using human fibrinogen (FCB) degraded with bromocyan as a stimulant (Perheidin et al .: Thromb Haemostas 48, pp.266-269 (1982)). The activity was measured according to the method of Palek et al. (Palek et al .: Biochemistry 28, pp. 7670-). 7679 (1989)). In the presence of 0.13 mM Glu-plasminogen, 0.7 mM chromophore S S-2251, 0.6 mM FCB, 5-10 ng / ml plasminogen activator standard, 0.05¾ tween 80, 0.1¾ BSA and 0.15 The reaction was performed in Tris-HCl (H7.5) containing M NaCl.

The activation of plasminogen correlates with the addition of brassmin and, consequently, with the change in absorbance per unit time of 405 nn (dOD / dt). The absorbance of the reaction solution was measured every 3 minutes, and the dOD / dt was measured from the rate of change. Further, the value of dOD / dt was replotted against the reaction time. From the slope of this straight line was calculated value of DMOD / dt / nin or fflOD / min ^2. The calculated results are expressed as the concentration of the brassminogen activator (g / ml or IU / 〃g) Geary and Curtis: mOD / standardized with Thromb Haemostas 53, pp. 134-136 (1985)) min ² (Wittwer et al .: Biochemistry 28, pp. 766 2-7669 (1989)).

The fractions were measured for the direct hydrolysis activity using the chromogenic substrate S-2288 and the amount of plasminogen activator antigen by the ELISA method using an anti-human tissue plasminogen activator polyclonal antibody.

In addition to the two major beaks, a type I monomer and a type II monomer, respectively, a third beak eluted with 0.5 M L-arginine was identified. No activity was observed after this beak. Killing example 1

The activity per unit antigen i was calculated by dividing the direct hydrolysis activity value by the amount of antigen. As a result, the specific activity for the chromogenic substrate S-2288 per unit antigen i was unchanged in all active fractions, regardless of the strength of the affinity for lysine sepharose.

Using FCB as a stimulant, the indirect activity of Glu-plasminogen on the chromogenic substrate S-2251 in the presence of fibrin was measured for each active fraction, and the activity per unit antigen amount is shown in Figure 1B. expressed. As a result, the specific activity of the type II monomer was larger than that of the type I monomer, and the specific activity of the second active fraction eluted with 0.5 ML-arginine was larger than that of the former two. .

From these results, it was found that a novel, highly specific activity of the plasminogen activator was eluted after the type II monomer. 1

10 Eve I monomer, type I I monomer and new active fraction are 1-20. Stored frozen in C. Test Example 2

Table 1 summarizes the direct hydrolytic activity and the indirect activity in the presence of fibrin of Eve I monomer, Eve II monomer and the new active fraction. Since there is a belief that the reactivity to the antibody varies depending on the modification such as sugar chain addition, the amount of antigen was determined using a polyclonal antibody, and the specific activity per unit amount of antigen was calculated based on the value.

The direct hydrolysis activity for the chromogenic substrate S-2288 showed almost the same value for all three plasminogen activators. Table 1. Comparison of the activities of plasminogen activator subtypes. Direct hydrolytic activity. Indirect activity under stimulating conditions.

Plasminogen mean standard deviation mean standard deviation activator

Type I single i-body 9.8 1.2 429.7 33.8

Type II monomer 8.5 1.0 710.8 100.4

Type I I dimer 9.0 0.8 1684.8 121.0

However, with indirect activity in the presence of fibrin, the new active fraction was 3.9-fold for evening Eve I monomer and 2.4 for type II monomer. The specific activity was twice as high, indicating that this novel active fraction was extremely useful in the treatment requiring thrombolysis. Test example 3

Analysis of the novel plasminogen activator was performed by SDS-polyacryl gel electrophoresis and immunoblotting. The SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (Laenunli: feature 227, pp. 680-685 (1970)). After electrophoresis, the proteins in the gel were electrically transferred to a blotting membrane (Imnoviron manufactured by Millipore). The plasminogen activator pand was detected using a persimmon anti-human brassminogen activator polyclonal antibody and an anti-raspberry polyclonal IgG conjugated with lipophilic phosphatase.

Figure 2 shows the results of electrophoresis of each fraction of the plasminogen activator eluted from the lysine sepharose affinity column with a gradient of L-arginine under non-conducting conditions and immunoblotting. Was. As is evident from this figure, the novel plasminogen activator active fraction contains a molecule representing a single i-form of the brassminogen activator i In addition to the band of 64,000 to 65,000 daltons, about 120,000 daltons It was confirmed that there was a new band indicating the molecule i of this.

The same analytical procedure was performed after passing each type I monomer, type II monomer and new active fraction with 2-2-mercaptoethanol, and it appeared in the new active fraction. The band of 120,000 daltons was found to be a single band showing the same molecular weight as the type II simple S form by passing through 2-merbutanol (see Fig. 3). Test example 4

Next, the novel active fraction was analyzed for plasminogen activator using a sugar chain-cleaving enzyme.

Each of the fractions of type I monoisomer, type II monomer and novel activity was dialyzed thoroughly against 50 mM Tris-HCl buffer (pH 7.5) containing 50 μl of EDTA, and the final fraction was collected. * SDS was added so that the degree became 0 ·. 95. After treatment with C for 2 minutes, Triton X-100 was added to a final concentration of 0.5¾ to neutralize SDS. To this was added PNGase having a final concentration of 32 U / ml, and the mixture was treated at 37 ° C overnight.

The results of immunoblotting after electrophoresis of type I monomer, type II monomer, and a novel active fraction treated with PNGase that cleaves N-linked sugar chains are shown in the fourth section. This is shown in Figure A. All active fractions showed the same molecule S (about 63,000 daltons) in the original state after sugar chain cleavage. This indicates that the type I monomer, the eve II monomer and the new activity under normal conditions This indicates that the difference in the molecular weight of each 1z fraction is due to the distance of the sugar chain. It was also found that all of these fractions had sugar chains added.

Prior to EndoH treatment, each active fraction was sufficiently dialyzed against 50 sodium peroxidate buffer (pH 5.5), and 2-mercaptoethanol was added to a final concentration of 0.1M. To this was added EndoH at a final concentration of 401 / ιη1, and the mixture was treated at 37 ° C overnight.

EndoH cleaves the oligomannose structure. Figure 4B shows the results of immunlotting of the type I monomer, the type II monomer, and the novel active fraction after EndoH treatment and after electrophoresis under normal conditions. . Enzymatic treatment reduced the molecular weight of all types of plasminogen activator. Enzyme treatment was carried out at 37 ° C overnight, and detection was performed by SDS gel electrophoresis followed by immunopro- duction.

These results suggest that the novel plasminogen activator has an oligomannose structure similar to the type I monomer and the type II monomer. From these results, the homolog of the plasminogen activator of the present invention is a novel, type II dimer having a much higher specific activity than previously known plasminogen activator, and is used for the treatment of thrombolysis. It turned out to be useful.

Claims

The scope of the claims

1. Dimerized human tissue brassminogen activator.

2. Dimerized human tissue plasminogen activator through disulfide bonds.

3. The human tissue plasminogen activator according to claim 1, wherein the cysteine residues at the 83rd position from the N-terminus are disulfide bonded.

4. The human tissue plasminogen activator according to claim 1, comprising a type II human tissue plasminogen activator.

5. When human-derived cell culture supernatant is applied to lysine sepharose column chromatography, the fraction eluted with L-arginine at a concentration of 0.25M or more has a molecular weight of 120 kilodaltons and is a brassminogen activator. Child.

6. The human tissue plasminogen activator according to claims 1 to 5, which is a main chain.

7. A therapeutic agent for treating thrombosis comprising the human tissue plasminogen activator according to claim 1 as a component.