JPH0569506B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a plasminogen activator. In recent years, diseases caused by blood clots have been highlighted as an important problem, and streptokinase, urokinase, etc. have been used to treat them. However, it is known that all of these methods not only achieve the objective of dissolving thrombosis, but also degrade coagulation factors and the like in circulating blood, resulting in a bleeding tendency. The reason for this is thought to be that streptokinase, urokinase, and the like have poor affinity with the thrombus to be dissolved, and degrade blood coagulation factors and the like in the circulating blood. Therefore, there has been a need to develop a thrombolytic agent that has a high affinity for blood clots and has fewer side effects such as bleeding tendency. Therefore, research is being conducted on plasminogen activators (hereinafter referred to as PA), which are said to dissolve blood clots and have few side effects, and presentations have also been made on this topic. Regarding human-derived PA, detailed studies have been conducted on enzymes obtained from endothelial cells and normal tissues such as the uterus, as well as tumor tissues and cultured cells thereof. (References include Cancer Research 40 933-938 (1980) by Wilson et al. and Journal of Biological Chemistry 256 7035-7041 (1981) by Ritsken and Collan). It has been known that normal tissue cells such as human endothelial cells and human uterine cells, as well as tumor cells such as human melanoma and breast cancer cells, produce PA. However, normal tissue cells eventually die, so they cannot be used for industrial production. Also. The conventional culture of tumor cells such as human melanoma and breast cancer cells is only by adhesion culture in bottles, etc., and the productivity of PA is extremely low, and it has not been possible to mass-produce it industrially. Therefore, the present inventors first conducted research to find tumor cells that could be cultured in suspension by agitation, and found that KYM-I (a gift from Morimasa Sekiguchi, Institute of Medical Science, The University of Tokyo), a cell isolated from human lateral strangulosarcoma, They succeeded in discovering and isolating a mutant strain that can float and proliferate from culture. This mutant strain is a mutant strain
It was named KYM-A. As a result of further research, this mutant strain KYM-A
It was found that if cultured, a significant amount of PA can be produced and accumulated in the culture solution. This PA is PA−
It was named KYM. Generally, it is thought that there are two main types of PA in humans. That is, one type is a urokinase type that has poor affinity for fibrin, and the remaining type is a so-called tissue plasminogen activator (TPA) type that shows a high affinity for fibrin. Melanoma cells are well known as tumor cell lines that produce TPA-type PA. Melanoma cells mainly contain TPA type PA
It is attracting attention because it only produces On the other hand, among other types of tumor cells, only breast cancer cells have been reported as an exception that produce only TPA-type PA. In addition, previous research has suggested that transverse strangulation sarcoma cells produce only urokinase-type PA. Urokinase-type PF cannot be detected in the culture solution of mutant strain KYM-A. Therefore, since the PA-KYM of the present invention is produced from human rhabdomyosarcoma cells, it is novel in its origin, and is recognized as a TPA-type PA based on its physicochemical properties. Mutant strain used for production of PA-KYM of the present invention
KYM-A is a mutant strain that was discovered through repeated isolation and selective culture from a culture of KYM-I cells isolated from human rhabdomyosarcoma.
- It is very distinctive by producing KYM in large quantities. Although this mutant strain KYM-A can be subcultured by suspension agitation culture, the deposit was not accepted at the Institute of Fine Technology. The properties of mutant strain KYM-A are as follows. 1. Individual cells have a highly refractive spherical shape. 2 Cells may exist singly as floating cells, but they may also form chains or spherical aggregates. The number of cells included in the aggregate is approximately 2
~100 pieces. 3. When cultured in a plastic shed or agitated culture in a tank, this mutant strain can mostly grow as floating cells. 4. This mutant strain can be subcultured by floating agitation culture. 5. This mutant strain transforms into adherent cells by conditioned medium, etc. The altered cells exhibit epithelial cell-like morphology. The conditioned medium is a culture supernatant of cells other than this mutant strain, which is obtained by culturing appropriate cells in an appropriate basal medium for 1 to 100 hours, and then removing cells and their fragments from the culture medium. be. As cells other than this mutant strain, for example, human hepatoma cells (HuH6-Cl5 strain, HuH7 strain) (Nakamura et al., Cancer Research, vol. 42 3858-3863 1982) can be used. In order to make this mutant strain adhesive, in addition to using the above-mentioned conditioned medium, it is necessary to use a medium containing fibronectin, or to replace the adhesive surface of Shearley with collagen, gelatin, poly-L-lysine, or egg white. Adhesive culture of this mutant strain becomes possible by treatment with basic proteins such as lysozyme. When 6 10 ⁶ cells of this mutant strain are transplanted subcutaneously into nude mice or into the sac of ALS-treated hamsters, they form a tumor. 7. Chromosomes When the number of chromosomes was determined according to the standard method of cytogenetics, the most frequent chromosome numbers were 46 and 47, and the distribution showed a slight width around these. Therefore, mutant strains
The chromosome number of KYM-A is similar to that of normal human diploid (chromosome number), regardless of the tumor origin.
46) was found to be relatively similar. 8 This mutant strain produces significant amounts of PA-KYM. The culture for producing PA-KYM of the present invention may be either adherent culture or floating agitation culture. Mutant strain KYM-A adheres to plastic shear and glass shear and proliferates.
For the purpose of producing PA-KYM, it is possible to perform adhesive culture using roller bottles or microcarriers. However, floating agitation culture is suitable for industrial production. For production of PA−KYM, mutant strain KYM
-A cells are preferably cultured in a serum-supplemented medium and replaced with a serum-free medium when the cells enter the stationary phase of growth. As a serum supplemented medium, RPMI
(manufactured by Flow), MEM (manufactured by Flow), or a mixed medium of both, with approximately 10% fetal bovine serum added. Also, as a serum-free medium, it can be used as a basic medium.
It is preferable to use RPMI, MEM, or a mixed medium of both, to which insulin, human transferrin, monoethanolamine, and serine acid are added (this four-component supplemented medium is hereinafter referred to as ITES-supplemented medium). For the purpose of producing PA-KYM from cell line KYM-A, the cell line is cultured in suspension on a plastic shear array, and when an appropriate number of cells is reached, suspension agitation culture using a spinner flask is started. Spinner flasks range from 100ml to 8000ml
A volume of ml is preferred. Also, the inoculated cell density is
10 ⁴ to 10 ⁵ cells/ml is preferred, and stationary phase is reached at a density of 5×10 ⁵ to 2×10 ⁶ cells/ml. The culture temperature is not particularly specified, but it is approximately 30°C.
~40°C is suitable, particularly around 37°C. In addition, the gas phase is 100% air or 5-10%
air containing carbon dioxide gas, or 5 to 100
Air containing % oxygen is preferred. Cultivation can be done batchwise, but once the cells have grown sufficiently, the culture medium can be continuously replaced at intervals of 1 to 4 days, and PA can be used for about 1 month after the start of culture.
- A culture medium containing KYM can be obtained. PM-KYM of mutant KYM-A cells is an enzyme that activates plasminogen to plasmin, and since it belongs to proteins, any general method applied to protein purification can be applied. In general, salting out, adsorption, affinity chromatography, ion exchange chromatography, molecular sieves, etc., and appropriate combinations thereof can be considered. Further, these purification processes can be carried out either continuously or batchwise, and the method may be selected in consideration of efficiency, operability, etc. in purification. A zinc chelate agarose column in which the PA-KYM-containing serum-free medium obtained by culturing was sufficiently buffered with a Tris-HCl buffer containing sodium chloride (containing Tween 80 and sodium nitride, which will also be added to all dry solutions below). load on. After loading, wash the column using the same buffer.
Thereafter, gradient elution is performed using the same buffer solution and a buffer solution containing imidazole. RA−
KYM activity is observed in the gradient moiety of imidazole. After collecting the active fractions, they are concentrated using polyethylene glycol and dialyzed against a phosphate buffer at 4° C. for 24 hours while exchanging the external solution. PA-KYM activity is observed in the dialysate fluid.
The dialyzed fluid obtained here is loaded onto a concanavalin A sepharose column that has been sufficiently buffered with the same phosphoric acid drying solution. After loading, wash the column with the same buffer. Subsequently, gradient elution is performed using a dry solution containing potassium thiocyanate and α-methylmannoside. PA-KYM activity is observed in the gradient elution portion of potassium thiocyanate and α-methylmannoside. After collecting the active fractions, they are concentrated to an appropriate amount using polyethylene glycol, and then dialyzed against physiological saline containing Tween 80 and sodium nitride at 4°C for 24 hours while exchanging the external dialysis fluid. After dialysis, a cloudy precipitate is formed in the dialysis solution, and this precipitate is collected by centrifugation. PA-KYM activity is mainly observed in the recovered precipitate. This precipitate is dissolved in a small amount of phosphate buffer containing potassium thiocyanate and centrifuged to remove insoluble matter, thereby obtaining a supernatant containing PA-KYM activity. The supernatant obtained here was loaded onto a Schhairdex (G-200) column that had been sufficiently buffered with a phosphate buffer containing potassium thiocyanate, and when developed with the same buffer, PA-
An effluent fraction is obtained, containing KYM activity. Next, the physicochemical properties of purified PA-KYM will be shown. a Product of human rhabdomyosarcoma cell mutant KYM-A. b. Molecular Weight The non-reduced substance measured by SDS-polyacrylamide gel electrophoresis has two closely spaced bands with molecular weights ranging from about 56,000 to 62,000. Furthermore, the reduced form of this substance measured by the same method has two bands at molecular weights of about 32,000 and about 36,000. c. Action and substrate specificity This substance is an enzyme protein that causes a fibrin dissolution reaction in the presence of plasminogen.
Because this reaction requires the presence of plasminogen, it belongs to a group of substances called typical plasminogen activators, and when compared to urokinase, it exhibits a much stronger binding ability to fibrin than urokinase. Furthermore, when the synthetic substrate S-2444 is used as a substrate, Km is 1.32×10 ^-3 M, and Vmax is
It is 0.046IU/min・ml. d Optimum PH The optimum PH is about 8-10 and the action curve is shown in FIG. e Stable PH Stable PH is about 5-11, and the % remaining activity is shown in FIG. f Range of suitable temperature for action The action temperature curve at 30-45°C is shown in Figure 3. g. Temperature resistance There is almost no loss of activity up to 50°C with heat treatment for 90 minutes, but residual activity decreases to less than 60% at temperatures above 60°C. The % remaining activity is shown in FIG. h Inhibition The residual activity of each inhibitor was examined at 0.1 mM, 1 mM, and 10 mM, and the results shown in Table 1 were obtained. [Table] Inhibition and activation were investigated, setting the activity of the sample amount without added drug as 100%. i Amino acid composition When this substance, which is an enzyme protein, was hydrolyzed with 6N hydrochloric acid and subjected to amino acid analysis, the results shown in Table 2 were obtained. Amino acid composition was expressed as a percentage of the total number of amino acid residues. [Table] was not quantified.
j Ultraviolet absorption spectrum The ultraviolet absorption spectrum of an aqueous solution of this substance is as shown in FIG. k Solubility in solvents The solubility in water and saline solutions such as phosphate buffer is approximately 50 μg/ml, and when preparing solutions with higher concentrations, the presence of a solubility promoter, such as 1.6 M potassium thiocyanate, is required. . It is insoluble in organic solvents such as ethanol and ether. l Properties of the substance The lyophilized specimen is a white powder. m Color reaction After performing SDS-polyacrylamide gel electrophoresis, a PAS reaction was performed, and a pink coloration characteristic of glycoproteins was exhibited, indicating that it was a glycoprotein. Furthermore, the fact that this substance showed affinity for concanavalin A-agarose resin suggested that it was a glycoprotein. n Isoelectric point When this substance was analyzed by chromatography in the presence of 8M urea, the isoelectric point of the main component was
The pH was 7.5 to 8.0, and the isoelectric point of the subcomponent was PH7.0 to 7.5, indicating that it was a mixture of weakly basic proteins. Next, we will show how to measure PA-KYM activity.
The method is the same as the conventional method for measuring PA activity. (i) Fibrin plate method (according to the method of Astratupu et al.) (Arch.Biochem.Biophys. 40 346
-351 (1952)) A fibrin plate containing plasminogen is prepared by allowing thrombin to act on an agarose suspension containing fibrinogen and plasminogen at 37°C. The fibrin plate has a hole with a diameter of 3 mm for adding a sample for activity measurement. A sample volume of 5 to 10 μm is added to the sample hole, and the added fibrin plate is placed in an incubator at about 37° C. and taken out after a certain period of time to measure the diameter of the distinct fibrin dissolution zone. The activity in the sample is reflected in the diameter of the fibrinolysis zone, so the activity is determined by comparing the diameters. (ii) PA-KYM activity measurement method that utilizes a two-step reaction using a synthetic substrate (S-2251). 50mM Tris containing 0.15M sodium chloride
Plasminogen and PA-KYM solution dissolved in hydrochloric acid buffer (PH7.4) [50mM Tris-HCl buffer solution containing 0.15M sodium chloride (PH
7.4)] and held at 37℃ for 10 minutes (first stage reaction). After holding for exactly 10 minutes, immediately
Add 0.33M lysine solution [0.15M Tris-HCl buffer containing 0.15M sodium chloride to stop the first stage reaction. Subsequently, a synthetic substrate solution [3 mg/ml of synthetic substrate S-2251 dissolved in 50 mM Tris-HCl buffer (PH7.4) containing 0.15 M sodium chloride] was used and kept at 37°C for 30 minutes. Colorimetric determination of plasmin content generated in step reaction (wavelength 405nm)
(second stage reaction). In addition, to stop the second stage reaction, immediately after holding it for exactly 30 minutes, add 2.5M
This is carried out by adding an acetic acid solution. PA-
KYM activity is measured by absorption at 405 nm. (iii) PA-KYM activity measurement method that utilizes a one-step reaction using a synthetic substrate (S-2444). 50mM Tris-HCl buffer containing 0.15M sodium chloride (PH
8.8) A synthetic substrate solution [synthetic substrate S-
A solution of 3 mg/ml of 2444 dissolved in 50 mM Tris-HCl buffer (PH 8.8) containing 0.15 M sodium chloride] was added, and after holding at 37°C for exactly 90 minutes, the reaction was stopped with a 25 M acetic acid solution. PA-
KYM activity is determined colorimetrically (wavelength 405 nm). The PA-KYM of the present invention can be effectively used to dissolve thrombus generated in blood. The PA-KYM of the present invention is preferably in a dosage form that can be administered intravenously. Examples of additives for the composition include stabilizers such as mannitrate, albumin, gelatin, and sodium bisulfite; PH regulators such as sodium hydroxide and sodium phosphate; and isotonic agents such as sodium chloride, mannitrate, and glucose. be able to. The dosage and administration method for clinical use of PA-KYM of the present invention is to administer 100-100,000 IU of this product as a solution containing the above additives.
It is used by intravenous injection, intravenous drip injection, intravenous drip injection, subconjunctival or retrobulbar injection, with appropriate adjustment depending on the symptoms and progress. Next, examples of the present invention will be shown. Example 1 Cultivation For culturing mutant strain KYM-A, 2.0 g of sodium bicarbonate and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were added per medium.
1.192 g, RPMI-1640 medium (Flow Laboratories, USA) containing 60 mg of kanamycin sulfate and heat-inactivated fetal bovine serum (KC Biological, USA) to a final concentration of 10%, or medium 1
MEM medium (Flow Laboratory, USA) containing 2.0 g of sodium bicarbonate, 60 mg of kanamycin sulfate, and heat-inactivated fetal bovine serum to a final concentration of 10%, or the above two types of media in a 1:1 ratio. Use a mixture of KYM-A cells are inoculated into a plastic shed, and when approximately 100 ml of cell suspension is obtained as a result of proliferation, floating agitation culture using a spinner flask is started. By gradually increasing the size of the spinner flask, floating agitation culture of 8 is possible. The initial inoculation density at each stage is 10 ⁴ to 10 ⁵ cells/ml, and grafting is performed when the density reaches 5×10 ⁵ to 2×10 ⁶ cells/ml. Example 2 Harvesting When the cell density reaches 5×10 ⁵ to 2×10 ⁶ cells/ml, the medium is changed to serum-free medium. The medium is 1
2.0 g of sodium bicarbonate per serving, N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
1.192g Kanamycin Sulfate 60mg, Insulin 8.5
mg, human transferrin (manufactured by Sigma) 1
mg, ethanolamine 4.6mg and selenite 13μ
g, RPMI-1640 medium containing 20 KIU/ml of aprotinin (Sigma). Suspension agitation culture was carried out in this serum-free medium for 1 to 4 days.
The process is repeated 10 to 10 times, and the obtained recovered liquid is subjected to purification. Example 3 Purification The recovered solutions obtained in Example 2 were combined to remove aprotinin, Tween 80 and sodium nitride.
Final concentration of 20KIU/ml, 0.01 and 0.02 respectively
Add so that it becomes %. (These three chemicals are added to all buffer solutions below at these concentrations.) This sample was purified by the following method. (a) Porath et al.'s method (Nature, Uol258 598−
599, 1975) containing 1M NaCl.
After sufficient buffering with mM Tris-HCl buffer (PH7.05), 30 ml of culture solution is loaded. After loading, flow 3000 ml of the same buffer to wash the column. No PA-KYM activity was observed in the passed culture solution or column washing solution. Subsequently, gradient elution is performed using 1500 ml of the same buffer and a buffer (PH7.3) containing 150 mM imidazole in the same buffer, and fractionation is performed at a rate of 15 ml per fraction. PA− for each fraction
When KYM activity was measured, PA-KYM activity was prominent mainly in fraction number 125. The active fractions are collected in a dialysis tube, sprinkled with polyethylene glycol 20,000 powder, and concentrated at 4°C. After concentration, 0.01M phosphate buffer (PH6.7)
Dialysis is performed at 4°C for 24 hours while exchanging the external solution. After dialysis, the solution in the tube is centrifuged (10,000 rpm, 10 min) to obtain a supernatant. (b) Prepare 130 ml of the solution containing PA-KYM obtained in this way in 0.01 M phosphate buffer (PH6.7).
Concanavalin A sepharose column (1 x 20 cm) sufficiently buffered with (Pharmacia Fine
Chemicals, Inc.). After loading, wash with 100 ml of the same buffer. Subsequently, elution is performed with an adhesion gradient formed from the same buffer (150 ml) and a buffer containing 0.6 M potassium thiocyanate and 3 M α-methylmannoside (150 ml). Fractions were collected in 3.5 ml portions, and PA-KYM activity was observed mainly in fraction number 32. After collecting this fraction, it was concentrated to 5 ml using polyethylene glycol 20,000, and this was heated at 40°C.
Dialyze against physiological saline for 24 hours while exchanging the external dialysis fluid. During dialysis, the enzyme solution becomes cloudy,
After centrifugation (15000 rpm, 30 minutes), PA-KYM activity is recovered in the precipitate fraction. At this stage, it was possible to remove mixed proteins, mainly proteins recognized as macromolecules, than with PA-KYM in the past. The precipitate obtained by centrifugation is dissolved in 2 ml of 0.01M phosphate buffer (PH6.7) containing 1.6N potassium thiocyanate.
After removing residues that cannot be completely dissolved by centrifugation, a supernatant containing PA-KYM activity is obtained. The resulting solution was diluted with 0.01M phosphate buffer (PH6.7) containing 1.6N potassium thiocyanate.
Sephadex G-200 sufficiently buffered with
(Pharmacia Fine Chemicals) column (1.6
×85 cm) and develop with the same buffer.
When the fractions were collected in 2.4 ml portions, PA-KYM activity was observed mainly in fraction number 35. As described above, a purified sample of PA-KYM was obtained using a four-step method including the fractional precipitation method.
Table 3 shows the degree of purification at each stage. [Table] Protein concentration was determined by the Lowry method, μ
Expressed in g/ml. A color reaction using the synthetic substrate S-2444 is carried out at 37° C. for 90 minutes using an appropriate volume (aml) of enzyme solution. Color development at 405nm in that case
If OD ⁴⁰⁵ = b, the indicated enzyme concentration C
is expressed as C=b×1.0/a. Specific activity is /. The degree of purification is 1.0 in the condition of the medium.
This figure shows the increase in specific activity at each stage. Example 4 Molecular Weight and Purity Regarding the purified sample obtained in Example 3, the molecular weight was determined and the purity was verified at the same time. First, the non-reduced sample was subjected to SDS polyacrylamide electrophoresis containing 10% acrylamide at room temperature for 18 hours at a constant current of 5 mA according to the method of Laemmli (Nature Vol. 227 680 (1970)). The gel thus obtained was mixed with 2.5%
Shake for 1 hour in Triton X-100 aqueous solution,
Remove SDS. This was then transferred to an agarose plate containing fibrin and plasminogen (Granelli Piperno and Reich, J.Exp.Med Vol.
148 223-234 (prepared according to (1978)) and observed after keeping at 37℃ for 1 to 5 hours, two dissolution zones with estimated molecular weights of 56,000 to 62,000 were observed. No dissolution zone was observed anywhere else. On the other hand, when plasminogen was not added to the agarose gel using a similar analysis method in which fibrin was layered on a plate, no dissolution zone could be observed, indicating that fibrin dissolution is dependent on plasminogen. did. In addition, 20 μg/g of the IgG fraction of anti-urokinase antiserum was added to the fibrin plate.
ml, the lysis zone is comparable to fibrin plates without added IgG. Therefore,
The two bands with molecular weights around 56,000 to 62,000 were shown to belong to TPA-type PA rather than urokinase-type plasminogen activator. The purified sample of PA-KYM (approximately 0.1 μg) obtained in Example 3 was either left unreduced or 5% 2
- reduced by addition of mercaptoethanol and separated by SDS-polyacrylamide gel electrophoresis in a manner similar to that described above. The gel thus obtained was processed using the method of Oakly et al. (Analytical).
Biochem Vol 105 361-363 (1980))
The contained proteins were stained. From the results obtained in this way, the non-reduced purified sample shows two bands around the molecular weight of 56,000 to 62,000, and the reduced sample shows two bands with a molecular weight of about 32,000 and about 62,000.
Two bands were seen at 36,000. Furthermore, no other notable bands were observed other than these bands, indicating that the purified sample had been purified to an almost pure state. The standard proteins with known molecular weights used for molecular weight measurement were phosphorylase b (94000), bovine serum albumin (67000), and ovalbumin (43000).
and carbonic anhydrase (30000). Example 5 Examination of affinity for fibrin CNBr-activated Sepharose 4B (Pharmacia
After swelling 10g of fibrinogen (Kabi) with 1mM HCl2 and washing, add 1g of fibrinogen (Kabi) with 0.5M NaCl/
Coupling buffer of 0.1M NaHCO ₃ (PH8.3) 200
ml for about 10 hours at room temperature to allow adsorption. Next, add this resin to 0.05M phosphate buffer (PH7.5) containing thrombin (2 units, bovine thrombin, Mochida Pharmaceutical).
Pour into 100 ml and keep warm at 37℃ for 10 minutes to remove fibrin
Sepharose resin was obtained. The resin thus obtained was filled into a minicoum (6 ml) and equilibrated with 0.05M phosphate buffer (containing 0.01% Tween 80, pH 7.5). This column was loaded with 1 ml of PA-KYM solution or urokinase solution. First, it was washed with 15 ml of 0.05M phosphate buffer (PH7.5), and each ml fraction was collected. Then, the mixture was developed with 25 ml of the same phosphate buffer containing 1.6 M potassium thiocyanate and fractionated into 1 ml fractions. The enzyme activity contained in the washing solution and the potassium thiocyanate solution was measured using the synthetic substrate S-2444 to determine the recovery rate.
As shown in Table 4, PA-KYM is not included in the washing solution and requires potassium thiocyanate for its elution, so it is evaluated to have a high affinity for fibrin.However, urokinase has poor affinity and is not included in the washing solution. The activity is recovered. [Table] [Table]

[Brief explanation of the drawing]

Figure 1 is a diagram showing the action curve of each PH in PA-KYM, Figure 2 is a diagram showing the residual activity of each PH, and Figure 3 is a diagram showing the residual activity of each PH.
The figure shows the action temperature curve, FIG. 4 shows the residual activity at each temperature, and FIG. 5 shows the ultraviolet absorption spectrum.

Claims (1)

[Claims] 1. A method for producing a plasminogen activator, which comprises culturing human rhabdomyosarcoma cell mutant KYM-A in floating agitation and collecting the plasminogen activator from the resulting culture solution.