JPS61249926A - Manufacture of tpa product derived from fibroblast cell - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention Dissolving accidentally generated thrombi by the body's fibrinolytic system is essential for maintaining blood flow. The dissolution of bran and impurities is catalyzed by an enzyme called plasmin, but this enzyme normally does not exist in the blood as it is, but when it is needed, it is generated by the action of plasminogen activator on blood plasminogen. be done. Diseases that impair this system cause thrombotic vascular occlusion, which is one of the major causes of death in modern society. Administration of a blasminogen activator enhances the activity of this impaired fibrinolytic system, promoting lysis of the occlusive thrombus. Therefore, a plasminogen activator that can be safely administered to humans is highly desirable.

A plasminogen activator named O-kinase has been known for many years to be present in trace amounts in human urine and has been isolated on a commercial scale. Extensive clinical trials have demonstrated the efficacy of urokinase, primarily for the treatment of pulmonary edema, and its use is approved in many countries. However, the supply is also limited. In addition, urinary
Injection of kinase activates plasminogen in the blood without being restricted mainly to the vicinity of the thrombus site, so there is a risk of causing undesirable side effects.

Studies of tumor-derived cells, transformed cells, and cells in response to co-carcinogens have revealed increased production of blasminogen activator compared to normal cells; “Markers” for oncogenic cells
It has been suggested that it can be used as

A recent line of melanoma cells has been used to produce sufficient amounts of plasminogen for laboratory characterization and for conducting some clinical trials.

This melanoma enzyme is immunologically related to the activator isolated from the human uterus, belongs to the class of tissue-type plasminogen activators (t-PA), and is immunologically distinct from the T-O kinase. It turns out that they are also structurally different. It was also found that 1-PA binds to fibrin clots and that this binding significantly increases the rate of plasminogen activation. Therefore, this activation of plasminogen by t-PA is localized to the thrombus.

However, since t-PA originates from cancer cells, there are some concerns regarding its safety during use.

DESCRIPTION OF THE INVENTION It has now been revealed that normal diploid cell lines can be cultured to produce large amounts of plasminogen activator.

Plasminogen activator biosynthesis can be increased without using carcinogens, co-carcinogens, or other reported inducers. In the conditions used in the water droplets, cells are first adapted to produce PA by culturing in medium supplemented with specific serum and by using vessels pre-coated with specific substances. After a 5-15 day adaptation period during which the cells grow normally, the medium is changed to serum-free medium, into which the cells secrete plasminogen activator continuously for several weeks.

It has been found that accumulation of plasminogen activator in the production medium can slow down (or even stop) the biosynthesis of this enzyme. Secretion must be kept at low concentrations, either by using large amounts of medium or by continuously changing the medium or by adsorbing the enzyme.

There were no changes in the karyotype or growth characteristics of the cells used in this method. Therefore, cells that have been used once can be reused.

The cells used in the droplets are normal human fibroblasts that have been shown to produce live polio and rubella vaccines. Such cells include, for example, 1MR-90, Wl-
38, HEL299, and GM-1604.

The serum and surface coating agent used for water droplets are well known and used by many researchers for cell culture.

They have never been reported to have oncogenic activity, transform cells, or interfere with PA biosynthesis. Horse serum, which is a suitable serum in the adaptation stage, was used mainly because it is cheap and available in multiple quantities. Pre-coating of the container with a polymer, specifically poly-lysine, is preferred as it improves the adhesion of the WII cells and increases the growth activity of the cells in low concentration media.

Once adapted, the cells biosynthesized and secreted blasminogen activator, and serum-free medium was optimal for this production stage. However, it is known that in order to maintain cell growth activity, it is necessary to supplement the medium with a small amount of peptone. Lactalbumin hydrolyzate is commonly used as a cell maintenance medium and can also be used in PA production. However, other additives (such as casein hydrolyzate or neopeptone) were equally or more effective.

Blasminogen activator produced in normal fibroblasts cultured by the method described herein is of the t-PA type and can be isolated by the same method as t-PA obtained from melanoma cells. The two enzymes exhibit the same behavior when precipitated with ammonium sulfate and when precipitated with Zn-chelated-Sephalometric column and Sephadex G-150.

It was found that the t-PA type product derived from line miscellaneous blast cells also reacted with an anti-t-PA monoclonal antibody produced in a rabbit and an anti-PA monoclonal antibody obtained from a hybridoma of mouse cells. . This fibroblast enzyme is
It was also found that it reacted with a voriclonal antibody directed against kinase, but did not react with a monoclonal antibody against urokinase.

Electrophoresis on polyacrylamide in the presence of SO8 shows that there are two active variants of fibroblastic t-PA that are electrophoretically similar to melanoma t-PA. could not be distinguished from the variant. melanoma t
Similar to -PA, this fibroblast t-PA can be obtained as a "single-chain" or "two-chain" form, depending on whether or not an inhibitor of plasmin is added during preparation. Electrophoresis in reduced conditions yields a species with a mobility corresponding to a molecular weight of about 70 ± 5 KO from the "single-chain" form, and about 35 from the "two-chain" form.
Two bands with a mobility of -40 KD were obtained.

1. Cell culture human diploid fibroblast ml11 1MR-90 strain, Wl-3
-IEL-299 strain was obtained from ATCC, and GM-1604 strain was obtained from NIGLS Human Genetics Mutant Cell Repository and NIA Engineering Cell Repository (NIGLSHljlan Gen( 3tiCHu
tant Ce1l ReDO3itOrV and
NtAAqingCell Repository).

The medium used was Dulbecco's modified Eagle's medium (DM
EM) and Hams medium F-12 in a 1:1 (vol/vol)
) is a mixture. 10% fetal bovine serum (Fe2) was added to the medium.
) or supplemented with horse serum (H8).

Cells were cultured at 37°C in an atmosphere of 5% CO□ air.

Upon receipt, cells were thawed and plated in plastic culture dishes using Fe2. When colonies were formed, the cells in each culture dish were trypsinized and divided into three new blood cells. Next, 13I1
Cells were cultured using two passages in between. At each subculture, trypsinization and separation were performed at a ratio of 1:2 to 1:4 (depending on the growth rate of 1B follicles).

2. Adaptation to PA production Cells were collected by trypsinization and plated in culture blood precoated with poly D-lysine at a concentration of approximately 1-2 x 10' cells per surface of the dish, using H8 medium as described above. Cultivation was continued in the same manner as above, with at least 2 to 4 subcultures.

3. 2-3 days after the last subculturing of the adaptation phase, the medium was removed, the cells were washed with DMEM (serum-free), and then DMEM supplemented with peptone was added. Medium changes began 48 hours after transfer to production medium. At each medium change, approximately half of the production medium was removed and replaced with fresh medium.

A sterile solution containing 10 μ/d of coating material (usually poly-louridine) was brought into contact with the surface to be coated. Approximately 0.1 ae of solution was used for the surface 1aR2 to be coated.

After 5-15 minutes, remove the liquid and rinse the surface with sterile double-distilled water.
Washed twice. The containers were used immediately after coating.

2. Quantification of plasmin activator Quantification was carried out by the fibrin-agar plate method.

National Institute of Biological Standards
Itute of Biolooical 5tand
The WHO standard product of urokinase was obtained from ards (London, UK) and used to calibrate the quantitative method.

3. Product Recovery [Fibroblast plasminogen activator was routinely precipitated from the medium by adding 111 ammonium to 60% saturation. 0.1% Tween 80 and 5
The precipitate was dissolved in 0,2M Tris buffer (EIH7,8) containing mHE DTA and dialyzed against the same buffer or other buffers as required for subsequent studies.

To obtain a "single-chain" type of activator, the final concentration is 25 or 50 KILJ/ml! AbOtinin was added so that

Cells were adapted for PA production, transferred to production medium, and PA activity in the medium was quantified. The results (Fig. 1a) showed that PAil1 degree increased initially, but after reaching a certain concentration, no more enzyme was produced. However, repeated changes of the medium continued enzyme production at a virtually constant rate.

To examine the negative effects of exogenous PA on PA biosynthesis, approximately 50 tJ#! of purified plasminogen activator was added.

As seen in Figure 1b, PA concentration did not increase in this culture, but normal production was seen in control cultures without exogenous PA. Therefore, exogenous PA
appears to almost completely block production of the enzyme by cells.

When the PA-containing medium taken from the cells was then passed through a silica bed, approximately 90% of the enzyme was adsorbed. The results (Figure 1C) show that the once-used medium lacking PA can be reused;
This shows that it has no effect on the PA production rate.

2.1 Stand upright! 1 Divide the same culture plate into two, and culture one part in a poly-lourisine-coated culture dish using H8 medium as described above, and the other part in an untreated culture dish and FC8 medium. Cultured. Finally, in both cases, replace the culture medium with production medium and P
produced A. In pre-adapted cells, 1a
A production curve similar to the one shown in the figure was obtained, and the average speed was approximately 60 LJ/d.
/ day. However, in Hoso 11 (Fig. 2), which was not adapted, the production rate of PA was very slow. Furthermore, PA production by cells that did not undergo this adaptation was not affected by repeated exchange of the used production medium with fresh medium (Figure 2).

To investigate whether it is possible to adapt with sera other than H8, cells were cultured in a medium supplemented with several types of sera according to the adaptation method described above. 4 after transfer to production medium
As a result of measuring the PAW degree after 8 hours, it was found that pre-coating the culture vessel with poly-lysine and the properties of the serum also influenced the adaptation stage, and that their effects were synergistic (first table). Therefore, when combined with horse serum, fresh Shinto brother serum, and especially precoating, 111 cells with high PA production ability were obtained.

90 pieces and 150 mg + in culture dish, and 24 x 2
Cell mass was accumulated by subculturing in 41 industrial petri dishes. All plastic dishes were pre-coated with poly-lolysine and H8 medium was used throughout. When the cells became aggregates, the medium was replaced with production medium. 48
After some time, the PA concentration reached 100 U/d and medium exchange was started as described above.

(:i) Roller bottle with a capacity of 2 liters, 30 liters per pin
It was precoated with poly-lourisine by rolling for 10-15 minutes using a solution of M1.

The bottles were then washed twice with double distilled water. Next is 1.5X10711 per pin! The bottles were seeded with pre-adapted cells using 200 d of H8 medium. After 48 hours, the medium was changed to production medium and PA activity was monitored. When PA production slows down (usually after 10-15 days)
and the medium was collected when the PA concentration was in the range of 50-80 U/d.

(if) Cytodex of 105 cells/d and 347 d on microcarriers
Culture was started using dex)n. Cells were allowed to grow until the beads were virtually covered with cells and the mm concentration reached 106/sat. The medium was then changed to production medium. Fourth
PA activity in the medium reached 24 Ll/d at the 8th day, and at the 58th day, the PA activity in the medium reached 24 Ll/d.
50U/a for the eyes! reached.

m Zn-chelated Sepharose (FIG. 3) The material obtained from the culture medium by ammonium sulfate precipitation was applied to a column of Zn-chelated Sepharose. All the PA was adsorbed, but some inert material passed through. When eluted with a concentration gradient of increasing imidazole concentration, a protein peak with virtually no PA activity appeared first, followed by a peak with PA activity. Approximately 80% of the PA activity was recovered in this peak.

(It) LL] (Figure 4) The material obtained from the Zn-chelated Sephas was passed through a column of Sephadex G-150.

First, a high molecular weight inactive protein appears, then a molecular weight of 5O
PA appeared as another peak at the elution volume corresponding to -100 KO.

(iii) Fibrin column (Figure 5) The substance obtained by ammonium precipitation was applied to a fibrin column (1 ml! bed) adsorbed on Celite, and the fibroblast gIPA
Most of it was adsorbed on the column and was eluted only when eluted with a high ionic strength eluent. The overall daily yield of activity was 70.3%, of which 78.5% was in both the salt eluted fractions and the remainder in the unadsorbed material.

Characterization (1) Fibroblasts PA were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of SDS (Figure 6). Contact replica on fibrin-agar gel (first row) or reaction with rabbit antibody raised against melanoma t-PA (“Western” plot)
(column 3), P by its fibrinolytic activity
A was detected.

For comparison, melanoma t-PA was also run (second and fourth columns). As is clear from the figure, the two types of PA showed similar electrophoretic patterns and immunoreactions.

(11) In order to estimate the molecular group, fibroblast 11111PA was reduced and carboxymethylated with radioactive iodoacetic acid. Analysis by 5DS-PAGE (Fig. 7) revealed that approximately 7
Single chain molecule with molecular weight of 2±5KD and 35-40K
In the O region, A chain and Bll were demonstrated.

(iii) Streptoblasts PA were biosynthetically radiolabeled by adding [-8]methionine into the production medium. This PA was then precipitated with an antibody against t-PA (melanoma). The precipitate was dissolved in the presence of mercaptoethanol and analyzed by 5O8-PAGE (Figure 8). This gel was cut into thin slices and measured to determine the location of the radioactivity. Small amounts of substances with very large molecules m and very small molecular weights were also non-specifically adsorbed to anti-t-PA and to an irrelevant antibody used as a control. The main components that specifically precipitated anti-t-PA were a single chain PA (approximately 70 KD) and two molecular chains (35-40 KO).

Table 1 Blasminogen activator secreted by human diploid lineoblasts grown under various conditions

[Brief explanation of the drawing]

Figure 1a shows PA activity in the medium, Figure 1b shows the effect of exogenous PA on PA biosynthesis, and Figure 1C shows P
Figure 2 shows the effect of medium adaptation on PA production, Figure 3 shows the isolation of PA by zn-e rated Sepharose, and Figure 4 shows the effect of PA production by gel permeation. Figure 5 shows the separation of PA by fibrin column, Figure 6 shows the electropherogram of PA,
Figure 7 shows the molecular weight of PA, and Figure 8 shows the SDS of PA.
- Shows analysis by PAGE.

Claims (11)

[Claims] (1) Approximately 72±5 when analyzed by SDS-PAGE
In a method for producing a fibroblast-derived TPA (plasminogen activator) type product exhibiting a single chain molecule with a molecular weight of KD and an A chain and a B chain of 35-40 KD,
Human diploid fibroblasts are prepared by culturing them for multiple passages in media containing mammalian or avian serum, fetal bovine serum and goat serum are excluded, and the cells are then conditioned for production of the desired product. The above-mentioned method is characterized in that the product is obtained by transferring the product to a suitable medium and culturing it. (2) Cells are human fibroblast cell line 1MR-90, W1-3
8, HEL-299 and GM-1604. (3) The method according to claim 1, wherein the cells are cultured and prepared on a substrate coated with poly-lysine. (4) The method according to any one of claims 1 to 3, which is produced in a peptone-containing medium. (5) The method according to any one of claims 1 to 4, which is produced in a medium containing lactalbumin hydrolyzate, casein hydrolyzate, or neopeptone. (6) The method according to any one of claims 1 to 5, wherein the adjustment is performed with a medium containing horse serum or newborn calf serum. (7) The method according to any one of claims 1 to 6, wherein the conditioning is performed for 5 to 15 days, and then the cells are transferred to a serum-free medium. (8) The method according to any one of claims 1 to 7, wherein the product is continuously removed and prevented from increasing beyond a predetermined concentration in the medium. (9) Fibroblast-derived T as described above and in the Examples
Method for producing PA-type products. (10) Any one of claims 1 to 9
A fibroblast-derived TPA-type product obtained by the method described in section. (11) Anti-TPA voriclonal antibody raised in rabbit and anti-TPA obtained from mouse cell hybridoma
Claim 1, which is reacted with a monoclonal antibody.
A product obtained by the method according to any one of paragraphs 1 to 9.