JPS62103021A - Physiologically active substance with cytotoxic action and its production - Google Patents

Abstract

NEW MATERIAL:A physiologically active substance [molecular weight: 40,000-60,000 (gel filtration chromatography); isoelectric point: 5.0+ or -0.5; nonadsorptive to sepharose binding with concanavalin A; sensitive to trypsin, chymotripsin and pronase, but nonsensitive to RNase; pH7.2, stable 4 deg.C, partially deactivated by treatment at 56 deg.C for 1hr, almost perfectly deactivated by 70 deg.C treatment; stable to 20hr treatment at 7.2pH, deactivated at 2pH, almost deacti vated by 10pH treatment]. USE:Remedy for cancers and tumors. It has direct cytotoxic action in vitro against mouse L929 cells, human liver-originating HuL-1 cells. PREPARATION:Animal fibroblasts are cultured in serum-containing medium and serumfree medium, and the objective physiologically active substance of cytotoxicity is obtained from the supernatant of the culture mixtures by combination of salting, centrifugation, dialysis or the like. The substance can be produced by means of a microorganism such as Escerichea coli through biotechnology.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel physiologically active substance having cytotoxic action and a method for producing the same.

[Conventional technology]

Cytotoxic substances derived from mouse fibroblasts that are closely related to the present invention include interferon β (hereinafter sometimes abbreviated as β-IFN) and carcinobreaker X (hereinafter sometimes abbreviated as CBX). Are known. And β-IFN has a molecular weight of 22,000 to 23,000,
Isoelectric point 6.5-8.0 (Ando, Kobayashi, Immunopharmacology Vol. 1.
1-111983 l Life Science Publishing), CBX
has a molecular weight of 12,000 to 17,000 and an isoelectric point of 4.3 to
7.3 glycoprotein (Japanese Unexamined Patent Publication No. 148826/1982)
l1) respectively.

[Problem that the invention seeks to solve]

As a cytotoxic substance derived from animal fibroblasts, the aforementioned β-
Although IFN and CBX are known, little is known about other cytotoxic substances, and at present, none of their properties have been determined.

[Means to solve the problem]

In view of these circumstances, the present inventors have conducted extensive research and have discovered that animal fibroblasts produce a physiologically active substance that has a novel cytotoxic effect, and have completed the present invention.

That is, the present invention relates to a physiologically active substance that is produced from animal fibroblasts, has a cytotoxic effect, and has the properties described below, and a method for producing the same.

(al Molecular weight is 40,000-60,000 (gel filtration chromatography), (bl Isoelectric point is 5.0±0.5, tel
FPLC-Mono Q anion exchange chromatography, eluting at 0.34-0.35 M saline concentration, non-adsorbent to +dl concanavalin A-conjugated sepharose, (e) sensitive to trypsin, chymotrypsin, proase, Insensitive to RNase, (fl pH
Stable at 4°C in 7.2 buffer, partially inactivated (66% inactivation) after 1 hour treatment at 56°C, almost completely inactivated at 70°C, fg) pH 7, 20 hour treatment Stable at pH 2
It is inactivated at pH 10, and most of it is inactivated (83% inactivated) at pH 10. It has a direct cytotoxic effect on mouse 929 cells and human liver-derived HuL-1 cells in vitro.

As cells producing the cytotoxic substance of the present invention, fibroblasts of mice, rats, rabbits, humans, etc. can be used, but mouse fibroblasts are preferably used, and these cells are stimulated with a stimulant. Alternatively, it can be produced by culturing without stimulation.

Any stimulant that can induce the cytotoxic substance of the present invention may be used, such as lectins such as concanavalin, phytohemagglutinin (PHA), etc.
, lipopolysaccharide (LPS), various antigens, and phorbol esters (for example, 12-O-tetradecanoylphorbol-13-acetate: PMA), which can be used alone or in combination. .

Furthermore, during cell culture, it is also possible to use a serum-containing medium or a serum-free medium.

Purification from the obtained culture supernatant is carried out by conventional methods generally used for protein separation, such as salting out, centrifugation, dialysis,
It can be carried out by combining methods such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, ultrafiltration and lyophilization.

That is, after concentrating the culture supernatant obtained in the present invention by ammonium sulfate salting out or ultrafiltration,
We perform gel filtration using a carrier that has passed through the separation of ~200,000 substances, and further perform preparative isoelectric focusing using FPLC-Mono, Q anion exchange chromatography, and Ultrodex. , can be separated and purified by affinity chromatography using immobilized concanavalin A. The cytotoxic substance of the present invention thus obtained is a fluorescent substance having the following properties.

(11 Molecular weight: The molecular weight is 40,000 to 60,000 as determined by gel filtration using a FPLC-Superose 12 (manufactured by Pharmacia) column (Figure 1).

(2) Isoelectric point: The isoelectric point is 5.0±0.5 by performing preparative isoelectric focusing (pH 3 to 9) using Ultrodenox (manufactured by LKB).

(3) Ion exchange chromatography: FPLC
-Mon.

By adsorbing on Q anion exchange chromatography (manufactured by Pharmacia) and performing a linear concentration gradient of 0-0.5 M sodium chloride in 0.01 M phosphate buffer (pH 7,2), 0.34-0 It is eluted at a salt concentration of .35M (Figure 2).

(4) Affinity chromatography: Non-adsorbable in concanavalin A-bound Sepharose (manufactured by Pharmacia) equilibrated with 0.0° C.M phosphate buffer (pH 7.2) containing 0.3M sodium chloride.

(5) Other properties (i) Enzyme treatment: Insensitive to ribonuclease treatment, but sensitive to trypsin, chymotrypsin, and proase treatment.

(ii) Thermal stability: 1) in H7,2 buffer at 4°C;
Stable for 1 hour treatment, but partially inactivated at 56°C (6
(6% deactivation), and almost completely deactivated at 70°C.

(iii) pH stability: Stable at pH 7 for 20 hour treatment, inactivated at pH 2, mostly inactivated at pH 10 (
83% inactivation).

(6) Cytotoxic effect: It has a cytotoxic effect on at least mouse L929 cells and human cultured cells (human liver HuL-1). However, the activity level of this substance is A.

Flick et al. J. Immunological
Methods + 68 +167-175 (1
984), and the unit of activity was 5.
The amount giving 0% cytotoxic effect is expressed as 1 unit (U).

Furthermore, as a means of producing this substance, in addition to a method of culturing animal fibroblasts, it is also possible to produce it using microorganisms such as Escherichia coli by genetic engineering means.

[Action and effect of the invention]

The cytotoxic substance of the present invention can be used as a therapeutic agent for cancer, tumors, etc., and as a reagent in the medical and pharmaceutical fields.

When the cytotoxic substance of the present invention is used as a medicine, it can be formulated according to ordinary formulation techniques, for example, by mixing the substance with carriers, excipients, diluents, etc.
It can be provided in the form of tablets, capsules, injections, etc.

〔Example〕

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 Mouse fibroblast L5 cells (mouse fibroblast L929
substrain; Terashima et al., J. Rad, Res., 12 (
2).

61-72. (1972)) 3.3 X I O'
cells/ml were cultured at 37°C for 72 hours in the presence of 10% serum or in the absence of serum, and the production of cytotoxic substances in the culture supernatant was measured. 300
U/ml, and in the absence of serum, an activity of 2000/ml was observed.

Example 2 Mouse fibroblast L5 cells were suspended in serum-free Ham's F12 medium and then placed in a 24-well microtest plate.
Planted at 3 x 10' pieces/ml, 72 with various stimulants
The cells were stimulated for a time, cultured, and the production of cytotoxic substances in the culture supernatant was measured. As a stimulant, 10 ng/ml of 12
-0-tetradecanoylphorbol-13-acetate (PMA) and 20 pg/m! Noribobolisancalide (LPS), 10 μg/ml phytohemagglutinin P (PHA-P), or 5 μg/ml concanavalin A (conA) were used in combination.

The results are shown in Table 1.

As is clear from Table 1, considerable activity is observed even without stimulation, but stimulation with ConA+PMA
A slight increase in activity production is observed.

Example 3 L5 cells were incubated at 37°C using Ham's F12 medium (serum-incompatible).
Rotation culture was carried out for 2 to 3 days. 1.4J of this culture supernatant was
50 with MIO membrane filter (manufactured by Amicon)
It was concentrated by ultrafiltration to 0 times. Next, gel filtration was performed using FPLC-Superose 12 (Pharmacia Fine Chemicals), and as shown in Figure 1, the activity peaks were found between opalpmin (OVA) and bovine serum albumin (BS).
A), the molecular weight is 40,000-60,0
It was 00.

The active fraction was further fractionated by FPLC-Mono Q anion exchange chromatography (manufactured by Pharmacia). The Mono Q column was equilibrated with 0.01M phosphate buffer (pH 7,2), and after sample addition and washing, elution was performed using a linear concentration gradient of 0 to 0.5M sodium chloride. The activity peak is 0 as shown in Figure 2.
．． It was eluted at a salt concentration of 34-0.35 M. The activity recovery rate at this stage was 6.4% compared to ultrafiltration concentration.
The specific activity was 7×10'U/■ protein.

Example 4 Mouse 929 cells and human liver HuL in the substance of the present invention
A comparative study was conducted regarding the cytotoxic effect on -1 cells.

As shown in Figure 3, the cytotoxic effect of this substance is
Although slightly weaker than that for mouse L929, human liver Hu
It also showed cytotoxicity against L-1 cells.

Example 5 Other properties of the substance of the present invention were investigated.

First, in the enzyme treatment, 100 p of ribonuclease
g/ml trypsin IQOug/11, chymotrypsin lOOμg/ml, proase 100μs/ml
were added to the substances of the present invention and treated at 37°C for 2 hours, followed by activity evaluation.

For thermal stability, a sample of about 400 U/ml was heated to 0.
0.01M phosphate buffer containing 14M salt (pH 7.2
) for 1 hour at 4°C, 56°C, and 70°C, and the remaining activity was measured.

Regarding pH stability, approximately 2500/ml samples were tested at pH 2 (Q, 02M glycine buffer), pH 7 (0, 1
O, 01M phosphate buffer containing 4M NaCl) and pH 1
After each treatment in 0.02M glycine buffer (0.02M glycine buffer) for 20 hours, the cells were dialyzed against 0.01M phosphate buffer (pH 7.2) containing 0.14M sodium chloride, and the residual activity was measured.

As a result, the cytotoxic substance of the present invention is insensitive to ribonuclease and sensitive to trypsin, chymotrypsin, and proase in enzymatic treatment, and in terms of thermostability, it is stable at 4°C but partially inactivated at 56°C. (66
% inactivation), almost completely inactivated at 70°C; pH stability: 1) stable at H7 but inactivated at pH 2; 1) mostly inactivated at HIO (83% inactivated) It turned out that.

Note that the cytotoxic substance of the present invention differs from conventional β-IFN and CBX in terms of molecular weight and pH stability.

[Brief explanation of drawings]

Figure 1 shows the elution pattern by gel filtration using FPLC-Superose 12 column, Figure 2 shows the elution pattern by FPLC-M
FIG. 3 shows the elution pattern of a linear concentration gradient of saline (0 to 0.5 M) using an ono Q column, and the cytotoxic effect on mouse 929 cells and human liver HuL-1 cells, respectively. In addition, in Figures 1 and 2, ←→ indicates cytotoxic activity (%), -
・- means absorbance 280 nm (0, D, zso),
-m- represents a linear concentration gradient due to common salt, respectively.

Claims (2)

[Claims] (1) A physiologically active substance that is produced from animal fibroblasts, has a cytotoxic effect, and has the properties described below. (a) Molecular weight is 40,000 to 60,000 (gel filtration chromatography), (b) Isoelectric point is 5.0±0.5, (c) FPLC-MonoQ anion exchange chromatography , eluted at a salt concentration of 0.34-0.35 M; (d) non-adsorbent to concanavalin A-conjugated sepharose; (e) sensitive to trypsin, chymotrypsin, proase, and insensitive to RNase; f) pH 7.2
(g) Stable at 4°C in a buffer solution of Stable, but at pH 2
(h) It has a direct cytotoxic effect on mouse L929 cells and human liver-derived HuL-1 cells in vitro. (2) A cytotoxic effect characterized by culturing animal fibroblasts in serum-containing and serum-free media to produce physiologically active substances having the following properties and collecting them from the culture supernatant. A method for producing a physiologically active substance having (a) Molecular weight is 40,000 to 60,000 (gel filtration chromatography), (b) Isoelectric point is 5.0±0.5, (c) FPLC-MonoQ anion exchange chromatography , eluted at a salt concentration of 0.34-0.35 M; (d) non-adsorbent to concanavalin A-conjugated sepharose; (e) sensitive to trypsin, chymotrypsin, proase, and insensitive to RNase; f) pH 7.2
(g) Stable at 4°C in a buffer solution of Stable, but at pH 2
(h) It has a direct cytotoxic effect on mouse L929 cells and human liver-derived HuL-1 cells in vitro.