JPS6067429A - Preparation of physiologically active substance - Google Patents

Abstract

PURPOSE:To produce a large amount of a physiologically active substance having anti-tumor activity, by transplanting carcinoma cells to a normal mammalian animal, administering an immuno-activating substance to the cancerous animal, administering a component originated from a specific bacterial cell, etc. to the animal, and separating the objective substance from the animal. CONSTITUTION:Carcinoma cells (e.g. RL-male 1) are transplanted in the skin of a normal mammalian animal (e.g. BALB/C more). An immino-activating substance (e.g. killed cell of Corynebacterium parvum, living cell of B.C.G, etc.) is administered to the animal e.g. by intravenous injection to effect the primary stimulation of the animal, and then the animal is administered with a component originated from bacterial cell represented by the bacterial LPS, or a synthetic polysaccharide or a synthetic lipopolysaccharide as a secondary stimulant e.g. by intravenous injection. The body fluid or blood collected from the animal is purified by the combination of the basic anion exchange chromatography, gel filtration, etc. to obtain the substance having a TNF-like activity in a state having high activity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a physiologically active substance having antitumor activity.

A variety of antitumor physiologically active substances obtained in serum or by cell culture have been known so far, and E, 8, Carsiyell, 1. ,J.
TNF (Tumor Necro) discovered by Old et al.
sisFactor). Particularly, a lot of research has been carried out on TNr' in recent years, and it is attracting a lot of attention because its specificity in attacking only tumor cells may be the key to conquering cancer.

TNF can be produced by giving a primary stimulation with B, C, G, etc. to mammals such as mice, and then giving a secondary stimulation with Norivolisasoccaride (LPS) such as endotoxin. Although it was thought to be a trace substance, recent research has shown that activated macrophages
□ (hereinafter referred to as "Mφ") from cell culture systems is also progressing. Research has been conducted on a simple VIJ method (Japanese Patent Application Laid-open No. 141785/1985) in which physiological saline is injected.

Furthermore, although it has been revealed that the main body of TNP is a glycoprotein, there are many theories regarding its molecular weight and other physical properties (Japanese Patent Laid-Open No. 140725/1983, etc.).
Although the physiologically active substances produced by the above production and purification methods are collectively called TNF, it has not yet been proven that they are exactly the same substance, and groundbreaking research has been awaited.

In view of the current situation described in -1-, the inventors of the present invention continued their intensive research and succeeded in discovering an innovative method for obtaining a substance with TNF-like action in large quantities and in a highly active state. The invention was completed.

The gist of the present invention lies in the fact that the animals that have been used for TNf' production have been changed from normal animals to tumor-bearing animals.

The configuration of the present invention will be explained in detail below.

In carrying out the present invention, first, syngeneic cancer cells such as R1 and male 1 are transplanted into a normal mammal, such as a BALB/C mouse, at a dose of about 1×10 61171 I, for example, into the skin by a conventional method. The cancer cells transplanted through this operation cause cancer tissue to colonize the transplanted site.

After this, Coryne-bac usually returns within 1 to 6 days.
A primary stimulus is given by intravenously administering killed bacteria, B, C, G, live bacteria, etc. at a dose of 250 to 2000 pg, preferably 500 μg or less, and a secondary stimulus is usually given after 8 to 15 days. A bacterial cell-derived component typified by bacterial LPS, a synthetic polysaccharide or a synthetic ribopolysaccharide, etc. in an amount of 1 to 50 μg, preferably about 5 μg or less, or Lipid A in an amount of 10 to 1000 μg. +rg
The drug is administered intravenously, preferably in a dose of about 300 μg or less.

Through these series of operations, hemorrhagic necrosis occurs in the animal transplanted cancer tissue, after which complete regression of the layer can be observed.

Therefore, for example, when implementing the present invention on cancer patients, C8 parvum killed bacteria, B,
C, G, live bacteria, etc. are administered to give a primary stimulus, and then, usually 8 to 30 days later, non-toxic Lipid is administered as a secondary stimulus.
By administering A and the like, it is possible to cause necrosis and regression of the cancer site.

According to the present invention, blood is collected from the heart of the animal by a conventional method 2 to 4 hours after the secondary stimulation, or at or before the onset of hemorrhagic necrosis in the animal's transplanted cancer tissue. It is preferable to collect blood by a method such as using a syringe or a syringe from an artery.

Furthermore, the present invention can be carried out by collecting body fluids other than blood, such as ascites, lymph fluid, etc., or by obtaining animal organs such as a tile, crushing them, and extracting them with physiological saline or the like. It can be implemented.

The solution or blood thus obtained can be purified by carrying out the following commonly used purification method in combination with appropriate Mi.

(1) Basic anion exchanger chromatography (2)
Gel filtration (3) Affinity chromatography with fixed cibacron blue or concanavalin A, etc. Slab electrophoresis with (7+) polyacrylamide gel In addition, in practicing the present invention, without collecting blood, at the time of LPS administration, Alternatively, it is also possible to apply a method in which a large amount of physiological saline is injected into the abdominal cavity of the animal after administration of Lps, and then the physiological saline is collected and purified.

The gist of the present invention is that by changing the subjects to which immunostimulating substances have been administered, which had previously been limited to normal animals, to tumor-bearing animals, surprising effects have been produced. The following stimulation method and method for purifying the target substance can be carried out by appropriately applying any method known so far.

It is known that the BALB/C mouse used in the present invention is less sensitive to LPS etc. than other animals. Generally, when obtaining physiologically active substances by conventional methods, it has not been possible to easily avoid death of animals due to LPS by carrying out one cycle, but according to the present invention, the low sensitivity characteristic of LPS can be avoided. By making the animal bear a tumor and administering a primary stimulant, the sensitivity to LPS etc. can be increased, and by administering a small amount of LPS etc., the target substance can be obtained without killing the animal.

In addition, when using other strains of mice or animals of other species, choose a dose that is lower than the commonly used LPS dosage that will not cause animal death, or choose a dosage that is lower than the commonly used LPS dosage, or choose a dosage that does not cause death of the animal, or By administering low-toxicity Libido A, the target substance can be obtained without killing the animal.

In addition, previous research has shown that the effect within Mφ activated by the primary stimulus is maintained for 7 to 30 days or more, so as long as the animal is kept alive, the effect of the primary stimulus can be maintained for more than 7 to 30 days. No reapplication is necessary.

Therefore, if a method of collecting the target substance without killing the animal using, for example, the intraperitoneal physiological saline injection method (Japanese Unexamined Patent Publication No. 141785/1985) is applied to carry out the present invention, appropriate recovery can be achieved. After a period of time, a similar target substance can be obtained by performing a secondary stimulation using LPS etc. without performing a primary stimulation, making it extremely easy to obtain the target substance. I can do it.

In testing the effects of the present invention, conventional methods commonly used up to now can be used. For example, the effect of the present invention can be seen by simultaneously measuring the 50% inhibitory activity against mouse-derived L-929 cells in the same system as the physiologically active substance obtained by the conventional method. According to this method, the physiologically active substance obtained according to the present invention exhibited an effect more than 4 times superior to that obtained by the conventional method in terms of dilution ratio.

EXAMPLES The present invention will be described in more detail below with reference to Examples.

Example I BAL, B/c mouse, male (male, 6 weeks old) 1
RL♂1 cancer cells (1×lO61 [IJ) were subcutaneously transplanted into 0 mice and left overnight. The next day C, 500 μg of parvun+killed bacteria is administered intravenously. LP525μ after 7 days
g is administered intravenously, and 2 hours after administration, whole blood is collected from the heart. Whole blood was collected and centrifuged to obtain 4.6 ml of serum.

50% of this serum against mouse-derived L-929 cells
The inhibitory activity was 160 (expressed as a dilution factor).

When a similar experiment was performed on BAL B/C mice to which no cancer cells were transplanted, only % or less activity was observed in terms of dilution ratio.

I"2

Claims (1)

[Claims] (1) Cancer cells are transplanted into a normal mammal, and then an immunostimulatory substance is administered to the tumor-bearing animal, followed by bacterial cell-derived components such as Noriboborosasocalide derived from Gram-negative bacilli or synthetic polysaccharides or synthetics. characterized by administering ribopolysaccharide etc. and then collecting an antitumor substance from the animal,
Method for producing physiologically active substances.