JPS62155221A - Kit for inducing tumor necrosis factor - Google Patents

Abstract

PURPOSE:To provide the titled kit containing a macrophage activation factor (MAF) and supernatant liquid of cultured Bordetella pertussis, dead cell thereof or their treated product and utilizable for the remedy and prevention of tumor. CONSTITUTION:The objective kit for inducing tumor necrosis factor (TNF) contains MAF and supernatant liquid of cultured Bordetella pertussis, dead cell thereof or their treated product. MAF can be prepared by injecting BCG to a mouse by intravenous injection, extracting spleen cell from the mouse about 3 weeks after injection, culturing the cell and filtering and sterilizing the supernatant liquid. The cultured Bordetella pertussis is obtained by culturing Bordetella pertussis in Bordet-Gengou medium added with serum, transplanting the cultured cell in a charcoal-containing semisynthetic medium and culturing the cell. Inactivated Bordetella pertussis (BPV) may be used in place of the dead Bordetella pertussis cell. The composition of one dose of the kit for an adult is shown in Table. EFFECT:Extremely large amount of TNF can be produced in a short time compared with conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a kit for inducing tumor necrosis factor, which contains a macrophage activating factor and a culture supernatant of Bordetella pertussis, killed cells, or a processed product thereof. The kit of the present invention is useful in the medical industry because it can be used for tumor treatment and prevention.

Prior Art Tumor necrosis fac
tor (hereinafter abbreviated as TNF) is
/l/ (Carswell) et al. [Proceedings of the National Academy of Sciences (Proc. Natl.

Acad, Sci,), USA 72.3666 (
1975)'].

TNF is usually used in advance as a briming agent (P agent) for Bacillus spp.
Calmette-Guerin (BCG)] or Propionibacterium acnes (Propio
nibacterium acnes (PA)
Administer E to mammals such as mice, rats, and rabbits,
After 2 to 3 weeks, Escherichia coli-derived lipopolysaccharide 1 was added as an eliciting agent (E agent).
de(LPS)) is produced in the serum.

Japanese Journal of Bacteriology 39.389 (1984) and 40, 285 (1985) include
FA was administered as a drug, and 11 days later Bordetella pertussis (B. pertussis) was administered as a drug E.
Hereinafter abbreviated as BP)] cell surface fraction (-L P S
It has been reported that TNF is produced in the serum by administering a drug (not including

Problems to be Solved by the Invention and Means for Solving It is desired to develop a drug that is more effective as an inducer of TNF. The present inventor used macrophage activating factor as a P agent.
gfactor (hereinafter abbreviated as MAF)],
It has been found that TNF can be efficiently produced by using a culture supernatant of Bordetella pertussis, killed cells, or a processed product thereof as agent E.

Components of the Invention The present invention provides a kit for inducing TNF, which includes MAF and a culture supernatant of Bordetella pertussis, killed cells, or a processed product thereof.

In the present invention, M AF is Fiddler (Fiddle).
r) et al.'s method [Cancer Research
Res,).

36, 3008 (1976) E (hereinafter referred to as MAF-1).
) and the Broken-Eiding of the National Academy of Sciences (Proc.

Natl, Acad, Sci,)ll5A 80.5
Examples include interferon-γ (hereinafter referred to as IFN-γ) produced by the method described in 842.1983. M.A.
For F-1, lung cells were prepared from a mouse that had been aged for about 3 weeks after intravenously injecting 4 x 10' BCG, and then transfected with concanavalin A agarose (Con^-Agarose; E4 LA).
After culturing for 2 days in RPM11640 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) containing (manufactured by BS, Inc.), centrifugation was performed twice for 10 minutes at 3,000 revolutions/min to collect the culture supernatant. The supernatant was concentrated approximately 10 times by ultrafiltration using a UK-10 filter (manufactured by Toyo Kagaku Sangyo Co., Ltd.) to a concentration of 0.45
Obtained by oversterilization using a mesh membrane filter (manufactured by Fuji Photo Film Co., Ltd.). In addition, the culture is 75
Using a CD tissue culture flask (Corning), add 1.5X lung cells to 20 ml of RPM11640 medium.
The test was carried out in a 5% CO2 incubator at 37° C. under conditions containing 10" concanavalin Δ and 2 to 4 mg of concanavalin Δ, and the mixture was stirred 4 to 5 times per day.

MAF-1 includes cytotoxic agents such as interferon and interleukin-2.

B. pertussis cultures were grown in Bordet Gengou medium (DI) supplemented with serum.
FCD) and cultured in charcoal-supplemented semi-synthetic medium [Cohen-Wheeler medium (Cohen-Wheeler).
Microbiology Handbook, Gihodo, 1952, 137
0 page) to which 1 g/l of activated carbon was added] and grown at 35°C.
, cultured for 2 days is used.

The supernatant was collected from a culture containing approximately 4 X 101a/ml between pertussis and approximately tripled! LiI bacteria, dead bacterial cells inactivated by heat treatment and formalin treatment, bacterial cell membrane components produced by known general methods such as the method of Tanabe et al. The purified product can be used as an E agent.

As the killed bacteria of Bordetella pertussis, inactivated Bordetella pertussis (hereinafter referred to as BPV) as shown in "Japanese Pharmacopoeia Explanation, Reduced Edition, Revised by Takushi, page E-187 (1981)" may be used.

The kit of the present invention contains the following composition for one dose of TNF induction for adults (average body weight: 60 kg).

MAF: 0.001-1mg IFN r: 0.001-1mg BPV: 4
X10'~4X1010 Bacillus pertussis culture 3x concentrate 20.5~5+++1 Bacillus pertussis membrane component: 0.001
When using the kit of the present invention for ~10 mg of humans, MAF or IFN-T as a P agent is injected (intravenously,
intramuscularly, intradermally, subcutaneously, intraserosally, at the tumor site) and culture supernatant of Bordetella pertussis as agent E within 6 hours.

Injection of killed bacterial bodies or their processed products (intravenous, intramuscular).

sarcastic, subcutaneous, intraserosal, tumor localized). With this prescription, TN can be delivered to the body in extremely large amounts in a shorter time than conventional methods.
F can be generated.

Animals, such as mice, rats, sheep, and cows.

It can also be prescribed to horses and other animals in the same way as Kamikita to generate TNF in the body.

Haranaka et al. [Int, J, Cancer 34.263 (
1984)) isolated TNF was MM46,! J
ethA, Ehrlich.

It has been reported that it exhibits antitumor effects against various tumors such as Sarcoma 180. It is expected that a significant amount of TNF produced in vivo by the use of the kit of the present invention will exert an antitumor effect.

The activity of TNF produced in a living body can be determined by the method of Ruff et al. [Infectious Immunology], which involves collecting blood from a living body and separating serum.

Immunol, ), 31.380 (1980)
).

106 units of TNF activity corresponds to 1 mg of protein.

Example 1 TNF production using MAF-1 as P agent and BPV as E agent: 4 C3H/Ha mice per group (12 weeks old, male).

Shizuoka Experimental Animals) was given a 1/4 diluted solution of MAF-1.
Inject 2ml intravenously, and 3 hours later, administer 4XIG as agent E.
', 4X10', 4XIO7, 4X10' BPV
was injected intravenously. After 2 hours, whole blood was collected from the mice and TNF activity was measured according to the method described above. The results are shown in Table 1.

Table 1 Also, as a P agent, the diluted solution of MAF-1 shown in Table 2 was used as Q,
TNF activity was measured according to the method described above using 4×10′ BPV as the 2ml SE agent.

The results are shown in Table 2.

Table 2 As a comparative example, 4 x 10' pieces of BPV were used as agent E.

Escherichia coli-derived LPS C Manufactured by Difco] 3
waste.

Oyohibishi Vanil (OK432. Manufactured by Chugai Pharmaceutical Co., Ltd.) 3
K E (Klinishe Einheit: I
The same procedure as above was carried out using KE (corresponding to 0.1 mg of dried bacterial cells).

Separately, 4 x 10' pieces of BCG (manufactured by Nippon BCG Manufacturing Co., Ltd.) were intravenously injected into mice as a P agent, and two weeks later, E
Escherichia coli-derived LPS3■ was intravenously injected as a drug, and 2 hours later, TNF activity in the serum was measured.

The results of both comparative examples are shown in Table 3.

Table 3 Example 2 T: r F N -r as P agent and BPV as E agent
Or TNF production when using BP@culture supernatant: except using 104 units of TFN-γ as the P agent and a diluted solution of BPV and BP culture soil as the E agent at the concentration shown in Table 4 at 0.2 ml per mouse. was carried out in the same manner as in Example 1. The results are shown in Table 4.

Table 4 Also, using IFN-γ at the concentration shown in Table 5 as the P agent and BPV4XIO'' as the E agent, T
NF activity was measured. The results are shown in Table 5.

Table 5 Example 3 Effect on MM46 intradermal transplantation tumor 106 MM46 tumor cells per group of 6 C3H/H
e Mouse (12 weeks old, male, Shizuoka experimental animal) abdominal skin (
intra dermal: id), and prescribed once each on days 3, 6, and 9. The prescription was to intravenously inject 2 ml of MAF-10, prepared in the same manner as in Example 1, as agent P, and 3 hours later, inject BPV2X10' as agent E.
This was done by intraperitoneally injecting the individual.

The size of the tumor mass [diameter -] increases over time. The antitumor effect was determined by measurement.

The results are shown in Figure 1. In the figure, * indicates the control, MU indicates the 3-day prescription, Δ indicates the 6-day prescription, and 0 indicates the 9-day prescription.

Further, a) indicates P<0.05, and b) indicates P<0.01.

Example 4 Effect on MM46 subcutaneously transplanted tumor MM46 tumor cells 4 x 10' cells per group of 6 C3H/H
It was subcutaneously transplanted into the inguinal region of a mouse (12 weeks old, male, Shizuoka experimental animal), and prescribed on the 9th day and 166th day after transplantation. The formulation was as follows: M prepared in the same manner as in Example 1 as a P agent.
The experiment was carried out by intravenously injecting 2 ml of AF-10, and 3 hours later, intravenously injecting 109 BPV4X as agent E.

The antitumor effect was determined in the same manner as in Example 3. The results are shown in Figure 2. In the figure, ・ is the control, MU is the 9th day, 0 is the 9th day and 1
The prescription for day 6 is shown. * indicates P<0.01.

As a comparative example, a conventional combination formulation with agents P and E and the formulation of the present invention were simultaneously carried out. That is, 2 ml of MAF-IQ prepared in the same manner as in Example 1 was injected intravenously as agent P, and 3 hours later, BPV4XlO' pieces and
In ○, 3KE of 432 and 3 μg of E. coli-derived LPS were intravenously injected. Separately, on the 3rd day of tumor transplantation, BCG was used as a P agent.
4x107 injections were administered intravenously, and another injection was given on the 14th day after transplantation. 1
On the day, 3 μg of Escherichia coli-derived LPS was intravenously injected as agent E to examine the therapeutic effect.

The therapeutic effect was determined by the tumor weight 30 days after tumor implantation.

The results are shown in Table 6.

Table 6 MAF-I BPV l 1.95±0.18 Significant differences between the present invention (MAF-1, BPV) and each group are a)
was P<0.001, b) was P<0.01.

Effects of the Invention Since the present invention can efficiently induce TNF in humans and animals, it can be used for tumor treatment and prevention. The TNF-inducing effect of the kit and method of the present invention is superior to that of the conventionally known combination of agent P and agent E, and is an unexpected effect.

[Brief explanation of drawings]

FIG. 1 shows the therapeutic effect of MM46 intradermal transplantation on tumors. FIG. 2 shows the therapeutic effect on VIM46 subcutaneously transplanted tumors.・-1/ Part L Figure Q 10 20 Tumors 11 Hidden Dredges (Figure 2 Summer Daily Comparison of 4 Tumors)

Claims (2)

[Claims] (1) Kit for inducing tumor necrosis factor containing macrophage activating factor and culture supernatant of Bordetella pertussis, killed cells or processed products thereof (2) Tumor necrosis in animals characterized by inducing tumor necrosis factor in the animal body by administering macrophage activating factor and B. pertussis culture supernatant, killed cells, or processed products thereof to the animal Factor induction method.