JP3230531B2 - IFN-γ-inducing active substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel substance having an interferon-γ-inducing activity obtained from a hemolytic streptococcal preparation (hereinafter referred to as a streptococcal preparation) OK-432 and a method for producing the same.

[0002]

2. Description of the Related Art It is known that OK-432 which is a streptococcal preparation has an ability to produce cytokines such as interferon (hereinafter abbreviated as IFN) and interleukin 2, and has an antitumor effect. The present inventors have previously described TS- which is a monoclonal antibody against OK-432.
1 antibody was successfully obtained, and using this, an analysis study of the tissue translocation of OK-432 was performed, and it was reported that IFN was induced and expressed by OK-432 in activated macrophages and NK cells (J .Biol.Response Mod., Vol.7, No
2.p212-228. (1988)).

[0003] However, the TS-1 antibody does not contain IFN.
It was not possible to determine in which fraction of OK-432 the inducibility was present. Therefore,
The present inventors have further studied a monoclonal antibody against OK-432, and as a result, succeeded in obtaining a novel monoclonal antibody against OK-432, the TS-2 antibody. An antibody, which indicates an IgM class or subclass;
OK-432 recognizes sugar chain antigens on the surface of bacterial cells, and
-432 was confirmed to be an antibody that reacts with the fraction capable of inducing IFN-γ, and a patent application was previously filed (Japanese Patent Application No.
No. 311441).

[0004]

SUMMARY OF THE INVENTION The present invention relates to the above TS
-2 Streptococcal preparation OK-4 using monoclonal antibody
An object of the present invention is to obtain a novel substance having IFN-γ-inducing activity from No. 32 and clarify its properties to provide a novel substance that can be expected as an antitumor agent.

[0005]

Means for Solving the Problems The present inventors have prepared affinity chromatography using the TS-2 monoclonal antibody obtained in the previous invention as a ligand, and using this, from OK-432 to Morrison's. Purifying an antigen recognized by the TS-2 from a glycolipid sample extracted with butanol by the method,
Furthermore, fractionation was carried out on a Sephacryl S-300 column, and the present invention was achieved by examining the glycolipid concentration, IFN titer, NK activity, and LAK activity of each fraction.

That is, the present invention relates to a streptococcus preparation OK-4.
OK-432 50KE dissolved in saline
Add 1-butanol and mix, collect the aqueous layer,
Add pronase to the aqueous layer solution, centrifuge and centrifuge.
Can be obtained from the collected glycolipid preparation, and have the following properties
An IFN-γ-inducing active substance having: (1) has a molecular weight of 70 kilodaltons; (2) binds to a monoclonal antibody produced by a hybridoma having accession number FERM P-11846;
IFN-γ induction ability is 56 ° C., heat treatment for 30 minutes, Pronase 0.2 mg / ml, 37 ° C., 1 hour treatment, Neuraminidase 0.5 IU / ml,
Not deactivated by treatment at 37 ° C. for 2 hours and treatment with 0.15 M NaCl-ammonia (PH11), and treatment with periodic acid 50 mM, treatment at 4 ° C. for 2 hours, and treatment with 0.2 M glycine-HCl (PH2.5) Deactivate with . 1-butanol was added to OK-432 50KE obtained by dissolving a streptococcal preparation OK-432 in physiological saline, and the mixture was mixed. The aqueous layer was collected, pronase was added to the aqueous layer solution, and the mixture was centrifuged. From the glycolipid preparation from which the supernatant was collected, the antigen substance was separated and purified by affinity chromatography using a monoclonal antibody produced by a hybridoma of Accession No. FERM P-11846 against OK-432 as a ligand. Then, the obtained purified sample was subjected to Sephacryl S-3.
The above-mentioned IFN is obtained by fractionating with a C.00 column to obtain a substance having a molecular weight equivalent to 70 kDa.
A method for producing a γ-inducing active substance.

Hereinafter, the present invention will be described in detail. I of the present invention
To obtain an FN-γ inducing active substance, first, OK-432
It is necessary to extract a glycolipid preparation (hereinafter referred to as OK-PS) having the ability to induce IFN production, NK and LAK activity confirmed in an earlier application (Japanese Patent Application No. 2-311441). . This extraction was performed as described in detail in the Examples.
rrison's method (THE JOURNAL OF BIOLOGICAL CHEMISTRY.
VOL.250.No8.P2911-2919 (1975)].

Next, affinity chromatography using a TS-2 monoclonal antibody as a ligand is used in order to further purify the OK-PS and bring it closer to the target substance of the present invention. As the TS-2 monoclonal antibody for producing this affinity chromatography, the TS-2 monoclonal antibody which was invented by the inventor first and which has been applied for a patent as Japanese Patent Application No. 2-311441 is used. That is, the monoclonal antibody TS-2 is a monoclonal antibody against the streptococcal preparation OK-432, shows the class and subclass of IgM, recognizes the sugar chain antigen on the surface of the bacterial body of OK-432, and recognizes the OK-432 It is characterized by reacting with a fraction capable of inducing γ-interferon.

The obtaining method is described in detail in the specification of the above-mentioned application, and it may be used. However, once the outline is briefly described, it was extracted from a mouse immunized intraperitoneally with OK-432. A spleen cell and a mouse-derived myeloma cell were subjected to cell fusion, and a hybridoma formed and OK-432 was obtained.
After screening a hybridoma having an antibody-producing ability against, a hybridoma clone obtained by cloning is cultured, and the target antibody is purified from the culture supernatant or the hybridoma clone is administered intraperitoneally to a mouse. And a method for purifying the target antibody.
The above hybridoma clone has been deposited with the Japan Institute of Microorganisms as Microorganisms Kenkyu No. 11846 (FERM P-11846).

Using affinity chromatography using TS-2 thus obtained as a ligand, O
When an antigenic substance recognized by TS-2 is separated and purified from K-PS, a purified sample (hereinafter referred to as OK-PAS) is obtained.

Then, the OK-PAS was converted to Sephacryl S-
Fractionation is performed using a 300 column, and the glycolipid concentration, IFN titer, NK and LAK activity are measured for each fraction. As a result, as shown in FIG. 1, it was confirmed that the fraction containing the target IFN-γ-inducing active substance was 8 fractions. Therefore, the target substance of the present invention can be obtained from the eight fractions.

The IFN-γ-inducing substance of the present invention contained in this fraction 8 has an IFN-γ-inducing activity as shown in FIG. 1, and has an antitumor effect in vivo as clearly shown in FIG. Substance. The molecular weight of the substance of the present invention can be determined by gel filtration [Immunological Experiment Procedure 10 (edited by the Japanese Society for Immunology) 3119-3142].
1981] was found to be 70 kilodaltons (kd). Further, when the physicochemical properties of the present invention were examined, the results shown in Table 1 were obtained.

[0013]

[Table 1]

[0014]

The present invention will be described below with reference to examples. In addition, the measuring method used in the Example was performed by the following method.

(1) Determination of glycolipid [0015] Uronic acid was determined by a carbazole sulfate reaction according to the method of Bitter et al. [Anal. Biochem. 4. P330 (1962)].

(2) IFN titer IFN activity was measured using human amniotic membrane-derived cells [Fogh, Lund, Pr.
oc. Soc. Exp. Biol. Med., 94. p532-537 (1957); FL
Cells] and vesicular stomatitis virus (VSV). IFN typing is anti-human IFN
-Α antibody (LeeBiomolecular Research Inc.), anti-human IFN-β antibody (Lce Biomolecular Research Inc.),
A neutralization test was performed using an anti-human IFN-γ antibody (ENDOGEN). That is, a mixed antibody of anti-human IFN-α antibody and anti-human IFN-β antibody adjusted to 500 U / ml with neutralizing activity or 200 μl of anti-human IFN-γ antibody was serially diluted to 800 μl of culture supernatant, which is an IFN sample. In addition,
The reaction was performed at 4 ° C. for 24 hours. Thereafter, the remaining IFN titer was measured.

(3) NK, LAK activity NK, LAK activity is measured by using human erythroblastic leukemia cells [Blood, 45, p32 (1975);
K-562 cells] and human Burkitt's lymphoma-derived cells [Cancer Res. 28. p1300-1310 (1
968); Daudi cells] was performed with ⁵¹ Cr transmigration method using. That is, 10 ⁶ target cells were transferred to 100 μCiNa ₂ Cr ₅₁ O.
After radiolabeling by reacting at 37 ° C. for 90 minutes at ₄
10 ⁴ pieces / hole in a 6-well microtiter plate
Implanted at a rate of 100 μl. Human peripheral blood mononuclear cells (PBMC) prepared with RPMI 1640 culture solution were added as effector cells at a ratio of 2 × 10 ⁵ cells / 100 μl and cultured at 37 ° C. for 4 hours.

The NK and LAK activities are expressed as% impairment, and were calculated from the following formula. % Disability = (count of each sample−count of background / total count−count of background) ×
100

Example 1 (Extraction of OK-432 Glycolipid Fraction (OK-PS)) The extraction method of OK-432 glycolipid fraction was the method of Morrison [THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 250, No.
8, P2911-2919 (1975)]. That is, 1 to 50 ml of OK-432 dissolved in 5 ml of physiological saline
-5 ml of butanol was added and mixed. After this, 35:00
After centrifugation at 0 × g for 20 minutes, the aqueous layer was collected. To this extraction solution was added pronase at 20 μg / ml.
The reaction was performed for 4 hours. After centrifugation at 35,000 × g for 20 minutes, the supernatant was collected. This extracted sample was added to a phosphate buffer solution [P
BS (-)] to give a glycolipid preparation (OK-PS).

Example 2 (Monoclonal antibody TS-2)
Preparation of female Balb / C mouse (6 weeks old) intraperitoneally with OK-43
2 (manufactured by Chugai Pharmaceutical) and 5 clinical units (KE) were administered for immunization. One week later, OK-43 was further injected intraperitoneally.
Two 5KEs were additionally immunized, and three days after the final immunization, spleen cells were aseptically removed from the mice to prepare splenocytes. The spleen cells and NS- which are Balb / c-derived myeloma cells
One cell was prepared according to the method of Kohler and Milstein [Eur. J. Immunol 6.
P511-519 (1976)], polyethylene glycol 4,
000 (manufactured by Wako Pure Chemical Industries), and cell fusion was performed in serum-free RPM11640 medium. As a result, 288 hybridoma supernatants were obtained. As a result of performing an ELISA search using the hybridoma culture supernatant, four hybridomas producing an antibody against OK-432 were obtained.

Cloning of the four screened hybridomas by the limiting dilution method was performed for 2-3 times.
The hybridoma clone was produced twice, producing an antibody against OK-432 and showing stable growth. The hybridoma was designated as TS-2, and was accorded an accession number to the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology.
ERM P-11846). The TS-2 hybridoma was cultured to produce the monoclonal antibody TS-2. Purification of the produced TS-2 antibody was performed using the hybridoma culture supernatant or the hybridoma in Bal.
b / c mice received 20 mM T from ascites administered intraperitoneally.
Sephacry using ris-HCl buffer (pH 8.0)
l S-300 (Pharmacia) column chromatography.

Example 3 (Separation / Purification of Antigen Substance Recognized by TS-2) The TS-2 monoclonal antibody prepared in Example 2 was used in 0.1%.
Dilute with 1M NAHCO ₃ buffer. The TS-2 solution and CNBr-activated Sepharose 4B (Pharmacia
) Was reacted at room temperature for 2 hours with stirring so that TS-2 was adjusted to 1 mg / ml. Gel was packed in a column to prepare an affinity column using a TS-2 monoclonal antibody as a ligand.

5 ml of the OK-PS solution (500 Mg / ml) obtained in Example 1 was added to the affinity column.
After reacting at room temperature for 2 hours, the eluate (0.15 M Na
Cl-ammonia PH11) was added thereto to elute the antigen bound to the TS-2 antibody to obtain a separated and purified sample of the antigenic substance (hereinafter referred to as OK-PSA). The extracted O
K-PSA was immediately dialyzed against PBS (-) to return to stable conditions.

Example 4 (Acquisition of target substance to be fractionated into OK-PSA) The OK-PSA obtained in Example 3 was separated into 25 fractions (3 ml per fraction) by Sephacryl S-300 column chromatography (Pharmacia). Fractionated. That is, the purified sample was applied to a Sephacryl S-300 column (16 mm × 360 mm / Pharmacia) in PBS (pH 7.2).
Elution was performed, and fractionation was performed at 3 ml / fraction to obtain 25 fractions.
For these fractions, the respective glycolipid quantification, IFN titer,
NK activity and LAK activity were measured. The results are shown in FIG. As apparent from FIG. 1, the peak of the glycolipid coincides with the peaks of the IFN titer and the NK and LAK activities in fraction 8, indicating that the IFN-γ-inducing substance is present in this fraction 8. confirmed.

Therefore, the target IFN-γ-inducing substance was fractionated from this fraction 8 according to a conventional method. The molecular weight of this substance was determined according to the gel mouth method (cited in the literature), and 100 kDa of Lentinan (Lentinan, Ajinomoto) and Schizophyllan (SPG, Kaken Pharmaceutical) 4 as molecular weight markers.
When measured using 5 kilodaltons, a special result of 70 kilodaltons was obtained. Further, when its physicochemical properties were examined, the results shown in Table 1 above were obtained.

Example 5 (Anti-Tumor Effect) Six week-old, female Balb / c nude mice, 2 × 1 intraperitoneally
After transplanting 0 ^seven human salivary gland cancer cell (HSG cells), it was divided the mouse into five increments following two groups. (1) Group administered with the substance of the present invention: 100 μg of the IFN-γ-inducing active substance of the present invention was intraperitoneally administered for 5 days every other day from the day after transplantation. (2) Control group: 0.2 ml of physiological saline was intraperitoneally administered every other day for 5 days from the day after transplantation.

FIG. 2 shows the relationship between the survival rate and the number of days elapsed from the date of HSG cell transplantation as a result of examining the survival days of the mice in the above groups (1) and (2). FIG. 2 shows that the survival time was prolonged in the group to which the substance of the present invention was administered, indicating that the substance of the present invention has an antitumor effect.

[0028]

EFFECT OF THE INVENTION The IFN-γ-inducing substance of the present invention can be obtained from the monoclonal antibody TS-2 from the streptococcal preparation OK-432.
A novel substance separated and purified by using IFN
-Has γ-inducing activity, and has the effect of exhibiting an antitumor effect in vivo.

[Brief description of the drawings]

FIG. 1 is a view showing glycolipid concentration, IFN titer, NK activity and LAK activity of each fraction obtained by fractionating OK-PSA with a Sephacryl S-300 column.

FIG. 2 is a graph showing the relationship between the survival rate and the number of elapsed days in each group of the substance of the present invention and a control example.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁷ Identification code FI C12P 21/08 C12R 1:91) (C12P 21/08 C12N 15/00 C C12R 1:91) 5/00 B

Claims (2)

(57) [Claims] 1. A streptococcal preparation OK-432 is added to physiological saline.
Add 1-butanol to the dissolved OK-432 50KE
Mix, collect the aqueous layer, and add it to the aqueous layer solution.
After adding pronase and centrifuging, the supernatant was collected and the glycolipid label was collected.
IFN-γ having the following properties:
Inducing actives. (1) has a molecular weight of 70 kilodaltons; (2) binds to a monoclonal antibody produced by a hybridoma having accession number FERM P-11846;
IFN-γ induction ability is 56 ° C., heat treatment for 30 minutes, Pronase 0.2 mg / ml, 37 ° C., 1 hour treatment, Neuraminidase 0.5 IU / ml,
Not deactivated by treatment at 37 ° C. for 2 hours and treatment with 0.15 M NaCl-ammonia (PH11), and treatment with periodic acid 50 mM, treatment at 4 ° C. for 2 hours, and treatment with 0.2 M glycine-HCl (PH2.5) Deactivate with . 2. 1-butanol is added to OK-432 50KE obtained by dissolving the streptococcal preparation OK-432 in physiological saline, and the mixture is mixed. The aqueous layer is collected, pronase is added to the aqueous layer solution, and the mixture is centrifuged. The accession number FERMP P for the OK-432 was obtained from the glycolipid preparation from which the Qing was collected.
The antigenic substance was separated and purified by affinity chromatography using a monoclonal antibody produced by the hybridoma of No. 11846 as a ligand, and the obtained purified sample was fractionated by a Sephacryl S-300 column. 2. The IFN-γ according to claim 1, wherein a substance having a molecular weight equivalent to 70 kilodaltons is obtained.
A method for producing an inducing active substance.