JPH04183398A - Ts-2 monoclonal antibody and its production - Google Patents

Abstract

PURPOSE:To develop a more improved pharmaceutical of OK-432 or a useful immunopotentiator by using a TS-2 monoclonal antibody reactive with a fraction having gamma-interferon induction ability of the OK-432. CONSTITUTION:A splenic cell extirpated from a mouse in which OK-432 is administered into the abdominal cavity and immunized is subjected to cell fusion to a myelomatous cell derived from a mouse and a hybridoma having an antibody productivity against the OK-432 is screened from the formed hybridoma. Cloning is then carried out to provide a hybridoma clone, which is subsequently cultured to purify the objective antibody from the culture solution supernatant or the hybridoma alone is administered into the abdominal cavity of a mouse to purify the objective antibody from the ascitic fluid thereof. Thereby, a TS-2 monoclonal antibody is produced. The resultant hybridoma capable of producing an antibody against the OK-432 is TS-2 (FERM-P 11846).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a TS-2 antibody, which is a novel monoclonal antibody against the streptococcal preparation Q K-432, and a method for producing the same.

(Conventional clamping technique) O K-432, a streptococcal preparation, has the ability to produce cytokines such as interferon (hereinafter abbreviated as IFN) and interleukin 2, and has the ability to produce natural killer (hereinafter referred to as N
It is known that it exerts an antitumor effect through the induction and activation of immunocompetent cells such as K cells, macrophages, and cytotoxic T cells.

On the other hand, it is also known to activate IFN, macrophages, or NK cells.

The present inventors previously succeeded in obtaining the TS-1 antibody, which is a monoclonal antibody against OK-432, and used this to conduct analytical research on the tissue migration of OK-432.
reported that it is expressed in macrophages and NKI cells induced and activated by K-432 (J, f3io
1. f? esponse Mod, Vol. 7. N
o2, p212-22B, (198B)).

(Problems to be Solved by the Invention) However, with the TS-1 antibody described above, it was not possible to determine which fraction of QK-432, which is a bacterial cell material, has IFNI conductivity.

The object of the present invention is to suppress IF\ production of OK-432,
An antibody that also suppresses NK and LAK activity,
O reacts with the fraction of K-432 that has IFN-inducing ability.
We have created a new monoclonal antibody against K-432, and using this antibody, we are developing a better formulation of OK-432 and searching for a new and useful immunostimulant.

[Means to solve the problem]

In order to solve the above problems, the present inventors conducted extensive research on monoclonal antibodies against OK-432, and as a result, succeeded in obtaining the TS-2 antibody, which is a novel monoclonal antibody against QK-432. The present invention was achieved by discovering that antibodies have various properties that solve the above problems.

That is, the present invention is a monoclonal antibody against the hemolytic streptococcal preparation OK-432, which exhibits the IgM class and subclass, recognizes the sugar chain antigen on the bacterial surface of OK-432, and is capable of inhibiting r-interferon of OK-432. TS-2 heptanoclonal antibody characterized by reacting with a fraction having inducibility, and OK-4 intraperitoneally to obtain the same.
A hybridoma clone obtained by cell fusion of flax cells excised from mice immunized with OK-432 and mouse-derived myeloma cells, screening for hybridomas capable of producing antibodies against OK-432 from the resulting hybridomas, and cloning. A method for producing an S-2 monoclonal antibody, which comprises culturing the antibody and purifying the antibody of interest from the culture supernatant, or administering the hybridoma clone intraperitoneally to a mouse, and purifying the antibody of interest from the ascites. It provides:

The present invention will be explained in detail below.

The TS-2 antibody of the present invention is a novel monoclonal antibody against the streptococcal preparation OK-432, and represents an IgM class and subclass. The present inventors confirmed that this antibody recognizes the sugar chain antigen on the surface of OK-432 cells using the method described later.

In addition, the TS-2 antibody almost completely suppressed γ-IFN production of OK-432, NK and LAK (-Lymphok
Ine activated killer) activity was also found to be suppressed.

Roughly speaking, Morrison's method described in the bond is OK-4.
The glycolipid sample (OK-PS) extracted from 32 was
production, NK and LAK activity induction ability,
It was found that the TS-2 antibody almost completely suppressed IFN production in OK-PS and also suppressed the induction of NK and LAK activities. On the other hand, the polysaccharide sample (Su-PS) contains IFN.
No production, NK or LAK activity induction ability was observed.

From these results, the TS-2 antibody of the present invention is OK-43
It was confirmed that IFN of No. 2 reacts with a fraction that has the ability to induce γ-IFN.

Next, the method for producing the TS-2 antibody of the present invention will be explained.

First, OK was administered intraperitoneally to a 6-week-old female Ba1b/c mouse.
-432 to 5 clinical units (Klinische
Einheit, hereinafter abbreviated as KE, KE is OK -
432 units, IKE contains 0.1 mg dry cocci (1
07-108>] and immunize.

One week later, OK-432 was injected intraperitoneally into this mouse.
Three days after the final immunization, the mouse is immunized with an additional dose of 5KE, and the glenoid is aseptically removed from the mouse to prepare the glenoid cells.

The obtained splenocytes and Ba1b/c mouse-derived myeloma cells, P3-NS-1-Ag4-1 (hereinafter abbreviated as N5-1) (K6hier G, Hilsteiri
C,,Eur,J,IllImuno l,vo
l 6. p511-519 (1976)) in polyethylene glycol 4000 according to a conventional method (the method of Sixhler and Milstein described above).

OK-4 in the hybridoma formed after cell fusion
Those having the ability to produce antibodies against 32 were screened using enzyme-linked immunosorbent assay (abbreviated as ELISA).

After screening, hybridomas producing anti-OK-432 monoclonal antibody were cloned two to three times by limiting dilution method.

In the present invention, ELISA and limiting dilution were carried out using conventional methods, such as those described in Iwasaki, Suzu, Monoclonal Antibody Hybridoma and ELISA, published by Kodansha (1983).

As a result, the obtained hybridoma producing antibodies against OK-432 was identified as D5-2 (Feikoken Bacterial Serial No. 1184).
No. 6).

The class and subclass of the antibody produced by this hybridoma are determined by using the secondary antibody as mouse immunoglobulin class and subclass-specific rabbit immunoglobulin (MoAb-3ub-Iso).
EL using typing kit (Bio-Rad)
Determined by ISA.

The anti-OK-432 monoclonal antibody was purified using 20 m) fTri.
5ephac using s-HCI buffer (pH 8,0)
rylS-300 (Phar-macia) Column O
? Do it with Togelahui.

Next, the measurement method used to confirm the function and properties of the TS-2 antibody of the present invention will be explained.

The tumor-bearing nude mice were 6 weeks old, male BaIb,
/C nude mouse dorsal subcutaneous t, -1x to7 H5
G cells [human salivary gland cancer cells: Shirasa, Sahara et al., Canc
er 48P745-752 (1981)), and 8-10. tumors developed. Tumor-bearing nude mice were used.

Indirect fluorescent antibody method: Frozen sections were prepared from tumors obtained by administering OK-432 < 2.5 KE) into tumor-bearing nude mice.
The reactivity of -432 and anti-OK-432 monoclonal antibody was investigated by indirect fluorescent antibody method. In this case, the monoclonal antibody used was biotinylated, and the secondary antibody was FIT.
CII-specific avidin was used.

To investigate the specificity of anti-OK-432 monoclonal antibody treated with iodic acid and pronase, frozen sections of tumors from nude mice treated with OK-432 were treated with periodic acid or pronase, and indirect fluorescent antibody method was performed. Periodic acid treatment is 50 m)! 10 mHTris-HCI buffer containing periodic acid (pH 7,4)
The reaction was carried out by reacting at 4°C for 2 hours at The mixture was reacted at 37°C for 1 hour.

Immunoelectron microscopy As a search for the reactivity of OK-432 and anti-QK-432't noclonol antibody, OK-432 and the otinized anti-OK
-432 antibody was reacted with t! A precipitate was formed.

After reacting this immunoprecipitate with Bell A oxidase-labeled avidin, dimethylaminoazobenzene < DAB.

The color was developed using Wako Tsumugi Co., Ltd.). After Epon embedding according to a conventional method, ultrathin sections were prepared and observed with a transmission electron microscope.

PBMC were prepared by specific gravity centrifugation using Ficol +-t+ypaque and diluted at a ratio of 1xlO-6/mj7.
The cells were cultured for 24 hours in RPMI 1640 medium containing 0% tallow serum.

OK-432 processing is OK -4320~0.1KE/'
This was done by adding m to the medium. Anti-OK-432 monoclonal antibody treatment was performed on OK-432 and MAblOμJ.
After reacting at 4°C for 11 hours, PBMC prepared at the above ratio was added and cultured at 37°C for 24 hours.

Thereafter, the IFN titer in the treated culture supernatant and the NK and LAK of the treated PBMC described in the next section were measured.

■ IFN titer measurement: IFN activity was measured using human amniotic membrane 11 (Fog).

Lund, Proc, Soc, Exp, Bi
ol, Hed,, 94. p532-537 (
1957): FL cells) and hydrolytic stomatitis virus (
A plaque reduction method using VSV) was performed.

The type classification of IFN is based on anti-human D-I FN antibody (LeeB
iomolecular Research Inc.
), anti-human β-IFN antibody (Lee Biomol
A neutralization test was conducted using anti-human r-IFN antibody (EN[)OGEN). In other words, the neutralizing activity is 500U/
Anti-human α-IFN antibody prepared in II and anti-human B-I
Mixed antibody of FN antibody or anti-human γ-IFN antibody 200
800 μm of culture supernatant, which is a serially diluted IFN standard
m and reacted at 4°C for 24 hours.

After this, the remaining IFNjJ value was measured.

Measuring method for NK and LAK activities: PBM treated with OK-432 and anti-OK-432MAM
To measure the NK and LAK activities of C, human erythroblastic white carcinoma cells (Bfood, 45,0
32 (1975): K-562 cells], LAK
Human Burkitt lymphoma-derived cells (Can
cerRes, 28. p1300-1310 (1
968): 51Cry using Daudi cells]
This was done using the t1 out method. That is, 106 target cells were io
oμC! N a2 Cr 5104 37°C190
After reacting for minutes and radiolabeling, the cells were implanted into a 96-well microtiter plate at a ratio of 104 cells/100 μm per hole. PBMC prepared in RPMI 1640 culture medium were used as effector cells, 2×10” cells/1
00 μm and cultured at 37°C for 4 hours.

The NK and LAK activities were expressed as % impairment ability and calculated using the following formula.

The extraction method for the glycolipid fraction of OK-432 is based on Horr i S.
On's method (THE JOURNAL OF BIOL
OGICAL CHEI41STRYVOL, 250.
NO8, P2911-2919 (1975) ) M
I went to semi-shite. That is, 5 ml of 1-butanol was mixed with OK-4325OKE dissolved in 5 ml of physiological saline. After this, it was centrifuged for 20 minutes at 35,0OOX (+), and the aqueous solution layer was collected.
It was added at 20 μg/ml and reacted at 37°C for 24 hours.

After centrifugation at 35,000XC+ for 20 minutes, the supernatant was collected. This extracted sample was added to a phosphate buffer solution (PBS (-)).
The sample was dialyzed to obtain a glycolipid sample (hereinafter referred to as OK-PS).

Polysaccharide fraction is 5treptococcus Pyoge
5lade method from nes^-3SU strain (JOURN
AL OF BACTERIOLOGY.

VOL, 90. NO3, P667 (1965))
A quasi-citrate-extracted Shita polysaccharide standard (hereinafter referred to as 5u-PS; manufactured by Chugai Pharmaceutical Co., Ltd.) was used.

〔Example) The present invention will be explained below with reference to Examples.

Example 1 <Production of hybridoma TS-2 and production of monoclonal antibody TS-2> OK-
432 (manufactured by Chugai Pharmaceutical) was administered for immunization.

One week later, the mouse was further immunized intraperitoneally with 5KE of OK-432, and 3 days after this final immunization, the mouse was aseptically inoculated ip? The viscera was removed and splenocytes were prepared. These flax cells and the N5 described in the description of the method for producing the TS-2 antibody mentioned above.
-1 cells by the method of Kohler and Mi 1stein (
Polyethylene glycol 4 according to the literature, supra)
,000 (manufactured by Wako Tsumugi Pharmaceutical), serum-free RPMI
Cell fusion was performed in 1640 medium.

As a result, 288 hybridoma supernatants were obtained.

As a result of performing an ELiSA search using this Hi1 ridoma culture medium, four Hi1 ridomas that produced antibodies against Q K-432 were obtained.

These four screened hybridomas were cloned two to three times by limiting dilution method, and Q
A hybridoma clone was obtained that produced antibodies against K-432 and exhibited stable growth.

The hybridoma was named TS-2, and the accession number was given to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the number was 11846.
No. (FERM P-11846').

Anti-mouse immunoglobulin class and subclass of the TS-2 antibody produced from this TS-2 are used as secondary antibodies.
υBrillas-specific rabbit immunoglobulin (MoAb-3u
When it was examined by ELISA using IT:Bio-Rad for b-Isotyping, it was found to be ICIM.

For purification of TS-2 antibody, hybridoma culture supernatant or hybridoma was intraperitoneally administered to Ba1b/c mice.
, 0) using 5ephacryl S-300 (5-
300 (performed with Phar column chromatography.

Example 2 (Properties of TS-2 antibody and antigen recognized by it) 2.5KE of OK-432! Frozen sections of tumors from tumor-bearing nude mice administered with Yamauchi were examined using the indirect fluorescent antibody method described above. As a result, OK-43 was determined by TS~2 antibody.
The presence of 2 was clearly observed.

The tumor-bearing nude mice used in the experiment were 6-week-old female B.
1 x 107 H5G cells were subcutaneously transplanted into the back of an a1b/c nude mouse, and approximately 1 month after transplantation, 8-10 m+ cells were transplanted.
The one that caused the development of tumors was used.

(2) Properties of antigen recognized by TS-2 antibody: O
After treating the tumor sections treated with K-432 with periodic acid and pronase, the reactivity with the TS-2 antibody was examined by indirect fluorescent antibody method. As a result, it was found that the OK-432 antigen recognized by the TS-2 antibody disappeared in the sections treated with periodic acid, but was not affected by the pronase treatment.

From this, it was confirmed that the antigen recognized by the TS-2 antibody was a sugar chain antigen. Furthermore, when the above-mentioned immunoelectron microscopy using the TS-2 antibody was performed, reactivity was observed on the surface of OK-432 cells, indicating that the TS-2 antibody recognized the surface antigen of OK-432. Ta.

The final concentration of O~0. IKE/1Ill O
K-432 was added to the medium and PBMC were cultured for 24 hours, and the IFN titer in the culture supernatant and the NK and LAK activities of the treated PBMC were measured. As shown in Figures 1 and 2, the IFN titer, NK and AK activities increased with the addition of OK-432 at 11 degrees, reaching a peak of 0.01KE/g.
It turned out to be tt.

Next, in order to determine the type of iFN induced by OK-432, the culture supernatant of OK-432-treated PBMC was treated with anti-α/β-IFN antibody and anti-γ-IFN antibody, and then When the IFN titer was measured using the method described above, anti-α/
Although β-IFN antibody treatment had almost no effect on IFN titer, anti-T-IFN antibody treatment significantly decreased it. These results are shown in Table 1. From these facts, I
FN was found to be primarily r-IFN.

Table 1 Hann-Whitney uTe5t:★:
P<0.01 then γ induced by OK-432
-The influence of TS-2 antibody on IFN, NK and LAK activities was investigated. That is, PBMCs were cultured for 24 hours in OK-432 treated with TS-2 antibody in advance, and the IFN titer in the culture supernatant and the NK and LAK activities of the treated PBMCs were measured by the method described above.

As a result, as shown in Table 2, γ-IFN induced by OK-432 was almost completely suppressed by treatment with the TS-2 antibody, and NK and LAK activities were also suppressed.

(Left below) Table 2 Effect of TS-2 antibody on IF-'N production, NK and LAK activities of OK132 Experiment 1. Experiment 2 used PBMC from different healthy individuals.

Example 4 (O of OK-PS extracted from OK-432
The reactivity of OK-PS extracted from K-432 according to the method of Morrison and 5u-ps extracted according to the method of SI ade with the TS-2 antibody was determined by ELISA.
(When I searched for it, reactivity was found for both.

Next, OK-PS and 5uPS IFN production, NK and LA
When we examined the ability to induce K activity, we found that Figs.
As shown in the figure, OK-PS is associated with IFN production, NK and LA.
5u-PS had the ability to induce K activity, but no induction ability was observed in 5u-PS.

Furthermore, we investigated the effects of TS-2 antibody on the ability of OK-PS to induce IFN production, NK and LAK activities, and found that
As shown in Table 3 &: IFN production is completely suppressed and NK
, LAK activity was also found to be suppressed.

By combining the above results, it was revealed that the monoclonal antibody TS-2 of the present invention reacts with the fraction of OK-432 that has r-IFN-inducing ability.

Table 3 OK-PS IFN cows, NK and 1. - Effect of Nann-Whitney U Te5t on AK activity: ★:
P<0.01. *: P<0.05.・：
P<0205 [Effect of the Invention] As explained above, the monoclonal antibody TS-2 antibody of the present invention is a novel monoclonal antibody against the streptococcal preparation OK-432, and is a monoclonal antibody of the IgM class,
This subclass recognizes the sugar chain antigen on the bacterial surface of OK-432. And the TS-2 antibody is OK
Since it reacts with the fraction of -432 that has T-IFN inducing ability, it can be used to detect TS-2 on OK-432.
By analyzing and purifying the antigen recognized by antibodies, it may be possible to obtain preparations that are more effective than those derived from OK-432, and new and more effective immunostimulants may also be developed. You can expect it.

[Brief explanation of drawings]

FIG. 1 is a graph showing the induction of IFN by OK-432, and FIG. 2 is a graph showing the induction of NK and LAK activities by OK-432. FIG. 3 is a graph showing IFN induction by OK-PS, and FIG. 4 is a graph showing NK induction by OK-PS. FIG. 5 is a graph showing the induction of LAK activity, and FIG.
FIG. 6 is a graph showing the induction of IFN by ps, and FIG. 6 is a graph showing the induction of NK and LAK activities by 5u-PS.

Claims (1)

[Scope of Claims] 1. A monoclonal antibody against streptococcal preparation OK-432, which indicates the IgM class and subclass,
-432 recognizes sugar chain antigen on the bacterial surface and OK-4
A TS-2 monoclonal antibody characterized by reacting with a fraction having the ability to induce γ-interferon of 32. 2. OK-432 was administered intraperitoneally. Splenocytes excised from immunized mice were fused with mouse-derived myeloma cells, and the resulting hybridomas were screened for hybridomas capable of producing antibodies against OK-432, followed by cloning. TS-2, which is characterized in that the hybridoma clone obtained by performing the above steps is cultured, and the target antibody is purified from the culture supernatant, or the hybridoma clone is intraperitoneally administered to a mouse, and the target antibody is purified from the ascites fluid. Method for producing monoclonal antibodies. 3. The method for producing a TS-2 monoclonal antibody according to claim 2, wherein the obtained hybridoma clone is TS-2 Kaikoken Bibori No. 11846. 4. The method for producing a TS-2 monoclonal antibody according to claim 2, wherein the mouse is a Balb/C mouse. 5. Mouse-derived myeloma cells contain p3-NS-1Ag4
3. The method for producing a TS-2 monoclonal antibody according to claim 2, wherein the TS-2 monoclonal antibody is -1. 6. Screening using enzyme-linked immunosorbent assay (ELISA method)
3. The method for producing a TS-2 monoclonal antibody according to claim 2, wherein the cloning is carried out by a limiting dilution method, and the purification is carried out by column chromatography.