JP3047926B2 - Antibody against mouse interleukin-6 receptor and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibody that specifically binds to mouse IL-6 receptor, a method for producing the same, and a use thereof.

[0002]

2. Description of the Related Art Interleukin-6 (BSF2 / below IL-)
6) is a protein that induces various important physiological activities by binding to the IL-6 receptor on target cells and is widely involved in cell proliferation and differentiation. Furthermore, it has been reported that abnormal production of IL-6 may be a causative factor of various autoimmune diseases (Kishimoto, Hirano, Ann. Rev.
Immunol., 6, p485, 1988). Antibodies to human IL-6 receptor that inhibit IL-6 action are expected as inhibitors of IL-6. In the process of developing an anti-human IL-6 receptor antibody, an anti-mouse IL-6 receptor antibody that inhibits IL-6 action is administered to a model mouse exhibiting autoimmune disease symptoms due to mouse IL-6 overproduction, It is essential to examine its effects.

[0003]

Accordingly, the present invention seeks to provide various types of antibodies against the mouse IL-6 receptor.

In order to achieve the above object, the present inventors have developed an anti-mouse IL-6 receptor antibody capable of specifically binding to mouse IL-6 receptor; a monoclonal antibody having the above-mentioned properties. Producing hybridomas;
Sensitizing a mammal with a mouse IL-6 receptor antigen, fusing immune cells from the mammal with a myeloma cell line,
And a method for producing a hybridoma, which comprises cloning a strain that recognizes mouse IL-6 receptor from the fusion strain; culturing the hybridoma, and collecting a monoclonal antibody that recognizes mouse IL-6 receptor from the culture. Producing an anti-mouse IL-6 receptor antibody; immunizing a mammal with a mouse IL-6 receptor antigen and collecting a polyclonal antibody recognizing the mouse IL-6 receptor from the animal. Provided are a method for producing an anti-mouse IL-6 receptor polyclonal antibody, and a method and a kit for measuring mouse IL-6 receptor comprising two kinds of monoclonal antibodies against mouse IL-6 receptor.

[0004]

DETAILED DESCRIPTION OF THE INVENTION

1. Antibody to mouse IL-6 receptor The antibody of the present invention specifically recognizes mouse IL-6 receptor, and includes a monoclonal antibody and a polyclonal antibody. The mouse IL-6 receptor recognized by the antibody of the present invention refers to either or both of a mouse soluble IL-6 receptor produced by genetic engineering and a natural mouse IL-6 receptor on a cell membrane. Monoclonal antibodies include those that competitively inhibit the binding of mouse IL-6 or human IL-6 to the mouse IL-6 receptor, and those that do not competitively inhibit this. As an example of the former, the hybridoma of the present invention
An example is an RS13 monoclonal antibody produced by RS13. An example of the latter is an RS15 monoclonal antibody produced by hybridoma RS15.

[0005] The immunogen used for the production of the antibody of the present invention may be a mouse engineered mouse soluble
Examples of the IL-6 receptor include natural mouse IL-6 receptor purified from mouse cells and mouse IL-6.
Cells expressing the receptor may be used. However, natural mouse IL-6 receptor and cells expressing the mouse IL-6 receptor are usually not efficient immunogens due to the low expression level of the mouse IL-6 receptor. However, once an antibody against the mouse IL-6 receptor is obtained, it can be used to efficiently use native mouse IL-6.
The -6 receptor can be prepared and used to produce more diverse antibodies.

[0006] Hybridomas can also be prepared in accordance with a conventional method (Immunological Experiment Procedures, p447, edited by the Japanese Society of Immunology, 1975). For example, a mammal such as a rat is immunized, spleen cells are obtained from the animal, and the spleen cells are fused with established myeloma cells. As myeloma cells,
Rat myeloma cell line YB2 / 0 (Dainippon Pharmaceutical) and the like can be exemplified. To produce a monoclonal antibody, the hybridoma cloned as described above is cultured, and the monoclonal antibody is collected from the culture supernatant. Alternatively, the hybridoma is transplanted into the abdomen of an animal,
It is also possible to obtain ascites from which monoclonal antibodies can be isolated.

The antibody in the hybridoma cell supernatant or the antibody in the ascites can be concentrated by a conventional method, for example, by ammonium sulfate salting out, and further subjected to affinity chromatography, for example, a carrier having soluble mouse IL-6 receptor immobilized thereon. Can be purified by affinity chromatography using Polyclonal antibodies can also be produced in a conventional manner, for example, by immunizing rats, rabbits, guinea pigs, goats and the like with the mouse soluble IL-6 receptor as an immunogen.

2. Chemical assay of mouse IL-6 receptor The chemical assay of mouse IL-6 receptor refers to the physiological concentration of mouse IL-6 receptor contained in mouse serum, urine, etc., and culture of mouse cells. Mouse IL in culture supernatant
It is intended to accurately and rapidly measure -6 receptor concentration. In the measurement method of the present invention, the mouse IL-6 receptor in the analysis sample is bound to an antibody against the mouse IL-6 receptor bound to the solid support, and the bound mouse IL-6 receptor is measured by a conventional method.

As the solid support, a commonly used support such as a microtiter plate, various types of beads, for example, plastics such as polystyrene and polypropylene, and inorganic materials such as metal ceramics can be used. Immobilization of the antibody is also performed according to a conventional method. For example, the antibody may be dissolved in a PBS solution at a concentration of, for example, 2 μg / ml, added to the wells of the plate at 100 μl each, and allowed to stand overnight.

The mouse IL-6 receptor bound to the solid support via an antibody against the mouse IL-6 receptor comprises:
It can be measured by an ordinary method. For example, an antibody against mouse IL-6 receptor different from an antibody against mouse IL-6 receptor bound to a solid support,
A rabbit-derived polyclonal antibody against the IL-6 receptor is bound thereto, and those labeled with an antibody that recognizes these antibodies are used. Examples of the labeling substance include enzymes such as alkaline phosphatase and horseradish peroxidase, and biotin.

[0011]

EXAMPLES The present invention will now be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Embodiment 1 FIG. Preparation of Rat Monoclonal Antibody Against Mouse IL-6 Receptor To prepare a rat monoclonal antibody against mouse IL-6 receptor, Japanese Patent Application No. 2-2158 was used as an immunogen.
Recombinant soluble mouse described in 86 specification
The IL-6 receptor was used. Immunization was performed as follows. 100 μg of soluble mouse IL-6 receptor per Wistar rat per week was dissolved in PBS.
Immunization subcutaneously three times in total.

Splenocytes from the immunized rats were fused with the parental rat myeloma cell line YB2 / 0 according to a conventional method using polyethylene glycol. Screening was performed as follows. 5 μg /
100 μl of a PBS solution containing ml of mouse IL-6 receptor was added to a 96-well microtiter plate per well, and the mixture was left overnight at 4 ° C. After washing, a 1% BSA-PBS solution was added, and the mixture was left at room temperature for 2 hours.

After washing, the culture supernatant of hybridoma (100 μl
l) was added and left at room temperature for 2 hours. For control, use myeloma cell line instead of hybridoma culture supernatant
YB2 / 0 normal culture supernatant was added. After washing, 100 μl of an anti-rat immunoglobulin antibody (2 μg / ml) bound to alkaline phosphatase as a labeling enzyme was added, and the mixture was allowed to stand at room temperature for 2 hours. After washing, 100 μl of alkaline phosphatase substrate (1 mg / ml) was added, and the mixture was allowed to stand at 37 ° C. for about 30 minutes.
It was measured with an immunoreader. FIG. 1 shows that the color development was significantly increased when the culture supernatant of RS13 and RS15 was added. This indicates that RS13 and RS15 are producing anti-mouse IL-6 receptor antibody.

Embodiment 2 FIG. Preparation of Guinea Pig Polyclonal Antibody Against Mouse IL-6 Receptor Soluble mouse IL-6 receptor dissolved in PBS was subcutaneously immunized 100 times per week per guinea pig, three times a week. One week later, whole blood was collected, and the antibody was purified by an ordinary method.

Embodiment 3 FIG. Measurement of IL-6 neutralizing activity of anti-mouse IL-6 receptor monoclonal antibody and polyclonal antibody of anti-mouse IL-6 receptor M1 cells were 1 × 10 ⁵ cells / ml, 0.2 ml / well at 40 ng / well.
The cells were cultured under conditions to which ml of human IL-6 and various concentrations of hybridoma culture supernatant or anti-mouse IL-6 receptor polyclonal antibody were added. After 60 hours, 0.75 μCi / well of tritiated thymidine was added,
After 6 hours, the cells were collected and the incorporated radioactivity was measured.

The data in FIG. 2 show that the RS15 antibody does not inhibit the action of IL-6, whereas the RS13 antibody and anti-mouse IL-6
This shows that the receptor polyclonal antibody inhibits the action of IL-6. This is because the RS13 antibody bound to mouse IL-6 receptor on M1 cells and inhibited the binding between human IL-6 and mouse IL-6 receptor. Accordingly, mouse IL- in which the RS13 antibody was genetically engineered
Natural type mouse on mouse cells as well as 6 receptors
It was shown to also bind to the IL-6 receptor.

Embodiment 4 FIG. Mouse IL-6 Receptor Chemical Assay 100 μl per well of PBS containing 2 μg / ml of RS13 antibody was added to a 96-well microtiter plate for 4 nights.
Left at ℃. After washing with PBS / 0.05% Tween20, 1
00 μl of 1% BSA was added and left at room temperature for 2 hours. P
After washing with BS / 0.05% Tween20, soluble mouse IL-6 receptor of a known concentration (Japanese Patent Application No. 2-215886) was added at 100 μl per well, and left at room temperature for 2 hours.

After washing with PBS / 0.05% Tween20, 2 μm
100 μl of PBS containing g / ml biotinylated RS15 antibody was added per well, and the mixture was left at room temperature for 2 hours. PBS / 0.05
After washing with 20% Tween 20, PBS / 0.05% Tween 20 containing avidin peroxidase diluted 2500 times was added and left at 37 ° C. for 1 hour. After washing with PBS / 0.05% Tween20,
Add a peroxidase substrate and leave at 37 ° C for about 30 minutes.
The color development at 15 nm was measured.

FIG. 3 shows that the color developed intensely with an increase in the mouse IL-6 receptor concentration in the sample, that is, the mouse IL-6 receptor concentration in the sample can be measured by this method.

[0020]

According to the method of the present invention, polyclonal antibodies and monoclonal antibodies specifically recognizing mouse IL-6 receptor and the method for chemically measuring mouse IL-6 receptor can be used to obtain extremely small amounts in the natural state. This makes it possible to analyze various properties of the mouse IL-6 receptor produced only in E. coli. This is of great significance in the study of solid generation and immune mechanisms, and in the development of therapeutic drug diagnostics and the like based on those results. Furthermore, an antibody that inhibits the biological action of IL-6 provided by the present invention may comprise IL-6
It is useful for developing therapeutic agents for various autoimmune diseases in which abnormal production of -6 is considered to be a causal factor.

Furthermore, the antibodies of the present invention are also useful for immunoassay of mouse IL-6 receptor.

[Brief description of the drawings]

FIG. 1 shows RS1 measured by the method shown in Example 1.
3, anti-mouse IL-6 in culture supernatant of RS15, YB2 / 0
Shows the relative amount of receptor antibody.

FIG. 2 shows RS13, measured by the method shown in Example 3.
Of anti-mouse IL-6 receptor antibody and anti-mouse IL-6 receptor polyclonal antibody in the culture supernatant of RS15,
Fig. 3 shows the inhibitory effect of IL-6 on induction of differentiation into M1 cells.

FIG. 3 shows color development of a plate when measured by the method described in Example 4.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // A61K 39/395 ABA C12N 5/00 B ABC 15/00 C (C12P 21/08 C12R 1:91) (72) Inventor Hiroshi Suzuki 967-1 Kashigaya, Ebina-shi, Kanagawa Prefecture (72) Inventor Kiyoshi Hokawa 7-37-17, Sagami-Ono, Sagamihara-shi, Kanagawa Prefecture (56) References JP-A-2-288898 (JP, A) JP-A-61 -275654 (JP, A) European Journal of Immunology, Vol. 19, (1989), p. 2107-2114 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 21/00-21/08 C12N 5/00-5/28 C12N 15/00-15/90 G01N 33/53 G01N 33 / 577 A61K 39/395 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (11)

(57) [Claims] 1. A rat anti-mouse IL-6 receptor monoclonal capable of specifically binding to a mouse IL-6 receptor.
Le antibody. 2. The method according to claim 1, which competes with mouse IL-6 or human IL-6 for binding to mouse IL-6 receptor.
The antibody according to 1. 3. The antibody according to claim 2 , which is RS13. 4. The antibody of claim 1 , wherein the antibody does not compete with mouse IL-6 or human IL-6 for binding to mouse IL-6 receptor. 5. The antibody according to claim 4 , which is RS15. 6. A hybridoma of a rat spleen cell and a rat myeloma cell that produces the monoclonal antibody according to claim 1 . 7. Lack by mouse IL-6 receptor antigen.
The door sensitized spleen cells of the rat are fused with myeloma cell lines of the rat, and mouse IL-6 from the fusion strain
The method for producing a hybridoma according to claim 6, wherein a strain that recognizes the receptor is cloned. 8. culturing the hybridoma of claim 6, preparation of rat anti-mouse IL-6 receptor monoclonal antibody and recovering a monoclonal antibody that recognizes the murine IL-6 receptor from the culture Method. 9. A method comprising contacting a solid support on which a rat anti- mouse IL-6 receptor monoclonal antibody is immobilized with an analysis sample to thereby convert the mouse IL-6 receptor in the analysis sample to the rat anti- mouse IL-6 receptor monoclonal antibody. A mouse anti- mouse IL-6 receptor monoclonal antibody different from a rat anti- mouse IL-6 receptor monoclonal antibody immobilized on the solid support. A method for detecting or measuring mouse IL-6 receptor, which is measured using a polyclonal antibody. 10. A kit for performing the method of claim 9 , wherein the kit comprises a rat anti- mouse IL-6 receptor model.
A kit comprising a noclonal antibody. 11. The rat anti- mouse IL-6 receptor
The monoclonal antibody is a rat anti-mouse IL-6 receptor
And characterized in that it is a chromatography monoclonal antibody RS13 or RS15
11. The kit according to claim 10, wherein