JPH0646877A - Monoclonal antibody that recognizes osteoclasts - Google Patents

Abstract

(57) [Summary] (Modified) [Structure] Monoclonal antibody to mouse osteoclasts and the fusion antibody-producing rat splenocytes immunized with mouse osteoclasts and mouse myeloma cells Hybridoma. The antibody has the following properties and is a monoclonal antibody belonging to the IgM class. (1) Reacts with tartrate-resistant acid phosphatase activity-positive bone tissue-derived cells and does not substantially react with tartrate-resistant acid phosphatase activity-negative bone tissue-derived cells; (2) mouse splenocytes, thymocytes, bone marrow cells Substantially does not react with. [Effect] Osteoclasts and osteoclast precursor cells can be specifically identified, fractionated or quantified from a mouse osteocyte population.
It can be used for the evaluation of new drugs developed for the purpose of suppressing various bone resorptions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody against mouse osteoclasts and a hybridoma producing the same. More specifically, the present invention relates to a monoclonal antibody produced by a hybridoma formed by the fusion of mouse splenocytes immunized with mouse osteoclasts and mouse myeloma cells and belonging to the IgM class having the following properties: ( 1) Reacts with multinucleate and mononuclear bone tissue-derived cells positive for tartrate-resistant acid phosphatase activity; (2) Substantially does not react with cells derived from bone tissue with negative tartrate-resistant acid phosphatase activity; (3) Spleen of mouse Substantially no reaction with cells, thymocytes or bone marrow cells.

[0002] In recent years, active attempts have been made to utilize monoclonal antibodies having high specificity as research, diagnostic and therapeutic means. The inventors of the present invention also completed the present invention as a result of earnest research for establishment of a monoclonal antibody having high specificity for mouse osteoclasts and establishment of a hybridoma producing this monoclonal antibody.

The anti-mouse osteoclast-rat monoclonal antibody provided by the present invention specifically identifies osteoclasts and osteoclast precursor cells from an osteoclast-containing mouse osteocyte population, and FACS (fluoresense). activated cell
These cells can be sorted using a sorter) or the like. The uniform osteoclasts and osteoclast precursor cells thus obtained provide an excellent means in a screening system for new drugs developed for the purpose of suppressing various bone resorptions. In addition, by directly or intravenously administering the monoclonal antibody of the present invention to mice, various bone metabolisms, that is, bone resorption-formation pathological animals with altered formation balance, and screening for new drugs effective for bone metabolic diseases Can be used for Furthermore, the monoclonal antibody provided by the present invention can also be used for quantification of osteoclasts and osteoclast precursor cells in an osteocyte population collected from a bone disease model mouse, and various bone metabolic diseases A more accurate determination of the effect of the drug on

[0004]

2. Description of the Related Art Bone is a dynamic tissue that has various physiological roles. In bone tissue, bone resorption and bone formation are performed while maintaining a certain balance in order to perform its physiological role, which is called bone remodeling. Osteoclast precursor cells are mononuclear cells that fuse to become multinucleated osteoclasts. Osteoclasts are multinucleated cells that are particularly responsible for bone resorption. As one means for studying the role and function of osteoclasts in detail, monoclonal antibodies against osteoclasts, markers on the surface, and functional molecules have been prepared and used for analysis.

Horton et al. [J. Cell Biol, vo
l.109, p.1817-1826 (1989); Exp.Cell Res., vol.19
5, p.368-375 (1991)], a giant cell tumor of human bone (g
Using iantcell tumor (also called osteoclastoma) as an immunogen, mouse monoclonal antibodies 23c6 and 13c2 against human osteoclasts have been obtained. These monoclonal antibodies recognize the vitronectin receptor αvβ3 expressed on osteoclasts and react with renal cells other than osteoclasts. It also cross-reacts with human, chicken, and rabbit osteoclasts among animal species. Furthermore, functionally, these monoclonal antibodies are known to inhibit the bone resorption effect of human and chicken osteoclasts. Similarly, as a monoclonal antibody against human osteoclasts, Chamber et al. [Calcif Tissue Int., V
ol.49, p.317-320 (1991)] is a human osteoclast tumor (ost.
Eoclastoma) as an immunogen, mouse monoclonal antibodies 211D and 312G have been obtained. Similar to 23c6 and 13c2, these monoclonal antibodies react with cells of the kidney except osteoclasts. However, since it does not show cross-reactivity with rabbit and rat osteoclasts, it is possible that it recognizes an epitope different from 23c6 and 13c2.

[0006] As a monoclonal antibody against osteoclasts of other animal species, Osdoby et al. [J. Cell Biol.
vol.100, p.1592-1600 (1985); J. Bone Mineral Re
s., vol.6, p.1353-1365 (1991)], using mouse osteoclasts as an immunogen, mouse monoclonal antibodies 121F, 35.
I got L and 75B. Of these, the 121F antibody recognizes a molecule of 91 to 96 Kd on the surface of chicken osteoclasts. Similarly, as a monoclonal antibody against chicken osteoclasts, Vaeaenaenen et al. [J. Bone Mineral
Res., Vol.6, p.1092-1097 (1991)] has obtained a mouse monoclonal antibody K20. This K20 antibody recognizes the 150 Kd molecule on chicken osteoclasts and reacts with renal cells in addition to osteoclasts. Functionally, it suppresses the bone resorption effect of chicken osteoclasts.

The characteristics of these monoclonal antibodies are that they react with renal cells in addition to osteoclasts, and that the 23c6, 13c2 (Horton) and K20 (Vaeaenaenen) antibodies suppress the bone resorption effect of osteoclasts. There is a point to do. However, none of the antibodies show cross-reactivity with mouse osteoclasts, and there is no report that a monoclonal antibody against mouse osteoclasts has been produced yet.

[0008]

[Means for Solving the Problems] As a result of various studies, the present inventors succeeded in creating a novel monoclonal antibody having high specificity for mouse osteoclasts and a hybridoma producing this monoclonal antibody. The present invention has been completed. The method for preparing the hybridoma and the monoclonal antibody of the present invention and the analysis of their properties will be described in detail below with reference to Examples.

[0009]

Example 1 Preparation of Mouse Osteoclasts The femurs and transbones of 10-day-old ICR mice were aseptically removed,
Α-MEM (α-Modified) containing 5% FCS (fetal calf serum)
Eagle's Medium) Suspend in culture medium. After allowing to stand for about 3 minutes to precipitate bone slices, etc., collect the cell suspension in the supernatant, centrifuge at 1,200 rpm for 5 minutes, add a new culture solution to the cell pellet, and add about 5 x 10 ⁶ cells. The cell concentration was adjusted to / 8 ml, parathyroid hormone (hereinafter abbreviated as PTH) was added to 10 ^-8 M, and 15 ml of this cell preparation liquid was added to a culture flask (75 cm ² bottom area) at 37 ° C. Incubate for 7 days in a 5% CO ₂ incubator. On the third day of the culture, the medium is replaced with a new culture medium containing PTH 10 ^-8 M. After culturing for 7 days, adherent cells were collected using a cell scraper. When the tartrate-resistant acid phosphatase activity (hereinafter abbreviated as TRAP), which is one marker of osteoclasts, was examined in the cells thus recovered, about 20% of the cells were positive. Also these TR
When the nuclei of AP-positive cells were stained with methyl green, a few cells (1% or less) were multinucleated cells having 6 to 7 nuclei, but the others were mononuclear cells. Furthermore, the bone resorption of these cells was confirmed as follows. That is, this cell 1 × 10
⁵ / well (96-well culture plate) 2 on ivory piece
After culturing at 37 ° C. in a 10% CO ₂ incubator for a day, the ivory pieces were stained with Coomassie Brilliant Blue, and 500 to 3,000 resorption cavities were observed on the ivory surface. This absorption activity was almost completely suppressed by eel calcitonin 10 ^-8 M. From these results, PTH 10 ^-8 M
It was considered that the TRAP-positive cells contained in the cells cultured for 7 days in A.c. contained osteoclasts.

[0010]

Example 2 Preparation of Splenocytes by In Vivo and In Vitro Immunization First, in vivo immunization was performed as follows. That is, 1 × 10 ⁷ cells prepared in Example 1 were mixed with 2 × 10 ⁸ pertussis killed cells, and the mixture was intraperitoneally administered to female Wistar rats every two weeks for immunization. After that, only 1 × 10 ⁷ cells prepared in Example 1 were intravenously administered, and 3 days later, the spleen was aseptically removed. To further sensitize, in vitro immunization was performed as follows. That is, ⁶ × 10 ⁶ cells prepared in Example 1
The cells were spread on a well culture plate, allowed to stand in a CO ₂ incubator for 30 minutes, and then washed three times with a culture medium to remove floating cells. When the TRAP activity of the cells attached to the plate was examined, about 70 to 80% of the cells were positive. Cells prepared by such a panning method can be used in vivo.
2.5 × 10 ⁷ cells / well were immunized with, mixed with 100 µg of muramyl dipeptide / well, and incubated at 37 ° C for 5 days for 5 days.
The cells were cultured and sensitized in a% CO ₂ incubator. After culturing, splenocytes were collected.

[0011]

Example 3 Preparation of Hybridoma RPMI-16, a cell culture medium commonly used in the art
Immunized rat spleen cells 1 × 10 ⁷ and mouse myeloma cells P3U1, 2 thoroughly washed with 40 culture medium [purchased from Nissui Pharmaceutical Co., Ltd.]
× 10 ⁶ pieces were mixed and centrifuged at 1,200 rpm for 5 minutes.
After separation, the splenocytes and P3U1 mixed cell group were thoroughly disentangled, and then 37 ° C polyethylene glycol 1540 with stirring.
[Wako Pure Chemical Industries, Ltd.] Add 0.5 g of a mixture of 4 g and 6 ml of RPMI-1640 culture solution over 1 minute, and then add 4 at 200-800 rpm.
Centrifuge for minutes. After separation, 37 ℃, RPMI-1640 culture solution 6
ml was gradually added with stirring for 5 minutes and centrifuged at 800 rpm for 5 minutes. After separation, the supernatant was discarded, and the cells were loosely loosened, and then HAT medium (RPMI-1640 medium was added with hypoxanthine 10 ^-4 M, thymidine 1.6 × 10 ^-5 M and aminopterin 4 × 10 ^-7 M). Medium) was added and gently suspended.
Add the suspension to a 96-well culture plate at 1 × 10 ⁵ cells / 200 μl /
Each well was dispensed and cultured at 37 ° C. in a 5% CO ₂ incubator for 7 to 10 days. A part of the culture supernatant was collected from the wells in which the fused cells grown in a colony were observed, and the antibody production was measured by the solid phase enzyme immunoassay using an antibody against the anti-rat antibody. Double staining by TRAP activity of cells and immunofluorescent antibody method described in Example 4 for wells in which antibody production was observed (double staining).
Method) to determine the reactivity to TRAP-positive cells,
Hybridomas in wells showing strong reactivity only with positive cells were cloned by the limiting dilution method, and clone 2G8-F10 (I
gM), 1F8-D12 (IgM) was selected as an antibody-producing hybridoma strain against osteoclasts.

The hybridoma strain producing clone 2G8-F10 is Rat-Mouse hybridoma / 2G8-F10, and the hybridoma strain producing clone 1F8-D12 is Rat-.
It is named Mouse hybridoma / 1F8-D12, and under the accession numbers of Micro-industrial Research Institute No. 13081 (FERM P-13081) and Micro-industrial Research Institute No. 13080 (FERM P-13080), respectively, it is a microbial industrial technology Deposited at the Institute.

[0013]

Example 4 Double Staining Method The double staining method is the method of Modderman et al. [Bon
e, vol.12, p.81-87 (1991)]. That is, 50 μl of the hybridoma culture solution was added to 5 × 10 ⁴ cells prepared in Example 1, and the mixture was incubated on ice for 1 hour. After the reaction, the plate was washed 3 times with phosphate buffered saline containing 0.1% sodium azide and 3% FCS (hereinafter abbreviated as staining buffer), and then goat antibody F (ab ') ₂ anti-mouse IgG (H + L)- FITC (Fl
uoresent-5-isothiocyanate) conjugate (Calt
Add 50 ml of 50 times diluted solution (purchased from Ag) and incubate on ice for 1 hour. After the reaction, the cells are washed three times with a staining buffer, and the cells are centrifuged at cytospin at 800 rpm for 5 minutes to prepare a cell sample on a slide glass. After fixing the cells with methanol, TRAP staining is performed. TRAP staining was 0.0 in the presence of 27 mM L [+]-tartaric acid.
Contains 5 mg / ml naphthol AS-BI phosphate.
It was carried out by incubating with a 1 M sodium acetate (pH 5.2) solution at 37 ° C. for 15 minutes. After TRAP staining, it was embedded in a cover glass with non-fluorescent glycerin and then observed with a fluorescence microscope.

[0014]

Example 5 Reactivity of 2G8-F10, 1F8-D12 Antibodies with Various Cells In the cells prepared in Example 1, 2G8-F10 antibody is an osteoclast and an osteoclast precursor cell TRAP activity positive. It strongly reacted only with polynuclear cells and mononuclear cells, and showed almost no reactivity with cells negative for TRAP activity. In addition, the 1F8-D12 antibody strongly reacted with TRAP activity-positive polynuclear cells and mononuclear cells, and showed reactivity with a part of TRAP activity-negative cells. Furthermore, these two antibodies did not react at all with mouse splenocytes, thymocytes and bone marrow cells.

[0015]

INDUSTRIAL APPLICABILITY By the monoclonal antibody provided by the present invention, osteoclasts and osteoclast progenitor cells can be specifically identified and sorted from a mouse bone cell population. The uniform osteoclasts and osteoclast precursors thus obtained provide a screening system for new drugs developed for the purpose of suppressing various bone resorptions. Further, by directly administering the monoclonal antibody of the present invention to mice, various pathological animals with altered bone metabolism balance can be prepared and used for screening new drugs effective for bone metabolic diseases.

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Claims (5)

[Claims] 1. A monoclonal antibody that specifically recognizes mouse osteoclasts and osteoclast precursor cells. 2. The monoclonal antibody according to claim 1, wherein the mouse osteoclasts and osteoclast precursor cells are tartrate-resistant acid phosphatase activity-positive cells. 3. An IgM produced by a hybridoma formed by the fusion of rat splenocytes immunized with mouse osteoclasts and mouse myeloma cells, which has the following properties:
Antibodies belonging to the class: (1) Reacts with tartrate-resistant acid phosphatase activity-positive polynuclear and mononuclear bone-derived cells; (2) Substantially reacts with tartrate-resistant acid phosphatase-negative bone tissue-derived cells No; (3) It does not react substantially with mouse splenocytes, thymocytes, and bone marrow cells. 4. The monoclonal antibody according to claim 1, which is 2G8-F10 or 1F8-D12. 5. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is 2G8-F10.