JPH0967400A - Monoclonal antibody, hybridoma producing the same antibody and its utilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new antibody for detection, etc., of cell having macrophage colony stimulation factor (M-CSFR), specifically binding to M-CSFR receptor (M-CSFR) capable of reacting with M-CSF and neutralizing its action. SOLUTION: This new monoclonal antibody against M-CSFR comprises specifically bind to microphage colony stimulation factor receptor (M-CSFR) having properties reacting with a macrophage colony stimulation factor (M-CSF) and neutralizing action of M-CSF on the differentiated and proliferated cell. The antibody is useful for simply detecting and measuring M-CSFR and the cell having M-CSFR with a high sensitivity and a high accuracy. The monoclonal antibody is obtained by mixing M-CSFR with an adjuvant, subcutaneously administering the mixture to a rat, immunizing the rat, collecting the spleen cell after carrying out additional immune, fusing the immunized cell with myeloma cell in HAT culture medium, culturing the fused cell, selecting a cell having antibody activity, cloning the cell and culturing the resultant hybridoma.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a macrophage colony stimulating factor receptor (hereinafter abbreviated as M-CSFR).
And an hybridoma producing the same. Furthermore, the present invention utilizes the antibody to produce mouse MC
A method for detecting SFRs or cells having the same and a macrophage colony stimulating factor (hereinafter referred to as M-CS) from bone marrow cells using the antibody and an antibody against mouse c-kit.
The present invention relates to a method for separating progenitor cells that proliferate in response to F).

[0002]

M-CSF was discovered as a factor that stimulates the production and functional expression of monocytes and macrophages, and then c
When the DNA was isolated, its entire structure was revealed [Kawasaki, E. et al. S. , Et al. , Sc
ience, 230 , 291-296 (1985), W.
ong, G.G. G. , Et al. , Science, 2
35 , 1504-1508 (1987)]. On the other hand, M-
CSFR has been shown to be the proto-oncogene c-fms [Sherr, C. et al. J. , Et al. , C
, 41 , 665-676 (1985)], and was found to be a receptor of about 170 kd having tyrosine kinase activity. It was also clarified that when homodimer M-CSF binds to M-CSFR, M-CSFR is autophosphorylated, tyrosine kinase is activated, and a signal is transmitted. Subsequent studies showed that the op mouse, a mutant mouse that develops marble disease,
-Point mutation of the CSF gene has been shown, and it has been clarified that the cause of marble disease is the lack of M-CSF activity [Yoshida, H .; , Et
al. , Nature, 345 , 442-444 (19).
90)]. It was also found that M-CSF has a fat metabolism promoting action [Shimano, H. et al. , Et al. ,
J. Biol. Chem. , 265 , 12865-12
875 (1990)].

From the above studies, it was found that M-CSF not only stimulates the production and expression of functions of monocytes / macrophages, but also has various functions such as differentiation / maturation of osteoclasts and promotion of fat metabolism. It was

However, there are many unclear points regarding the role of M-CSF in the actual bone marrow. One of the reasons for this is that M-CSF deficiency extremely inhibits the development of the bone marrow cavity that supports bone marrow hematopoiesis as seen in op mice and suppresses hematopoiesis. There is a point that cannot be clearly made. To solve this problem, M- in adult bone marrow hematopoiesis
In order to clarify the functions of CSF and M-CSFR, it is important to prepare an antibody that inhibits the function of M-CSFR and analyze its expression and function in normal bone marrow. Moreover, it is considered that the generation of blood cells is regulated by the differentiation stage and lineage-specific expression of growth factor receptors, but among these growth factor receptors, those expressed in undifferentiated blood cells are c-kit [Og
awa, M .; , Et al. , J. et al. Exp. Med. ,
174 , 63-71 (1991)]. We will examine in detail which cells respond to M-CSF in the process of differentiation from these c-kit positive cells to macrophages, which are mature cells, or separate these cells and use them for studying functional expression. Requires an antibody against M-CSFR.
Furthermore, antibodies against M-CSFR can be a powerful weapon for studies on bone formation and fat metabolism.

[0005]

DISCLOSURE OF THE INVENTION The object of the present invention is MC
M-CSF characterized by reacting specifically with SFR
To provide a monoclonal antibody against R and a hybridoma producing the same. It is also an object of the present invention to provide a method for detecting M-CSFR or cells having it. Still another object of the present invention is to provide M-CS.
It is intended to provide a method for isolating progenitor cells proliferating in response to M-CSF from bone marrow cells using an antibody against FR and an antibody against c-kit.

[0006]

By using the antibody of the present invention, cells having M-CSFR or soluble M-CS can be obtained.
It is possible to provide an immunodetection method capable of easily detecting and measuring FR with high sensitivity and high accuracy. The antibody of the present invention can also be used for research to elucidate the mechanism of blood cell differentiation and proliferation. Furthermore, since the antibody of the present invention is specific to M-CSFR, a method for specifically purifying M-CSFR by a method such as affinity chromatography is also provided.

The antibody of the present invention serves as an immunizing antigen of mouse M-
It can be manufactured using CSFR. More specifically, for example, a fused cell (hyb) of a mammalian plasma cell (immune cell) immunized with the above immunogen and a mammalian plasmacytoma cell
(Ridoma, hybridoma), and a clone which produces a desired antibody that recognizes mouse M-CSFR is selected from this, and the clone can be produced by culturing the clone.

The mouse M-CSFR as an immunogen used in the above method is not particularly limited as long as it is a mouse M-CSFR, and any known mouse M-CSFR is known.
Mouse cell line having, for example, mouse monocyte-derived cell line J774.1 (ATCC, TIB-67) or mouse myeloblast cell line NFS60 [Holmes, K
L. , Et al. , Proc. Natl. Aca
d. Sci. USA. 82 , 6687-6691
(1985)]. Furthermore, cells or cell fractions of cells or the like that have been introduced with the mouse M-CSFR gene and have become capable of expressing mouse M-CSFR, or a preparation and gene set obtained by purifying mouse M-CSFR from these cell fractions. It may be any of mouse M-CSFR, a chimeric protein of M-CSFR and another protein, and a peptide having a partial amino acid sequence of mouse M-CSFR, which are produced according to the replacement technique. According to the method described in WO94 / 28160, mouse M-CSFR or M-CS
A chimeric protein of FR and another protein is preferably used as an immunogen, and a chimeric protein of the extracellular portion of mouse M-CSFR and the constant region of immunoglobulin H chain is particularly preferable. Since this chimeric protein specifically binds to the protein A column, it can be easily purified. Furthermore, since immunoglobulin is more stable in blood than other proteins, it is expected to be more stable in blood than when this chimeric protein is administered to animals. If so, it is considered that the antibody can be efficiently produced. The mammal immunized with the immunizing antigen in the above method is not particularly limited, but it is preferable to select it in consideration of compatibility with plasmacytoma cells used for cell fusion, generally, rat, Armenian hamster. Etc. are advantageously used.

Immunization can be carried out by a general method, for example, by administering the above-mentioned immunizing antigen to mammals by intravenous, intradermal, subcutaneous or intraperitoneal injection. More specifically,
The immunogen is preferably administered to a mammal several times every 2 to 30 days, optionally in combination with a conventional adjuvant, so that the total dose will be about 100 to 500 μg / rat. As the immune cells, it is preferable to use spleen cells extracted about 3 days after the final administration.

Further, as the other parental cell, the mammalian plasmacytoma cell to be fused with the above-mentioned immune cell, various known cells such as P3X63Ag8 (ATC) can be used.
C, TIB-9), P3X63Ag8.653 (ATC
C, CRL-1580), P3X63Ag8U. 1
(ATCC, CRL-1597), P3 / NS1 / 1-
Ag-1 (ATCC, TIB-18), Sp2 / 0-A
g14 (ATCC, CRL-1581) and the like, and Y3-Ag1.2.3 (ATCC, CRL-16) in rats.
31) and the like myeloma cells can be used.

The fusion reaction between the above-mentioned immune cells and plasmacytoma cells can be carried out by a known method, for example, the method of Milstein et al. [Meth. Enzymol. , 73 , 3-46 (19
81)] and the like. More specifically, the above fusion reaction is performed by using a conventional fusion promoter such as polyethylene glycol (PGE) and Sendai virus (HV).
J) and the like in a normal medium, and an auxiliary agent such as dimethylsulfoxide can be added to the medium, if necessary, in order to further enhance the fusion efficiency.

The ratio of immune cells to plasmacytoma cells used is the same as in the usual method. For example, it is usual to use about 1 to 10 times more immune cells than plasmacytoma cells.
Examples of the medium at the time of the fusion reaction include various types usually used for the growth of plasmacytoma cells, for example, RPMI1640 medium, MEM medium, and other types used for this kind of cell culture. Fetal bovine serum (FC
It is advisable to remove serum supplements such as S). For the fusion, a predetermined amount of the above immune cells and plasmacytoma cells is mixed well in the above medium, preheated to about 37 ° C., and a PEG solution,
For example, if the average molecular weight is about 1000 to 6000,
It is usually carried out by adding to the medium at a concentration of 30 to 60 w / v% and mixing. Thereafter, a desired hybridoma is formed by repeating the operation of sequentially adding an appropriate medium, centrifuging and removing the supernatant. Isolation of the desired hybridomas obtained can be carried out using conventional selection media such as H 2
It is carried out by culturing in AT medium (medium containing hypoxanthine, aminopterin and thymidine). The H
Culturing in AT medium may be carried out for a time sufficient to kill cells (unfused cells etc.) other than the target hybridoma, usually for several days to several weeks. The hybridoma thus obtained is subjected to a search for a desired antibody and a monocloning by an ordinary limiting dilution method.

The target antibody producing strain can be searched by, for example, ELIS.
Method A [Engvall, E. , Meth. Enzymo
l. , 70 , 419-439 (1980)] and radioimmunoassay (RIA) methods, which are generally used for detecting antibodies [Meth. Enzymol. ,
92 , 147-523 (1983)] and the M60 of NFS60 cells or bone marrow cells.
-M-CSF by inhibition test of biological activity of M-CSF such as inhibition of CSF-dependent growth and flow cytometry
A binding test to R-expressing cells is performed in combination. The above immunogens can be used for this search.

The thus obtained hybridoma which produces the desired monoclonal antibody recognizing mouse M-CSFR can be subcultured in an ordinary medium, and
It can be stored in liquid nitrogen for a long time.

The desired antibody can be collected from the above hybridoma by culturing the hybridoma according to a standard method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal having compatibility with the hybridoma and proliferating it to obtain ascites. Method etc. are adopted. In general, the former method is suitable for obtaining highly pure antibodies, and the latter method is suitable for mass production of antibodies.

The monoclonal antibody of the present invention obtained as described above has a specific reaction with mouse M-CSFR. Furthermore, the monoclonal antibody of the present invention has the following properties. a) Molecular weight: about 160,000 b) Subclass of antibody: IgG2a, κ c) M-CSF has an activity of inhibiting phosphorylation of M-CSFR. d) M-CSF has an activity of inhibiting the action of growing an M-CSF-dependent cell line. e) M-CSF has an activity of inhibiting the action of inducing macrophages from bone marrow cells. The antibody of the present invention is an antibody that strongly recognizes mouse M-CSFR, and such an antibody is suitable for detecting cells having mouse M-CSFR. The antibody of the present invention also recognizes human M-CSFR. From the above, M-CSFR was measured by using the monoclonal antibody of the present invention in a sandwich assay consisting of an antibody labeled with radioactive or enzyme and an immobilized antibody, or in flow cytometry with a fluorescent dye-labeled antibody. can do. For example, after reacting cells with the antibody of the present invention, FITC-labeled anti-rat IgG as a secondary antibody
Antibodies were added and the cells were subjected to M-
The presence or absence of CSFR can be detected.

Using the antibody of the present invention, mouse M-C can be analyzed by a conventional immunological assay such as a sandwich assay.
The SFR or cells with it can be detected. Furthermore, since the antibody of the present invention can inhibit the action of M-CSF, studies for elucidating the mechanism of differentiation and proliferation of macrophages, studies for differentiation of hematopoietic stem cells into osteoclasts [Kodama, H. et al. , Et al. , J. et al. Exp.
Med. , 173, 1291-1294 (1991)]
Study of arteriosclerosis thought to be involved in phenotypes and macrophages [Ishibashi, S. et al. , Et a
l. , J. et al. Biol. Chem. , 265 , 141
09-14117 (1990)] and the like.

[0018]

EXAMPLES Examples will be given below to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 Purification of mouse M-CSFR-human IgG chimeric protein: (1) Cloning of mouse M-CSFR cDNA and cloning
Us M-CSFR-human IgG chimeric protein expression vector
Construction of the base sequence reported as cDNA of mouse M-CSFR [Rothwell, V. M. & Rohrsc
heider, L .; R. Oncogene Res. ,
1 , 311-324 (1987)], the following 2
Various kinds of primers were synthesized with a DNA synthesizer. 5'CCTCGAGTATAGGAGTTGGGGGCCT
CCTC 3 '(SEQ ID NO: 1 in the sequence listing) and 5'CGGGATCCCTCATCGGGGAGCTGC
TTG3 '(SEQ ID NO: 2 in the sequence listing)

20 pmpl of each of the above primers, J77
4.1 cDNA obtained from mRNA of cell was 0.2μ
g in a 0.5 ml microcentrifuge tube and add 20 mM
Tris-HCl buffer (pH 8.3), 1.5 mM MgC
l ₂ , 25 mM KCl, 100 μg / ml gelatin, 50 μM each dNTP, 4 units Each reagent is added so as to be Taq DNA polymerase, and the total amount is 50 μl. Perkin-Elmer under the conditions of denaturing DNA at 94 ° C. for 1 minute, primer annealing at 62 ° C. for 2 minutes, and primer extension at 72 ° C. for 3 minutes.
Using a Cetus DNA thermal cycler, 30
Cycle reaction was performed. The synthesized DNA is a restriction enzyme Xh.
It was digested with oI and BamHI, and electrophoresed on a 1% agarose gel, and a DNA fragment of about 1.5 kb containing the nucleotide sequence of the extracellular region of mouse M-CSFR was prepared by a conventional method (Molecular Cloning. Cold.
Spring Harbor Laboratory.
New York. ).

On the other hand, B. Human C donated by Seed
D4-human IgG chimeric protein expression vector, CD4R
g [Aruffo, A .; , Et al. Cell, 6
1 , 1303-1313 (1990)] with restriction enzyme Xh
It was digested with oI and BamHI, and a DNA fragment containing the hinge region (H) and constant region portions (CH2 and CH3) of the human IgG1 gene was electrophoresed on a 1% agarose gel for separation and purification. This DAN fragment and the DNA fragment containing the nucleotide sequence of the extracellular region of mouse M-CSFR obtained by the above operation were ligated with T4 DNA ligase.

Escherichia coli (MC1061 / P3 strain) was transformed with the ligated DNA according to a standard method, and a plasmid DNA was prepared from the obtained transformant. Next, this plasmid DNA was digested with restriction enzymes XhoI and BamHI to confirm that the desired mouse M-CSFR DNA fragment was incorporated. (The plasmid is called CDMmfmsIg). FIG. 1 shows a construction diagram of a mouse M-CSFR-human IgG chimeric protein expression vector CDMmfmsIg for expressing animal cells.

(2) Mouse M-CSFR-in animal cells
Expression and Preparation of Human IgG Chimeric Protein CDMmfmsIg obtained in (1) above was added with 50 mM Tris-HCl buffer (pH 7.5), 400 μg / ml D
EAE Dextran (Pharmacia) and 100μ
It was added to RPMI1640 medium containing M chloroquine (Sigma) at 4 μg / ml. On the other hand, COS-1 cells (ATCC, which were grown to 50 to 70% confluence in RPMI1640 medium containing 10% fetal bovine serum using a dish having a diameter of 10 cm).
CRL-1650) was washed once with PBS, and 4 ml of the DNA mixture obtained above was added to each dish to give 5%.
It was cultured at 37 ° C. under the condition of CO ₂ . After 4 hours, cells were washed with PBS and 1% Nutridoma-NS
D-MEM / F- containing (Boehringer Mannheim)
25 ml of 12 medium (Gibco) was added, and the culture was further continued.

After 4 days, the culture supernatant was collected, and the culture supernatant was mixed with PB.
PROSEP-A (Bioproces) equilibrated with S
Sing). The column was washed with PBS to remove unadsorbed substances. Then, it was eluted with a 10 mM hydrochloric acid solution, and 1/10 volume of 0.2 M sodium phosphate buffer (pH 8.0) was added to the eluate to neutralize it. Thus, a purified mouse M-CSFR-human IgG chimeric protein was obtained.

Example 2 Preparation of Monoclonal Antibody: The mouse M-CSFR-human IgG chimeric protein obtained in Example 1 was used as an antigen for mouse M-CSFR.
That is, 500 μg of mouse M-CSFR-human IgG chimeric protein was mixed with complete Freund's adjuvant and subcutaneously administered to Wistar rats. 50 μg mouse M-CSFR prepared with physiological saline after about 30 days
-Human IgG chimeric protein was administered intravenously.

After 4 days, splenocytes were prepared and subjected to a conventional method [Sud.
o, T. , Et al. Proc. Natl. Ac
ad. Sci. USA, 90 , 9125-9129.
(1993)] and fused with P3X63Ag8.653 cells (ATCC, CRL-1580). Fused cells in 10% fetal calf serum (FCS), 100 units / m
l penicillin, 100 μg / ml streptomycin,
RPMI16 containing 50 μM 2-mercaptoethanol
Suspend in 40 medium and add 100 to 96 well plate.
The cells were added at a ratio of μl / well and cultured at 37 ° C. in a 5% CO ₂ incubator.

After selecting the hybridoma cells in HAT medium, the supernatant was screened by two different assay systems.

The first test was to determine whether the antibodies in the culture supernatant of hybridomas inhibit the M60-dependent growth of NFS60 cells. NFS60 cells were washed twice with phosphate buffer (PBS), 10% FCS and 50%.
RPMI 1640 containing μM2-mercaptoethanol
Suspend in medium. 1x10 cells in a 24-well plate
Add ⁵ cells / 0.5 ml / well, and then add 100 μl of the culture supernatant of the hybridoma. After culturing for 1 hour, M-CS
Add F. After culturing for 2 days, the number of cells was counted to examine the M-CSF-dependent growth inhibitory activity in the hybridoma culture supernatant.

In the second test, the antibody in the hybridoma culture supernatant is derived from NFS60 cells or monocytes derived from J774.
The hybridoma supernatant was reacted with the above cells to determine whether or not they bind to the M-CSFR of 1 cell (ATCC, TIB-67), stained with FITC-labeled anti-rat Ig antibody as a secondary antibody, and subjected to flow cytometry. Analyzed in.

The culture supernatant of about 1200 wells of the hybridoma was screened by the above-mentioned method, and one cell line producing an antibody against the mouse M-CSFR of interest was detected. Cloning was repeated by the limiting dilution method to obtain the desired antibody-producing clone. The clone (AFS98
-1) is FE at the Institute of Biotechnology, Institute of Biotechnology, AIST
Deposited as RMBP-15105.

Example 3 Characteristics of antibody obtained from AFS98 (hereinafter referred to as AFS98): (1) Subclass of antibody The subclass of the above antibody determined using a rat antibody subclass detection kit (Zymed) is (IgG2a,
κ).

(2) After culturing the molecular weight hybridoma in a serum-free medium, ammonium sulfate salting out was performed twice, and purification was performed using an anti-rat IgG antibody-bound Sepharose 4B column. After that, SDS-polyacrylamide gel electrophoresis was performed and stained with Coomassie Brilliant Blue, no bands other than AFS98 were observed. Molecular weight of the antibody obtained by this analysis (sum of molecular weight of heavy chain and light chain is taken as molecular weight of antibody)
Was about 160 kd.

(3) Crossreactivity It was examined whether or not AFS98 reacts with the chimeric proteins of various receptors produced by the method for producing the chimeric proteins shown in Example 1 and human IgG. As a result, AFS98 was found to be mouse M-CSFR-human IgG.
Reacts strongly with the chimeric protein, human M-CSFR-human I
It was found to react weakly with the gG chimeric protein. Further, mouse PDGFα receptor-human IgG chimeric protein, mouse flk1-human IgG chimeric protein, mouse flk2-human IgG chimeric protein, mouse IL-7
Receptor-human IgG chimeric protein and mouse gp
It was shown not to cross-react with the 130-human IgG chimeric protein.

(4) Neutralizing activity It was examined whether colonies formed from bone marrow cells in response to hematopoietic factors are inhibited by AFS98.

Femurs were collected from C57BL mice and subjected to a conventional method [Benner, R. et al. , Et al. , In Imm
unological Methods. Vol.
2, (Lefkovits, I. & Pernis,
B. , Ed. ) 247-261, Academic P
less, New York. (1981)], bone marrow cells were prepared.

Next, the bone marrow cells were passed through a stainless mesh to remove the cell clumps, transferred to a dish having a diameter of 100 mm (Corning Co.), and allowed to stand at 37 ° C. under 5% CO ₂ for 1 hour for adhesiveness. Cells were removed. Further α-ME
Washed twice with M (Gibco), 20% FCS, 1% deionized bovine serum albumin (Sigma), 1% methylcellulose, so that the cell concentration becomes 2 × 10 ⁴ cells / ml.
The cells were suspended in α-MEM containing 0.1 mM 2-mercaptoethanol and various hematopoietic factors and various concentrations of AFS98, added to 1 ml per dish (Falcon) having a diameter of 35 mm, and cultured under the conditions of 37 ° C. and 5% CO 2. .
Each hematopoietic factor was mouse IL-3: 100 units / ml, mouse GM-CSF: 100 units / ml, mouse M-CS.
F: added at a concentration of 10 ng / ml. .

These hematopoietic factors and various concentrations of AFS9
When the number of colonies formed per 2 × 10 ⁴ bone marrow cells by the combination of 8 was examined on the 7th day after culturing, as shown in FIG. 2, only the colonies formed by M-CSF were inhibited by AFS98. Was done.

From the above results, it was found that AFS98 specifically neutralizes the action of M-CSF. Also 10 ng
The concentration of AFS98 that completely inhibits the colony-stimulating activity of M-CSF / ml was 0.1 μg / ml.

Example 4 M-CS by flow cytometry using AFS98
FR detection: (1) M-CSFR positive cells using flow cytometry
Of the mouse myeloblast cell line FDC-P2, NFS60 (M-
Showing CSF-dependent proliferation) and monocyte-derived cell line J77
React 4.1 with AFS98 and use FIT as a secondary antibody
When stained with a C-labeled anti-rat IgG antibody and analyzed by flow cytometry, the results were NFS60 and J774.
1 was clearly positive. However, FDC-P2 was negative.

(2) Mice using flow cytometry
Detection of M-CSFR Positive Cells in Bone Marrow Cells The proportion of M-CSFR positive cells in normal mouse bone marrow cells was analyzed by flow cytometry. AFS98 is FIT
It was used after being labeled with C or biotin. Femurs were collected from C57BL mice and subjected to a standard method [Benner, R. et al. ,
et al. , In Immunological M
methods. Vol. 2, (Lefkovits,
I. & Pernis, B .; , Ed. ) 247-26
1, Academic Press, New Yor
k. (1981)], bone marrow cells were prepared. ACK4, which is an anti-c-kit antibody (a marker for undifferentiated hematopoietic cells) obtained by labeling AFS98 labeled with FITC with bone marrow cells and biotin, [Ogawa, M. et al. , Et al. ,
J. Exp. Med. , 174 , 63-71 (199
1)], stained with phycoerythrin-avidin (Becton Dickinson) and analyzed by flow cytometry.

AF in which bone marrow cells were labeled with biotin
Ma, a monocyte-based marker labeled with S98 and FITC
c1 [Springer, T .; , Et al. , Eu
r. J. Immunol. 9 , 301-306 (197)
9)] or AFS98 and FIT labeled with biotin
C-labeled B cell marker B220 (RA3-
6B2) [Coffman, R .; L. Immunol.
Rev. , 69 , 5 (1986)] or AFS98 labeled with biotin and FITC-labeled erythroid marker Ter119 [Ikuta, K. et al. , Et a
l. , Cell, 62 , 863-874 (1990)].
Were subjected to flow cytometry analysis by staining with phycoerythrin-avidin.

As a result, 1.9% of c-kit positive M-CSFR negative cells, 4.8% of c-kit negative M-CSFR positive cells and 0.4% of c-kit positive M-CSFR positive cells. And Mac1 positive M-CSFR negative cells 15.2%, Mac1 negative M-CSFR positive cells 1.
5% and Mac1 positive M-CSFR positive cells were 3.2%. In addition, B220 positive M-CSFR negative cells are 2
4.5%, B220 negative M-CSFR positive cells 4.1
%, B220-positive M-CSFR-positive cells were 0.1%, and Ter119-positive M-CSFR-negative cells were 46.6.
%, Ter119 negative M-CSFR positive cells were 4.4.
%, Ter119 positive M-CSFR positive cells 0.3%
Met. From the above results, M-CSFR was 3.2% in monocyte cells, 0.1% in B cell cells, 0.3% in erythroid cells, and 0.4% in undifferentiated cells. It was shown to be expressed. In addition, M-CSFR-positive cells were about 4 to 5% in the bone marrow.

Example 5 Detection of cell surface antigen recognized by AFS98: 4 × 10 ⁷
1 J774.1 cell with 1 ml of 0.15 M NaCl
And 0.5 mg / ml sulfo-NHS-bio
The cells are suspended in 0.1 M Hepes buffer (pH 8.0) containing tin (Pierce) and reacted for 30 minutes at room temperature with occasional slow stirring to label the cell surface protein with biotin. The cells were washed 3 times with RPMI1640 medium, 1% Triton X-100, 1 mg / ml.
pefablocSC, 1 μg / ml leupe
ptin, 1 μg / ml pepstatin, 0.1
μg / ml aprotinin, 5 mM EDTA,
Suspend in 50 mM Tris-HCl buffer (pH 7.2) containing 150 mM NaCl (hereinafter referred to as IP) and suspend in ice for 3
The cells were lysed at 0 minutes. Dynabeads (Dyna beads in which goat anti-rat IgG was bound to the cell lysate after centrifugation)
(Company 1) was added and reacted overnight at 4 ° C. The Dynabeads were removed with a magnet, the supernatant was divided into two equal parts, and the goat anti-rat IgG-binding Dynabeads having AFS98 adsorbed on one side and the rat anti-mouse IL-on the other side as a control antibody.
4 antibody (11B11) (Pharmingen) adsorbed goat anti-rat IgG-bound Dynabeads was added and reacted at 4 ° C. for 1 hour. After washing the beads 5 times with IP, add sample buffer for electrophoresis to the beads, and heat to 100 ° C.
Was treated for 5 minutes and electrophoresed on an 8% SDS-polyacrylamide gel under reduction. After electrophoresis, the protein was transferred to a nitrocellulose membrane (Amersham). The nitrocellulose membrane was blocked with Block Ace (Dainippon Pharmaceutical Co., Ltd.), and the membrane was washed with 50 mM Tris-HCl buffer (pH 7.4) containing 0.05% Tween 20 and 0.15 M NaCl (hereinafter referred to as T-TBS). After washing, the streptavidin-horseradish peroxidase complex (Amersham) was added to T-TBS for 300 times.
Immerse in 0 times diluted solution. Allow to react for 1 hour, T-TB
After washing with S, the protein transferred to the membrane was detected by the ECL detection system of Amersham. As a result, the control antibody 11B11 does not precipitate any protein in J774.1 cells, but AFS98 has a molecular weight of about 170 k.
It was shown to precipitate the protein of d from J774.1 cells. This approximately 170 kd protein is mouse M-
It was in agreement with the molecular weight of CSFR.

Example 6 Identification of a protein recognized by AFS98 and having a molecular weight of about 170 kd: When M-CSF binds to M-CSFR, M-CSF
It is known that R is phosphorylated. In order to clarify whether the protein having a molecular weight of about 170 kd recognized by the antibody of the present invention, AFS98, is M-CSFR, it was examined whether the protein of about 170 kd was phosphorylated by M-CSF.

J774.1 cells containing 10% FCS in R
Cultured in PMI1640 medium, washed 3 times with PBS, F
Further culture is continued in RPMI 1640 medium without CS. 16 hours later, after washing with RPMI1640 medium, J
774.1 cells 25 to make 1 × 10 ⁷ cells / ml
mM Hepes buffer (pH 7.2), 0.1 mM Na
_The cells were suspended in RPMI1640 medium containing ₃ VO ₄ . 1X
107 J774.1 cells were treated with 0.1 μg of human MC
37 with or without SF (R & D Systems)
Incubated at ° C. 10 minutes later, cells were ice-cooled to 1 mM N
It was washed 3 times with PBS containing a ₃ VO ₄ . 2 mM cells
The cells were lysed by suspending them in IP containing Na ₃ VO ₄ , 10 mM sodium pyrophosphate and 100 mM NaF (hereinafter referred to as V-IP), and placing them on ice for 30 minutes. After centrifugation, goat anti-rat IgG-bound Dynabeads (Dynal) was added to the cell lysate, and the mixture was reacted overnight at 4 ° C. Remove Dynabeads with a magnet and add AFS9 to the supernatant.
Goat anti-rat IgG binding Dynabeads adsorbed with 8 were added and reacted for 1 hour, and after washing the beads 5 times with V-IP,
Add the sample buffer for electrophoresis to the beads, and add 100
After treatment at 5 ° C for 5 minutes, electrophoresis was performed on 8% SDS-polyacrylamide gel under reduction. After electrophoresis, the protein was transferred to a nitrocellulose membrane (Amersham) and the anti-phosphotyrosine antibody 4G10 (Upstate B) was used.
Western blotting was performed using iotechnology. Detection of proteins that react with 4G10 is E
CL system (Amersham) was used.

As a result, J cultured in the presence of M-CSF was
Approximately 170 k precipitated from A. 774.1 cells by AFS98
It was found that the protein of d was phosphorylated, and the protein precipitated by AFS98 from J774.1 cells cultured in the absence of M-CSF was not phosphorylated. From this, it was strongly suggested that the protein of about 170 kd is M-CSFR that is specifically phosphorylated by M-CSF, and it was revealed that AFS98 is an antibody that recognizes M-CSFR.

Example 7 Separation of M-CSF-Proliferating Cell Population from Bone Marrow Cells: AFS98 Antibody of the Invention and Anti-mouse c-
Using a kit antibody, ACK4, it was investigated what kind of cell population in bone marrow cells would form colonies in response to M-CSF.

Bone marrow cells of C57BL / 6 mice were reacted with biotin-labeled ACK4 and FITC-labeled AFS98, and subsequently stained with phycoerythrin-avidin. The population was divided into positive, c-kit positive MCSFR negative and c-kit positive MCSFR positive populations. 10 of each of these fractionated cells
00, using 10000 unfractionated bone marrow cells,
The ability of these cells to form colonies induced by M-CSF was examined.

As shown in FIG. 3, c-kit negative MC
The SFR-positive cell population did not form colonies with M-CSF, and c-kit positive M-CSFR negative and c-kit
A positive M-CSFR positive cell population formed colonies with M-CSF. The number is 10 times higher in the c-kit positive M-CSFR negative cell population than in the c-kit positive M-CSFR positive cell population. In addition, the number of cells forming colonies with M-CSF in the c-kit positive M-CSFR positive cell population is twice as large as that of bone marrow cells that do not fractionate cells.

From the above, M-CSF in bone marrow cells
It was shown that the cell population having a high ability to form macrophage colonies in response to c-kit positive M-CSFR negative cell population, and then c-kit positive M-CSFR positive cell population.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, a monoclonal antibody specific to M-CSFR is provided, and immunity capable of easily detecting and measuring M-CSFR and cells having M-CSFR with high sensitivity, high precision and easily. Assay methods are provided. Further, since the antibody of the present invention has a property of neutralizing the action of M-CSF on cells functionally expressed in response to M-CSF, studies on differentiation of bone marrow cells into macrophages, hematopoietic stem cells to osteoclasts are performed. Research into differentiation, research into qualitative metabolism,
A method for studying the role of M-CSF signaling in studies such as arteriosclerosis is provided. Further provided is a method for separating and purifying monocyte / macrophage progenitor cells by separating cells using the antibody of the present invention and the term c-kit antibody.

[0052]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCTCGAGTAT GGAGTTGGGG CCTCCTC

SEQ ID NO: 2 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CGGGATCCTC ATCGGGGAGC TGCTTG

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a method for constructing a vector expressing a chimeric protein consisting of mouse M-CSFR and human IgG.

FIG. 2 is a view showing that AFS98, which is a monoclonal antibody of the present invention, inhibits the differentiation and proliferation induction of macrophages from bone marrow cells by M-CSF.

FIG. 3 is AFS98 which is the monoclonal antibody of the present invention and A which is a monoclonal antibody against mouse c-kit.
FIG. 2 shows the reactivity of M-CSF of a cell population separated from bone marrow cells using CK4 using a cell sorter.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location G01N 33/566 C12N 5/00 B 33/577 9162-4B 15/00 C // (C12P 21 / 08 C12R 1:91)

Claims (6)

[Claims] 1. A specific binding to a macrophage colony stimulating factor receptor, which has a property of neutralizing the action of macrophage colony stimulating factor on cells that differentiate and proliferate in response to macrophage colony stimulating factor. A monoclonal antibody against the macrophage colony stimulating factor receptor. 2. The monoclonal antibody according to claim 1, which has the following characteristics. a) Molecular weight: about 160,000 b) Subclass of antibody: IgG2a, κ c) Macrophage colony stimulating factor binds to macrophage colony stimulating factor receptor and has an activity of inhibiting the action of phosphorylating macrophage colony stimulating factor receptor. d) The macrophage colony stimulating factor has an activity of inhibiting the action of growing a macrophage colony stimulating factor-dependent cell line. e) It has an activity of inhibiting the action of macrophage colony stimulating factor to induce macrophages from bone marrow cells. f) It has an activity of binding to mouse and human macrophage colony stimulating factor receptors. 3. A hybridoma producing the monoclonal antibody according to claim 1 or 2. 4. A hybridoma AFS98-1 (FERM
The hybridoma according to claim 3, which is BP-15105). 5. A macrophage colony stimulating factor receptor characterized by using the monoclonal antibody according to claim 1 or 2 in an immunoassay or flow cytometry using an antibody labeled with a radioactive substance, an enzyme or a fluorescent dye. How to measure. 6. A monocyte / macrophage progenitor cell is isolated by separating c-kit positive macrophage colony stimulating factor receptor negative cells using the monoclonal antibody according to claim 1 or 2 and the monoclonal antibody against c-kit. Method of separation and purification.