JPH03297391A - Mouse monoclonal antibody mab710f and production thereof - Google Patents

Abstract

NEW MATERIAL:An antibody having a function capable of inducing differentiation of pre-monocyte cell and useful in detection of surface antigen participating in the differentiation of human bone marrow based cell strain. USE:The above-mentioned antibody is useful as a surface marker of a bone marrow based cell and useful in detection of surface antigen 710F having extremely important role in differentiation of bona marrow based cell and studies of differentiation of bone marrow based cell or adhesion mechanism of the above-mentioned cell. PREPARATION:A bone marrow based cell strain is cultured in the presence of phorbol 12-millistate-13 acetate and then the above-mentioned compound is removed by cleaning and then re-cultured and the resultant bone marrow based cell strain is injected into a mouse abdominal cavity. Then immnospleen cell obtained from the above-mentioned mouse is fused with mouse mielloma cell and an antibody capable of producing the aimed antibody from the resultant hybridoma is selected and cloned and then cultured.

Description

[Detailed Description of the Invention] C. Industrial Application Field] The present invention is useful for detecting surface antigens of human myeloid cell lines and peripheral monocytes, which play an important role in the differentiation of promonocytic cells. The present invention also relates to a novel mouse monoclonal antibody MAb710F having the function of inducing differentiation of said cells, and a method for producing the same.

(Prior Art) Immune system cells are known to be generated from multipotent stem cells through two main differentiation routes: lymphoid cell lineage and myeloid cell lineage. Of these, research has progressed significantly in recent years regarding the differentiation of the former lymphocyte lineage, and much is being elucidated, but regarding the latter differentiation of the myeloid cell lineage, there are still many areas that are unclear. However, this remains a research topic that will require further efforts in the future.

That is, for example, the human promyelocytic leukemia cell line 1-fL
- phorbol 12-myristate 1 for 60 cell lines
It has already been discovered that reagents such as 3-acetate (hereinafter abbreviated as PMA) induce differentiation toward macrophages (for example, Huberman et al.
, (1979) Proc, Nat, Acad, Sci.
, USA 76 p. 1293-1297), but due to this fact, the mechanism by which differentiation of the cells is induced remains unclear.

[Problem to be solved by the invention]

Under the above-mentioned circumstances, the present inventors were conducting repeated research on the differentiation of myeloid cell lineages, and discovered that HL-6
It was discovered that the results are improved when cells such as cell lines adhere to the surface of the culture dish. That is, H L treated with PMA
It has been known for some time that the -60 cell line adheres weakly to the surface of a culture dish, but it has been discovered that under certain conditions it adheres extremely strongly and that morphological changes occur in the cells. I discovered it. Further research on this point revealed that such a phenomenon was caused by the specific antigen 710F discovered by the present inventors, and that this antigen plays an extremely important role in the differentiation of myeloid cell lines. It turned out that it has. As a result of further research on these, we succeeded in obtaining a novel monoclonal antibody MAb710F that specifically binds to the antigen, and the collaboration between this new antibody and PMA causes the above-mentioned improvement in cell adhesion and morphological changes. It was discovered that this was induced, and the present invention was developed.

[Means to solve the problem]

That is, the present invention provides mouse monoclonal antibody MAb710F, which is an antibody that has the function of inducing the differentiation of promonocytic cells and is an antibody used for detecting a surface antigen involved in the differentiation of human myeloid cell lines. , and a myeloid cell line were cultured in the presence of phorbol 12-myristate-1-13 acetate (PMA) in a medium, the PMA was washed away, and the resulting PMA-treated myeloid cell line was cultured again. Injected intraperitoneally into a mouse, then take out immune tile cells from the mouse, fuse these with a mouse myeloma cell line to prepare hybridomas, and then screen among these hybridomas for hybridomas that produce the antibody of interest, The present invention provides a method for producing the mouse monoclonal antibody MAb710 F, which comprises performing cloning according to a standard method, culturing the obtained hybridoma, producing an antibody therefrom, and then purifying the antibody of interest.

The cell line used as a raw material in the present invention is a myeloid cell line, and among these, HL-60, THP-1, and U937 cell lines are preferred, and HL-60 cell line is particularly preferred.

Next, the mouse monoclonal antibody MAb710 of the present invention
The method for producing F will be explained using an example in which HL-60 cells are used as a raw material.

First, HL-60 cells are grown in a medium, such as Hymedium (H
y Medium) 606 medium in the presence of PMA.

The obtained PMA-treated HL-60 cells are injected into the peritoneal cavity of a mouse, and after a certain period of time, the immunopilot cells are removed. Hybridomas are prepared by fusing these splenocytes with mouse myeloma cells.

Next, this hybridoma is screened for a hybridoma that produces a target antibody that has the effect of strengthening cell adhesion to a culture dish by the method detailed below, and cloned by a standard method.

The obtained hybridoma is further cultured to produce an antibody, which is purified to obtain the desired mouse monoclonal antibody MAb710F.

The characteristics of the mouse monoclonal antibody MAb710F of the present invention are derived from the antigen 710F that specifically binds thereto. Therefore, in order to understand the characteristics of the antibody of the present invention, it is necessary to investigate the characteristics of the antigen. Various analysis methods, namely observation by phase contrast microscopy and scanning electron microscopy, ELISA method, flow cytometry, radioactive iodinated labeling, biosynthetic labeling, immunoprecipitation method, 5DS-polyacrylamide gel electrophoresis method, enzyme It is preferable to perform multiple digestion methods. Including these points, the production method, action and effect of mouse monoclonal antibody MAb710F of the present invention, and its antigen 71
0F and the like will be specifically and detailedly explained in the following embodiment.

〔Example〕

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

Example 1 (1) Raw material cells and their preservation and preparation: Cell line P3LI 1. HL-60゜TH
P-1, U937, MOLT4 and [) aud
RPM116 i supplemented with penicillin 1100ti/d, streptomycin 1001J/d, glutamine 2o+H, and 10% fetal bovine serum (Hyclones)
40 medium (manufactured by Flow Laboratory).

On the other hand, peripheral blood mononuclear cells are treated with Ficoll pack (Ficol pack).
It is separated from heparin-treated human venous blood by centrifugation (manufactured by Pharmacia LKB Biotechnology) and cultured in RPM11640 medium containing 10% FBS.

(2) Preparation of monoclonal antibody: Mouse monoclonal antibody MAb 710F is a human promyelocytic leukemia cell treated with PMA, HL-
Prepared by immunizing mice with 60 cells.

That is, first, HL- at a concentration of 5×105 cells/rIi
60 cells in serum-free, albumin-free Hymedium (H
y Hedium) 11 in 606 medium (manufactured by Koshiiri)
Incubate at 37°C for 1 hour in the presence of 00n/d PMA.

Next, the cells were washed several times with the medium to remove PMA, and the cells were cultured at the same temperature for 24 hours at 8°C. This PM
A treatment) Approximately 107 IL-60 cells were added to BALB/
c Mice are injected intraperitoneally three times every two weeks.

Three days after the final injection of R, immune tile cells were taken out from the mouse, and the cells and P3UI cells (mouse myeloma cell line) were incubated with Galfre et al.
re 266 (1977) P550-552]
Fuse according to the protocol.

The obtained hybridomas are cultured in Hymedium 606 medium in a feeder layer of mouse mast cells using a 96-well plate.

Next, in order to screen for hybridomas producing the antibody of interest in the culture supernatant of the hybridomas, the mode of inhibition of cell aggregation or adhesion of PMA-treated HL-60 cells to the culture dish was observed.

That is, approximately 10" HL-60 cells treated with PMA were suspended in each individual's Hymedium 606 medium in a 96-well plate, and then 50" of the culture supernatant of the aged hybridoma was suspended.
μg was added to each person, followed by culturing for 24 to 48 hours and observation using a phase contrast microscope. As a result, some of the control cells aggregated or slightly adhered to the plastic wall, but some of the hybridoma supernatants obtained by the above preparation method clearly suppressed cell aggregation. It was observed that some had increased adhesion to food and dish surfaces.

In addition, one of the supernatants that has the inhibitory ability contains antibodies, anti
- It was recently discovered that it contains ICAM-1. What is noteworthy is that a substance was found in the hybridoma supernatant that did not suppress cell aggregation or recruitment, but instead induced cells to strongly adhere to the surface of the culture dish. .

A hybridoma producing an antibody having such a function was subcloned by repeating the limiting dilution method, and the obtained subclone was named HB710F. This new hybridoma HB710F has been submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, with accession number No. 11407.
(FERN P-11407). MAb 710 F produced from the above subclone can be purified from ascites by ammonium sulfate precipitation and DEAE-Sephacel chromatography.

The isotype of the MAb was determined using an enzyme-linked immunosorbent assay (ELI) using an anti-mouse isotype antibody (manufactured by Gym Laboratory).
Determined by SA).

(3) Differentiation induction by PMA and MAb710F
L-60 cells were washed three times with Hymedium 606 medium, suspended in the same medium to a concentration of 106 cells/d, and then cultured with PMAloo nMrd in a GO2 incubator for 1 hour. After 3 washes, the cells were 105
Resuspend 100μ of the suspension in cells/rrdl medium.
l in a microtiter plate at 0.2 μg/rtd.
1 of MAb 710 F.

As a result, cell aggregation and weak adhesion to the culture dish surface were observed in HL-60 cells cultured with PMA, whereas HL-60 cells cultured with both PMA and MAb 710 F showed cell aggregation and weak adhesion to the culture dish surface. It was confirmed by phase-contrast microscopy that some of the samples had significantly increased adhesion to the culture dish surface.

Particularly in the latter case, after 24 hours of culture, it was clearly observed by scanning electron microscopy that the cells had spread over the dish surface and were firmly attached.

In addition, regarding the increased adhesion of the cells, ED
This is also evidenced by the fact that attempts to detach attached cells from the surface of the culture dish using a TA/trypsin mixture were unsuccessful.

As for changes in cell morphology, it was observed that cells changed from spherical to microvilli-like, spindle-like, and ratuffle-like. or,
The hybridoma supernatant exhibiting the above activity was completely adsorbed to anti-mouse immunoglobulin antibodies bound to Sepharose beads, suggesting that it is an immunoglobulin rather than a cytokine-like activity.

The monoclonal antibody having the IC1M isotype produced from the hybridoma described above and purified from the supernatant containing it was named MAb710F.

Co(7)MAb710F antibody at 20-200nQ/l
When used within the range of ll1, H
It has been confirmed that the adhesion of L-60 cells is improved.

(4) Antigen recognized by MAb710F: Flow cytometry (flow cytometry) was performed using the method described below.

5X10'' cells were incubated with 0.3% bovine serum albumin and 0.3% bovine serum albumin.
Phosphorus containing 02% NaN3! ! 2 in buffer solution (PBS)
Wash twice and dilute in 100 μM of PBS at 4°C.

Incubate with 0.5-1.0μ9 antibody for 30 minutes. The cells are then washed three times and stained with Fab fragments of rabbit anti-mouse immunoglobulin labeled with fluorescein isodiocyanate for 30 minutes at 4°C. In the case of two-color analysis, the cells are further stained with an antibody labeled with phycoerythrin (manufactured by Becton Dexon).The stained cells were analyzed with Faxter (FAC3TAR, manufactured by Becton Dexon).

As a result, it was confirmed that 710 F antigen was expressed in all of the myeloid cell lines shown above.

It was also found that PMA treatment increased its expression level, and the effect was more pronounced in THP-1 cells than in HL-60 cells.

In addition, general (macrophage-specific Leu-
It was also found that the 710F antigen was detected on peripheral blood mononuclear cells such as those expressing M3 and 1-eu15 surface antigens.

On the other hand, peripheral blood lymphocytes were only slightly stained with MAb 710 F.

The above results indicate that 710 F antigen is specific to macrophage cells.

(5) While researching the differentiation-inducing ability of MAb710F and the role of PMA, MOL
It was found that T-4 cells were morphologically unresponsive to treatment with PMA and MAb 710 F, yet expressed the 710 F antigen on the cells. On the other hand, to induce morphological changes in myeloid cell lines, the corresponding antigen 7
It has been found that it is not sufficient that MAb710 F binds to 10 F, and that pretreatment of the cells with PMA is essential. However, the antigenic expression level of 710F does not change dramatically with PMA treatment, and these findings suggest that the sensitivity of cells to MAb may be due to something else rather than the amount of antigen expressed on the cell surface. I found out that it worked.

That is, PMA modifies the 710F antigen (e.g., via phosphorylation) and at the same time binds to MAb710F, thereby transmitting a signal to the cytoplasm that promotes cell attachment and cytoskeletal remodeling for the MAb710F antibody of the present invention. It is presumed that it provides a function to encourage Therefore, the most important point in the present invention is that MAb 710 F of the present invention is useful for inducing differentiation of myeloid cells through the above-mentioned mechanism.

(6) Production of MAb710F antibody and 710F antigen
Scientific characteristics: The characteristics and functions (effects) of MAb710F antibody are 710
Derived from F antigen. Therefore, in order to understand the characteristics of MAb 710 F, it is necessary to investigate the biochemical properties of the 710 F antigen.

(6)-■ Plate 1 treated with PMA and MAb710F for comparison.
- When 11 Rg/d of GRGDS peptide, which represents the normal recognition site of integrin receptors, was added to IL-60 cells, there was no effect on cell adhesion.Similarly, collagen type II or IV, fibronectin, vitronectin , laminin, GRI[b-IVa,
No such inhibitory effect was observed with antibodies against extracellular matrix or cell adhesion molecules such as CR3.

Furthermore, anti-LFA-1 and anti-ICAM-1 antibodies do not have the same effect of inducing cell adhesion as the antibodies of the present invention. Furthermore, CA-1gM discovered by Takahashi et al. does not have the same effect as the antibody of the present invention, and is also distinguished from fibronectin and gotronectin.

107 HL-60 cells were washed 3 times with PBS and treated with Bolton and Hunter reagent (Bolto) by the specified method.
n and Hunter Reagent: A
The cell surface is radioactively iodinated with a product manufactured by Mershan.

(b) Biosynthetic label: HL-60 cells pretreated with PMA as described above were labeled with
After culturing for an hour, the 107 cells were washed three times with PBS, suspended in 5II of Hymedium 606 medium supplemented with 10% methionine in a T25 flask, and labeled with 300 μG of [35S]methionine for 6 hours. In some cases, 1 μg/rrti of tunicamycin may be added to the cells. After labeling, the cells are washed three times with complete medium, and RIPA lysis buffer (1% Triton-100, 1%
Sodium deoxycholate, 0.1% SDS, 10n+
H-tris-HCL, 0.5M NaCl
, 2mN phenylmethylsulfone fluoride 988
．． Lyse cells at 0) for 1 hour on ice or at room temperature.

Then 35,000 rp using a Beckman rotor.
n+.

Insoluble matter is removed by centrifugation at 4°C for 30 minutes. The resulting lysate was subjected to immunoprecipitation.
Store in aliquots at 5°C.

Immunoprecipitation of 710F antigen was performed by J. Biol, Chei.
263 (1988) P806-811'/
, by the method described in the paper by KOI/alla et al.

MAb710 F is protein-A-Sepharose (P
The antigen in the extract of HL-60 cells was immobilized with rabbit anti-mouse immunoglobulin (Mils) on rote in-A-3epharose (Pharmacia LKB Biotechnology) and radioiodinated with this. Molecules were immunoprecipitated.

Next, the precipitate obtained by this immunoprecipitation was
e 227 (1970) psao~685 Lae
5DS- by the method described in the paper by mlllli
It was analyzed by PAGE method and observed by fluorography.

As a result, it was confirmed that the immunoprecipitate molecules migrated in a broad band with a relative molecular weight of 35,000 to 70,000 under reduction staining in the 5DS-PAGE method.

Furthermore, when the immunoprecipitate was examined after biosynthetic radiolabeling with [35S]methionine as described above, 35,0(
) It was confirmed that the relative molecular weight was 70,000.

(d) The acetate-based chemical immunoprecipitate was digested using Glycobeptitapi F (manufactured by Boehringer Mannheim) under the specified staining conditions.

This a! The prime-treated immunoprecipitate had increased electrophoretic mobility and became a single band.

Furthermore, when biosynthetic labeling was performed in the presence of tunicamycin, the electrophoretic profile of the immunoprecipitate was found to be similar to that treated with glycopeptiter t''F.

From these results, the precursor molecular species before addition of sugar chains are 24,
It can be seen that the homogeneous molecule with a relative molecular weight of 500 becomes a mature molecule through heterogeneous N-glycosylation.

Note that MAb710 F reacts with individual antigens that are not N-glycosylated, which may indicate that the peptide contains an epitope that transmits a differentiation signal to the cytoplasm.

In any case, as a result of various investigations and analyses, the 710 F antigen that we discovered and specified was found to be a novel antigen.

Example 2 The THP-1 cell line treated with PMA and MAb 710F in the same manner as in Example 1 strongly adhered to the plastic dish used for culture. Moreover, it exhibited a dendritic appearance in terms of morphology.

U937 and macrophage-like cell lines showed strong cell adhesion when treated with PMA and MAb 710 F as in Example 1.

However, in MOLT-4 cells (T cell line), both PMA alone and PMA-MAb710 F treatment induced IL-
No such effect was observed on 60 cells. Da
udi cells (B cell lineage) also have the above-mentioned M
No effect of Ab-7101T monoclonal antibody was observed.

These results revealed that the cell lines that respond to PMA-MAb710 F treatment are limited to myeloid cell lines.

(Effect of the invention) The novel surface antigen 710F discovered and specified by the present inventor is
As is clear from the above explanation, it is an antigen that is useful as a surface marker for myeloid cells and plays an extremely important role in myeloid cell differentiation.

Therefore, the novel mouse monoclonal antibody MA of the present invention
Since b710F is an antibody that specifically binds to the 710F antigen, it is useful for detecting the antigen and at the same time
It works together with MA to improve the adhesion ability of myeloid cells and also helps to reconstruct the cell frontier, and together they have the important function of inducing the differentiation of myeloid cells. It is a useful heptadonal antibody.

In addition, the mouse monoclonal antibody MAb710 of the present invention
Naturally, F is useful for research on the differentiation of myeloid cells or the adhesion mechanism of these cells by utilizing its functions and effects.

Claims (1)

[Scope of Claims] 1. A mouse antibody that has the function of inducing differentiation of promonocytic cells and is used for detecting a surface antigen involved in the differentiation of human myeloid cell lines. Monoclonal antibody MAb710F. 2. Immunize mice with a myeloid cell line treated with phorbol 12-myristate 13-acetate, fuse the immunized splenocytes with mouse myeloma cells, and then screen and clone the resulting hybridomas. The mouse monoclonal antibody MAb710 according to claim 1, which is produced by culturing the hybridoma after carrying out
F. 3. Myeloid cell lines are HL-60, THP-1 and U93
The mouse monoclonal antibody MAb710F according to claim 2, which is a cell line selected from 7 cell lines. 4. Mouse monoclonal antibody MAb710F according to claim 3, wherein the myeloid cell line is an HL-60 cell line. 5. After culturing the myeloid cell line in the presence of phorbol 12-myristate-13-acetate in a medium, washing and removing the phorbol 12-myristate 13-acetate,
The myeloid cell line obtained by culturing again is injected into the peritoneal cavity of a mouse, and then immunized splenocytes are taken out from the mouse and fused with a mouse myeloma cell line to prepare hybridomas. According to claim 1, the method comprises: screening hybridomas that produce the antibody of interest, performing cloning according to a standard method, culturing the obtained hybridomas, producing antibodies from the hybridomas, and then purifying the antibody of interest. A method for producing mouse monoclonal antibody MAb710F. 6. Myeloid cell lines are HL-60, THP-1 and U93
6. The method for producing mouse monoclonal antibody MAb710F according to claim 5, wherein the cell line is selected from 7 cell lines. 7. The method for producing mouse monoclonal antibody MAb710F according to claim 6, wherein the myeloid cell line is an HL-60 cell line. 8. The method for producing mouse monoclonal antibody MAb710F according to claim 5, wherein the medium is Hymedium 606 medium. 9. Mouse monoclonal antibody MAb710F according to claim 5, wherein the mouse used for immunization is a BALB/c mouse.
manufacturing method. 10. Mouse monoclonal antibody MAb710 according to claim 1, wherein the mouse myeloma cell line is P3UI cell line.
Manufacturing method of F.