A monoclonal antibody is disclosed capable of specifically reacting with the human mesothelial cell but not with other normal cells and human tumor cells, and therefore applicable in diagnosis of cancers.

BACKGROUND OF THE INVENTION 
The present invention relates to a monoclonal antibody capable of 
specifically reacting with the human mesothelial cell and to a method of 
producing the monoclonal antibody. The present invention is useful in 
diagnosis of cancers such as cytological diagnosis of expectoration for 
lung cancer, cytological diagnosis of pleural effusion for lung cancer and 
cytological diagnosis of ascitic fluid for various abdominal cancers. 
It is known that in lung cancer, cancer cells desquamate into sputum and/or 
pleural effusion, and that in various abdominal cancers, cancer cells 
desquamate into ascitic fluid. Therefore, it is important in diagnosis of 
cancer to detect cancer cells desquamating into sputum and/or pleural 
effusion, or ascitic fluid. Diagnosis and differentiation of cancer cells 
are generally carried out by pathologists. Sputum, pleural effusion and 
ascitic fluid contain many kinds of normal cells, and above all it is 
difficult to distinguish cancer cells from mesothelial cells and 
macrophages. 
Recently, many monoclonal antibodies having specificity for cancer cells 
have been produced by hybridoma technique, and application of the 
monoclonal antibodies to cytodiagnosis have been made. Since some of the 
monoclonal antibodies react with macrophages and/or mesothelial cells or 
some of them react only with particular cancer cells, it is not easy to 
carry out cytodiagnosis of cancer by use of the monoclonal antibodies. 
Especially, it is difficult for even a pathologist to distinguish cancer 
cells from mesothelial cells. 
Accordingly, a monoclonal antibody which specifically reacts with the 
mesothelial cell but not with cancer cells, if available, would be useful 
in the cytodiagnosis of cancer. No monoclonal antibodies and antisera 
being capable of specifically reacting with the mesothelial cell have been 
know so far. 
The present inventors have first produced hybridomas between spleen cells 
of a mouse immunized with human mesothelial cells and murine myeloma cells 
and selected a hybridoma producing a monoclonal antibody which is capable 
of specifically reacting with human mesothelial cells but not with various 
human cancer cells. Further, the present inventors have made cytological 
diagnosis of expectoration, pleural effusion and ascitic fluid from 
patients with cancer using the monoclonal antibody of the present 
invention has a great significance in clinical pathology, and have now 
completed the present invention. 
SUMMARY OF THE INVENTION 
The present invention provides a monoclonal antibody of the IgG class 
capable of specifically reacting with human mesothelial cells but not with 
other normal human cells and human tumor cells. 
DESCRIPTION OF THE INVENTION 
The monoclonal antibody according to the present invention is obtained by 
fusing spleen cells of a mouse immunized with human mesothelial cells, 
with murine myeloma cells to prepare hybridomas, selecting from among the 
resulting hybridomas a hybridoma clone producing the required monoclonal 
antibody, and cultivating the selected hybridoma in a suitable culture 
medium or intraperitoneally administering the selected hybridoma to a 
mouse thereby to cause hybridoma cell propagation in the ascitic fluid in 
the mouse, followed by separation of the product antibody from the culture 
medium or the ascitic fluid as the case may be. 
A method of producing the monoclonal antibodies according to the invention 
is described in detail below. 
(1) IMMUNIZATION OF ANIMAL AND PREATION OF ANTIBODYPRODUCING CELLS 
Mice between 3-10 weeks of age, preferably 8-week-old mice, are immunized 
with human mesothelial cells, to cause such mice to prepare 
antibody-producing cells in the spleen, lymph node and peripheral blood. 
The immunization is performed generally by administering human mesothelial 
cells (5.times.10.sup.5 to 5.times.10.sup.6 cells per animal), together 
with an appropriate adjuvant (e.g. Freund's complete adjuvant, or aluminum 
hydroxide gel plus B. pertussis vaccine) to the mice subcutaneously, 
intravenously or intraperitoneally. Thereafter, the same antigen 
administration is repeated 2 to 5 times at 1 to 2 week intervals. Three to 
seven days after each immunization, the blood is sampled from the 
eyeground venous plexus and the serum of each sample is tested to 
determine whether it reacts with human mesothelial cells by the 
immunocytochemical staining given hereinafter. 
Immunocytochemical staining: 
A mesothelial cell suspension (1.times.10.sup.7 cells/ml) in PBS comprising 
1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate and 7.65 g 
of sodium chloride in 1 l of distilled water (pH 7.2) is distributed into 
the wells of a 12-well micro-titer slide glass (Flow Laboratories, CAT No. 
60-412-05) coated with egg white albumin in an amount of 5 .mu.l per well 
and dried over cooled air for 5 to 10 minutes. 
Then, acetone cooled at -20.degree. to -30.degree. C. is added to each well 
and the slide glass is allowed to stand for 10 minutes. After drying for 2 
to 3 minutes, endogenous peroxidase is inactivated by immersion in 3% 
hydrogen peroxide in methanol solution for 30 minutes. After washing well 
with PBS, 20 .mu.l of antiserum or monoclonal antibody solution (1 to 20 
.mu.g/ml) is distributed into each well as the first antibody, and the 
slide glass is allowed to stand for 30 minutes to 2 hours at room 
temperature and washed well with PBS. Then, 20 .mu.l of a biotin-labeled 
rabbit anti-mouse immunoglobulin (10 .mu.g/ml) is distributed into each 
well as the second antibody, and the glass is allowed to stand at room 
temperature for 30 minutes and then washed well with PBS. 
Avidin-biotin-perioxidase complex (ABC reagent, product of Vector) is 
added to the wells, and the glass is allowed to stand at room temperature 
for 30 minutes and then washed well with PBS. Color is developed by the 
addition of diaminobenzidine as the substrate solution for peroxidase, and 
the glass is observed with a microscope. 
The procedure subsequent to ABC reagent is made by the method recommended 
by Vector. When the target cell is various tumor cells or normal cells 
instead of mesothelial cells, the immunocytochemical staining is conducted 
in the same manner except for changing a cell fixed on a slide glass as 
the first step. In preparation for cell fusion, human mesothelial cells 
are intraperitoneally administered to the immunized mice in a dose of 
5.times.10.sup.5 to 5.times.10.sup.6 cells per animal 3 to 4 days prior to 
the fusion treatment. The spleens are then extirpated and the spleen cells 
are prepared for fusion. 
That is, the spleen is cut into fragments in MEM (product of Nissui 
Pharmaceutical), loosened up with forceps, and centrifuged at 1,200 rpm 
for 5 minutes. The supernatant is discarded, and the sediment are deprived 
of erythrocytes by treatment with Tris-ammonium chloride buffer (pH 7.65) 
for 1-2 minutes, washed three times with MEM, and used as the spleen cells 
for fusion. 
Mesothelial cells are obtained from intine of thorax and of abdominal 
cavity on autopsies. Cells are collected from the surface of the intine 
and washed with PBS two or three times. Alternatively, mesothelial cells 
are obtained from the homogenates of the cardiac vesicle are washed with 
PBS and centrifuged at 1,200 rpm for 5 minutes, and then the pellet is 
suspended in PBS. 
(2) PREATION OF MYELOMA CELLS 
A mouse-derived established myeloma cell line is used. Suitable examples 
are the 8-azaguanine resistant mouse (BALB/c-derived) myeloma cell lines 
P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology 81, 
1-7 (1978)], P3-NSI/1-Ag41 (NS-1) [European J. Immunology, 6, 511-519 
(1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269-270 (1978)], P3-X63-Ag8 653 
(653) [J. Immunology, 123, 1548-1550 (1979)] and P3-X63-Ag8 (X63) [Nature, 
256, 495-497 (1975)], all of which are commercially available. The passage 
of these cell lines is performed in 8-azaguanine medium [normal medium 
prepared by adding, to RPMI-1640 medium, glutamine (1.5 mM), 
2-mercaptoethanol (5.times.10.sup.-5 M), gentamicin (10 .mu.g/ml) and 
fetal calf serum (FCS; product of CSL) (10%), with further supplementation 
with 8-azaguanine (15 .mu.g/ml)]. The cell line selected for cell fusion 
should be transferred to normal medium 3 to 4 days before fusion to ensure 
the cell count of not less than 2.times.10.sup.7 on the day of fusion. 
(3) CELL FUSION 
The antibody-producing cells immunized in (1) and the myeloma cells 
obtained in (2) are washed well with MEM or PBS and mixed in a cell number 
ratio of antibody-producing cells:myeloma cells in the range of 5:1 to 
10:1 and then subjected to centrifugation (1,200 rpm, 5 minutes). The 
supernatant is discarded and the cell sediment is loosened up. With 
stirring at 37.degree. C., a mixture of 2 g of polyethylene glycol 1,000 
(PEG-1,000), 2 ml of MEM and 0.7 ml of dimethylsulfoxide is added in an 
amount of 0.2-1 ml per 10.sup.3 antibody-producing cells, and MEM is added 
until the whole volume is made 50 ml after several additions of 1-2 ml of 
MEM at 1 to 2 minute intervals. After centrifugation (900 rpm, 5 minutes), 
the supernatant is discarded and the cell sediment is loosened gently. To 
the cells is added 100 ml of normal medium (RPMI-1640 with 10% FCS). The 
cells are gently suspended in the medium with a measuring pipette. 
The suspension obtained is distributed, in 1 ml-portions, into the wells of 
a 24-well incubation plate. Incubation is carried out in a 5% CO.sub.2 
incubator at 37.degree. C. for 24 hours. HAT medium [normal medium 
supplemented with hypoxanthine (10.sup.-4 M), thymidine 
(1.5.times.10.sup.-5 M) and aminopterine (4.times.10.sup.-7 M] is added to 
the incubation plate (1 ml per well) and incubation is conducted for a 
further 24 hours. Thereafter, 1 ml of the culture supernatant is discarded 
and the same volume of fresh HAT medium is added at 24-hour intervals for 
2 days. The incubation in the CO.sub.2 incubator at 37.degree. C. is 
continued for 10-14 days. 
In those wells in which grown fused colony-forming cells are found, 1 ml of 
the supernatant is discarded and the same volume of HT medium (HAT medium 
minus aminopterine) is added, followed by medium replacement with fresh 
portions of HT medium at 24-hour intervals for 2 days. 
After 3 to 4 days of cultivation in HT medium, a portion of the culture 
supernatant is collected and assayed for antibody titer relative to human 
mesothelial cells by the above-mentioned immunocytochemical staining. In 
like manner, the reactivities with normal human cells or tumor cells are 
also determined, and those wells in which selective reactivity with human 
mesothelial cells are selected. 
Cloning is repeated twice by limiting dilution technique and those clones 
for which high antibody titer value are stably obtainable relative to 
human mesothelial cells, are selected as anti-human mesothelial cell 
monoclonal antibody-producing hybridoma cell lines. 
(4) PREATION OF MONOCLONAL ANTIBODY 
Eight- to ten-week-old female BALB/c mice treated with pristine 
[intraperitoneally administered with 0.5 ml of 2, 6, 10, 
14-tetramethylpentadecane (pristane) and fed for 2-weeks] are 
intraperitoneally injected with the anti-human mesothelial cell monoclonal 
antibody-producing hybridoma cells obtained in procedure (3) above at a 
dose of 2-4.times.10.sup.6 cells per animal. In 10-21 days, the hybridoma 
cells produce ascites carcinoma in the mice. The ascitic fluid is 
collected from such mice, centrifuged (3,000 rpm, 5 minutes) to remove 
solids, subjected to salting out with 50% ammonium sulfate, dialyzed 
against 0.04M phosphate buffer (pH 8.0) supplemented with 0.03M NaCl, and 
passed through DE52 .RTM. (product of Whatman) column. An IgG fraction is 
collected and used as a purified monoclonal antibody. 
The isotype of the antibody is determined by Ouchterlony's method (double 
immunodiffusion) [Seibutsukagaku Jikkenho (Methods in Experimental 
Biochemistry), vol. 15, Introduction to Experimental Immunology, p. 74, 
Gakkai Shuppan Center, 1981]. 
The quality of protein is estimated by the Folin's method, followed by 
calculation based on the absorbance at 280 nm [1.4 (OD.sub.280) 
approximately corresponds to 1 mg of immunoglobulin per ml]. 
The monoclonal antibody thus obtained is effective in cytological diagnosis 
of expectoration for lung cancer and cytological diagnosis of pleural 
effusion for lung cancer and cytological diagnosis of ascitic fluid for 
various abdominal cancers by the above-mentioned immunocytochemical 
staining.

Certain specific embodiments of the present invention are illustrated by 
the following examples. 
EXAMPLE 1 
(1) PREATION OF ANTIBODY-PRODUCING CELLS 
Eight-week old female BALB/c mice (Shizuoka Agricultural Cooperative 
Association for Laboratory Animals) were intraperitoneally administered 
and immunised with human mesothelial cells (2.times.10.sup.6 cells per 
animal) as an antigen, together with aluminium hydroxide gel (2 mg per 
animal) and killed B. pertussis vaccine (Chiba Serum Institute; 
1.times.10.sup.9 cells per animal) as an adjuvant. The same antigen 
administration was repeated 3-5 times at a dose of 2.times.10.sup.6 cells 
per animal at 1 to 2 week intervals without an adjuvant. From among these 
immunized mice, those mice whose antisera intensely reacted with human 
mesothelial cells were selected, and spleen cells were prepared from such 
mice and submitted to cell fusion. 
(2) PREATION OF MYELOMA CELLS 
The 8-azaguanine-resistant murine myeloma cell line P3-U1 was cultivated in 
normal medium to thereby secure not less than 2.times.10.sup.7 cells at 
the time of cell fusion, and submitted to cell fusion as a parent strain. 
(3) HYBRIDOMA PRODUCTION 
The spleen cells and myeloma cells obtained in (1) and (2), respectively, 
were used in a ratio of 5:1 and subjected to fusion according to the 
procedure previously described. After cultivation in HAT medium at 
37.degree. C. in 5% CO.sub.2 incubator for 14 days, fused cells were 
selected, and after change of the medium to HT medium, cultivation was 
continued. Based on the results of anti-human mesothelial cell antibody 
titer determination, active wells were selected, and after change of the 
medium to normal medium, cloning was repeated twice by the limiting 
dilution technique. The clone for which antibody titer value was stably 
obtainable relative to human mesothelial cells was selected as anti-human 
mesothelial cell-producing hybridoma cell line KM-277. The hybridoma cell 
line KM-277 has been deposited with the European Collection of Animal Cell 
Cultures, Great Britain, as of October 23, 1986 as ECACC No. 86102303 
under the Budapest Treaty. 
(4) MONOCLONAL ANTIBODY PURIFICATION 
Eight-week-old female BALB/c mice treated with pristane were 
intraperitoneally injected with the hybridoma cell line KM-277 obtained in 
(3) at a dose of 4.times.10.sup.6 cells per animal. In 10-21 days, the 
hybridoma produced ascites carcinoma. The ascitic fluid was collected from 
ascitic fluid-bearing mice (5 to 10 ml per animal), centrifuged to remove 
solids (3,000 rpm, 5 minutes), subjected to salting out with 40% ammonium 
sulfate, dialyzed against 0.04M phosphate buffer supplemented with NaCl 
(0.03M), and passed through a DE52 .RTM. (product of Whatman) column (bed 
volume: 50 ml) at a flow rate of 20 to 30 ml/hr. An IgG fraction was 
collected and used as the purified antibody. 
(5) ANTIGENIC SPECIFICITY OF KM-277 
The specificity of the thus-obtained monoclonal antibody KM-277 for three 
human mesothelial cell samples, two human peripheral lymphocyte cell (PBL) 
samples and various cancer cell lines was investigated using 
immunocytochemical staining. 
The results are shown in Table 1. 
TABLE 1 
______________________________________ 
Staining level by 
Sample or Cell line KM-277* 
______________________________________ 
Mesothelial cell sample A 
++ 
B + 
C ++ 
PBL sample A - 
B .+-. 
Myeloma cell line SK-Ly-18 
- 
T cell leukemia cell lines HSB-2 
- 
MOLT-3 - 
Lung cancer cell line PC-10 
- 
PC-7 - 
PC-13 - 
PC-6 - 
Gastric cancer cell line MKN-1 
- 
KATO-III - 
Pancreatic cancer cell line HPAF 
- 
Malignant melanoma cell line SK-28 
- 
Fetal lung cell line L-132 
- 
Fetal skin cell line Detroit 551 
- 
______________________________________ 
*Staining level observed with microscope. 
As shown in Table 1, KM-277 was very specifically reactive with the human 
mesothelial cell but not with PBL and the various cancer cell lines. 
EXAMPLE 2 
In this example, cells in sputum samples and pleural effusion samples 
derived from three patients with lung cancer, and cells in ascitic fluid 
samples derived from four patients with gastric cancer were stained by the 
immunocytochemical staining using KM-277. The mesothelial cells in smeared 
preparations (smear) were stained and cancer cells in all the preparations 
were not stained. Therefore, cancer cells can be distinguished from 
mesothelial cells in smears.