JPH022393A - Anti-human ovarian carcinoma monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:An antibody belonging to the class IgG, reactive to mucinous ovarian adenocarcinoma, serous ovarian carcinoma, lucidus ovarian carcinoma, mammary cancer and uterine cancer, having a protein as an antigen determinant and specifically bonding to a protein of the ascites of a patient of ovarian carcinoma having a molecular weight of >=1,000,000. EXAMPLE:Anti-human ovarian carcinoma monoclonal antibody KOC-708. USE:A diagnostic and remedy for the above cancers. Reacting with the above cancers at a high rate. PREPARATION:A hybridoma capable of producing the antibody KOC-708 [e.g., hybridoma strain KOC-708 (FERM BP-1762)] is cultured or administered to a mouse to form ascites and the objective antibody is separated from the cultured product or the ascites.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anti-human ovarian cancer monoclonal antibody and a method for diagnosing and treating cancer using the same.

INDUSTRIAL APPLICABILITY The present invention can be used for pathological diagnosis and treatment of ovarian cancer, breast cancer, and uterine cancer, and can be used in the fields of diagnostic agents and medicine.

Ovarian cancer is one of the most difficult cancers to detect, along with pancreatic cancer.

Since the ovary is a 2g organ located deep in the body, there are few good diagnostic methods. It is also difficult to distinguish between tumors. Among ovarian cancers, regarding serous ovarian adenocarcinoma,
Tumor marker CA-125 has been reported [Journal of Clinical Investigation (J.
Cl1n,! nvest, ) 68, 1331 (19
81)), its clinical usefulness is being confirmed, but its specificity for serous ovarian adenocarcinoma is extremely high, making it ineffective for diagnosing mucinous ovarian adenocarcinoma. Carcinoembryonic antigen (carcinoembryonic antigen) is a marker for mucinous ovarian adenocarcinoma.
ionic antigen (CE8)], but the positive rate is low and it is difficult to say that it is necessarily practical. Monoclonal antibodies that are reactive with mucinous ovarian adenocarcinoma and mucinous ovarian gland)1÷, monoclonal antibodies that are reactive with all ovarian cancers, including serous ovarian adenocarcinoma, in immunohistological analysis. is known [Cancer Research]
cer Res,) 60, 787 (1!185)
, (1985), Journal of National
Cancer Institute (,J,N,C,!,
) 76, 781 (1!386)) is a monoclonal antibody that reacts with all ovarian cancers, including serous and mucinous ovarian adenocarcinoma, and also with breast cancer and uterine cancer. No sexual antibodies are known.

Problems to be Solved by the Invention If there is an Ift clonal antibody that reacts with ovarian cancer in general, and also with breast cancer and uterine cancer, it would be extremely useful in the diagnosis and treatment of cancer in the field of obstetrics and gynecology. be.

Means for Solving the Problems The present inventors have demonstrated that monoclonal antibodies produced by hybridomas established using a high molecular weight fraction of ascites of human ovarian cancer patients as an immunogen can be used to target ovarian cancer cell lines and ovarian cancer tissues of various histological types. The present inventors have discovered that the present invention reacts with uterine cancer and breast cancer tissues at a high rate, and is useful for pathological diagnosis and treatment of ovarian cancer, uterine cancer, and breast cancer, and has completed the present invention.

The present invention involves creating a hybridoma by fusing mouse tile cells immunized with a high-molecular fraction of ascites from a human ovarian cancer patient with mouse myeloma cells, and selecting a monoclonal antibody that is reactive against human ovarian cancer. Then, the hybridoma is cultured in a medium or administered to a mouse to cause ascites cancer in the mouse, and the 2'
Anti-human ovarian cancer monoclonal antibodies obtained by collection from A'B feed or ascites are provided.

The rn clonal antibody of the present invention belongs to the IgG class,
It is reactive to mucinous ovarian adenocarcinoma, succulent ovarian adenocarcinoma, clear cell ovarian cancer, breast cancer, and uterine cancer, and is a molecular m-molecule in ascites of ovarian cancer patients.
,000,000 or more, and recognizes the protein as an antigenic determinant.

As a specific example of the monoclonal antibody of the present invention, hybridoma cell line KOC-708 (FCIIM [1
1'-1762) was produced by Zuro KOC.
-708 is mentioned.

The method for producing the clonal antibody of the present invention will be explained in detail below.

[Animal Immunization and Preparation of Antibody-Producing Cells Mice aged 3 to 10 weeks, preferably 8 weeks, are immunized with the polymeric fraction of ascites from a human ovarian cancer patient, and the animal's limbs, lymph nodes, and peripheral lymph nodes are inoculated. Antibody-producing cells in the blood are produced 'a. It is preferable to use mice to be immunized that have been pretreated with human normal range 2: to make them immune tolerant. The immunization method involves injecting the animal subcutaneously, intravenously, or intraperitoneally with an appropriate adjuvant, such as Freund's complete adjuvant (Co
Freund's Adjuvant)
Aluminum hydroxide gel and Bordetella pertussis vaccine, etc.] together with the polymer fraction of ascites of human ovarian cancer patients (10 to
500 μg/mouse). Thereafter, the antigen is administered 2 to 5 times every 1 to 2 weeks. Blood was collected from the fundus venous plexus on days 3 to 7 after each immunization, and the enzyme immunoassay method (ELI) was used to demonstrate that the serum reacts with human ovarian cancer.
SΔ): Igaku Shoin 1976] etc.

Enzyme immunoassay: 96 large EIA plates [Flow Laboratories (
(manufactured by Flow Laboratories)], membrane components of one normal or tumor cell tissue (10 to 10% protein content)
l, o o μg/ml containing membrane fragments) at 100
Dispense ~200μtl/well, leave it at 4℃ for 1-2 nights, remove the supernatant, and add resin water or PBS.
(Ninatrium phosphate 14.83g, potassium phosphate 0.21g 1 table salt 7. (i5g, distilled water 1f, p) 1
After washing thoroughly with 7.2), dispense 100-200 μl/well of 1% BSΔ (bovine serum albumin)-P)33 and leave it at 4°C for 1-2 nights to separate the remaining proteins on the plate. The binding residues were blocked. After that, the B.S.A.
- After discarding the PBS and thoroughly washing with resin water or PBS, use the BSΔ-PI3S"C diluted sample as the first antibody (mouse serum, hybridoma jiζ culture supernatant, mr+"l Ip, and sexual antibody)', r l
Dispense OOμl/well and incubate at 4°C for 1t. 2M NaC once with resin water! After washing six times with the solution,
Rabbit anti-mouse immunoglobulin Ig as second antibody
G-peroxidase conjugate [manufactured by Dako (DheKO),
100μQ of 100 times diluted solution from Kyowa Medex
Dispense into each well and leave at room temperature for several 1d for 2 hours.

After washing with PBs, add 550 mg of ABTS2!
(a solution prepared by adding 1 μQ/ml of hydrogen peroxide to a solution dissolved in 1 solution (pH 4, 2) If immediately before use), and the color development is measured by the absorbance of OD 4151111. At this time, ascites of an ovarian cancer patient Mice that strongly react to the high-molecular fraction are used as human ovarian cancer-immunized mice and as a source of antibody-producing cells for the production of No. 1 hybridomas.

When using cells themselves as antigens for enzyme immunoassay, Falcon
Culture target cells in 3072 plates, 0.25%
Add geltaraldehyde-PBS, leave at room temperature for 1 to 2 hours, wash thoroughly with PBS, and add 1% BSA-P.
Add 100 to 200 μQ of I35, leave to stand for 2 hours, wash thoroughly with resin water or PBS, and use the plate to measure antibody titer in the same manner as using a general antigen-coated plate.

For cell fusion, 3 to 4 days before fusion treatment, immunized mice were treated with 1O of the high molecular weight fraction of ascites from human ovarian cancer patients.
~400 μg/mouse is administered into the llu cavity, IIQI visceral cells are removed, and lung cells are prepared. Shred the innards in MEM (manufactured by Hinaga Pharmaceutical Co., Ltd.), loosen with a bottle set,
Centrifuge at 0 rpm for 5 minutes, discard the supernatant, and add Tris-ammonyl chloride (+1 solution) (p fl 7.65
) for 1 to 2 minutes to remove red blood cells, washed three times with MEM, and provided as hepatocytes for fusion.

The polymer fraction of ascites from a human ovarian cancer patient is prepared as follows.

That is, after thawing ascites from a patient with papillary gland lid among serous ovarian adenocarcinomas that had been frozen and preserved at -80°C, 3,000
Centrifugation was performed at 0 rpm for 10 minutes, and the supernatant from which solid JF2 was removed was passed through a Cellulofine GCI, -2000SF (Seikagaku Corporation) column to collect the high molecular fraction with a molecular weight of 000.000 or more. It is collected and used as a high molecular fraction of ascites from ovarian cancer patients.

In addition, both fractions around the molecule h1,150,000 are collected and used as a low molecular fraction.

(2) Preparation of myeloma cells As myeloma cells, established cell lines obtained from mice are used. For example, °, 8-azaguanine-resistant mice (B
ΔLB/c derived) myeloma cell line P3-X638g8-U
l(P3-Ul)C Current Topics in Microbiology and Immunology! (Curr.
ent Topics in Microbiolog
y and Immunology -1) ] C European Journal of Immunology (Eur.
aρean J.

1mmunology) 6. 511-519
(1976)], SP 210-Ag
14 (SP-2) C Nature (Naire)
276, 269-270 (1978)), P.
3-X63-Ag8653 (653) C Journal of Immunology (J, Immunology)
123.1548-1550 (1979) ), P
3-X 63-Ag 8 (X63) [Fuichi +
-(Nature) 25[i, 495-497
(1975):1 etc. are used. These cell lines were grown in 8-azaguanine medium 1 [PM! - Glutamine (1,5rr+M) in 1640 medium.

2-mercaptoethanol (5x IO-5M).

The cells are subcultured in a normal medium containing dientamicin (10 μg/ml) and fetal bovine serum (FC3, manufactured by C3L) (10%), and a medium containing 8-abguanine (15 μg/ml). Passage into normal medium 3-4 days before cell fusion to ensure a cell number of 2X10' or more on the day of fusion.

(3) The antibody-producing cells immunized in cell fusion (1) and the myeloma cells obtained in (2) were thoroughly washed with MEMJ7~ or PBS, and the number of cells was determined to be: antibody-producing cells; myeloma cells -5~ Mix to 1oll and centrifuge (1.20 rpm, 5 minutes)
After that, the supernatant was discarded, and the precipitated cell group was thoroughly loosened, and then 2 g of polyethylene glycol 1000 (PEG-1,000), MEM was added at 37°C while stirring.
A mixture of 2 ml and dimethyl sulfoxide 0.7 ml
, 2-1 ml/IQ3 antibody-producing cells, add 2 ml of MEMI several times every 1-2 minutes, then add MEM to bring the total volume to 50 ml. Centrifugation (900r
pm, 5 minutes), discard the supernatant, gently loosen the cells, and add normal medium (RPM+-1 (i40.FC3IO
%) and gently suspend the cells by suctioning and blowing with a volumetric pipette.

This ufQ solution is dispensed at 1 ml/well into a 24-well culture plate, and cultured at 37° C. in a 5% CO2 incubator for 24 hours. 1 ml/well of HAT medium in N culture plate [hyboxanthin (10-'?v4) in normal medium,
Thymidine (medium supplemented with 1,5X 1 ('I-'M) and mitubzolin (/lX10-'M))] was added,
Incubate for an additional 24 hours. After that, every 24 hours for 20 days,
On culture? +”t l m Discard l and newly create the same amount of 11
Add 8 Tjz~ ground, C02 incubator AA 37℃
Culture for ~14 days at lO.

For holes where fused cells that have grown in colonies are observed, discard the double 1 +++ l and add IIT medium (HΔ'F
The same amount of the medium (from which aminopterin has been removed) is added, and the medium is converted to HT medium every 24 hours for the next 2 days.

After culturing in H'r medium for 3 to 4 days, a portion of the culture supernatant is taken and the antibody titer against the macromolecular fraction of ascites from a human ovarian cancer patient is measured by the enzyme immunoassay method described above. At this time, the reactivity with human normal cell tissue or its membrane components is also measured using the same method, and those that specifically react with human ovarian cancer cell tissue or its 11 components are selected. For the holes that strongly reacted with fur tissue or its membrane components and did not react with human normal cell tissue or its membrane components, cloning was repeated twice by limiting dilution method to stabilize the human ovarian tissue. human ovarian cancer cells 1
A hybridoma strain in which a strong antibody titer was observed in the tissue or its membrane components is selected as an anti-human ovarian cancer monoclonal antibody-producing hybridoma strain.

(4)! Preparation of r'-clonal antibodies Bristan treatment [0.5 ml of 2,6,10,14-tetramethylbentatecan (rrisLane) was administered intraperitoneally;
2 to 4 XIO' cells/mouse of the anti-human ovarian cancer monoclonal antibody-producing hybridoma cells obtained in (3) are injected intraperitoneally into 8- to 10-week-old nude female mice kept for 2 weeks. The hybridoma turns into ascites cancer in 10 to 21 days. Ascites was collected from this mouse and centrifuged (3,000
Orpm for 5 minutes) to remove solids, salted out with 50% ammonium sulfate, and added with 0.04M phosphate buffer (p
H8,0,0,03MNaCj! After dialysis with D
Passed through a column of E 52 (Whatman contract),
Both IgG components are collected and used as a purified clonal antibody.

Determination of antibody isotypes was performed by Ouchterlony (Oucht.
erlony) Method (Double Immune Expansion 11 Method) (Introduction to Immunology Experiments, Biochemistry Experiment Methods 15, published by Gakkai Publishing Center, 74
Page, 1981).

The amount of protein was quantified using the Folin method and the absorbance at 280 nm [1,4(OD2so)-Itonoglobulin I+
p [ng/ml].

The specificity of the obtained l't clonal antibody was determined by its reactivity with normal or tumor tissue or its membrane components derived from various human Ili organs obtained from multiple specimens, and by culture of various human normal or tumor cells. The reactivity with cell lines or human fetal cell culture lines or their membrane components, and the reactivity with conventionally known carcinoembryonic antigens (e.g., CEΔ) can be determined using cumulative immunoassay, immunohistochemical determination, or CABC method. ), etc., and in all measurement methods, cancer other than human ovarian cancer is
Choose something that doesn't react as much as possible.

(5) Antigen analysis When carrying out the enzyme-linked immunosorbent assay and immunohistochemical standard methods described above, antigens (polymer fraction of ascites of ovarian cancer patients, ovarian cancer JΔ cultured cell line, oval carcinoma tissue) were analyzed using neuraminidase ( nc
uraminidase).

After pretreatment with an enzyme such as protease or a reagent such as periodic acid, the reaction with a monoclonal antibody is avoided, and the difference in reactivity between the original antigen and the monoclonal antibody without such treatment is determined. The chemical properties of the antigen (the chemical properties of the antigenic site recognized by monovalent antibodies) were clarified. That is, if antigenicity disappears by neuraminidase treatment, it is assumed that sialic acid is involved in the antigenic determinant, if it disappears by protease treatment, protein, and if it disappears by periodic acid treatment, it is assumed that sugar chains are involved in the antigenic determinant. .

The Western plotting method used in the examples is the method of Tovein et al. [Proceedings of the
National Academy of Science (Pro
c, Natl, 8 cad.

Sci.) 76, 4354 (1979)] was used. That is, the macromolecular fraction of ascites of ovarian cancer patients or
The low molecular weight fraction was electrophoresed using SDS polyacrylamide gradient gel (4-20%) (DEFCO!!), and the resulting gel was superimposed on a nitrocellulose membrane, and the electrophoretic transfer/plotting device ( 112 (4j liquid C3g/j)l-1)su(hydroxymethyl)aminomethane, 144g/i! 10 in glycine, 20% methanol, pI-18,3]
Electricity is applied to the membrane at 40 V (ioomA) below ℃ to transfer the protein to the membrane. After the nitrocellulose membrane is air-dried, it is blocked with a 3% gelatin solution. After washing t1+, it is immersed in the Vibrid-7 culture supernatant and kept at room temperature. Incubate for 2 hours with rabbit anti-mouse I and nog iff purine I.
gG-peroxidase conjugate [manufactured by DAKO>, sold by Kyowa Medex.

400x dilution solution] and allowed to react at room temperature for 2 hours.

After washing, add peroxidase coloring solution (100 ml of Tris-HCl buffer saline (TI3S) [2.42 g of Lis(7-do-t1-xymethyl)aminomethane),
29.24g of salt.

60 μ of 30% hydrogen peroxide in 1 part distilled water, pH 7.51
The solution containing C was immersed in 20 ml of chilled methanol in HRP Color Development Reagent (manufactured by BIO-RAD) [a mixture of a solution containing iomH and a solution containing iomH at room temperature] while shielded from light to develop color. to stop the reaction.

Pathological diagnosis using monoclonal antibodies is performed by immunohistochemical staining [ABC method described in [Enzyme antibody method] (Watanabe J Table-1, edited by Nakane Ho, Gakugei Kikaku, 1985), page 106].

Examples of the present invention will be shown below.

Example 1゜(1) Preparation of immunogen - Of the ovarian adenocarcinomas frozen at 80°C,
After melting the ascites of a patient with papillary adenocarcinoma, 3.00 Orpm,
Centrifuge for 10 minutes and remove 8 ml of supernatant to remove solids.
, 5M NaCl! 10mM phosphate buffer (pH 7) containing
Cellulofine GCL-2000SF (manufactured by Seikagaku Corporation) with a bed volume of 750 m1 equilibrated with , 2)
Kara!・The polymer fractions (void fractions; fraction numbers 41 to 50) with molecules ml, 000,000 or more were collected, and this was used as the polymer fraction of ascites of a human ovarian cancer patient. Cell [1 fine GCL2000 S
The elution pattern of I'' is shown in FIG.

(2) Preparation of antibody-producing cells Human normal lung membrane components (100 μg protein/mouse) were added to the
It was administered intravenously to newborn BALB/c mice (manufactured by Shizuoka Experimental Animals) within 4 hours.

After a week had elapsed, the high molecular weight fraction 10 (jμg (protein equivalent) of 7 mice prepared in (1) of human ovarian cancer patients was given to mice with aluminum hydroxide Udokel 2 mg/mouse, killed Bacillus pertussis vaccine lx I O/ It was administered intraperitoneally with the animals.Hereafter, 1 to 2
Every other week, 100 μg of the same antigen (protein equivalent) for 7 animals
Immunized ~5 times. Among these immunized mice, mouse 4 immunized mice were selected whose anti-component 11'7 strongly reacted with the macromolecular fraction of ascites of human ovarian cancer patients, and hepatocytes were prepared from these mice. Subjected to fusion.

(3) Preparation of mouse myeloma cells The 8-abguanine resistant mouse myeloma cell line P3-11 was cultured in a normal medium, and 2×10′ or larger cells () were used as a parent strain for cell fusion.

(4) Preparation of hybridoma The hepatocytes and myeloma cells obtained in (2) and (3) were combined into 5:
1), fused as described above, (cultivated in ΔT medium at 37°C for 14 days under CO25% to select fused cells), changed to IT medium and further cultured, human ovary We measured the antibody titer against cancer, selected an active hole, changed it to a normal JS site, repeated the cloning twice, and determined that it was human by enzyme-linked immunosorbent assay and immunohistological determination method (ΔBC method). Human eggs do not react at all with normal cells and tissues! !
The hybridoma line KOC-708, which produces monoclonal antibodies reactive with cancer, was selected.

(5) Purification of monoclonal antibodies Hybridoma strain KOC708 obtained in (4) was injected intraperitoneally into 8-competent nude chick mice treated with Bristane at 4×10′ cells/mouse.

After 10-21 days, the hybridoma turned into ascites cancer. Collect ascites from mice with ascites (5-10ml/
solid matter was removed by centrifugation (3,000 rpm, 5 minutes). Salted out with 40% 0% ammonia sulfate, 0.04M phosphoric acid 1)2tJj solution (p)-18,
After dialysis with 0,0,03M NaCj+), DE
52 (manufactured by WhaLman) (Betvoluno・
50 m1) was passed through the column at a flow rate of 20 to 30 m1/hour to collect both IgG and f+'i! j! 'r'-clonal antibody.

(fi) The specificity of KOC-708.
The reactivity W properties of No. 08 are shown in Table 1.

Measurements were performed by enzyme immunoassay (ELISA) as follows.

A place to target the macromolecular fraction of ascites from ovarian cancer patients. 96-hole plate for enzyme immunoassay) (Linbro!!
A) IOμg/ml egg E fm patient 111! Add 50μQ/well of water polymer fraction, 2118f at 37υ
It was left to stand overnight at ill or 4°C and fixed to the bottom of the plate hole. After washing with PBS, add 10% PBS containing IO% FC3.
Dispense 0 μQ/well to protect active residues on the plate.

After washing with PBS, 50μQ of first antibody (KOC-708)
/ hole and left at 37°C for 1 to 2 hours or at 4°C overnight to react with the target antibody. 0.05%7ween2
The plate was washed five times with 13BS containing 0 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) to remove unreacted antibodies. Peroxidase-conjugated rabbit anti-mouse immunoglobulin (Miles) was used as the second antibody.
-Ycda: 200fff dilution) 50μQ/well was added and reacted at 37°C for 1 hour. 0.05%Twee
After washing with n-20-containing 1'BS five times, it was washed three times with resin water. Start the reaction by adding 50 μQ/well of BTS to
The reaction was stopped by adding 50 μQ/well of 5% sodium lauryl sulfate in water.

When targeting cultured cells: Culture the cells in JΔ culture 9G hole plate (1, i sieve "manufactured by Company 0"), and when they reach confluence, use the above ovary 11
; The reaction was carried out in the same manner as in the case of the polymer fraction of condensate from 4 patients and 1. However, the reaction conditions for both the first antibody and the second antibody are at room temperature at 30°C.
After color development, the reaction was stopped by transferring the reaction solution to a 96-well plate for analysis.

In the case of antigen CEΔ, the same procedure as in the case of tissue was performed except that CEA was used instead of the polymer fraction of ascites of an ovarian cancer patient. In either case, /1190nm is the reference wavelength and 415
The absorbance at nm was measured.

As shown in Table 1, KOC-708 reacted with various histological types of ovarian cancer at a high rate. However, it does not react with CEA, indicating that it is different from an anti-CEA antibody.

Example 2 Formalin-fixed, paraffin-embedded tissue sections of various normal adult, fetal organs and cancer tissues sliced into five rhinoceros using a microtome were fixed on glass slides coated with egg albumen gamma rubumin, deparaffinized with xylene, and then treated with alcohol. It was made hydrophilic in stages with water. 5 minutes with resin water, 0.3
Endogenous peroxidase was blocked by standing at room temperature for 30 minutes in methanol containing %H,C)a.

Next, the sections were washed with PBS''Q for 20 minutes, and then allowed to stand for 20 minutes at room temperature in diluted normal horse serum. After washing, the oocyte milk diluted biotinylated antibody [biotinylated heron anti-1gG antibody (
(Manufactured by Vector)] was allowed to incubate for 330 minutes, and after further washing,
Avidin-biotin-peroxidase complex (Vector Co., Ltd.) was reacted for 30 minutes. After thorough washing, peroxidase substrate [0.02% + (0.1
M) 0.1% diaminohenzidine detrahydrochloride (diaminobcnzidine LeLrahyd) adjusted with a mild lysate solution (pH 7,2).
rochloride)] for 22 minutes, and the reaction was stopped in water cooling. After hematoxylene staining, the specimen was dehydrated with alcohol-water 41 and xylene, fixed with Canada balsam, and examined under a microscope. The results are shown in Table 2.

As shown in Table 2, KOC-708 reacted strongly with ovarian cancer tissue at a high rate. Moreover, KOC-708 also reacted strongly against uterine cancer and breast cancer at a high rate. Normal/fetal tissues (heart, pancreas, stomach,
Although some cells reacted very weakly with some cells (large intestine, kidney, thyroid, brain, coccyx, liver, small intestine, etc.), there was no reaction at all with other tissues.

Example 3! In order to analyze the antigen recognized by the n-clonal antibody KOC-708, a polymer fraction of ascites from an ovarian cancer patient was treated with the following various enzymes and reagents, and then its reactivity with KOC-708 was examined.

Fermentation-+: and reagents trypsin (GrBCO, 2.5% solution) in PBS, 0.25% neuraminidase (Boehringer Mannheim), 0.1M acetate buffer (pH 4,5) - 3mM C
aCA, medium 0. lU/m α-L-fucosidase (Boehringer Mannheim, Inc.) 0,1 in 0.1M phosphate buffer (p!(6,3))
U/ml Propase (manufactured by Sigma) 10 U/ml in 0.1M phosphate buffer (pH 7,2)
m1 NalO4 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) PBS [1150mM Polymer fraction of ovarian cancer patient 1 condensate ('fFL white amount lOμg
/ ml) was dispensed into ErA plates (manufactured by Linbro) at 5 uQ/well, and left overnight at 4°C.
Wash 3 times with BS and add 200 μQ of 1% BSΔ-PBS.
/ well: (Leave at room temperature for 0 minutes to 2 hours, PBS
After washing three times with water, 50 μQ/well of the above enzyme solution or reagent solution was dispensed and reacted at 37° C. for 1 hour. Then 5 on PBS
Wash it twice! p-O-ionic antibody KOC-708 (1
0 μff/ml) was dispensed at 50 μσ/well and left at 4° C. overnight.

After washing 5 times with Tween-20-PBS (Twecn-20 manufactured by Wako Junkuwa Kogyo Co., Ltd.), peroxidase 4 antibody mouse IgG (manufactured by Dako Co., Ltd., sold by Kyowa Medex, 400-fold diluted solution) was added. Add 50μQ/well, react for 2 hours at room temperature, wash 5 times with 'l'we en-20-PBS, add 100μQ of Al3TS substrate solution, and incubate for 30 minutes.
After a minute of reaction, the absorbance at 415 nm was measured.

As shown in FIG. 2, antigenicity was abolished by trypsin and protease treatment. From this result, monoclonal antibody KOC-708 uses protein as an antigenic determinant.
= 1.

Example 4 KOC-7 was determined by the Western blotting method described above.
The reactivity with the high-molecular fraction and low-molecular fraction of ascites from a patient with 08 ovarian cancer was investigated. The results are shown in Figure 3. KOC-
708 reacted with proteins with molecules of more than m,000.000 in the high molecular fraction.

Effects of the Invention According to the present invention, monoclonal antibodies useful for pathological diagnosis and treatment of ovarian cancer, breast cancer, and uterine cancer are provided.

[Brief explanation of the drawing]

The m1 diagram is the cell f1 Fine GCI-2000 of Example 1.
The elution pattern of SF is shown. FIG. 2 shows the results of antigen recognition analysis of monoclonal antibody KOC-708. The figure shows neuraminidase 0, IU/
ml, mouth contains α-17-fucosidase 0. lU/ml. ○
is trypsin 0.25%, mu is protease l0LI/
ml, △ is Naro. 50mM treatment is shown. Figure 3 shows the results of examining the reactivity of monoclonal antibody KOC,708 using the Western blotting method in Example 4. Δ) Amido black staining, B) KOC-
708 immunostaining. In both △) and B), ■ and ■ are molecular weight markers (myosin: 200 Kd, β-power °
Lactosidase: I16.25Kd, Funas fluorylase B: 92.5Kd, Bovine serum albumin:
66.2 Kd, ovalbumin: 45 Kd), ■ is a high molecular fraction of ascites of an ovarian cancer patient, and ■ is a low molecular fraction of ascites of an ovarian cancer patient. Patent applicant (+02) Kyowa Hakko Kogyo Co., Ltd. Fig.

Claims (5)

[Claims] (1) Belongs to the IgG class, has reactivity to mucinous ovarian adenocarcinoma, serous ovarian adenocarcinoma, clear cell ovarian cancer, breast cancer, and uterine cancer, and has a molecular weight of 1,000,000 in ascites of ovarian cancer patients.
An anti-human ovarian cancer monoclonal antibody that specifically binds to the above proteins and whose antigenic determinant is a protein. (2) The anti-human ovarian cancer monoclonal antibody KOC-708 according to claim (1). (3) Hybridomas capable of producing anti-human ovarian cancer monoclonal antibody KOC-708 are cultured in a medium or administered to mice to form ascites, and the anti-human ovarian cancer monoclonal antibody is injected into the culture or ascites. A method for producing anti-human ovarian cancer monoclonal antibody KOC-708 by accumulating KOC-708 and collecting it from the culture or ascites. (4) A hybridoma producing the monoclonal antibody according to claim (1). (5) Hybridoma strain KOC-708 (FBRMBP
-1762).