JPH0424996B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a monoclonal antibody against human ovarian cancer and a hybridoma producing the same. More specifically, the present invention relates to hybridomas produced by the fusion of antibody-producing cells immunized with human ovarian cancer cells and myeloma cells, and which belong to the class of IgG ₁ having the following properties: Regarding monoclonal antibodies: (1) Reacts with mucinous cystadenocarcinoma cells, endometrioid carcinoma cells, and mesonephroid carcinoma cells; (2) Reacts with serous cystadenocarcinoma cells and mucinous cystadenocarcinoma cells (3) Does not react with cells of uterine body cancer other than ovarian cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, colorectal cancer, liver cancer, and seminoma; (4) Ovarian, brain, heart, lung , does not react with normal tissue cells of the liver, stomach, intestines, pancreas, kidneys, uterus, fallopian tubes, and testicles; (5) Does not degrade with trypsin. Furthermore, the present invention relates to hybridomas formed by the fusion of antibody-producing cells immunized with human ovarian cancer cells and myeloma cells and producing monoclonal antibodies belonging to the class of IgG ₁ having the following properties: : (1) Reacts with mucinous cystadenocarcinoma cells, endometrioid carcinoma cells, and mesonephroid carcinoma cells; (2) Does not react with serous cystadenocarcinoma cells and mucinous cystadenocarcinoma cells; (3) Ovarian Does not react with cells other than tumors such as uterine body cancer, lung cancer, breast cancer, stomach cancer, pancreatic cancer, renal cell cancer, colorectal cancer, liver cancer, and seminoma; (4) Ovarian, brain, heart, lung, liver, stomach, intestine , does not react with normal tissue cells of the pancreas, kidneys, uterus, fallopian tubes, and testicles; (5) Does not degrade with trypsin. It is well known that in recent years, efforts have been made to utilize highly specific monoclonal antibodies as diagnostic and therapeutic tools. The present inventors also completed the present invention as a result of intensive research to establish a monoclonal antibody with high specificity for human ovarian cancer and to establish a hybridoma that produces this monoclonal antibody. did. Ovarian cancer is a type of gynecological cancer that is not as common as uterine cancer, but because it is an intra-abdominal tumor, early diagnosis is often difficult and it is characterized by a poor prognosis. Therefore, in order to improve the treatment results of ovarian cancer, it is necessary to establish a reliable early diagnosis method and perform appropriate treatment.Recently, Bast et al.
Bhattacharya et al.
and Milstein, C.: Eur. J. Immunol., 6, 511−.
519, 1976) to create a monoclonal antibody (hereinafter abbreviated as MoAb) against ovarian cancer-related antigens, and published basic results for diagnosing and treating ovarian cancer using immunological methods. did. first,
Bast et al. created MoAbOC-125 for cultured cells of human serous cystadenocarcinoma and analyzed it using a fluorescent antibody method. reported that it is a MoAb against cancer-related antigens (J.Clin.
Invest., 68, 1331-1337, 1981). However, Bast
In a second report by Kabawat et al., OC-125 reacted with endometrioid carcinoma, mesonephric carcinoma, and benign serous cystadenocarcinoma as well as with serous cystadenocarcinoma, but did not react with mucinous cystadenocarcinoma. Therefore, we reported that OC-125 is a MoAb against a surface antigen common to three types of ovarian tumors: serous, endometrioid, and mesonephric (Am.J.Clin.
Pathol., 79, 98-104, 1983). on the other hand,
Bhattacharya et al. created MoAb ID3 and ID5 using an extract of a poorly differentiated cystadenocarcinoma that could not be distinguished as serous or mucinous as an immunogen. these are,
In an absorption experiment using Solidphase RIA, it reacted only with mucinous cystadenocarcinoma and did not react with other organ cancers at all.
reported that it was a MoAb (Cancer Research,
42, 1650-1654, 1982). But these
All MoAbs have the disadvantage that they react with normal tissue, malignant tumors other than ovarian cancer, and benign tumors of the ovary, but do not react with mesonephric cancer. As a result of various studies, the present inventors succeeded in creating a new MoAb that is highly specific for mucinous cystadenocarcinoma, which is the most common type of ovarian cancer, and a hybridoma that produces this MoAb. The present invention has now been completed. The method for preparing the hybridoma and MoAb of the present invention will be described in detail below. In the following explanation, the cancer cells used as immunogens for producing antibody-producing hybridomas and the various cells used to examine the reactivity of the MoAb of the present invention with each cell are all human cells. . First, a suitable animal is immunized with cancer cells. As the immunogen, ovarian cancer cells such as mucinous cystadenocarcinoma cells, endometrioid carcinoma cells, and mesonephric carcinoma cells can be used. These cancer cells are not limited to specific strains. The animal to be immunized may be a mouse, a murine animal such as a rat, or other animals, but mice are usually preferred. Specifically, for example
BALB/C mice are inoculated several times at intervals of several days to several weeks with mucinous cystadenocarcinoma cells, their homogenates, or antigens collected therefrom. The amount of inoculation is 1
Preferably, 10 ⁵ to ^{10 7} cells are used per animal at a time. Thereafter, the spleen is removed from the mouse, and antibody-producing cells (spleen cells) are obtained by centrifugation. Since these cells do not have the ability to proliferate, they are fused with cells that have the ability to self-propagate. Myeloma cells are particularly preferred as cells with self-propagation ability. It is preferable to use myeloma cells from the same species as the animal from which the antibody-producing cells were obtained, and it is also preferable to select cells that do not produce antibodies. A solution (or suspension) of antibody-producing cells and myeloma cells containing a cell fusion agent such as polyethylene glycol
In addition, perform cell fusion. The ratio of antibody-producing cells to myeloma cells used is preferably about 5:1 in cell number ratio. The obtained fused cells were separated by limiting dilution method,
A hybridoma in which the separated fused cells are grown, the antibodies produced in each well are reacted with various cell tissues, etc. by a known method, such as a fluorescent method or an enzyme antibody method, and the desired antibody is produced from the result. can be selected. The selected hybridoma can be cultured in a culture medium and the desired MoAb can be collected from the supernatant using a conventional method. MoAb can also be grown as a tumor and recovered from the serum or ascites of nude mice. The MoAb thus obtained belongs to the IgG ₁ class and has a molecular weight of approximately 150,000. This MoAb is not degraded by trypsin. The inventors named this MoAb MoAb4C7. As shown in the examples described later, the present invention
MoAb4C7 was found to be positive in 76.9% of mucinous cystadenocarcinomas, 85.7% of endometrioid carcinomas, and 81.8% of mesonephric carcinomas; No reaction was observed with ovarian tumors, other organ cancer tissues, or normal tissues. Therefore, the MoAb4C7-producing hybridoma of the present invention is a MoAb-producing hybridoma directed against an antigen present in mucinous cystadenocarcinoma. The fact that MoAb4C7 reacted with endometrioid carcinoma indicates that cancer-related antigens specific to mucinous cystadenocarcinoma also exist in endometrioid carcinoma.
The fact that MoAb4C7 reacted with mesonephric carcinoma indicates that cancer-associated antigens specific to mucinous cystadenocarcinoma are also distributed in mesonephric carcinoma. That is, the present invention
The antigen recognized by MoAb4C7 is a surface antigen present on the surface of mucinous cystadenocarcinoma cancer cells, the surface of endometrioid carcinoma cancer cells forming glandular cavities, and the surface of mesonephric carcinoma cancer cells. Since the MoAb of the present invention can be used to detect several types of ovarian cancer, it can be said to be an extremely useful antibody when considering clinical application. That is, the present invention
MoAb4C7 can also be used for diagnostic purposes, such as by measuring the antigen concentration in body fluids using methods such as radioimmunoassay, or by imaging the radioactivity of MoAb labeled with a radioactive isotope.
Using this MoAb4C7, highly accurate diagnosis of mucinous cystadenocarcinoma can be made. In addition, the MoAb of the present invention can be used for treatment using MoAb alone targeting cancer tissues that carry the corresponding antigen, or for missile therapy using a MoAb-anticancer drug complex that uses MoAb as a means for specifically delivering an anticancer drug to cancer tissues. It can also be used. The indirect enzyme anti-enzyme method used in the Examples and Experimental Examples of the present invention was carried out as follows according to the method of Tsuji (Immunology Experimental Procedures, X, 3161-3164, 1981). After mincing cancer cells, 0.1% trypsin (Sigma) and 0.1% collagenase (Sigma)
The cells were reacted at 37° C. for 2 hours in a solution containing 20% of the cellulose, and the cells were made into single cells using a stainless steel mesh (S-200 Nagata Rika), which were then washed and suspended with PBS to serve as sample cells. Micro ELISA plate U 2001 pretreated with 0.25% glutaraldehyde (Wako Pure Chemical Industries)
(Dynateck Lab.) Add ¹⁰⁴ sample cells to each well, centrifuge at 160G for 10 minutes, and then fix and dry by adding 99% ethanol. Next, 50μ of hybridoma culture supernatant was added and reacted at room temperature for 30 minutes, washed with PBS, and then used as a second antibody.
biotinyl antimouse IgG (Vector Lab.12.5μg/
ml), remove 50μ, react at room temperature for 30 minutes, and then wash with PBS. Next, horseradish
peroxidase Avidin D (Vector Lab.2μg/ml)
After adding 50μ of
Wash with PBS, perform color reaction for 5 minutes at room temperature using 50μ of orthophenylenediamine (0.2) g/ _ml , 0.15M citrate buffer, PH4.0) containing 0.05% _H2O2 , Add 50 _μ % _H2SO4 ,
Automatic microphotometer (Corona MTP12)
Measure with. In addition, the indirect fluorescent antibody method was carried out as follows according to the method of Takada (Journal of the Academy of Military and Medical Sciences 7, 91-99, 1982). Cultured cells and tissue sections placed on microslide glass (Matsunami Glass) were soaked with 99% ethanol for 5 minutes.
Fix for 5 minutes and wash with PBS (physiological phosphate buffer) for 5 minutes. MoAb on cultured cancer cells or tissue sections
Remove 50μ of (200μ/ml) and incubate at room temperature for 30 minutes, being careful not to dry. PBS
After washing with biotinyl antimouse as the second antibody
IgG (Vector Lab.12.5μg/ml) 50μ and 30μg at room temperature
Incubate for minutes and wash with PBS. Finally, FITC
Conjugated Avidin D (Vector Lab.10μg/ml)
After reacting with 50μ of 100 μg of chlorine for 5 minutes at room temperature and thoroughly washing with PBS, the cells were sealed in 90% glycerol and 10% PBS and observed with a fluorescence microscope (Nikon VFD-R). Example: Preparation of monoclonal antibody against mucinous cystadenocarcinoma The immunogen was prepared by cutting into small pieces mucinous cystadenocarcinoma cells (hereinafter referred to as HOVA-1) collected from a nude mouse transplant strain established from mucinous cystadenocarcinoma. An amount equivalent to 1×10 ⁶ cells was added to twice the amount of RPMI-1640 and homogenized, and the total amount was used. The above-mentioned amount of OVA-1 homogenate was intraperitoneally injected into 6-week-old male BALB/C mice (Charles River) four times every week, and the spleens were removed from the mice after 3 months. The cell fusion was carried out according to the method of Watanabe et al. (Immunological Experimental Procedures, 2963-2967, 1978) and Takada's method (Journal of the Medical College of Japan, 7, 91-99, 1982). That is, after cutting the removed spleen into small pieces, it was passed through a stainless steel mesh (S200 Nagata Rika).
0.7% in the sediment obtained by centrifugation at 1500 rpm and 200 G.
Add 10ml of NH ₄ Cl to remove red blood cells, and add RPMI−1640.
Live cell suspension obtained by washing twice with 3.9×10 ⁷ cells/22
Mouse myeloma cell line P3×63−Ag8−U1 (hereinafter referred to as
P3U1 obtained by washing P3U1 twice with RPMI-1640, 7.8 × 10 ⁶ pieces/2.5 ml (5:1),
It was centrifuged at 2000 rpm and 200G for 10 minutes. After thoroughly stirring the precipitated cells, 1 ml of RPMI-1640 containing 42.5% (w/v) polyethylene glycol (PEG1000, Sigma) at 37°C and pH 7.4 was gradually added over 1 minute while rotating. Cell fusion was performed. One minute after the reaction, RPMI-1640 was gradually added to bring the total volume to 30 ml, and the cell fusion was completed. 2000rpm,
RPMI containing 10% fetal bovine serum after centrifugation at 200G
-1640, add 100ml to make a cell suspension.
0.2 ml was dispensed per well of a Falcon micro culture plate (3042) and cultured at 37°C in a 5% CO ₂ incubator. After 24 hours, half of the supernatant was replaced with HAT medium (hypoxanthine, aminopterin, thymidine 10%, fetal bovine serum) every two days.
On the 10th day, remove the supernatant and check for the presence or absence of antibody production using the enzyme antibody method described below, and use the limiting dilution method to reduce the number of hybridomas in wells that were positive for antibody production to 0.3 to 0.6 per well. It was adjusted to Initially, HT (hypoxanthine, thymidine, 10% fetal bovine serum) was used as the medium, and 5 x 10 ⁵ BALB/C mouse thymocytes/well were added as a feeder layer. Next, RPMI with 10% fetal bovine serum
Replaced with 1640 times ground. Cloning using this limiting dilution method
Approximately 500 types of antibody-producing cells were created by conducting the experiment several times. Among these, a nude mouse passage transplant strain of mucinous cystadenocarcinoma (OVA-
1) MoAb4C7-producing hybridoma 1 that reacts with
I got the seeds. Mass culture of the obtained hybridomas was carried out by increasing the amount of hybridomas from 1 well to 5 wells, then to 24 wells (Falcon 3008), and finally
Culture was performed using Falcon tissue culture flasks (3013, 3024). γ collected by salting out the culture supernatant with 50% saturated ammonium sulfate
- Lyophilize the globulin fraction and
MoAb4C7 was obtained. Both the MoAb-producing hybridoma and the dried MoAb were stored frozen at -80°C. Experimental Example The reactivity of the monoclonal antibodies obtained in the above examples to various cells was investigated. (1) Reactivity to various human ovarian cancers, etc. MoAb4C7 obtained in the example was dissolved in PBS at a concentration of 200 μg/ml, and its reactivity to ovarian tumors, mainly ovarian cancer, was examined by indirect fluorescent antibody method. The results were as shown in Table 1. That is, the monoclonal antibody of the present invention
MoAb4C7 was detected in 10 of 13 cases (76.9%) of mucinous cystadenocarcinoma, 6 of 7 cases (85.7%) of endometrioid carcinoma, and 9 of 11 cases of mesonephric carcinoma, including cancer cells in ascites.
81.8%) showed a positive reaction. On the other hand, all 12 cases of serous cystadenocarcinoma and 6 cases of mucinous cystadenocarcinoma showed no response. MoAb4C7 has no effect on unclassifiable cancer other than the four types of epithelial ovarian cancer mentioned above, dermoid cystic carcinoma, embryonal carcinoma, Krukenberg tumor, solid teratoma, germinoma, granulosa cell tumor, etc. No reaction was shown.

[Table] (2) Reactivity to various human cancers other than ovarian cancer The reactivity is as shown in Table-2. MoAb4C7 showed no reactivity to the various cancers mentioned above.

[Table] (3) Reactivity to various normal human tissues When indirect fluorescent antibody assay using MoAb4C7 was performed on adult and 14-week-old and 15-week-old fetal normal tissues, it was found that ovaries, brain, heart, and lungs were detected. No reaction was observed in any tissues including the liver, stomach, intestines, pancreas, kidneys, uterus, fallopian tubes, and testicles. Display the results -
Shown in 3.

【table】

Claims (1)

[Scope of Claims] 1. A hybridoma formed by the fusion of mouse splenocytes immunized with human ovarian cancer cells and mouse myeloma cells, and belonging to the IgG ₁ class with the following properties: Single clone antibody belonging to: (1) Reacts with mucinous cystadenocarcinoma cells, endometrioid carcinoma cells, and mesonephric carcinoma cells; (2) Does not react with serous cystadenocarcinoma cells and mucinous cystadenocarcinoma cells (3) Does not react with cells of uterine body cancer other than ovarian cancer, lung cancer, breast cancer, stomach cancer, pancreatic cancer, renal cell carcinoma, colorectal cancer, liver cancer, seminoma; (4) ovary, brain, heart, lung, Does not react with normal tissue cells of the liver, stomach, intestines, pancreas, kidneys, uterus, fallopian tubes, and testicles; (5) Not degraded by trypsin.