JPH0217160B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to monoclonal antibodies useful in the diagnosis and treatment of human lung cancer and the like. (Prior Art) Various studies have been conducted on monoclonal antibodies against cancer based on the method of Kohler et al., and many attempts have been made to utilize them in the diagnosis of human cancer. (Problem to be solved by the invention) At present, many attempts have been made to create monoclonal antibodies for diagnosis of human lung cancer, but a monoclonal antibody with practical value in terms of specificity has not yet been found. do not have. (Means for Solving the Problems) The present inventors have completed the present invention as a result of intensive research into practical monoclonal antibodies useful for tissue diagnosis of human cancer, particularly lung cancer. That is, the present invention provides: ``(1) Monoclonal antibody NE-25, which exhibits an antigen-antibody reaction with a protein antigen with a molecular weight of 25 x 10 ³ daltons present in human small cell lung cancer, and belongs to the IgG ₁ isotype and has the following properties. 1. Reacts with small cell lung cancer, glioma, and neuroblastoma in humans. 2. Reacts with normal tissue of human nerves, thyroid gland, and adrenal gland. 3. Reacts with human lung, esophagus, stomach, small intestine, large intestine, and kidney. (2) It has an antigen-antibody reaction with a protein antigen with a molecular weight of 35 × 10 ³ daltons present in human normal epithelial cells and epithelial-derived cancer cells, and has the following properties.
Monoclonal antibody PE-35 belonging to the IgG ₁ isotype. 1 Reacts with human lung cancer, stomach cancer, colorectal cancer, breast cancer, and hepatobiliary tract cancer. 2. Does not react with human glioma, neuroblastoma, sarcoma, or lymphoma. 3 Reacts with normal epithelial tissues of human lung, thyroid gland, esophagus, stomach, small intestine, and large intestine. ”. Note that the cells, tissues, etc. mentioned in the following description refer to human cells, tissues, etc. unless otherwise specified. The immunoglobulin (Ig) isotype of the NE-25 antibody and PE-35 antibody of the present invention is IgG ₁ .
Also, the antigen that NE-25 antibody reacts with (NE-25 antigen)
is present in small cell lung cancer, and the NE-25 antigen is a protein antigen with a molecular weight of 25×10 ³ Daltons. on the other hand,
The antigen that PE-35 antibody reacts with (PE-35 antigen) exists in normal epithelial cells and epithelial-derived cancer cells, and
The 35 antigen is a protein with a molecular weight of 35 x 10 ³ Daltons. Regarding the reactivity of NE-25 antibody to various cultured cell lines, it was positive for most cell lines for small cell lung cancer, and almost all cells for neurological tumors (glioma and neuroblastoma). The strain shows positive results. Regarding the reactivity of the NE-25 antibody to various tumor tissues, it was positive in most cases of small cell lung cancer, and in almost all cases of neurological tumors (glioma and neuroblastoma). The test results are positive for lung squamous cell carcinoma, lung adenocarcinoma, and lung large cell carcinoma in most cases.
Regarding the reactivity of NE-25 antibody to normal tissues, reactivity was observed with nerves, thyroid, adrenal glands, etc.
No reactivity is observed with the lungs, esophagus, stomach, small intestine, large intestine, kidneys, etc. On the other hand, regarding the reactivity of PE-5 antibody to various cultured cell lines, small cell lung cancer, squamous cell lung cancer,
Most cell lines were positive for lung adenocarcinoma;
Most cell lines are positive for gastric cancer, colorectal cancer, pancreatic cancer, hepatobiliary tract cancer, breast cancer, and renal cancer, but almost all cell lines are positive for neurological tumors (glioma and neuroblastoma), sarcoma, and lymphoma. The cell line shows negative results. Regarding the reactivity of PE-35 antibody to various tumor tissues, it was positive in most cases for lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, hepatobiliary tract cancer, and breast cancer, and it was positive for neurological tumors (glioma and nerve cancer). Almost all cases show negative results for sarcoma (blastoma) and lymphoma. Regarding the reactivity of the PE-35 antibody to normal tissues, reactions were observed with the epithelium of the bronchus, esophagus, stomach, small intestine, and large intestine, as well as with the thyroid gland. Based on its reactivity in normal and tumor tissues, the NE-25 antibody is thought to recognize differentiation antigens that are expressed during differentiation into nerve cells and endocrine cells, whereas the PE-35 antibody is pan-epithelial. Both antigens were found in most small cell lung cancers, which are considered to be lung cancers that showed a reaction to antigens, which can be called sexual antigens, and showed differentiation into neuroendocrine cells. NE-25 antibody and
PE-35 antibody is a useful antibody for immunohistological examination of lung cancer. The monoclonal antibody of the present invention can be prepared using known methods such as the method of Kohler and Milstein [Nature 256, 495-
497 (1975)] and the method of Ueda et al. [Proc Natl Acad
Sci USA785122-5126 (1981)]. For example, the monoclonal antibody of the present invention can be produced as follows. Immunizing an animal such as a mouse or rat with an antigen recognized by the monoclonal antibody of the present invention (e.g., lung cancer cells, neurological tumor cells, etc.) and obtaining antibody-producing cells from the immunized animal;
This is fused with myeloma cells, and from the resulting hybridomas, hybridomas that produce the monoclonal antibody of the present invention are selected, cultured, and the antibodies are recovered. Immunization methods, fusion methods, selection of hybridomas, recovery of antibodies, etc. can be performed by known conventional methods. More specifically, the monoclonal antibody of the present invention can be produced, for example, as follows. First, mice are immunized with lung cancer cells, neurological tumor cells, etc. The animal to be immunized is not limited to mice, but murine animals such as rats or other animals may be used; however, it is usually preferable to use mice. The mice for this immunization include BALB/c mice, BALB/c mice and other mouse strains.
F1 mice etc. are used. Mice are inoculated with lung cancer cells and the like several times every few days to several weeks. Thereafter, the spleen is removed from the mouse, and splenocytes (including antibody-producing cells) are collected using a standard method. It is preferable to use myeloma cells from animals of the same species, and when mouse splenocytes are used as the fusion partner, mouse myeloma cells are used. Specifically, MOPC-21, NS/1 [Nature,
256, 495-497 (1975)], SP2/o-Ag14
[Nature, 277 131-133 [(1979)], S194/5,
XXO.BU.1 [J.Exp.Med.148.313-328 (1978)] etc. are used. Splenocytes and myeloma cells are mixed at a ratio of 1:1 to 10 pairs, and fusion is performed using, for example, NaCl (about 0.85%), dimethyl sulfoxide [10 to 20% (v/v)], and polyethylene glycol with a molecular weight of 1000 to 6000. in a phosphate buffer (PH7.2-7.4) containing Fusion is carried out, for example, by incubating both cells at 35-37°C for 1-3 minutes. The selection of hybridomas is, for example, hypoxanthine (1.3-1.4 mg/dl), aminopterin (18-1.4 mg/dl),
20 μg/dl), thymidine (375-400 μg/dl), streptomycin (50-100 μg/ml), penicillin (50-100 units/ml), glutamine (3.5-4.0
g/) and a basal medium containing beef tallow serum (10-20%) to select cells that will grow. As the basal medium, RPMI1640 medium, Eagle's MEM medium, etc., which are commonly used for culturing animal cells, are used. Preferably, hybridoma cloning is carried out at least three times using the limiting dilution method. In selecting a hybridoma that produces the antibody of the present invention, the reactivity of the secreted antibody is examined, and a hybridoma that produces an antibody having the same reactivity as above is selected, and the antigen molecule recognized by the hybridoma is confirmed to have the above-mentioned molecular weight. This can be done by checking. If the hybridoma is cultured in the same manner as normal animal cell culture, the antibody of the present invention will be produced in the medium. For example, 2 to 5 x ¹⁰⁶ hybridoma cells were treated with streptomachine (50 to 100 μg/ml), penicillin (50 to 100 units/ml), glutamine 3.5 to
4.0g/), contains beef tallow serum (10-20%)
Using 10-20ml of RPMI1640 medium, 95% in a flask.
By culturing in the presence of % CO ₂ -5% O ₂ at 35 to 37° C. for 3 to 7 days, antibodies are gradually accumulated in the culture solution. In addition, hybridoma cells were transferred to pristane-treated nude mice or BALB/
c) The antibody of the present invention can be accumulated in the ascites fluid by transferring to the peritoneal cavity of mice and proliferating. That is, these mice were intraperitoneally injected with pristane (2, 6, 10, 14tetramethy1).
pentadecane (manufactured by Aldrich, USA) 0.5-1ml
Inject 5-10x into the peritoneal cavity 2-3 weeks later.
Transplant 10 ⁶ hybridoma cells. Usually 7
After ~10 days, ascites will accumulate and be collected. When the molecular weight of the monoclonal antibody of the present invention was measured by immunoprecipitation using ¹²⁵ I-labeled small cell lung cancer SCLC-SA, the NE-25 antigen exhibited 25 × 10 ³ daltons, and the PE-35 antigen exhibited 35 × 10 ³ Daltons. Immunoprecipitation reaction: small cell lung carcinoma culture line SCLC-SA2 as target cells
×10 ⁷ pieces at 0.5mci in the presence of 200μg of iodogen
Label with Na ¹²⁵ I. These labeled cells were mixed with 0.1-1% (v/v) NP.
-40 to solubilize the cell extract (1 to 10
×10 ⁵ cpm) monoclonal antibody (1-10 μl)
was reacted with 5 to 20 μl of rabbit anti-mouse immunoglobulin (Cappel, USA) as a secondary antibody for 30 minutes at 4°C, and further incubated with staphylococcus at 4°C for 1 hour. Immunoprecipitates are prepared by reacting with Cotchus aureus (CowanI strain) and analyzed by SDS-polyacrylamide gel electrophoresis. The biological activity of the monoclonal antibody of the present invention is shown in the Examples. Mouse-mixed hemadsorption test used in the examples
assay, M-MHA] method, the presence or absence of adhesion of lipophilic cells to target cells is observed in a microplate (Falcon, 3040) with monolayer cell culture or suspended cells attached, according to the method described below. As the lipophilic cells, those obtained by reacting sheep red blood cells with a mouse anti-sheep red blood cell antibody and then reacting them with a rabbit blight mouse immunoglobulin serum are used. Mouse mixed hemocyte adsorption test [M-MHA]: M-MHA is the original method of Espmark and Fagreus (Acta Pathol Microbiol Scand Suppl 154 258
−262, 1962). The method for producing indicator red blood cells is as follows. Washed sheep red blood cell 2% suspension and an equal volume of 1000-fold diluted mouse anti-sheep red blood cell pore body (prepared by hyperimmunizing BALB/c mice with sheep red blood cells) were reacted at 24°C for 45 minutes, and after washing, the 2% suspension was washed again. An equal volume of 200-fold diluted rabbit anti-mouse immunoglobulin (Cappel, USA) was reacted at 24°C for 45 minutes as a suspension, then washed twice and made into a 2% suspension again, which is called M-MHA.
used as indicator red blood cells. As a means of the M-MHA testing method, the cells to be detected are hybridoma cell culture supernatants or hybridoma cell-inoculated BALB/c mice or
BALB/c-derived nude mouse ascites and 45 at 24℃
After reacting for 1 minute and washing off the antibody, react with 0.2% indicated sheep erythrocytes at 24°C for 45 minutes. After washing briefly once with phosphate buffered saline (PBS), rosette formation of indicated erythrocytes was observed under an optical microscope. Judgment is made based on the presence or absence of. In addition, in the Examples, staining of various tissues for selecting the monoclonal antibody of the present invention and staining of various tissues with the monoclonal antibody of the present invention were performed using Hsu, S,
M, et al.'s method (J. Histochem, Cytochem29, 577
580, 1981) by acetone fixation and staining of frozen sections using the avidin-biotin-peroxidase complex method (ABC method). In other words, frozen sections of lung cancer tissue, etc., containing 10% normal pig serum.
After treatment with PBS for 30 minutes, the mixture was reacted with a solution containing the antibody at room temperature for 2 hours, and further reacted at -4°C overnight. After washing with PBS for 15 minutes, biotinylated anti-mouse immunoglobulin (7.5 μg/ml) was added.
Treated for 30 minutes. After washing this with PBS for 15 minutes,
Treated with avidin DH-bithionated peroxidase complex for 30 minutes at room temperature. This was washed with PBS for 15 minutes, and then reacted with a diaminobenzidine solution (50 mg diaminobenzidine, 0.006% _H2O2 , Tris buffer PH7.6) for 5 _to 10 minutes. After staining the cell nucleus with hematoxylin, it was mounted in a conventional manner and examined under a microscope. (Example) Example 1 (Manufacture of PE-35 antibody) Eight-week-old female BALB/c mice were initially incubated with 5 x ¹⁰⁶ small cell lung cancer culture lines (small cell lung cancer culture lines).
Immunized subcutaneously with a cell population consisting of a 1:1 mixture of SCLC-SA and SCLC-SM, and one month later, immunized with 1 x 10 ⁷ cells each intraperitoneally twice at 2-week intervals. I went there. Furthermore, lung small cell carcinoma culture line
Both SCLC-SA and SCLC-SM were strains established at the Aichi Cancer Center. Three days after the final immunization, the spleen was removed and passed through a stainless steel mesh to prepare a cell suspension. This 1.0 x 10 ⁸ splenocytes and 2.0 x 10 ⁷ 8
−Azaguanine-resistant myeloma cells P3−NS1−
Mix Ag4/1 (NS/1) and add 1ml to the sediment after centrifugation.
47.5% polyethylene glycol (average molecular weight
4000) and stirred gently for 2 minutes. After washing, the cell mixture was suspended in RPMI medium containing 10% fetal bovine serum (complete RPMI medium) and plated in a 96-well microculture plate at a ratio of ¹⁰ cells per well.
It was dispensed in ml portions. After 24 hours, hypoxanthine
100μM, aminopterin 0.4μM, thymidine 16μM
0.1 ml of complete RPMI medium (HAT medium) containing After starting the culture, collect 0.1ml of the culture supernatant twice a week and add it to the HAT.
0.1 ml of medium was added. After 14 days, fused cells began to appear in the wells. After that, the culture supernatant from each well was removed, and it was confirmed whether antibodies that react with small cell lung cancer were produced using the M-MHA method described above, and hybridomas in wells that showed positive antibody production were collected using limiting dilution. Cloning was repeated three times. That is, cells were diluted to 50 cells/ml or 10 cells/ml, dispensed in 0.1 ml portions into 96-well microculture plates pre-seeded with feeder cells, and diluted with HT medium (complete containing 100 μM hypoxanthine and 16 μM thymidine). RPMI medium) for two weeks. When one hybridoma colony was formed in one well, it was taken out as a clone. From these clones, by the above-mentioned M-MHA method and ABC method,
Hybridoma clones secreting antibodies having reactivity to each cell and tissue shown in the PE-35 antibody column were selected. The antibodies produced by the hybridomas obtained in this way were used as PE-35.
It was named an antibody. This hybridoma was intraperitoneally administered to mice and propagated by the following method to mass-produce PE-35 antibody.
That is, mice were intraperitoneally injected with 0.5 ml of pristane.
Thereafter, 5×10 ⁶ hybridoma cells were transplanted into the peritoneal cavity two weeks later, and ascites fluid was collected 10 days later. Example 2 (Production of NE-25 antibody) 5 x 10 ⁶ neuroblastoma cells (#1134) obtained from surgical materials were subcutaneously injected into 8-week-old female BALB/c mice, and 1 month later. more than 2 weeks apart
Immunization was performed by injecting 1×10 ⁷ cells into each peritoneal cavity. Three days after the final immunization, the spleen was removed and passed through a stainless steel mesh to prepare a cell suspension. These 5 x 10 ⁷ splenocytes and 1 x 10 ⁷
Mix NS/1, perform cell fusion and cloning in the same manner as in Example 1, and use the M-MHA method and ABC method described above to determine the reactivity to each cell and tissue shown in the NE-25 antibody column. Hybridoma clones secreting antibodies with
The antibody produced by the hybridoma thus obtained was named NE-25 antibody. This hybridoma was cultured in the same manner as in Example 1, and antibodies were mass-produced. Example 3 Each cancer and normal tissue or cell, NE-25 antibody, PE
In order to investigate the reaction with the M-35 antibody,
Tests were conducted using the MHA method and ABC method. The results are shown in Tables 1, 2 and 3.

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[Table] Example 4 In order to know the immunoglobulin isotype of the monoclonal antibody of the present invention, the monoclonal antibody of the present invention was incubated with each mouse Ig isotype (IgA, IgM, IgG ₁ , IgG _2a , IgG _2b ,
Precipitation reactions were performed on rabbit antiserum against IgG ₃ ). As a result, the monoclonal antibody NE-
Both 25 and PE-35 were found to be IgG ₁ . Example 5 When the reactivity against the small cell lung cancer culture strain used in Example 1 was examined by the M-MHA method, NE-
In both cases, the reactivity of the 25 antibody and the PE-35 antibody did not change when treated with neuraminidase, and the reactivity was inactivated by treatment at 100°C for 5 minutes. The above results indicate that both the NE-25 antibody and the PE-35 antibody recognize protein antigens. Example 6 NE-25 antigen and PE-
When the molecular weight of 35 antigen was measured, NE-25 antigen was 25
^x103 daltons, and PE-35 antigen is 35 x
Presented with 10 ³ Daltons. (Effects of the Invention) The monoclonal antibody of the present invention is particularly useful for immunohistological differentiation between small cell carcinoma and non-small cell carcinoma of lung cancer. It is also an antibody that can be applied to the treatment of small cell lung cancer.

Claims (1)

[Claims] 1. An antigen-antibody reaction with a protein antigen with a molecular weight of 25× ¹³³ daltons present in human lung cancer,
Monoclonal antibody NE-25 belonging to the IgG ₁ isotype with the following properties. 1 Reacts with human lung cancer, glioma, and neuroblastoma. 2. Reacts with normal human nerve, thyroid, and adrenal tissues. 3. Does not react with normal tissues of human lung, esophagus, stomach, small intestine, bulge, and kidney.