JPH0152400B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention has the following properties: ``The antigenic determinant that reacts with and recognizes glycoprotein antigens with a molecular weight of 1 million or more (gel filtration method) is a sugar chain with sialic acid at the end.
A monoclonal antibody belonging to the gM class. (1) Reacts with human gastric cancer, human colon cancer, human pancreatic cancer, human breast cancer, human lung cancer, human bile duct cancer, human uterine cancer, and human esophageal cancer. (2) Reacts with normal human submandibular gland, human proximal tubular epithelium, human bronchial glands, human squamous epithelial keratinized layer, human pancreatic islets of La (human pancreatic islets of Langerhans), human hepatocyte membranes, and human duodenal glands. do. (3) Reacts with human intestinal metaplastic gastric mucosa. (4) Does not react with normal human prostate, human bile duct epithelium, and human pancreatic duct epithelium. ”. Now that the production of monoclonal antibodies using cell fusion methods is being established, many researchers are attempting to produce new antibodies that recognize cancer-related antigens that are useful in cancer diagnosis and treatment. As a result of various studies on monoclonal antibodies, the present inventors discovered a monoclonal antibody that reacts with a high rate with various types of cancer, including human gastric cancer, and completed the present invention. Note that all cells, tissues, etc. mentioned in the following description are human cells, tissues, etc. unless otherwise specified. Among the many monoclonal antibodies reported to date, the monoclonal antibody CA19-9 developed by Koprowski et al. is being put into practical use for serodiagnosis of cancer. This monoclonal antibody CA19
The antigenic determinant recognized by -9 is Sialylated-
It has also been reported that Lewis ^a . The antigenic determinant recognized by the monoclonal antibody of the present invention is also a sugar chain having a sialic acid residue at the end. However, when the same specimen was immunostained with both and compared, some specimens reacted strongly with the antibody of the present invention but did not react with CA19-9, and some specimens did the opposite. Comparing the reactivity of both antibodies in normal human tissues, the monoclonal antibody of the present invention reacted with liver cell membranes, renal proximal tubular epithelium, pancreatic islet cells, and squamous epithelial keratinized layer. CA19-9 did not react with these. Furthermore, CA19-9 reacted with the prostate, bile duct epithelium, and pancreatic duct epithelium, but the monoclonal antibody of the present invention did not react. Therefore, both recognize different antigenic determinants. The monoclonal antibody of the present invention reacts with gastric cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer, cholangiocarcinoma, uterine cancer, and esophageal cancer, as shown in the Examples below. The monoclonal antibody of the present invention reacts with normal submandibular glands, proximal tubular epithelium, bronchial glands, squamous epithelial cornified layer, pancreatic islets, hepatocyte membranes, and duodenal glands. The monoclonal antibodies of the invention react with intestinal metaplastic gastric mucosa. The monoclonal antibody of the present invention can be used in normal prostate glands,
It does not react with bile duct epithelium or pancreatic duct epithelium. When gastric cancer tissue was treated with neuraminidase, a sialic acid degrading enzyme, the reactivity of the monoclonal antibody of the present invention to the gastric cancer tissue disappeared. This indicates that sialic acid is present in the antigenic site (antigenic determinant) recognized by the monoclonal antibody of the present invention. Furthermore, when gastric cancer tissue was treated with periodic acid, the reactivity of the monoclonal antibody of the present invention to the gastric cancer tissue disappeared. This means that the antigenic site (antigenic determinant) recognized by the monoclonal antibody of the present invention
indicates that it is a sugar chain. The molecular weight of the antigen with which the monoclonal antibody of the present invention reacts is 1 million or more (gel filtration method). Furthermore, the antigen with which the monoclonal antibody of the present invention reacts has the ability to take up glucose amine, and therefore this antigen is a glycoprotein. That is, the antigen with which the monoclonal antibody of the present invention reacts is a glycoprotein with a molecular weight of 1 million or more (gel filtration method), and the antigenic determinant recognized by the monoclonal antibody of the present invention is a sugar chain having sialic acid at its terminal end. . The monoclonal antibody of the present invention belongs to the gM class, and its L chain is a K chain. The antigen that reacts with the monoclonal antibody of the present invention is often present in large amounts not only in cancer tissues but also in the serum of patients with gastric cancer, colon cancer, and pancreatic cancer. The monoclonal antibody of the present invention can be produced, for example, as follows. That is, an animal such as a mouse or a rat is immunized with an antigen recognized by the monoclonal antibody of the present invention (the antigen may be isolated and used, but cancer cells containing the antigen or a homogenate thereof may also be used). , obtain antibody-producing cells from the immunized animal, fuse these with myeloma cells, clone the resulting fused cells, select the fused cells that produce the monoclonal antibody of the present invention, and culture them to produce the antibody. to recover. Immunization methods, fusion methods, selection of fused cells, etc. can be carried out by conventional methods. More specifically, the monoclonal antibody of the present invention can be produced, for example, as follows. First, mice are immunized with cancer cells such as gastric cancer cells. The animal to be immunized is not limited to mice, but murine animals such as rats or other animals may be used, but it is usually preferable to use mice. Also, homogenates or isolated antigens thereof may be used instead of cancer cells. For example, BALB/C mice are inoculated with cancer cells or their homogenate or isolated antigen several times at intervals of several days to several weeks. The amount of inoculation is 1
Preferably, 10 ⁵ to 10 ⁸ cells are used per animal at a time. Thereafter, the spleen is removed from the mouse and subjected to centrifugation to obtain antibody-producing cells. 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 of animal. Furthermore, it is preferable to select myeloma cells that do not produce antibodies. Cell fusion is performed by mixing antibody-producing cells and myeloma cells with a cell fusion agent such as polyethylene glycol. The ratio of antibody-producing cells to myeloma cells used is 2:1 to 10:
It is preferable to set it to 1. The obtained fused cells are separated by limiting dilution method, and after the separated fused cells are grown, the antibodies produced in each well are treated with various types of antibodies using known methods such as fluorescent antibody method or enzyme antibody method. Hybridomas that produce the desired antibody are selected from the reaction results with cell tissues and the like. It is also possible to culture selected hybridomas in an incubator and obtain antibodies from the supernatant, but in vivo, for example, by injecting hybridomas into the peritoneal cavity of a (nude) mouse and growing it as a tumor within the body of a (nude) mouse, it is possible to obtain antibodies from the supernatant fluid. ) It is also possible to collect antibodies from mouse serum or ascites fluid. Cancer cells such as gastric cancer cells can be used as cells used as immunogens to produce the monoclonal antibodies of the present invention, but these cancer cells are not limited to specific strains. The antibody-producing cells used to produce the monoclonal antibodies of the present invention are B cells, and B cells circulate in the body but accumulate in the spleen, etc., so it is preferable to remove the spleen and use it, but it is not necessarily the spleen. It is sufficient to use a portion where many B cells exist. In addition to the above-described method, the monoclonal antibody of the present invention can also be produced as follows. That is, a cancer tissue, such as a gastric cancer tissue, containing an antigen that reacts with the monoclonal antibody of the present invention is subcutaneously transplanted into an athymic mouse, such as a BALB/C nu/nu nude mouse. As the cancer tissue to be transplanted, for example, several pieces of 2 to 3 mm square cancer tissue are used. 1 to 1.5 months after the cancer tissue was transplanted, when the maximum diameter of the tumor had grown to about 1 cm, a mouse with a thymus gland, such as BALB/
Lymphocytes (T cells and B cells) contained in the spleen of a C nu/+ nude mouse are intraperitoneally administered to the aforementioned tumor-bearing mouse (an athymic mouse bearing a tumor approximately 1 cm in diameter). It is sufficient to administer the amount equivalent to the amount of lymphocytes contained in the spleen of one mouse. One month after administration, tumors in tumor-bearing mice shrink or disappear. The spleen (antibody-producing cells) of mice whose tumors have shrunk or disappeared is removed and fused with myeloma cells. The fusion method, subsequent selection of fused cells, culture of the selected fused cells, and recovery of the monoclonal antibody of the present invention can be carried out in exactly the same manner as described above. Since the antigen with which the monoclonal antibody of the present invention reacts often appears in the serum of cancer patients, the monoclonal antibody of the present invention can be used for serodiagnosis of cancer patients. Particularly in the case of patients whose serum contains a large amount of the antigen recognized by the monoclonal antibody of the present invention, it can be used for therapeutic effects and recurrence prediction. Furthermore, by adding a radioactive isotope to the monoclonal antibody of the present invention, it can be used for cancer localization diagnosis, or by combining an anticancer agent and the monoclonal antibody of the present invention, it can be used for missile therapy. Furthermore, by using the monoclonal antibody of the present invention for lesions that are histologically difficult to distinguish from cancer, it may be possible to differentiate the lesions. Example 1 (1) Production of monoclonal antibody Nude mouse transplanted human gastric poorly differentiated adenocarcinoma (St-4) was cut into 2-3 mm pieces and BALB/C
The tumor was implanted subcutaneously into nu/nu (thymic nude mice), and approximately one and a half months later, the maximum diameter of the tumor was 10 mm. On the other hand, the spleen of one BALB/C nu/nu + nude mouse was cut into small pieces, passed through a stainless steel mesh, and made into a cell suspension using 0.5 ml of physiological saline. BALB/C nu/nu) with a tumor of approximately 10 mm.
was administered intraperitoneally. The tumor disappeared one month after administration. So St-
0.2ml of the homogenate from step 4 in BALB/C nu/
It was administered intraperitoneally to nu. Three days later, the spleen was removed from the mouse. The cell fusion method was carried out according to the method of Watanabe et al. (Immunology Experimental Procedures, 2963-2967, 1978). That is, the removed spleen was cut into small pieces, passed through a stainless steel mesh, and centrifuged at 1500 rpm and 200 G. To the resulting sediment, 50 ml of 0.7% NH ₄ Cl was added to remove red blood cells, and the pieces were washed twice with RPMI-1640. Mouse myeloma cells ( ^P3-
X63−Ag8−U1) (hereinafter referred to as P3U1) with RPMI
2×10 ⁷ P3U1 cells (5:1) obtained by washing twice with -1640 were mixed and centrifuged at 2000 rpm and 200 G for 10 minutes. After thoroughly loosening the precipitated cells, 45
% (W/V) polyethylene glycol 4000
(Merck & Co.) at 37℃, PH7.4 RPMI−
1 ml of 1640 was added and treated for 8 minutes. After 1 minute of reaction, gradually add RPMI1640 to the total amount.
Cell fusion was completed by reducing the volume to 40 ml. 1000rpm,
RPMI containing 10% fetal bovine serum after centrifugation at 100G
Add 40ml of −1640 to make a cell suspension at 37℃.
Cultured in an incubator filled with 5% _CO2 . 24 hours later,
Replace with HAT medium (hypoxanthine, aminopterin, thymidine 10% fetal bovine serum),
The culture was dispensed into a Costar micro culture plate in an amount of 0.2 ml per well. On the 10th day, the supernatant was removed, the gastric cancer tissue was fixed in formalin, and the paraffin sections were stained with enzyme antibody method to check for antibody production.The hybridomas in the wells that showed positive antibody production were counted per well.
Cloning was performed using the limiting dilution method so that the number of cells was 0.6. Initially, HT (hypoxanthine, thymidine, 10% fetal bovine serum) was used as the medium.
BALB/C nu/+ mouse thymocytes at 5×10 ⁵ /well were added as a feeder layer. then 10
The medium was replaced with RPMI-1640 medium supplemented with % fetal bovine serum. Cloning by limiting dilution method was performed twice. In addition, for large-scale culture, increase the amount of hybridoma from 1 well to 5 wells, then to 24 wells (Falcon3008), and finally to Falcon tissue culture.
Add 0.1% _NaN3 to the supernatant obtained from flask culture at 4°C.
Saved at. (2) Selection of the monoclonal antibody of the present invention and staining of various tissues with the monoclonal antibody. Staining of various tissues for selection of the monoclonal antibody of the present invention and staining of various tissues with the monoclonal antibody can be carried out by the method of Hsu, SM, etc. (J.
Histochem.Cytochem., 29, 577-580, 1981)
The analysis was carried out by formalin fixation and staining of paraffin sections using the adivin-biotin-peroxidase complex method according to . That is, after fixing with 10% formalin and embedding in paraffin, which is widely used, thinly sectioned human gastric cancer tissues, other human cancer tissues, and normal human tissues were deparaffinized.
Treated with methanol containing 0.3% H ₂ O ₂ for 20 minutes. After washing with phosphate buffered saline (PBS), the cells were treated with PBS containing 10% normal pig serum for 30 minutes. Next, the mixture was reacted with a solution containing the antibody at room temperature for 2 hours, and further reacted at 4°C overnight. and
After washing with PBS for 15 minutes, the cells were treated with biotinylated anti-mouse immunoglobulin (7.5 μg/ml) for 30 minutes. After washing this with PBS for 15 minutes, it was treated with avidin DH-biotinylated peroxidase complex for 30 minutes at room temperature. After washing this with PBS for 15 minutes, it was 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. (3) Results We examined the reactivity of the antibodies produced in 78 out of 400 wells, and found that the antibodies reacted with gastric cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer, cholangiocarcinoma, uterine cancer, and esophageal cancer, and also with normal jaw cancer. It reacts with the inferior glands, proximal tubular epithelium, bronchial glands, squamous stratum corneum, pancreatic islets, hepatocyte membranes, and duodenal glands, and also reacts with intestinal metaplastic gastric mucosa, but normal prostate, bile duct epithelium, One hybridoma strain was selected that produced a monoclonal antibody that did not react with pancreatic ductal epithelium. Using the monoclonal antibodies of the present invention produced by the selected hybridomas, reaction tests with various cancer tissues or normal tissues were conducted according to method (2) above. (A) Table 1 shows the reactivity test results of the monoclonal antibody of the present invention against various cancer tissues.

【table】

[Table] As shown above, 100% for colorectal cancer and pancreatic cancer;
60.7% of gastric cancers, 81.5% of breast cancers, and 94.1% of lung cancers, especially adenocarcinomas, reacted with the monoclonal antibody of the present invention. Positive cases of adenocarcinoma often have mucus,
It strongly reacted with the monoclonal antibody of the present invention at the cell membrane. In addition, positive cases of squamous cell carcinoma showed a strong reaction only in the stratum corneum. Other bile duct cancer 1
The reaction was positive in 1 out of 3 cases, 2 out of 3 uterine cancer cases, 1 out of 1 esophageal cancer case, and 1 out of 2 renal cancer cases. (B) Table 2 shows the reactivity test results of the monoclonal antibody of the present invention with respect to various normal tissues. Table-2 Reactivity with normal tissue Normal tissue Number of positive cases/number of samples Submandibular gland 5/5 Proximal tubular epithelium 5/5 Bronchial gland 10/10 Squamous epithelium stratum corneum 10/10 Pancreatic islets 10 /10 Hepatocyte membrane 10/10 Duodenal gland 5/5 Normal tissue Number of positive cases/number of samples Prostate 0/5 Bile duct epithelium 0/5 Pancreatic duct epithelium 0/10 Pancreatic acinus 0/10 Brain 0/5 Nervous tissue 0/10 Smooth muscle 0/10 Striated muscle 0/10 Adipose tissue 0/10 Connective tissue 0/10 Blood vessels 0/10 Lymph nodes 0/10 Normal stomach mucosa 0/10 Large intestine mucosa 0/10 Small intestine mucosa 0/10 Spleen 0 /5 Thyroid 0/5 Breast gland 0/10 Testicle 0/3 Bladder mucosa 0/5 Bone 0/5 Pelvis 0/5 Cartilage 0/5 Furthermore, the monoclonal antibody of the present invention is effective against intestinal metaplasia and gastric mucosa. 11 out of 25 cases responded. Example 2 In (2) of Example 1, avidin-biotin-
Gastric cancer tissue samples were stained with neuraminidase prior to peroxidase complex staining.
It was treated with 0.2 U/ml at 37°C for 2 hours. Meanwhile, another gastric cancer tissue sample was treated with 0.5% periodic acid in the same manner.
It was treated at 37°C for 1 hour. When a reaction test of the monoclonal antibody of the present invention to the two gastric cancer tissues thus obtained was conducted in the same manner as in Example 1, staining disappeared in both cases. From the above, it can be seen that the recognition site (antigenic determinant) of the monoclonal antibody of the present invention is a sugar chain in which sialic acid is present at the end. Example 3 In order to determine the immunoglobulin class of the monoclonal antibody of the present invention, a test was conducted using the monoclonal antibody of the present invention, various anti-mouse Ig sera, and agar gel precipitation reaction. The monoclonal antibody of the present invention is anti-mouse IgM
Although it showed a clear precipitation line with serum and anti-mouse K chain serum, it did not react with any serum against IgG, IgA, IgD, IgE, or λ chain, indicating that the monoclonal antibody of the present invention is an IgM K-type immunoglobulin. found. Example 4 (1) 1×10 ⁷ hybridomas producing the monoclonal antibody of the present invention obtained in Example 1 were intraperitoneally administered to BALB/C nu/+ mice after treatment with pristane. One week later, about 5 ml of ascites was collected and subjected to gel filtration using Sepharose CL-6B. A fraction that reacts with IgM was obtained from each fraction by the Ouchterlony method in the same manner as in Example 3, and this was used as a purified antibody. Purified antibodies were prepared using the method of Guesdon et al. (J.
Histochem.CYtochem.27, 1131-1139, 1979)
Biotinylated according to. (2) On the other hand, 5 ml of pancreatic cancer patient serum was added to Sepharose CL-
Gel filtrate with 6B column and separate each fraction.
Dispense 0.1 ml into a 96-well microtiter plate. After 24 hours, block each well with 5% bovine serum albuminated PBS, add 5 μg/ml of the biotinylated antibody prepared in section (1), and add avidin-biotin. -Peroxidase complex method was performed. The reaction was carried out in 0.1M citric acid buffer (PH
4.5) by adding 0.015% H ₂ O ₂ , and the reaction results were measured at OD450 using DYnatech Autoreader.
(MR580). The antigen recognized by the monoclonal antibody of the present invention was detected near Vo, and its molecular weight was 10 ⁶ Daltons or more. Example 5 H ³ -glucoseamine was added to the culture medium of Kato (gastric signet ring carcinoma) at a concentration of 15 μCi/ml, cultured for 3 days, the culture supernatant was taken, and free H ³ -glucose amine was extracted by dialysis. was eliminated. When this supernatant liquid was passed through an affinity column using the monoclonal antibody of the present invention, an antigen having ^H3 radioactivity was obtained. This indicates that the antigen with which the monoclonal antibody of the present invention reacts is a glycoprotein. Example 6 Sera from normal people, gastric cancer patients, pancreatic cancer patients, and colorectal cancer patients were measured using the monoclonal antibody of the present invention by the solid-phase sandwich method of enzyme immunoassay. In contrast, 3 out of 7 cases with gastric cancer patient serum, 4 out of 8 cases with pancreatic cancer patient serum, and 8 with colorectal cancer patient serum
Three of the cases were strongly positive.

Claims (1)

[Claims] 1. The antigenic determinant that reacts with and recognizes a glycoprotein antigen with a molecular weight of 1 million or more (gel filtration method) is a sugar chain with sialic acid at the end, and has the following properties:
A monoclonal antibody belonging to the gM class. (1) Reacts with human gastric cancer, human colon cancer, human pancreatic cancer, human breast cancer, human lung cancer, human bile duct cancer, human uterine cancer, and human esophageal cancer. (2) Reacts with normal human submandibular glands, human proximal tubular epithelium, human bronchial glands, human squamous epithelial keratinized layer, human pancreatic islets, human hepatocyte membranes, and human duodenal glands. (3) Reacts with human intestinal metaplastic gastric mucosa. (4) Does not react with normal human prostate, human bile duct epithelium, and human pancreatic duct epithelium.