JPS60178826A - Monoclonal antibody, its preparation, and antigen - Google Patents

Abstract

PURPOSE:To obtain the titled antibody, by fusing a myeloma cell with an antibody-producing cell immunized with a specific antigen existing in the human gastric cancer cell, cloning the resultant fused cell, and selecting and culturing the fused cell capable of producing the antibody of human gastric cancer. CONSTITUTION:A myeloma cell is fused with an antibody-producing cell immunized with the antigen existing in the human gastric cancer cell, having a molecular weight of >=1,000,000 (by gel-filtration method) and extractable with physiological saline water and 1M perchloric acid solution. The obtained fused cell is cloned, and the fused cell capable of producing the antibody of human gastric cancer is selected. Cultivation of the cell gives a monoclonal antibody belonging to the class of IgM. The antibody is reactive with the human gastric cancer, especially human scirrous cancer of the stomach, with the absorptive epithelium of the normal tissue of human colon, and with the human pancreatic cancer and human colonic cancer in the form wherein positive cells are found sporadically. The antibody enables the early detection of scirrous cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monoclonal antibody that reacts with an antigen contained in gastric cancer.

More specifically, the present invention is directed to the following: ``An antigen contained in human gastric cancer with a molecular weight of 1 million or more (gel filtration method) that undergoes an antigen-antibody reaction with an antigen extracted with physiological saline and 1M perchloric acid solution, and contains sialic acid. A monoclonal antibody that belongs to the IgM class and has the following properties.

(1) It also reacts with human gastric cancer, especially human hard gastric cancer.

(2) Reacts with the absorptive epithelium of normal human colon tissue.

(3) It reacts with human pancreatic cancer and human colon cancer in the form that positive cells are found here and there.

2. Antibody-producing cells immunized with an antigen contained in human gastric cancer with a molecular weight of 1 million or more (gel filtration method) extracted with physiological saline and 1M perchloric acid solution are fused with myeloma cells. A method for producing a monoclonal antibody, which comprises cloning a fused cell, selecting a fused cell that produces an antibody against human gastric cancer, and culturing the same.

3. An antigen contained in human gastric cancer that has a molecular weight of 1 million or more (gel filtration method) and can be extracted with physiological saline and 1M perchloric acid solution. ”.

Rigid gastric cancer, which accounts for approximately 10% of gastric cancers in Japan, is the type of gastric cancer with the worst prognosis. Early detection of this type of gastric cancer is difficult with the currently commonly used diagnostic methods for gastric cancer, and the development of a new diagnostic method is desired.

The present inventor investigated monoclonal antibodies that react with gastric cancer, particularly hard gastric cancer, and as a result discovered the monoclonal antibody of the present invention and completed the present invention.

In the following explanation, the cancer cells used as immunogens for the production of antibody-producing hybridomas and the various tissues used to examine the reactivity of the monoclonal antibodies of the present invention with each tissue are all human tissues. It is.

Anti-colon cancer monoclonal antibody 19-9 (CancerRes, 42.4820) reported by Kobroski et al.
-4823.1982) is reported to also react with hard gastric cancer. However, when the same specimen was immunostained with the monoclonal antibody of the present invention and the 19-9 antibody and compared,
A certain sclerosing cancer specimen strongly reacted with the monoclonal antibody of the present invention, but did not react at all with the 19-9 antibody. Therefore, both recognize different antigenic determinants. Furthermore, the monoclonal antibody of the present invention showed stronger reactivity to infiltrated gastric cancer cells.

The monoclonal antibody of the present invention is a monoclonal antibody that reacts with a new gastric cancer-related antigen.

Conventionally, there were no seven clonal antibodies that reacted with human rigid gastric cancer at a high rate, but the monoclonal antibody of the present invention
It is an excellent drug that responds to LF gastric cancer at a high rate. or,
Another feature of the present invention is that it reacts with signet ring cell carcinoma and poorly differentiated adenocarcinoma at a high rate when classified by histological type of gastric cancer.

The monoclonal antibody of the present invention reacts not only with hard gastric cancer but also with early gastric cancer at a high rate, as shown in the Examples below. It also reacts with intestinal metaplastic mucosa of the stomach, but in this case, positive cells (one stained cell and one cell that reacted with the monoclonal antibody of the present invention) are found only here and there infrequently. Furthermore, the monoclonal antibody of the present invention can be applied to the stomach, esophagus, lung, thyroid, myocardium, liver, pancreas, kidney, pulp, small intestine,
It does not react with the normal tissues of the prostate and Fumaru, but it does react with the absorptive epithelium of the normal tissues of the large intestine. It also reacts with pancreatic cancer tissue and colon cancer tissue, but in both cases, positive cells are clustered here and there.

It only exists in

There is no change in the reactivity of the monoclonal antibody of the present invention to the hard gastric cancer tissue whether or not the tissue is treated with neuraminidase, a sialic acid degrading enzyme. Therefore, the antigen recognized by the monoclonal antibody of the present invention No sialic acid is present at the site.

The molecular weight of the antigen with which the monoclonal antibody of the present invention reacts is 1 million or more (gel filtration method), and the antigen can be extracted with physiological saline or 1M perchloric acid solution.

The monoclonal antibody of the present invention belongs to the IgMO class and has a molecular weight of about 900,000.

The method for producing the monoclonal antibody of the present invention can be carried out, for example, as follows. That is, an animal such as a mouse or rat is immunized with the antigen of the present invention (cells containing the antigen of the present invention, such as gastric cancer cells can be used), antibody-producing cells are obtained from the immunized animal, and these and myeloma cells are immunized. The fused cells are fused, the resulting fused cells are cloned, and fused cells that produce antibodies against human gastric cancer are selected, and the fused cells are cultured to collect the antibodies. The immunization method, the fusion method, the selection of fused cells, etc. can be performed by the method described in Section 7: Misaki, and specifically, for example, by the method described below.

The monoclonal antibody of the present invention is formed by fusion of antibody-producing cells immunized with the antigen of the present invention, such as cells that react with the monoclonal antibody of the present invention, such as gastric cancer cells, and myeloma cells. Produced by hybridomas.

More specifically, the monoclonal antibody of the present invention can be produced, for example, as follows.

First, mice are immunized with gastric cancer cells. The animal to be immunized is not limited to mice; murine animals such as rats or other animals may be used, but mice are usually used.

For example, BAJ and B/C mice are inoculated with gastric cancer cells, homogenates thereof, or antigens collected therefrom several times at intervals of several days to several weeks. The inoculation amount is 105 per animal per time.
Preferably, ~10 cells are used. Thereafter, the lungs are 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 combined with cells that have the ability to self-propagate. Myeloma cells are particularly preferred as +tlll Jll having 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 adding antibody-producing cells and myeloma cells to a solution (or suspension) containing a cell fusion agent such as polyethylene glycol. The ratio of antibody-producing cells to myeloma cells used is preferably 5:1 to 10:1 in cell number ratio. The obtained fused cells are separated by the limiting dilution method, and after the separated fused cells are grown, the antibodies produced in each well are isolated from various cell tissues using known methods such as fluorescent antibody method or enzyme antibody method. Hybridomas that produce the desired antibody are selected from the results. Although it is possible to culture the selected hybridoma in an incubator and obtain antibodies from the supernatant, the hybridoma is injected into the peritoneal cavity of a nude mouse, grown as a tumor in the body of the nude mouse, and then incubated with nude mouse blood/i11 or It is also possible to use a method of collecting antibodies from ascites.

Gastric cancer cells such as Jh cells can be used as immunogens to produce the monoclonal antibodies of the present invention. These gastric cancer cells are not limited to specific strains, and gastric cancer cells are
Either of these can be used.

The antibody-producing cells used to produce the monoclonal antibodies of the present invention are I3 cells, and 13-al cells circulate in the body, but accumulate in the lungs, etc., so it is preferable to remove the lungs and use them, but this is not necessary. It does not have to be the lung either, and a part where a large number of 13 cells are present may be used.

The monoclonal antibody of the present invention can be used as a material for a diagnostic agent for gastric cancer, and the monoclonal antibody of the present invention and an anticancer drug can be combined for use in mizile therapy.

In addition, especially in hard gastric cancer, the mucosal lesions are relatively small, and X-rays,
Although it is said that early detection using an endoscope or the like is difficult, early detection becomes possible by using the monoclonal antibody of the present invention.

Example 1゜(1) Production of monoclonal antibodies Human gastric cancer strain (St-1'
5) was homogenized at a ratio of 10% in phosphate buffered saline and mixed with the same amount of Freund's complete adjuvant.
The same amount was administered intraperitoneally to C mice, two more times after one month, and then one more time after three months. For the final immunization, 0.1 ml of homogenate alone was intraperitoneally administered (2 months after the previous immunization), and 3 days later, the lungs were removed from the mice.

The method of cell fusion is the method of Watanabe et al.
．． 2963-2967.1978).

That is, the extracted lung was cut into small pieces, passed through a stainless steel mesh, and centrifuged at 500 rpm and 200 G. 0.7% N1-L and CI were added to 50 ml of the washed liquid to remove red blood cells. 2.8 x 108 cells/20 ml of lung cell suspension obtained by washing twice with I'LPMi-1640
Mouse myeloma cells (P3-X63-Ag8-Ul)
(referred to as Li Ding 1' 3 U I) is PMI -1,
I'3U16.4X10 washed twice with 640
6 pieces/2m/! (5:1) and 2000 rp
m, and centrifuged at 200 G for 10 minutes. After the precipitated cells were thoroughly loosened, they were washed with 45% (W/V) polyethylene glycol 4000 (Merck & Co., Ltd.) at 37°C, pH 7.
, 4 ILi) M ], -1640, 1 penalty was added and processed for 8 minutes.

One minute after the reaction, Jul) MI 1640 was gradually added to bring the total volume to 30 m6, and the cell fusion was completed. 2000rpm
, PM containing 10% tallow serum after centrifugation at 200 G? , -1640,100ml to make a cell suspension, ii'alcon m1cro culture
0.2 per well of plate (3042)
The cells were aliquoted and cultured at 37°C in a 5% CO2-filled incubator. After 24 hours, transfer half of the supernatant to I-IA every 2 days.
, and replaced with T medium (hyboxanthin, aminopterin, thymidine, 10% tallow serum).

On day 10, remove the supernatant, fix the gastric cancer tissue in formalin, and stain the paraffin section with an enzyme-linked antibody method to confirm the presence or absence of antibody production. The limiting dilution method was used so that the number of samples was 0.3 to 0.6. The medium was initially HT (
Hyboxanthin, thymidine, and 10% tallow serum) were used, and thymocytes of BALB/C mice (5×10′/well) were added as a feeder layer. Next 1
0% beef tallow blood 6! The medium was replaced with RfJMI-1640 medium supplemented with I.

Cloning by limiting dilution method was performed twice.

In addition, for mass culture, the amount of hybridomas in one well can be increased to 5 wells and 24xIV (Falcon 3008), and finally the li'alcon tissue
cultureflask was used. 0.1% NaN3 was added to the supernatant obtained by flask culture and stored at 4°C.

(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 are carried out in 1
- (su, S-Mo et al. method (J-His-1och
em, Cytocltcm, 29. 577-58
0.1981) by formalin fixation and staining of paraffin sections using the avidin-biotin-peroxidase complex method. That is, human gastric cancer tissues, other human cancer tissues, and normal human tissues that were fixed in 10% formalin, which is widely and commonly used, were cut into paraparaffin sections, deparaffinized, and then treated with methanol containing 0.3% L-I202 for 20 minutes. Processed for minutes. Then phosphate buffered saline (PB)
After washing with S), the cells were treated with PBS containing 10% tallow serum for 30 minutes. Next, it was reacted with a solution containing the antibody at room temperature for 1 hour. After washing with P 13 S for 15 minutes, biotinylated anti-mouse immunoglobulin (75 μg/
me) for 30 minutes. This was washed with 1) +38 for 15 minutes and then treated with avidin I) H-biotinylated peroxidase complex for 30 minutes at room temperature. After washing this with P138 for 15 minutes, diaminobenzidine solution (
50 rng diaminobenzidine, 0.006% H2O2
, Tris buffer plI7.6) for 5110 minutes. After staining the cell nucleus with hematoxylin, it was mounted in a conventional manner and examined under a microscope.

(3) Results The reactivity of the produced antibodies was examined in 28 out of 288 wells, and one hybridoma strain producing the monoclonal antibody of the present invention was selected from among them.

Reaction tests of the monoclonal antibody of the present invention with various cancer tissues or normal tissues were conducted according to the method described in (2) above.

Table 1 shows the reactivity test results of the monoclonal antibody of the present invention against hard gastric cancer and early gastric cancer.

Table 11 Reactivity of the monoclonal antibody of the present invention with hard gastric cancer tissue and early gastric cancer tissue 1 Advanced gastric cancer Hard gastric cancer (marked cell carcinoma) 10-1-+
2〃 (Poorly differentiated adenocarcinoma) 10-1-+ 3,0004 Advanced gastric cancer Hard gastric cancer (Poorly differentiated adenocarcinoma) +5 #
(Spotted fine j bubble a) +l- 6〃 (Poorly differentiated adenocarcinoma) -1-1- 7〃 (Spotted fine 1 carcinoma) ++1- 8 # (Poorly differentiated adenocarcinoma) + 9 - 10 - 11 n pr +10+ 12 +++ 13 ++1- 14 + 1, Tan16' -1-118 Early gastric cancer (well-differentiated adenocarcinoma) -19- 20+-1-+ 21'-t-++ 22-124 + 125 126 (Fine dotted M cancer) -1-1-+27 〃 + 28 (Poorly differentiated adenocarcinoma) +''- 29- 30 (Intermediated adenocarcinoma) ++1 31 Tan 32 - Note: per case One tissue section tested 4-+→-More than 50% of cancer cells stained + 5 to 50% of cancer cells stained 10 Less than 5% of cancer cells stained 1 No cells stained As mentioned above, 15 out of 17 cases of hard gastric cancer (88%) reacted with the monoclonal antibody of the present invention.Of these, more than half of the cancer cells in 9 cases, 6 cases of poorly differentiated adenocarcinoma and 3 cases of marked cell carcinoma, were stained. In both cases, the cytoplasm was strongly stained.The two cases in which no positive cells were found (cases 1 Nu9 and 1 ()) were poorly differentiated adenocarcinomas with a small proportion of cytoplasm.

Regarding early gastric cancer, 11 out of 15 cases (73%) reacted with the monoclonal antibody of the invention.

Among the 15 cases, 8 cases were well-differentiated adenocarcinomas (shown in Table 1).
Positive cells were found in 6 cases.

(13) The monoclonal antibody of the present invention reacted with intestinal metaplastic mucosa of the stomach in 10 cases and 9 seconds, but only a few positive cells were found here and there. Goblet cells or metaplastic progenitor tubes in intestinal metaplasia surrounding cancer tissues were stained in rare places.

(q Table 2 shows the results of reaction tests of the monoclonal antibody of the present invention on various normal tissues.

Table 29 Reactivity of the monoclonal antibody of the present invention with various normal tissues Number of samples Number of positive reactions Stomach 50 Esophagus 20 Lung 20 Thyroid 1 () Heart muscle 10 Tile 50 Liver 20 Pancreas 50 Kidney 20 Pith 1 0 Small Intestine 20 Prostate 10 Fumaru 10 The monoclonal antibody of the present invention reacts with the absorptive epithelium of the large intestine. That is, in the reaction test of the monoclonal antibody of the present invention against normal colon tissue, the absorptive epithelium was strongly stained in 6 out of 6 cases. In addition, colonic calyx IY and lII cysts were stained in 1 out of 6 cases.

0)) In the reaction test between the monoclonal antibody of the present invention and pancreatic cancer tissue, 9 out of 9 cases reacted. However, in all cases, positive cells were only found here and there infrequently.

In the reaction test between the monoclonal antibody of the present invention and colon cancer tissue, reactions occurred in all 5 out of 5 cases.

However, in all cases, positive cells were only found here and there infrequently.

(IJ) Table 3 shows the reactivity test results of the monoclonal antibody of the present invention against advanced gastric cancer tissues other than hard gastric cancer.

Table 3: Progressive gastric cancer (differentiated adenocarcinoma) of the monoclonal antibody of the present invention. When the reactivity against 8 (stomach cancer) was examined, both of the 2 cases were rated "-10"-.

Example 2 In (2) of Example 1, neuraminidase 0.0. pl-1 by I U/me
6,4, Stubborn gastric cancer tissue was treated at room temperature for 1 hour. A reaction test of the monoclonal antibody of the present invention to the thus obtained hard gastric cancer tissue was conducted in the same manner as in Example 1, and
No change in staining properties was observed due to Neela ξnidase treatment.

On the other hand, when we examined the reactivity of the 19-9 antibody (Kobroski et al., sialic acid exists in the antigen site recognized by the 19-9 antibody) to hard gastric cancer tissues, those that were not treated with neuraminidase were stained. However, the treated ones were not stained.

From the above, it can be seen that sialic acid is not involved in the antigen site recognized by the monoclonal antibody of the present invention.

Example 3 When we measured the antigen that reacts with the monoclonal antibody of the present invention in 8 human and tomato gastric cancer lines that had been passaged in nude mice, we found that the well-differentiated adenocarcinoma line contained the most antigen. The antigen was purified from the supernatant of this homogenate.

By January 52 column chromatography, the antigen to which the monoclonal antibody of the present invention reacts was found to have a NaCl concentration of 0.
It was found in the 18-0.28M fraction. The antigen in this fraction was detected at around 0 by gel filtration using 5epharose CL-613, and its molecular weight was 106 daltons or more.

This partially purified antigen formed a sedimentation line in an agar gel with the monoclonal antibody of the present invention, but not with anti-CEA and anti-N ("A").

SI) In S-PAGE, the antigen slightly migrated within the gel (1 + 1-0, (19) L) regardless of the presence or absence of 2-mercaptoethanol treatment.

Furthermore, the partially purified antigen could be extracted with physiological saline. Furthermore, this antigen could be extracted with a 1M perchloric acid solution.

Example 4 In order to determine the immunoglobulin class of the monoclonal antibody of the present invention, a test was conducted using the monoclonal antibody, anti-human ig serum, and agar gel precipitation reaction.

This monoclonal antibody showed a clear sedimentation line in anti-Human IgM hemolysis, but it was
It did not react with any serum of Ig1, and this monoclonal antibody was 1gM.
It turned out to be an immunoglobulin.

Patent applicant Akira Seki Patent applicant: Nippon Kayaku Co., Ltd.

Claims (1)

[Claims] 1. Human gastric cancer has a molecular weight of 1 million or more (gel filtration method) and undergoes an antigen-antibody reaction with an antigen extracted with physiological saline and 1M perchloric acid solution, and does not contain sialic acid. A monoclonal antibody belonging to the IgM class that reacts with non-specific antigenic sites and has the following properties. (1) Reacts with human gastric cancer. (2) Reacts with the absorptive epithelium of normal human colon tissue. (3) It reacts with human pancreatic cancer and human colon cancer in the form that positive cells are found here and there. 2. Fusing myeloma cells with antibody-producing cells immunized with an antigen contained in human gastric cancer with a molecular weight of 1 million or more (gel filtration method) extracted with physiological saline and 1M perchloric acid solution, A method for producing a monoclonal antibody, which comprises cloning the obtained fused cells, selecting fused cells that produce antibodies against human gastric cancer, and culturing them. 3. An antigen contained in human gastric cancer that has a molecular weight of 1 million or more (gel filtration method) and can be extracted with physiological saline and 1M perchloric acid solution.