JPS61249999A - monoclonal antibody - Google Patents

Abstract

PURPOSE:The titled cellular surface antigen recognition type antibody, produced by a hybridoma prepared by using a cellular strain (MKN-45) derived from a gastric low differentiation type glandular cancer as an immunization source, having a light chain of IgG2a and capable of exhibiting positive reaction to cancer cells, e.g. lung cancer, colonic cancer, gastric cancer, etc. CONSTITUTION:A monoclonal antibody obtained by fractionating a cell extract of an established cancer cell (MKN-45) derived from a gastric low differentiation type glandular cancer by gel filtration, mixing the resultant high-molecular fraction having 700,000-1,500,000 molecular weight with complete Freund's adjuvant, immunizing an animal, e.g. mouse, with the mixture, collecting a cell producing an antibody, carrying out cell fusion with a myelomatous cell of an animal of the same species, cloning the resultant cell to give a cloned hybridoma, transplanting the resultant cloned hybridoma to the abdominal cavity of an animal, e.g. BALB/C mouse, to produce the aimed antibody capable of exhibiting the positive reaction to cancer cells of lung cancer (PC-3), colonic cancer (Colo201), gastric cancer (MKN-45, or KATO-III), hepatic cancer (HEK) and nasopharyngeal cancer (KB) and recovering and purifying the antibody from the ascites.

Description

Detailed Description of the Invention [Technical Field] The present invention provides a cell line derived from gastric poorly differentiated adenocarcinoma (MKN-45).
This invention relates to the production of hybridomas produced as immunogens to produce monoclonal antibodies with specific properties.

[Prior art]

It can be said that the ultimate goals of cancer research are the search for substances that exhibit anticancer and anticancer effects and the early detection of cancer, that is, the establishment of early diagnostic methods. To date, various drugs, treatments, and reagents have been developed for cancer, but all of these affect not only cancer cells but also normal tissues and cells, and no matter how effective they are, they At present, its use is severely restricted due to side effects.

Immune reactions (antigen-antibody reactions) are highly specific, but with conventional polyclonal antibodies, no matter how many times the absorption procedure is repeated, very minor antigen determination, such as a subset of lymphocytes, can occur. It was difficult to recognize what was distinguished by the groups. Milstein (
[Kohler, G. and Milstein, C.:
Nature (Kδhler, G, and Mils
tein, c, : Nature) 256+495
(1975)) broke through this barrier by obtaining monoclonal antibodies that specifically recognized cancer-specific antigens on cancer cells or cancer-related antigens, thereby eliminating cancer without damaging normal tissue. It is expected that only cells can be specifically eliminated. Further, diagnostic agents or test reagents using monoclonal antibodies do not have cross-reactivity with normal serum components, and are thought to be able to detect cancer-related antigens and cancer-specific antigens with high sensitivity.

[Problem that the invention seeks to solve]

The present invention provides monoclonal antibodies that specifically react with specific cancer antigens.

Furthermore, the present invention provides a reagent for detecting an antigen against the above-mentioned specific cancer.

[Means for solving problems]

The present invention consists of a monoclonal antibody that specifically reacts with gastric cancer, particularly gastric cancer secreted antigens.

The monoclonal antibody of the present invention is produced by so-called cell fusion. That is, a fusion hybrid is formed between an antibody-producing cell and a myeloma cell, and this hybrid is cloned to produce an antibody that exhibits specificity for the cancer cell (WI, specific antigen having the above characteristics). Produced by selecting clones. The operation is
The method is conventionally known except that the following cells are used as immunization cells.

Just follow the method.

Antibody-producing cells include, for example, lung cells, lymph node cells, and lymph node cells from animals immunized with antigens obtained from established cancer cell lines.
An example of the cancer cell line 0, which is a B-lymphocyte, is a cancer cell line derived from a low differentiated adenocarcinoma (MKN-45).

Examples of animals to be immunized include mice, rats, horses, goats, and rabbits.

Antibody-producing cells are produced, for example, as follows.

That is, the gastric cancer-derived cell line MKN-45 (poorly differentiated adenocarcinoma) was destroyed by sonication, etc., and centrifuged (e.g., 10,000
0 to 20,000 OOG for 10 to 60 minutes) to obtain a cell extract, and this supernatant was filtered using a gel filtration carrier capable of separating substances with a molecular weight of 100,000 to 2 million (e.g., Sephadex, Sephacryl, Sepharose, A molecular sieve is used to separate the high molecular weight fraction into both high molecular weight fractions and low molecular weight fractions. The thus obtained polymer fraction having a molecular weight of approximately 700,000 to 1,500,000 is treated with, for example, complete Freund's adjuvant.
After mixing with dCo+wplete adjuvant),
Used for animal immunization. For immunization, inject approximately 1.5 x 10S to 10” cells subcutaneously, intramuscularly, or intraperitoneally into the animal.
It is carried out by administering the equivalent portion/time once or twice a week for 3 to 7 weeks. Approximately 3 to 5 days after the final immunization, antibody-producing cells are collected from the immunized animal.

As myeloma cells, those derived from mice, rats, humans, etc. are used. Preferably, the antibody-producing cells and bone marrow cells are derived from the same species of animal.

Cell fusion has been described, for example, by G., Kahler.
r) [Nature 256.495
．． (1975)) or a method analogous thereto. At this time, 30-50% polyethylene glycol (average molecular weight 1,000-4,000
) for about 1 to 3 minutes at a temperature of 30 to 40°C.

Cells obtained by cell fusion are subjected to cloning. That is, the cells are cultured in, for example, a microplate, the antibody titer in the culture supernatant of wells in which proliferation is observed is measured by, for example, an enzyme antibody method, and then cloning is performed by, for example, a limiting dilution method to obtain a clone. get. This clone is transplanted into the peritoneal cavity of a BALB/C mouse that has been previously administered pristane, and
Thirty days later, ascites containing a high concentration of monoclonal antibodies is collected and assayed. The obtained clones are then subjected to screening for clones that produce the desired monoclonal antibody. Screening is followed up by enzyme-linked immunosorbent assays and the like.

The monoclonal antibodies produced by the selected clones can be collected using ammonium sulfate fractionation and PE, which are conventionally known methods for purifying IgG.
This can be easily achieved by applying G fractionation, ethanol fractionation, and anion converters.

〔Effect of the invention〕

The monoclonal antibodies obtained by the present invention are thought to be specific to adenocarcinomas, recognize cell membrane surface antigens, and do not react with normal tissue-derived cultured cells, red blood cells, white blood cells, or normal tissues, and are cancer-specific. It is presumed that it recognizes specific antigens. That is, the monoclonal antibody of the present invention can be used for tumor imaging and conjugated with an anticancer drug.
Clinical applications such as targeting therapy are expected.

Example (1) Preparation of cancer-related antigen for immunization: Gastric cancer cell line (MKN-45 strain) was disrupted by ultrasonication and centrifuged (15.0 OOG, 30 minutes) to obtain a cell extract. . This supernatant was gel-filtered using a Sepharose 4B column and separated into a high molecular fraction and a low molecular fraction.

A high molecular weight fraction having a molecular weight of about 700,000 to 1,500,000 was mixed with complete soloin adjuvant and then immunized into mice once a week for 5 weeks.

Four days after the final immunization, mouse lungs were removed and used for the following cell fusion.

(2) Cell fusion and cloning: The above mouse tile cells and mouse myeloma P3U1
r, Top, Microbiol, Immuno
l, ) 81, 1. (1978)) at a ratio of 4:1, and the method of Kohler et al. [Immunological Methods (Academic Press), New York (Immunological Methods)]
(Acadesic Press), NewYor
k, 391.1979)) with some modifications, 42.6
% polyethylene glycol (average molecular weight 1,000)
Cell fusion was performed by reacting for 2 minutes using .

These cells were implanted into a 96-well microplate, and HA
After culturing in T medium (Table 1) for 9 to 14 days, HT medium (Table 1)
), and after it became possible to culture in a flask (25d), it was cultured in D-MEM medium (Table 1). The antibody titer in the culture supernatant of wells in which proliferation was observed was measured by enzyme antibody method, and the desired hybridoma was cloned from appropriate wells by limiting dilution method.

i.e. 2 per well in a microtiter plate.
5,000 mouse peritoneal exudate cells were implanted and then D
- Hybridomas were diluted with MEM medium to a total of 1015.2.5.1 1015.2.5.1 hybridomas, and the diluted hybridomas were inoculated into microtiter plates at a rate of 0.1 and cultured. After 4 days, add 0.1 ml of D-MEM medium, and then add 1 ml of D-MEM medium every 4 to 7 days.
Half of the culture medium was replaced. Colonies visible to the naked eye were formed 10 to 20 days after the start of culture, and a clone strain was obtained.

(Margin below) Table 1 (3) Screening method Two clones of the obtained hybridomas producing the desired monoclonal antibody were screened as follows.

(b) Explanation of the method Enzyme antibody method was performed as follows.

Samples were added to microplates coated with antigens (various cancer cell lines, partially purified cancer-related antigens, or normal cells), incubated at 37°C for 1 hour, washed, and peroxidase-labeled anti-mouse immunoglobulin (IgG + I gA +
IgM) rabbit antibody was added, and the mixture was further reacted at 37°C for 1 hour. After washing the unreacted labeled antibody, add 0-phenylenediamine solution and react at room temperature for 30 minutes.
The reaction was stopped by adding M sulfuric acid, and the absorbance at 490 nm was measured.

Using this method, the reactivity with various cells was investigated. The cross-reactivity with leukocytes is determined by B-galactosidase (8-Galac).
tosiclase)-labeled antibody was used. In addition, cross-reactivity with human red blood cells was examined using the PHA method using a mixture of human type A, B, and O red blood cells. carcinoembryonic antigen (Carc)
inoembryonic antigen) (C
Cross-reactivity with E A) was determined using CEA-sensitized blood cells.
I went with method A.

In order to investigate whether the monoclonal antibody recognizes the MKN-45 secreted antigen or the cell membrane antigen, the monoclonal antibody and MK
It was investigated whether reactivity with N-45 cells themselves was inhibited.

Inhibition test using enzyme antibody method (Inh
The specific method of ibition Te5t) is as follows. That is, the supernatant of the hybridoma is subjected to titration using an enzyme antibody method, and an appropriate dilution rate is determined based on the titration. Next, M
A 5- to 25-fold concentrated KN-45 culture supernatant was used as a stock solution, and an equal volume of each was diluted 1:5, 1:5" ... 1:5" to an appropriately diluted hybridoma supernatant, Incubate for 1 hour at 37°C. Then, an assay was performed using a conventional enzyme antibody method (target: MKN-45).

(b) Screening flow Primary screening: Target cell (MKN-45)
The enzyme antibody method using normal-derived cells (Flow 2000) was positive for MKN-45 and Flow
Wells negative for 2000 were selected.

Secondary screening: In addition, we tested negative in all assay systems using other normal-derived cell lines, red blood cells, and white blood cells.
Select ll.

2 to 3 cell lines selected in tertiary screening
The culture supernatant was cloned twice and the culture supernatant was used to clone many cancer-derived cells.
In addition to examining the reactivity with ll 1ine, whether the antigen recognizes a secreted type or a cell membrane type antigen is determined by an inhibition test using an enzyme antibody method.

(4) Collection and purification of monoclonal antibodies (a) The cloned strain obtained by the above screening was used in BA after 4 weeks of age to which 0.5@Z/bristane was previously administered.
Inject 2.0-3. OX
10? cel+/mouse was transplanted, and ascites containing a high concentration of monoclonal antibodies was collected 10 to 14 days later.

This ascites was diluted 5 to 10 times by adding 0.9% NaCj2 solution, then ammonium sulfate was added to a concentration of 40%, and the precipitate fraction was collected. After dissolving this precipitated fraction in as small a volume as possible of 0.9% NaCl solution,
The Cjt solution was used as an external solution for dialysis.

After the dialysis, high performance liquid chromatography (TS-G
etl G-30005W) to obtain an IgM fraction, which was used as a purified monoclonal antibody.

(b) This Lorne strain can be grown in a BSA-containing serum-free medium. That is, the cells were grown in a serum-free medium containing 0.5% BSA (RITC55-9 medium), and the culture supernatant was collected. Add ammonium sulfate to this supernatant to a concentration of 40%, separate the precipitate fraction, and add 0.9%
After adding NaCl solution and dissolving it, ammonium sulfate was further added to a concentration of 40% and the precipitate fraction was collected. Add this precipitate fraction to as small a amount as possible of 0.9% NaCj! After dissolution with a solution, 0.02 M physiological phosphate buffer was used as an external solution for dialysis. After the dialysis, this solution was again passed through a Sepharose 4B column bound with anti-fetal bovine serum antibody (rabbit) and then added to a DEAE-Cellulofine column.
Column chromatography was performed.

The first peak of DEAE-Cellulofine chromatography was used as purified monoclonal antibody.

(5) Properties of monoclonal antibodies The properties of the monoclonal antibodies produced by the clones thus screened are shown in Tables 2 and 3. Immunoglobulin classes were assayed by the Ophthalony method.

The enzyme antibody method used in the present invention was developed by Kennet.
net) et al. [monoclonal antibody (
Blennium Press) New York London, 37
6, (1980)), the ELIZA method [Enzyme Linkto] uses cells as they are.
Immunosorbent assay (Enzy+ae Li
nkedImmunosorbent As5ay))
(hereinafter abbreviated as CELISA).

(Margin below) Procedural amendment cap button

Claims (1)

[Claims] A monoclonal antibody produced by a hybridoma produced using a gastric poorly differentiated adenocarcinoma-derived cell line (MKN-45) as an immunogen and having the following characteristics: (1) Ig class (light chain): IgG_2_a (2) Recognized antigen type: Cell surface antigen (3) Reactivity with cancer cells: Shows positivity for the following cancer cells. Lung cancer (PC-3), colon cancer (Colo201), stomach cancer (
MKN-45), gastric cancer (KATO-III), liver cancer (HE
K), nasopharyngeal carcinoma (KB)