JPH042238B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical field] The present invention relates to a cell line derived from gastric poorly differentiated adenocarcinoma (MKN
-45) as an immunogen and produced by a hybridoma with specific characteristics. [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 them 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 its 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, occurs. It was difficult to distinguish between groups. Monoclonal antibodies developed by Milstein et al. [Ko¨hler, G. and Milstein, C.: Nature 256 ,
495, (1975)] breaks down this barrier,
It is expected that by obtaining monoclonal antibodies that specifically recognize cancer-specific antigens or cancer-related antigens on cancer cells, it will be possible to specifically eliminate cancer cells without causing damage to normal tissues. 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. [Problems to be Solved by the Invention] 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 the Problems] The present invention consists of a monoclonal antibody that specifically reacts with gastric cancer, particularly with cell membrane surface antigens of gastric cancer. 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 the hybrid is cloned to produce an antibody that exhibits specificity for the cancer cell (i.e., a specific antigen having the above characteristics). Manufactured by selecting. The operation may be carried out in accordance with conventionally known methods, except for using the following cells as immunization cells. Antibody-producing cells are, for example, splenocytes, lymph node cells, B-lymphocytes from animals immunized with antigens obtained from established cancer cell lines. An example of the established cancer cell line is a cancer cell line derived from poorly differentiated gastric adenocarcinoma (MKN-45). Animals to be immunized include mice, rats, horses,
Examples include goats and rabbits. Antibody-producing cells are produced, for example, as follows. That is, gastric cancer-derived cell line MKN-45
(poorly differentiated adenocarcinoma) is destroyed by ultrasonication, centrifuged (e.g. 10,000-20,000G, 10-60 minutes) to obtain a cell extract, and this supernatant is used to Molecular sieving is performed using a gel filtration carrier capable of separating substances (eg, Cephadex, Cephacryl, Sepharose, Biogel, etc.) to separate the substance into a high molecular fraction and a low molecular fraction. The polymer fraction with a molecular weight of approximately 700,000 to 1,500,000 obtained in this manner is, for example, treated with Freund Complete adjuvant (Freund Complete Adjuvant).
adjuvant) and then used for animal immunization. For immunization, administer approximately 1.5 x 10 ⁵ to 10 ⁸ cells per subcutaneous, intramuscular, or intraperitoneal injection once or twice a week.
This is done by administering 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 animals of the same species. Cell fusion has been described, for example, by Gee and Köhler (G.
Ko¨hler) [Nature 256 , 495,
(1975)] or a method similar thereto. At this time, using 30-50% polyethylene glycol (average molecular weight 1000-4000),
The reaction is carried out at a temperature of ~40°C for about 1 to 3 minutes. Cells obtained by cell fusion are subjected to screening. That is, the cells are cultured, for example, in a microplate, the antibody titer in the culture supernatant of the well where proliferation is observed is measured, for example, by an enzyme antibody method, and further, the cells are cloned by, for example, a limiting dilution method. to get a clone. For example, this clone is transplanted intraperitoneally into BALB/C mice that have been previously administered with pristane, and then
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. Recovery of monoclonal antibodies produced by selected clones can be easily achieved by applying conventionally known IgG purification methods such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, and anion converters. Ru. [Effects of the Invention] The monoclonal antibody obtained by the present invention is considered to be specific for adenocarcinoma, recognizes cell membrane surface antigens, and is compatible with normal tissue-derived cultured cells, red blood cells, white blood cells, and normal tissues. does not react, and is presumed to recognize cancer-specific antigens. That is,
The monoclonal antibody of the present invention can be used for tumor imaging and conjugation with anticancer drugs.
targeting therapy
It is expected to have clinical applications in the field. Example (1) Preparation of cancer-related antigen for immunization: Gastric cancer cell line (MKN-45 strain) was disrupted by ultrasonication and centrifuged (15000G, 30 minutes) to obtain a cell extract. Transfer this supernatant to Sepharose.
Gel filtration was performed using a 4B column to separate 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 Freund's adjuvant and then immunized into mice once a week for 5 weeks. Four days after the final immunization, the mouse spleen was removed and used for the following cell fusion. (2) Cell fusion and cloning: The above mouse splenocytes and mouse myelomer ×
63.653 [Journal of Immunology (J.
Immunol.) 123 , 1548, (1979)] in a ratio of 4:1, and the method of Kohler et al.
Press), New York (Immunological
Method (Academic Press), New York;
391, 1979)] with some modification, and using 42.6% polyethylene glycol (average molecular weight 1000).
Cell fusion was performed by reacting for minutes. The cells were planted in a 96-well microplate and cultured in HAT medium (Table 1) for 9 to 14 days.
Transfer to HT medium (Table 1) and add flask (25
cm ² ), D-MEM
Cultured in 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. Briefly, 25,000 mouse peritoneal exudate cells were seeded per well in a microtiter plate, and then 10, 5, 2.5, 1
Dilute the hybridoma to 0.1ml/0.1ml in a microtiter plate.
They were transplanted and cultured. After 4 days, 0.1 ml of D-MEM medium was added, and half of the medium was exchanged once every 4 to 7 days. Colonies visible to the naked eye were formed 10 to 20 days after the start of culture, and a clone strain was obtained.

[Table] (3) Screening method The obtained hybridomas were screened for clones producing the desired monoclonal antibody as follows. (b) Description of the method The enzyme antibody method was performed as follows. Add the specimen to a microplate coated with antigen (various cancer cell lines or partially purified cancer-related antigens or normal cells), react at 37°C for 1 hour, and after washing, add peroxidase-labeled anti-mouse immunoglobulin (IgG + IgA + IgM) rabbit antibody. , and further reacted at 37°C for 1 hour. After washing the unreacted labeled antibody, add O-phenylenediamine solution and react at room temperature for 30 minutes, then add 2M sulfuric acid to stop the reaction.
Absorbance at 490 nm was measured. Using this method, the reactivity with various cells was investigated. The cross-reactivity with leukocytes is B-galactosidase (B-
Galactosidase)-labeled antibody was used. others,
The cross-reactivity with human erythrocytes is human A, B,
Type O red blood cells were mixed and examined using the PHA method.
Carcinoembryonic antigen
Cross-reactivity with (CEA) was determined by the PHA method using CEA-sensitized blood cells. To investigate whether the monoclonal antibody recognizes the MKN-45 secreted antigen or the cell membrane antigen, the monoclonal antibody was incubated with MKN-45 culture supernatant to inhibit its reactivity with the MKN-45 cells themselves. I checked to see if it works. The specific method of inhibition test using enzyme antibody method is as follows.
It is as follows. That is, the supernatant of the hybridoma is subjected to titration using an enzyme antibody method, and an appropriate dilution ratio is determined based on the titration. Next, the MKN-45 culture supernatant was concentrated for 5 to 25 cultures and used as a stock solution.
1:5, 1:5 ² ... 1:5 ⁿ dilutions were added in equal amounts to appropriately diluted hybridoma supernatants, and incubated at 37°C for 1 hour. Then, it is assayed using a conventional enzyme antibody method (target: MKN-45).
I went there. (b) Screening flow Primary screening: Target cells (MKN-45) and normal-derived cells (Flow
Wells that were positive for MKN-45 and negative for Flow 2000 using enzyme-linked immunosorbent assay (2000)
Selected. Secondary screening: Further, select wells that are negative in all assays using other normal-derived cell lines, red blood cells, and white blood cells. Tertiary screening: The cell lines selected above are cloned two to three times, and the culture supernatant is examined for reactivity with many cancer-derived cell lines, and whether it recognizes secreted or cell membrane antigens. This is determined by an inhibition test using an enzyme-linked antibody method. (4) Collection and purification of monoclonal antibodies (a) BALB/C mice (male) after 4 weeks of age to which 0.5 ml/mice of pristane was previously administered using the cloned strain obtained by the above screening.
Transplant 2.0 to 3.0×10 ⁷ cells/mouse into the peritoneal cavity of 10
~14 days later, ascites containing a high concentration of monoclonal antibodies was collected. This ascites was diluted 5 to 10 times by adding 0.9% NaCl solution, then ammonium sulfate was added to a concentration of 40%, and the precipitate fraction was collected. This precipitated fraction was dissolved in as small a volume of 0.9% NaCl solution as possible, and then dialyzed using 0.9% NaCl solution as an external solution. After dialysis, high performance liquid chromatography (TSK-Gell G-3000SW) was performed to detect IgG.
Fractions were obtained and used as purified monoclonal antibodies. (b) This clone strain can be grown even in a BSA-containing serum-free medium. i.e. 0.5
%BSA-containing serum-free medium (RITC55-9 medium), and the culture supernatant was collected. Add ammonium sulfate to this supernatant to a concentration of 40%, separate the precipitate fraction, add 0.9% NaCl solution to dissolve it, and then add ammonium sulfate to a concentration of 40% to separate the precipitate fraction. A fraction was collected. This precipitate fraction should be collected in as small a quantity as possible.
After dissolving with 0.9% NaCl solution, it was dialyzed using 0.02M physiological phosphate buffer as an external solution. After the dialysis was completed, this solution was again passed through a Sepharose 4B column bound with fetal serum antibody (rabbit) and then added to a DEAE-Cellulofine column for column chromatography. The first peak of DEAE-cellulofine chromatography was used as purified monoclonal antibody. (5) Characteristics of monoclonal antibodies The characteristics of the monoclonal antibodies produced by the clones thus screened are as shown in Tables 2 and 3. Immunoglobulin classes were assayed using the Ouchterlony method. The enzyme antibody method used in the present invention is the ELIZA method, which uses cells as they are, according to the method of Kennett et al. [Enzyme Linked Immunosorbent]
Assay)] (hereinafter abbreviated as CELISA).

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Claims (1)

[Claims] 1. 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: Ig class (light chain): IgG _2a recognition Antigen type: Cell surface antigen Reactivity with cancer cells: Shows positivity for the following cancer cells. Lung cancer (PC-3), colon cancer (COLO-201), gastric cancer (MKN-45), stomach cancer (KATO-), liver cancer (HEK), nasopharyngeal cancer (KB).