JPH042239B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Field of Application] The present invention provides a well-differentiated esophageal cancer-derived cell line (TE-
The present invention relates to a monoclonal antibody with specific characteristics produced by a hybridoma prepared using 1) as an immunogen. [Prior Art] 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 determinations, such as sublets between lymphocytes, occur. It was difficult to distinguish between groups. Milsltein
[Ko¨hler, G. and Milstein, C.: Nature]
256, 495, (1975)] aims to break through this barrier by producing monoclonal antibodies that specifically recognize cancer-specific antigens or cancer-related antigens on cancer cells, thereby causing damage to normal tissues. It is expected that it will be possible to specifically eliminate only cancer cells. 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 comprises a monoclonal antibody that specifically reacts with esophageal cancer, particularly with cell membrane antigens of esophageal 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 specific 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. As cancer cell lines, well-differentiated esophageal cancer-derived cancer cell lines (TE
-1) is exemplified. Animals to be immunized include mice, rats, horses,
Examples include goats and rabbits. Antibody-producing cells are produced, for example, as follows. That is, well-differentiated esophageal cancer-derived cell TE-1
destroyed by ultrasonication, etc., and centrifuged (e.g., 10,000
~20,000G, 10-60 minutes) to obtain a cell extract, and transfer this supernatant to a gel filtration carrier capable of separating substances with a molecular weight of 100,000 to 2,000,000 (e.g., Cephadex, Cephacryl, Selfallose, Biogel, etc.) ) to separate into a high molecular fraction and a low molecular fraction. The molecular weight thus obtained is approximately 700,000 ~
The 1.5 million polymer fraction is used for animal immunization after being mixed with Freund Complete Adjuvant, for example. Immunization is administered subcutaneously, intramuscularly, or intraperitoneally to animals with approximately 1.5×10 ⁵ ~
It is performed by injecting the equivalent of 10 ⁸ cells/time.
After the first immunization, immunization is performed three times approximately every 3 to 5 weeks,
A final immunization will be performed approximately 3 months later. 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 myeloma cells are derived from the same species of animal. Cell fusion has been described, for example, by G. Galfre et al. [Nature 266 , 550,
(1977)] 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 for clones that produce the desired monoclonal antibody. That is, the cells are cultured in, for example, a microplate, and the antibody titer in the culture supernatant of wells in which proliferation is observed is measured by, for example, an enzyme antibody method, and wells producing appropriate antibodies are determined. get. Clones are obtained from such wells by further cloning, for example, by the limiting dilution method. This clone is transplanted intraperitoneally, for example, into a BALB/C mouse that has been previously administered pristane, and 10 to 14 days later, ascites fluid containing a high concentration of monoclonal antibodies is collected and assayed. Recovery of monoclonal antibodies produced by selected clones can be easily achieved by applying conventional immunoglobulin purification methods such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, and anion converters. be done. [Effects of the Invention] The monoclonal antibody obtained by the present invention specifically reacts with esophageal cancer cells, recognizes cell membrane surface antigens, does not react with normal tissue-derived cultured cells or normal tissues, and is effective against cancer cells. It is presumed that it recognizes a specific antibody. That is, the monoclonal antibody of the present invention is expected to have clinical applications in tumor imaging, conjugation with anticancer drugs, and targeting therapy. Example (1) Preparation of cancer-related antigen for immunization: Esophageal cancer cell line (TE-1 strain) was disrupted by sonication and centrifuged (15000G, 30 minutes) to obtain a cell extract. This supernatant was gel-filtered using a Cellallose 4B column and separated into a high-molecular fraction and a low-molecular fraction. After mixing, mice were immunized three times approximately every month, and a final immunization was performed three months later. 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 myeloma
P3U1 [Current Topics in Microbiology and Immunology (Curr.
Top.Mic-robiol.Immunl.) 81 , 1, (1978)]
The method of Koehler et al. [Immunological Method (Academic Press), New York (Immunological Method (Academic Press),
New York, 391, 1979)] with some modifications,
45% polyethylene glycol (average molecular weight
4000) for 2 minutes to perform cell fusion. 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;
Next, in D-MEM medium, 10, 5, 2.5, 1 piece/
Dilute the hybridoma to 0.1ml,
This was inoculated into microtiter plates in 0.1 ml portions and cultured. After 4 days, add D-MEM medium.
0.1 ml was added, and half of the medium was exchanged once every 4 to 7 days thereafter. 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 oxygen antibody assay was performed as follows. Add the specimen to a microplate coated with antigen (various cancer cell lines or partially purified cancer-related antigen or normal cells), react at 37°C for 1 hour, wash, and add peroxidase-labeled anti-mouse immunoglobulin (IgG + IgA-IgM) rabbit antibody. was added, and the reaction was further carried out at 37°C for 1 hour. After washing and removing unreacted labeled antibodies, an O-phenylenediamine solution was added, and the reaction was allowed to proceed at room temperature for 30 minutes. After that, 2M sulfuric acid was added to stop the reaction, and the absorbance at 490 nm was measured. Using this method, the reactivity with various cells was investigated. Cross-reactivity with carcinoembryonic antigen (CEA) was determined by the PHA method using CEA-sensitized blood cells. In order to investigate whether the monoclonal antibody recognizes the TE-1 secreted antigen or the cell membrane antigen, the reactivity of the monoclonal antibody with the TE-1 cells itself was inhibited using the TE-1 culture supernatant. 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, a culture supernatant of a hybridoma is subjected to titration using an enzyme antibody method, and an appropriate dilution ratio is determined based on the titration. Next, the TE-1 culture supernatant was concentrated 5 to 25 times and used as a stock solution.
1:5, 1:5 ² ...1:5 ⁿ dilutions are added in equal amounts to appropriately diluted hybridoma culture supernatants, and incubated at 37°C for 1 hour. Then, assay using a conventional enzyme antibody method (target: TE-1).
I went there. ₍ b) Screening process Primary screening: TE-
Wells positive for CCD 45-SK and negative for CCD _45- SK were selected. Secondary screening: All negative wells were selected in an assay system using other normal-derived cell lines. Tertiary screening: The cell lines selected above are cloned two or three times, and the culture supernatant is examined for reactivity with many cancer-derived cell lines, and whether secreted or cell membrane antigens are detected. This recognition is determined by an inhibition test using an enzyme-linked antibody method. (4) Collection and purification of monoclonal antibodies: (a) The cloned strain obtained by the above screening was administered to BALB/C mice (male) after 4 weeks of age, which had been previously administered 0.5 ml/mice of pristane.
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.
An IgM fraction was obtained and used as a purified monoclonal antibody. (b) This clone strain can be grown even in a BSA-containing serum-free medium. i.e. 0.5%
The cells were grown in a serum-free medium containing 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, 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 precipitated fraction should be collected in a small amount of 0.9
% NaCl solution, and then dialyzed against 0.02M physiological phosphate buffer as an external solution. After dialysis, add to DEAE-cellulofine column,
Column chromatography was performed. DEAE
- The first peak of cellulofine chromatography was used as a 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-linked antibody method used in the present invention is based on the method of Kennett et al. [Monoclonal Antibodies (Plenium Press) New York London, 376 , (1980)].
Immunosolvent Atsei (Enzyme)
This is the Linked Immunosorbent Assay (ELISA) method (hereinafter abbreviated as CELISA). [Table] [Table] [Table] For CELISA reactivity, + indicates the degree of measurement specimens that are determined to be positive. - indicated negative.
OD ₄₉₀ value in CELISA method -: 0 to 0.049 ±: 0.050 to 0.099 +: 0.100 to 0.399 ++: 0.400 to 0.699 +++: 0.700 to

Claims (1)

[Claims] 1. A monoclonal antibody produced by a hybridoma produced using a well-differentiated esophageal cancer-derived cell line (TE-1) as an immunogen and having the following characteristics: Ig class: IgM Recognizing antigen type: Cell surface antigen Reactivity with cancer cells: Shows positivity for the following cancer cells. Esophageal cancer (TE-1), Esophageal cancer (TE-2), Esophageal cancer (TE-3), Esophageal cancer (TE-5), Esophageal cancer (TE-7), Esophageal cancer (TE-10), Lung cancer ( PC−
3), endometrial cancer (ISHIKAWA), gastric cancer (MKN-28), gastric cancer (KATO-).