JPS6245599A - Monoclonal antibody - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a strain derived from moderately differentiated squamous cell carcinoma of the lung (PC-
10) This invention relates to a monoclonal antibody with a specific N character that is one step smaller than a hybridoma produced using the same as an immunogen.

(Prior Art) It can be said that the ultimate goals of cancer research are the search for anticancer substances that do not exhibit tumor opening effects, and the early detection of 1θ1, 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 although they are apparently effective drugs, 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 the subset between lymphocytes, occurs. It was difficult to recognize what was distinguished by the groups. Milstein (
Monoclonal antibodies developed by Milθtej, n) La [Kohler, G. and Milstein, C.
Stein, C.: Nature) 256°495 (1975)) aims to break through this barrier by obtaining monoclonal antibodies that specifically recognize cancer-specific antigens or cancer-related antigens on cancer cells. It is expected that cancer cells can be specifically eliminated without damaging 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 an antigen detection reagent for the above-mentioned specific layer.

(Means for Solving the Problems) The present invention consists of a monoclonal antibody that specifically reacts with a cell membrane surface antigen of moderately differentiated squamous cell carcinoma of the lung.

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 to 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 that subfertocytes are used as immunizing cells.

Antibody-producing cells include, for example, tile cells, lymph node cells, and lymph node cells from animals immunized with antigens obtained from established cancer cell lines.
3 + lymphocytes.

An example of the established cancer cell line is a cancer cell line derived from moderately differentiated squamous cell carcinoma of the lung (PC-10).

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

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

That is, a cell line derived from lung/moderately differentiated squamous cell carcinoma (P
C-10) by ultrasonication and centrifugation (example +
10,000 to 20,000 G for 10 to 60 minutes) to obtain a cell extract, and this supernatant was
Molecular sieving is performed using a gel filtration carrier (eg, Sephadex, Sephacryl, Sepharose, biogel, etc.) capable of separating tens of thousands of substances, and the product is separated into a high-molecular fraction and a low-molecular fraction. The thus obtained polymer fraction with a molecular weight of approximately 700,000 to 1,500,000 is treated with, for example, a complete 7° indoor adjuvant (
F'rθundComplete Ad, juvant
) and then used for animal immunization. Immunization is administered subcutaneously, intramuscularly or intraperitoneally to the animal with approximately 1.5X105~1
It is carried out by injecting the equivalent of 08 ce'l'l/time, and after the first immunization, immunization is given three times about every 3 to 5 weeks, and the final immunization is given 1 to 3 months later. Approximately 3~ from the final immunization
Five days later, antibody-producing Al1 cells are collected from the immunized animals.

As the bone marrow l1llj 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 can be achieved, for example, by G., Galfer (G.

Ga1fre) (Nature)
266, 550. (1977)) or a method analogous thereto. At this time, 30 to 5
0 polyethylene glycol (average molecular iii 1.
100 to 4,000) at a temperature of 30 to 40°C for about 1 to 3 minutes.

The 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, to identify wells producing appropriate antibodies. obtain. Cloning is further performed from such wells by, for example, limiting dilution to obtain clones. This clone is transplanted intraperitoneally, for example, into a BALB/C mouse that has been previously administered with pristane, and 10 to 30 days later, ascites containing a high concentration of monoclonal antibodies is collected and assayed.

Monoclonal antibodies produced by selected clones can be easily recovered by applying conventionally known immunopropurine purification methods such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, and anion exchanger. achieved.

(Effect of the invention) Monoclonal antibodies obtained according to the present invention.

It is thought to be specific to lung/moderately differentiated squamous cell carcinoma, recognizes cell membrane surface antigens, and does not react with normal tissue-derived cultured cells, red blood cells, white blood cells, or normal tissues, and is a sand 1-specific antigen. It is assumed that the system recognizes the

That is, the monoclonal antibody of the present invention can be used as a diagnostic agent, tumor imaging, as an antitumor agent alone, and as a conjugate with an anticancer agent.
Targeting the
It is expected that clinical applications such as ``rapy'' will be found.

Example (1) Preparation of cancer-related antigen for immunization: Established lung cancer cell line (PC-10 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.

After mixing the high molecular fraction with a molecule h1 of approximately 700,000 to 1,500,000 with complete Flobier adjuvant, it was given to mice every 3 weeks.
After multiple immunizations, a final immunization was given two 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 tile cells and mouse myeloma
and the method of K6hxer et al. Immunological Academic Press New York (Immunological Method
(Academic Press), New Yo
rk, 391° (1979)) with some modifications, 42
．． 6% polyethylene W glycol (average molecular weight 1000)
Multi-cell fusion was performed by reacting for 1 minute using .

These cells were implanted into a 96-well microplate, and HA
T medium (Table 1) Texo ~14 days 111 [after
Transfer to HT medium (Table 1) and add flask (25 cm
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.

That is, 92 cells per well in a microtiter plate.
5,000 mouse peritoneal exudate cells were implanted and then KD
- The hyperdomas were diluted with MEM medium to a ratio of 10.5.2.5.1 cells/previously grown, and cultured in a microtiter plate.

After 4 days [D-ME:M medium't O, 1xi was added, and after that, half of the medium was exchanged once every 4 to 7 days. After starting culture 1
Colonies visible to the naked eye were formed in 0 to 20 days, and a clone strain was obtained.

Table 1 (a) D-MEM medium: Dulbecco's modified Eagle medium (Dulbecco's
modified Eagle's Medium (
(manufactured by Hinaga Pharmaceutical Co., Ltd.)) with the following added calories.

Additives: (Final concentration) (Ingredients) 10% horse serum (FloW) 3001t
lf/l di-glutamine 1107/17/l
Ndium Pill (-T1001, U/-
Nijirin 100μυhiro Streptomycin 3.51/l
Glucose 3.7g/1HcLHCO3 (b) HAT medium: Add the following additives to D-MEM medium 1X10M Hypoxanthine 4X10-7M
Aminopterin 1.6 x 10 M
Thymidine←→I (T medium: HAT medium minus aminopterin (3) Screening method The obtained hybridomas were screened for clones producing the desired monoclonal antibody as follows.

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

Samples were added to a microplate coated with antigen (various cancer cell lines or partially purified cancer-related antigens or normal cells), incubated at 37°C for 1 hour, and washed with peroxidase-labeled anti-mouse immunoglobulin (gG + IgA + IgM).
) Rabbit antibody was added and the mixture was further reacted at 37°C for 1 hour. After washing and removing unreacted labeled antibodies, 0-phenylenediamine solution was added, and the mixture was allowed to react 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. Note that a β-galactosidase-labeled antibody was used for cross-reactivity with leukocytes. Other cross-reactivity with human red blood cells, h) A, B, 0
Type red blood cells were mixed and examined using the PHA method. Cross-reactivity with carcinoembryonic antigen (CEA) was determined by the PHA method using CEA-sensitized blood cells.

To determine whether the monoclonal antibody recognizes the PC-IQ secreted antigen or the cell membrane surface antigen, the monoclonal antibody was incubated with the PC-10 cells themselves using the PC-10 culture supernatant. We investigated whether reactivity was inhibited.

Inhibition test (Inhi) using enzyme antibody method
The specific method for (Te5t) is as follows. That is, a hybridoma culture supernatant is subjected to titration using an enzyme antibody method, and an appropriate dilution ratio is determined based on the titration.

Next, the PC-IQ culture soil was concentrated 5 to 25 times and used as a stock solution at 1:5, 1:52...1. :
Equal volumes of the 5n dilutions were added to appropriately diluted hybridoma culture supernatants, and incubated at 37°C for 1 hour. Then, analysis was performed using a conventional enzyme antibody method (target: PC-10).

(r2) Screening flow Primary screening: Target cell
cell) (PC-10) and normal-derived cells (F'lO
Wells that were positive for PC-IQ and negative for F'lOW-2000 were selected by enzyme-linked immunosorbent method using F'lOW-2000).

Secondary screening: Further, select wells that are negative in all analysis systems using other normal-derived cell lines, red blood cells, and white blood cells.

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, we also determined whether the antigen recognizes a secreted type or a cell membrane surface type.
Inhibition test using enzyme antibody method
ition Te5t).

(4) Collection and purification of monoclonal antibodies (a) Clone strains obtained by the above screening were preliminarily purified to zero
．． B A after 4 weeks of age when 5 mU of Zubristan was administered
LB/C mouse (male) intraperitoneally 2.0-3. OX
107cθ11 mice were transplanted, and ascites containing a high concentration of monoclonal antibodies was collected 10 to 14 days later.

Add 0.9"I=NaCj' solution to this ascitic fluid and make 5 to 1
After diluting the mixture 0 times, ammonium sulfate was added to give a dilution of 40 degrees, and the precipitate fraction was collected. This precipitated fraction should be collected in as small a quantity as possible with 0.9 NaC1! After dissolving with liquid, 0.
Dialysis was performed using 90% NaCl solution as an external solution. After completion of dialysis, high-speed liquid O”T Togelafy (TSK-Gell G-
30003W) to obtain a 9M fraction, which was used as a purified monoclonal antibody.

Naka) This loan strain can also be grown in a BSA-containing serum-free medium. That is, the cells were grown in Q, 54BSA-containing serum-free medium (R Engineering TC55-9 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 and dissolving the NaCl solution, ammonium sulfate was further added to a concentration of 40 tl, and the precipitate fraction was collected. This precipitated fraction was collected in a small amount of 0.9
After dissolution with aC1 solution, dialysis was performed using 0.02 M physiological phosphate buffer as an external solution.

After the dialysis was completed, this solution was added to a DEAFJ-Cellulofine column and column chromatography was performed.

(5) Characteristics of monoclonal antibodies F'ffl of the clones thus screened
The properties of the monoclonal antibodies 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.
[Monoclonal Antibodies (Frenium Press), New York, London 5th.

376, (1980)) using the ELISA method [Cellular Enzyme
Linkedou immunolubento assay (Cθuul
ar EnzymθLinked Immunosor
bent As5ay ) ) (hereinafter abbreviated as Cll5A).

Table 2 Table 3 Reaction specificity of anti-PC-IQ monoclonal antibody The CELISA reactivity in the above table is indicated by the degree of 61 regular samples determined to be positive for the reaction. - indicated negative. C.E.
The 0D49o value in the L engineering SA method is less than 1:0.05,
±: 0.05 or more and less than 0.1, 10: 0.1 or more and 0.4
It was less than

Agent: Patent attorney (st07) Takashi Sasaki (2 others)
7 Procedural amendment November 1, 1985? Day

Claims (2)

[Claims] (1) Lung/moderately differentiated squamous cell carcinoma-derived strain (PC-10
) is a monoclonal antibody produced by a hybridoma that has the following characteristics as an immunogen. [1] Ig class (subclass): IgM(k) [2]
Recognized antigen type: Cell membrane surface antigen [3] Reactivity with cancer cells: Shows positivity for at least lung cancer (PC-10) cells. (2) The monoclonal antibody according to claim (1), which exhibits reactivity with cancer cells and is also positive with esophageal cancer (TE-3) cells.