JPH0662889A - Antobody, cell system and medicine - Google Patents

Abstract

PURPOSE:To provide a monoclonal antibody which is an antibody against neutral saccharide chain and has reactivity especially to carcinoma of the stomach. CONSTITUTION:A monoclonal antibody produced from cell line MKN74 of carcinoma of the stomach as an immunogen shows reactivity to saccharide chain of glycoprotein having >=1,000,000 Da molecular weight especially of cell line MKN74 of carcinoma of the stomach and has an Ig class of IgM(kappa).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody capable of specifically binding to a cancer-related sugar chain antigen, a hybridoma cell line capable of producing the same, and a pharmaceutical composition using the monoclonal antibody. is there.

[0002]

2. Description of the Related Art Monoclonal antibodies that specifically recognize the presence of cancer cells are considered to be very effective in application to diagnosis and treatment of cancer. In addition, it is also known that changes in the structure of sugar chains produced by cells occur as cells become cancerous [Bulletin Du Cancer (Bulletin
du Cancer), Paris, 70, 118 (1
983)], and monoclonal antibodies against sugar chains of cancer cells have been actively produced. Aswell and Morel [Advance of Enzymology (A
dvance of Enzymology), No. 41
Vol. 99 (1974)], which led to the hypothesis that the presence of sialic acid is essential for the long-term existence of glycoproteins in the circulating blood, and therefore its application to in vitro diagnosis of cancer. Most of the monoclonal antibodies produced for the purpose were those for sialic acid-binding type sugar chains.

However, when these antibodies are applied to in-vivo diagnostic agents for cancer and anti-cancer agents, there is a drawback that the antibodies are trapped by many antigens existing in the circulating blood for a long time, and it is difficult to reach the cancerous part efficiently. From this, it is predicted that antibodies against neutral sugar chains, which are known to rarely appear in circulating blood or do not exist for a long time, are likely to reach the cancerous part without being trapped by the antigen in circulating blood. . This is also considered in gastric cancer.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an antibody against a neutral sugar chain, particularly a monoclonal antibody having reactivity with gastric cancer.

[0005]

The present inventors have completed the present invention as a result of extensive studies to solve the above problems. That is, the present invention is a monoclonal antibody having the following characteristics. a) A gastric cancer cell line MKN74 or a carbohydrate extracted therefrom was used as an immunogen to sensitize a mammal, and a hybridoma was obtained by fusing immune cells of the mammal and myeloma cells, and the hybridoma was extracted from the cancer cell line MKN74. A monoclonal antibody produced by a hybridoma screened with a soluble glycolipid fraction. b) The Ig class is IgM (κ). c) Reacts with gastric cancer cell line MKN74. d) Reactivity with gastric cancer cell line MKN74 with a glycoprotein sugar chain having a molecular weight of 1,000,000 Da or more.

The present invention is also a hybridoma cell line capable of producing the above monoclonal antibody. Furthermore, the present invention is a pharmaceutical composition containing the above monoclonal antibody as an active ingredient. The present invention will be described in detail below.

In the present invention, it is desirable to use cancer cells MKN74 or saccharides such as glycolipids or glycoproteins extracted from cancer cells MKN74 as antigens for immunizing mammals. It can be obtained by means. For example, the culture conditions for the cancer cells MKN74 are not particularly limited.

[0008] Further, as a method for obtaining a sugar such as a glycolipid or a glycoprotein from a cell, Young et al.
H. ) And Hakomori H., Journal of Biological Chemistry (The)
Journal of Biological Ch
), 246, 1192 (1971)
, Etc., and as a method for obtaining by organic synthesis, Sato et al. [Sato (Sato), Ito (Ito Y.), Nukada (Nukada T.), Nakahara (Y.) and Ogawa (Ogawa). Ogaw
a T.A. ), Carbohydrate Research (Carb
hydrate Research), Volume 167,
197 (1987)]. This may be used for immunity or the like.

Hybridomas are produced by so-called cell fusion. For example, immunizing an animal with an antigen
Cancer cells MKN74 were treated with phosphate buffered saline (hereinafter, PBS).
It is carried out by intraperitoneally injecting 2 to 5 × 10 ⁷ animals per 3 times every 3 days to 3 months. At this time, as animals to be immunized,
Examples include mice, rats, horses, goats, rabbits and the like.

The antibody-producing cells of the immunized animal are fused with myeloma cells, and the myeloma cells used are, for example,
Those derived from mouse, rat and human are preferred. Cell fusion can be performed, for example, by the method of Milstein et al. [Koh (Koh
ler G.L. ) And Milstein
C. ), Nature, Volume 256, 4
Page 95 (1975)]. That is, 30% to 60% polyethylene glycol (average molecular weight 1
000-4000) at a temperature of 30 ° C to 40 ° C for about 1 to 3 minutes.

The monoclonal antibody produced by the hybridoma thus obtained is screened by a solid phase enzyme immunoassay or the like using the neutral glycolipid fraction extracted from the cancer cell line MKN74 to recognize the cancer cell MKN74. A hybridoma cell line capable of producing a monoclonal antibody can be obtained. Further, the hybridoma cell line can be cultured to obtain the desired monoclonal antibody from the cells or the culture supernatant. The conditions for culturing the hybridoma are not particularly limited, and ordinary methods can be adopted. Further, as a method for recovering the antibody from the culture supernatant or the like, an ordinary method such as salting out or column chromatography can be adopted. In this way, a monoclonal antibody reactive with the neutral sugar chain antigen of gastric cancer cells can be obtained at a high rate. In the present invention, the monoclonal antibodies MC3-3 and MKA22, and the hybridomas MC3-3 and MKA22 that produce the same were obtained as described above.

These monoclonal antibodies have Ig class IgM (κ) and show reactivity with the gastric cancer cell line MKN74, and the molecular weight of the gastric cancer cell line MKN74 is 100.
It shows reactivity with glycoprotein sugar chains of 10,000 Da or more.

The pharmaceutical composition of the present invention can be obtained using the above monoclonal antibody as an active ingredient. Examples of the pharmaceutical composition of the present invention include therapeutic agents and diagnostic agents. For example, in order to apply the antibody as an in-vivo diagnostic agent, it is desirable to label the antibody, but the labeling method and means are not limited at all, and known methods and means can be used. Further, a radioactive isotope or the like is preferably used for labeling the antibody. For example, as a radioactive substance, ¹¹¹ In, ¹³¹ I, ⁹⁹ Tc or the like is bound to a monoclonal antibody by a conventional method. However,
The labeling substance should not be limited to the above substances.

In the present invention, in order to apply the antibody as a carcinostatic agent, the antibody is not only a natural antibody but also a toxin or a carcinostatic agent.
It can be used chemically or in the form of liposome bound to a radioisotope, etc., but these methods and means are not limited at all, and known methods and means can be used. As the toxin, for example, ricin A chain, diphtheria toxin, gelonin, saponin and the like are used. Further, as the anticancer agent, for example, mitomycin C, actinomycin D, bleomycin and the like are used. Also,
¹³¹ I, ⁹⁰ Y, ¹⁸⁵ Re and the like are used as the radioisotope.

The antibody used in the present invention may be not only a natural antibody but also an artificial antibody prepared by using the above antibody as a starting material and a protein chemical method. An example of an artificial antibody produced using a protein chemistry method is a fragment such as Fab that can be prepared by cleaving the antibody molecule with pepsin.

[0016]

INDUSTRIAL APPLICABILITY The monoclonal antibody recognizing the neutral sugar chain of the gastric cancer cell obtained by the present invention is expected to be applied as an in-vivo diagnostic agent for gastric cancer and an anticancer agent.

[0017]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. (1) Preparation of Neutral Glycolipids from Cancer Cells 50 g of gastric cancer cell line MKN74 was crushed with a warning blender and then 200 ml of isopropyl alcohol / n-
Extraction operation with a hexane / distilled water (55:20:25 V / V / V) solution was performed twice. The extract was dried with an evaporator and then chloroform / methanol / distilled water (4:
Fractionation (Folch 2: 1 V / V / V) solution
'S partition), the upper layer was again dried with an evaporator and dialyzed against distilled water. The thus obtained fraction of glycolipids having relatively long sugar chains is DEAE-
Using Sephadex A25, neutral sugar fraction (chloroform / methanol / distilled water (30: 60: 8 V /
V / V) and acidic sugar fraction (chloroform / methanol / 0.8M aqueous sodium acetate solution (30: 6)
(0: 8 V / V / V)). (2) Animal sensitization method: colon cancer cell line MKN74 was added to PB
Suspended in S and injected intraperitoneally into Balb / c mice, day 0 5
× 10 ⁷ pcs, 7th day 5 × 10 ⁷ pcs, 21st day 5 × 10 ⁷
The mice were immunized according to the above schedule, and 3 days after the final immunization, the mouse spleen was removed and used for the following cell fusion. (3) Cell fusion and cloning: 10: the above mouse splenocytes and mouse myeloma cells SP2 / 0-Ag14.
Cell fusion was performed by mixing at a ratio of 1 and reacting with 50% polyethylene glycol (average molecular weight 3000) for 2 minutes.

The cells were seeded in a 96-well microtiter plate and cultured in HAT medium for 7 to 14 days. The culture supernatant of the proliferated cells was screened by the ELISA method, and the cells in the positive wells were cloned.

That is, as a result of the ELISA, the cells in the wells in which the culture supernatant was positive were changed to mouse thymocytes (1 x 10
⁷ / ml) in RPMI1640 medium containing 1 /
0.2 ml was added to each well of a 96-well microtiter plate. Colonies that were visible to the naked eye were formed 7 to 14 days after the start of culture. The colonies thus obtained were similarly screened and cloned to obtain MC3-3 and MKA22 single clone strains which were positive for the target antigen. (4) Screening method Regarding the hybridoma, the screening of the clones producing the desired monoclonal antibody was carried out as follows.
It was performed by the ISA method. (A) Coating method of antigen to 96-well microtiter plate: The neutral glycolipid of gastric cancer cell line MKN74 obtained by the method described in Example (1) was added with 5 μg / ml of phosphatidylcholine. It was adjusted to 1 μg / ml with ethanol containing 2.5 μg / ml cholesterol. 20 μl of the prepared glycolipid ethanol solution was dispensed into each well and dried on a hot plate at about 37 ° C.
Overn at 4 ° C using PBS (containing 5% BSA)
Block at light or room temperature for about 2 hours,
It was used for ELISA after preventing nonspecific reaction. (B) ELISA method: 96 prepared by the method (a)
A sample was added to the well microtiter plate, reacted at room temperature for 1 hour and washed, and then a peroxidase-labeled anti-mouse immunoglobulin (IgG + IgM) goat antibody was added, and further reacted at room temperature for 1 hour. After the unreacted labeled antibody was removed by washing, 2,2 'azinodi- [3-ethylbenzthiazolinesulfonic acid] diammonium salt (ABTS) solution was added and reacted at room temperature for 10 minutes, then 0.5 M oxalic acid was added. The addition reaction was stopped and the absorbance at 415 nm was measured.

Thus, the hybridoma MC3-
3, MKA22 was obtained. By culturing these hybridomas, monoclonal antibodies MC3-3, MK
A22 was obtained. (5) Immunoglobulin class: monoclonal antibody MC
3-3, the immunoglobulin class of MKA22 was tested as IgM (κ) by the Octaloni method. (6) Reactivity with gastric cancer cell line MKN74: (a) Coating of gastric cancer cell line MKN74 onto 96-well microtiter plate Method: Cultured gastric cancer cell line MK
N74 was suspended in PBS at 1 × 10 ⁶ / ml, and 50 μl of the suspension was dispensed into each well of a 96-well microtiter plate coated with poly-L-lysine in advance.
After centrifuging at 00 rpm and leaving it at room temperature for 1 hour, 0.2
Add 50% glutaraldehyde in PBS to each well
It was further dispensed in liters. After standing at room temperature for 1 hour, PBS
Wash twice with PBS and use PBS (containing 5% BSA) at 4 ℃
Were used for ELISA after blocking for about 2 hours at Overnight or at room temperature to prevent nonspecific reaction. (B) ELISA method: 96 prepared by the method (a)
Monoclonal antibody MC3-3 or MKA22 was added to the well microtiter plate, reacted at room temperature for 1 hour and washed, and then peroxidase-labeled anti-mouse immunoglobulin (IgM) goat antibody was added, and further reacted at room temperature for 1 hour. It was After washing and removing the unreacted labeled antibody, 2,2′-azinodi- [3-ethylbenzthiazolinesulfonic acid] 2
After adding ammonium salt (ABTS) solution and reacting at room temperature for 10 minutes, 0.5M oxalic acid was added to stop the reaction, and the absorbance at 415 nm was measured to confirm the reactivity with gastric cancer cell line MKN74. It was (7) Extraction of glycoprotein of gastric cancer cell line MKN74 To 10 g of gastric cancer cell line MKN74, 40 ml of distilled water was added, and the mixture was pulverized with a warning blender, and 42 ml of supernatant was obtained by centrifugation. After adding the same amount of 2M perchloric acid (hereinafter referred to as PCA) to this, stirring was continued at 4 ° C for 1 hour,
Centrifugation gave 82 ml of supernatant. The obtained supernatant was neutralized with 6N KOH, the precipitate was removed by centrifugation, and then dialyzed against distilled water. The PCA soluble fraction derived from the gastric cancer cell line MKN74 thus obtained was purified by high performance liquid chromatography using the following TSK-gel G4000SW _XL (manufactured by Toso Co., Ltd., trade name). (8) T of PCA soluble fraction derived from gastric cancer cell line MKN74
Purification by high performance liquid chromatography using SK-Gel G4000SW _XL (manufactured by Toso Co., Ltd.) The PCA soluble fraction obtained by the method of (7) was concentrated to 1 ml by ultrafiltration, PSK was added to TSK gel G4000SW _XL connected to a HPLC device manufactured by Co., Ltd.
Elute with S at a flow rate of 1 ml / min, 0.5 mi
Fractions were collected every n to obtain a fraction eluted in the void. That is, a glycoprotein having a molecular weight of 1,000,000 or more derived from the gastric cancer cell line MKN74 was obtained. (9) Measurement of glycoprotein derived from gastric cancer cell line MKN74 (a) Monoclonal antibody MC3-3 or MKA22
Immobilization: 3 μg / ml of monoclonal antibody dissolved in 0.1 M sodium carbonate buffer (pH 9.6) in each well of an untreated microtiter plate (96 well Nunc plate, manufactured by Intermed) 200 μl of antibody MC3-3 or MKA22 solution was added and incubated at 4 ° C. overnight. Then, remove the solution from each well and add PBS.
After washing three times with a solution containing 0.04% tween-20 (hereinafter PBS-T), a PBS solution containing 0.1% bovine serum albumin (hereinafter BSA-P) was prepared.
300 μl of BS) was added to each well, blocking treatment was performed at 4 ° C., and the wells were stored.

(B) Preparation of horseradish peroxidase (HRP) -labeled antibody: HRP solution (5 mg / m) dissolved in 0.3 M sodium bicarbonate buffer (pH 8.1)
0.1 ml of 1% 1-fluoro-2,4-dinitrobenzene in ethanol was added to 1) and reacted at room temperature for 1 hour. 1.0 ml of 0.06 M sodium periodate was added to the solution and reacted for 30 minutes. Unreacted sodium periodate was added with 0.16M ethylene glycol 1.
After removing by adding 0 ml, it was dialyzed against 0.01 M sodium carbonate buffer (pH 9.5). Next, 5 mg of monoclonal antibody MC3-3 or MKA22 was added to give 5
Reacted for ~ 6 hours. 5 mg of sodium borohydride was added and left at 4 ° C. overnight. Then, in order to remove unreacted sodium borohydride, a 10 mM sodium phosphate buffer solution (pH: 0.85% sodium chloride) was added.
Dialysis was performed against 7.1) at 4 ° C. with stirring overnight. The reaction product was purified by high performance liquid chromatography using TSK-Gel G4000SW (manufactured by Toso Co., Ltd.) to obtain an HRP-labeled antibody.

(C) Quantification of glycoprotein antigen in the sample: The monoclonal antibody MC3-3 immobilized microtiter plate prepared by the method described in (a) in this example was returned to room temperature and then BSA- After washing with PBS solution, (8)
The gastric cancer cell line-derived glycoprotein antigen obtained by the method of
20 μl of a sample that had been double-diluted with, and 100 μl of the labeled MC3-3 monoclonal antibody prepared by the method described in (b) of this Example were added to each well. After incubating at 37 ° C. for 60 minutes as it was, the solution was removed and washed with a BSA-PBS solution three times.
It contains 1.2% 2,2-azinodi- (3-ethylbenzthiazoline sulfate) -diammonium salt (ABTS) and 0.01% hydrogen peroxide (H ₂ O ₂ ) 0.1.
A substrate solution consisting of M citrate buffer (pH 4.1) was added to each well in an amount of 200 μl, and the enzyme reaction was carried out at room temperature for 30 minutes, and then a 200 mM oxalic acid solution was added in an amount of 100 μl to stop the enzyme reaction. A similar reaction was performed using a monoclonal antibody MKN22-immobilized plate and a labeled MKA22 monoclonal antibody.

For each well of the above microtiter plate, the absorption intensity at a wavelength of 415 nm and a control wavelength of 492 nm was measured by an automatic microtiter plate reader (East Sotho).
It was measured by Mfg. Co., Ltd., MPR-A4, trade name).

The results are shown in the table. As you can see from the table,
When either of the monoclonal antibodies MC3-3 or MKA22 was used, the absorbance decreased as the dilution ratio increased, and these monoclonal antibodies showed that the gastric cancer cell line MKN7.
It was shown that the glycoprotein having a molecular weight of 1,000,000 or more obtained from Example 4 was recognized.

[0025]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 5/20 G01N 33/577 B 9015-2J // C12N 15/06 G01N 33/574 B 9015- 2J (C12P 21/08 C12R 1:91)

Claims (3)

[Claims] 1. A monoclonal antibody having the following characteristics: a) A gastric cancer cell line MKN74 or a carbohydrate extracted therefrom is used as an immunogen to sensitize a mammal, and fusion of the immune cell of the mammal with a myeloma cell. And a monoclonal antibody produced by the hybridoma screened with the neutral glycolipid fraction extracted from the cancer cell line MKN74. b) The Ig class is IgM (κ). c) Reacts with gastric cancer cell line MKN74. d) Reactivity with gastric cancer cell line MKN74 with a glycoprotein sugar chain having a molecular weight of 1,000,000 Da or more. 2. A hybridoma cell line capable of producing the monoclonal antibody of claim 1. 3. A pharmaceutical composition containing the monoclonal antibody according to claim 1 as an active ingredient.