JPS61104783A - Hybridoma producing anti-human carcinoma monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide the titled hybridoma obtained by the fusion of a tumor cell and the B-lymphocyte cell of an animal immunized with human carcinoma cell, essentially inert to the normal cell, reactive specifically with the carcinoma cell of plural organs, and reacting independent to the degree of differentiation of the carcinoma cell. CONSTITUTION:A B-lymphocyte antibody-producing cell is prepared by immunizing an animal with a human carcinoma cell, and the produced cell is fused with a tumor cell to obtain a hybridoma. A hybridoma producing an anti-human carcinoma monoclonal antibody having the above characteristics is selected from the produced hybridoma. The hybridoma is cultured, and the objective antihuman carcinoma monoclonal antibody is separated from the culture product. Since the monoclonal antibody reacts specifically with carcinoma cells and inert to normal cell, it can be utilized for the detection of cancer cell in the diagnosis of cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a hybridoma that produces a monoclonal antibody that specifically reacts with human cancer cells.

[Conventional technology]

Cell fusion technology [Nature,
256.459-497, (1975)), various monoclonal antibodies have been produced using this technique. Among them, various monoclonal antibodies against cancer have been created, and their applied research is progressing [GANN, 75.485 (1984), Proceedings of the National Academy of Sciences]
・Of the Sciences of the United・
Stiso Op América (Proc, Natj, ^
cad.

Sci, USA), 75.3405 (1978),
Proceeding Inc. 1 of the National Academy of Sciences of the United States of America (Proc, NatiA
cad, sci, USA).

76.1438 (1979), Science
nce), 212.53 (1981), etc.].

However, the monoclonal antibodies against human cancer that have been created so far have also reacted with some normal cells. Therefore, the practicality of using monoclonal antibodies for cancer diagnosis has been a problem.

Furthermore, Japanese Patent Application Laid-Open No. 59-128397 describes a monoclonal antibody against human gastric cancer. This monoclonal antibody specifically reacts with well-differentiated gastric cancer cells, but does not react strongly with undifferentiated cancer cells.

It should be noted that there is no example of creating a monoclonal antibody using TMK-1 (SC-6JCK nude mouse transplanted human gastric poorly differentiated adenocarcinoma-derived cells) as an antigen.

As mentioned above, various monoclonal antibodies are known, but monoclonal antibodies that do not substantially react with normal human cells, react with cancers in multiple organs, and react regardless of the degree of differentiation of cancer cells. is not known.

[Problem that the invention seeks to solve]

In order to solve the problems of conventional monoclonal antibodies against human cancer as described above, the present invention has been developed to provide monoclonal antibodies that do not substantially react with normal cells but react with cancers in multiple organs.
Furthermore, the present invention aims to provide a hybridoma that produces a monoclonal antibody that reacts regardless of the degree of differentiation of cancer cells.

[Means for solving problems]

In order to create a hybridoma that produces a monoclonal antibody having the above-mentioned characteristics, the present inventors used cell fusion technology to obtain a hybridoma, and by screening the hybridoma, a monoclonal antibody having all the above-mentioned characteristics was obtained. We succeeded in selecting a hybridoma that produced this, and completed this invention.

Therefore, this invention provides an anti-inflammatory drug that is obtained by fusion of tumor cells with B lymphoid cells of an animal immunized with human cancer cells, and which does not substantially react with normal cells but specifically reacts with cancer cells in multiple organs. This invention relates to a hybridoma that produces human cancer monoclonal antibodies.

[Specific explanation]

(1) A monoclonal body hybridoma The monoclonal antibody-producing hybridoma according to this invention is produced by: ■ preparing antibody-producing cells by immunizing an animal with human cancer cells; and ■ fusing these cells with tumor cells. 1. Select a hybridoma that produces an anti-human cancer monoclonal antibody having the above-mentioned characteristics. Then, (1) this hybridoma is cultured, and (2) the desired anti-human cancer monoclonal antibody can be obtained from the culture. In addition,
The above-mentioned general method itself is publicly known, for example, Japanese Patent Publication Nos. 58-45407 and 1983-128397, and Journal of Immunological Men's (J.
, Immunol, Methods), 39 285~
308 (1980), etc.

■ Preparation of antibody-producing cells Antibody-producing cells are prepared by immunizing an animal with an antigen and then extracting lung cells using a known method [for example, Methods in ENZYMOLOGY].
).

73.14 (1981) Academic Press] can thus be prepared. It can also be prepared by sensitizing B lymphoid cells with an antigen in vitro [see Japanese Patent Application No. 59-212578].

In the present invention, the mouse 1111'! Antigen TM inside
After administering K-1 (SC-6-JCK nude mouse transplanted human gastric poorly differentiated adenocarcinoma-derived cells) (initial immunization), the immunization procedure (boosting immunization) was performed twice every 10 days (repeat, starting from the final immunization). Three days later, lung cells were removed from the animals, and antibody-producing cells were prepared.

The above TMK-1 is 5C-6-JCK (cancer clinical, nail,
1605-1612 (1981)).

(2) Cell fusion This is a step in which the antibody-producing cells prepared above and 1@tumor cells are fused in the presence of a fusion agent to obtain a hybridoma that can be subcultured. Such cell fusion operation is performed by Nature (
Nature) (supra), Journal of Immunological Men (J, Immonon.

Methods) (above), bottom ``monoclonal antibody-''
Hybridoma and ELISA JP 50-60 can be performed with reference to Kodansha Scientific, etc.
In the case of the present invention, the above antibody-producing cells and tumor cells P3-X
63-Ag*U+ (Pxul) (Methods in ENZYMOL
OGY), 73.3-46 (1981)) was carried out in RPMI-1640 medium in the presence of about 40% polyethylene glycol (PEG).

■ Selection of hyperdoma To select a desired hyperdoma, first use the P used in the present invention:
+01 tumor cells, HAT medium (RPMI-1640 medium containing hypoxanthine, aminopterin and thymidine) [Scie
nce), 145.709 (1964)) [Methods in Enzymology (Methods in
ENZYMOLOGY), Mutual, 16-18 (1981)
).

Next, the hybridomas obtained in the above procedure are further cultured, and hybridomas producing the desired antibody are selected by analyzing the culture supernatant (confirming the presence of antibodies t=>). methods, agglutination reaction methods, radioimmunoassay methods, etc., but the simple ones are usually ELIS eight methods [Hens in Enzymology (M
eth, Enzymol L 70.419-439
(1980) etc.].

■ Cultivate hybridomas in a growth medium [e.g. RPM] according to the conventional method.
I-1640 medium (10-20% fetal calf serum (FCS)
))] [Methods in Enzymology (Methods in EMZYMO)]
, 73.42-43 (1981)), or by intraperitoneally administering the hybridoma to an experimental animal (mice, rat, etc.) in which these cells can proliferate and culturing it in vivo [Men in Enzymology (1981)]. Methods
in EMZYMO-LOGY), 73.43-4
4 (1981)).

■ Recovery of antibodies Antibodies can be collected from culture supernatants when hybridomas are grown in growth medium according to conventional methods, or from ascites of animals when hybridomas are grown in vivo in experimental animals. For example, it can be recovered according to the ammonium sulfate fractionation method). Furthermore, purified samples with higher antibody titers can be obtained by gel filtration, ion exchange chromatography, affinity column chromatography, or an appropriate combination of these methods. Bodies (MONOCLONAL ANTI)
BODIES) J405 (1980), published by Blenheim Press].

The specificity of the created monoclonal antibody can be confirmed according to the ELISA method described above using a system in which cancer cells are immobilized. It is also possible to use a tissue specimen of cancer cells and a commercially available kit. In the present invention, the ABC method [Journal of Histochemistry and Cytochemistry
Em, Cytochem), 29+577 (1981
)). Note that the ABC method can be easily carried out using the Vectorstin ABC kit.

The results are shown by specific data in Examples 2-4.

As shown in Table 3 (Example 3) for monoclonal antibodies derived from hybridomas 9A3.20D11 and 21A4, which are representative examples of hybridomas of the present invention, these monoclonal antibodies hardly react with normal cells, and this property is This is particularly noticeable in the 9A3-derived monoclonal antibody and the 20D11-derived monoclonal antibody.

As shown in Table 2 (Example 2), the monoclonal antibodies produced by the hybridomas of this invention universally react with gastric cancer cells of a wide range of tissue types, regardless of the degree of differentiation. This property is particularly remarkable in 9A3-derived monoclonal antibodies and 20D11-derived monoclonal antibodies.

Furthermore, as shown in FIG. 4 (Example 4), the monoclonal antibody of the present invention can be used not only for gastric cancer but also for cancers of other organs, such as lung cancer, breast cancer, old island cancer, esophageal cancer, duodenal cancer, bile duct cancer, and urinary organ cancer. It can respond to multiple cancers, including skin cancer, colon cancer, and salivary gland cancer.

(3) Human voice monoclonal body The anti-human cancer monoclonal antibodies obtained in this way are purified into high antibody titer samples as necessary, and then used for cancer diagnosis (pathological diagnosis, radiological diagnosis, etc.). ) can be used for Also,
It could also be applied to treatment methods (missile therapy) in which chemotherapeutic agents act only on cancer cells in combination with chemotherapeutic agents conventionally used to treat cancer [BRITISH MEDICAL JOURNAL]
OLIRNAL), Dish, 461-462 (1982)
, Ibid., Fallen L11447 (1982)).

(Effects of the Invention) Monoclonal antibodies produced by the hybridoma of the present invention specifically react with cancer cells and do not react with normal cells, so they can be used for cancer diagnosis. That is, the hybridoma of the present invention can be used for cancer diagnosis. When using a monoclonal antibody produced with a labeled substance (enzyme, fluorescent substance, etc.) bound to it, the antibody specifically reacts with cancer cells. Furthermore, when these monoclonal antibodies are bound to metal proteins (such as ferritin) or other substances that interfere with electron beams, it is possible to detect cancer cells using an electron microscope.

Since the monoclonal antibodies produced by the hybridomas of the present invention react with cancer cells in multiple different organs, there is no need to prepare antibodies for each organ.

Further, the antibody of the present invention has a wider range of applicability than conventional antibodies because it reacts with cancer cells regardless of the degree of differentiation of cancer cells, that is, it also reacts with undifferentiated cancer cells.

The monoclonal antibody derived from hybridoma 9A3 and the monoclonal antibody derived from hybridoma 20D11 of the present invention particularly exhibit the above-mentioned characteristics.

This monoclonal antibody is particularly suitable for use in diagnosing gastric cancer because it reacts relatively strongly with gastric cancer cells and does not substantially react with normal cells other than neutrophils and macrophages.

S1uIk+1) Cell PreparationBa1b/c mice were injected with human gastric poorly differentiated adenocarcinoma TMK-
Immunization was performed by intraperitoneally administering 10" of 1111 cells, and booster immunizations were given twice at 10-day intervals after the initial immunization. Lung cells were aseptically removed from the mice on the third day after the final immunization. The cells were loosened and suspended in RPMI-1640 medium, and then filtered through a nylon mesh to prepare a suspension of mouse tile cells (2 x 10' cells, 7 ml).

On the other hand, the mouse tumor cell PsU1 (
A suspension (RPMT-1640 medium) was prepared according to a conventional method.

(2) Cell fusion The mouse tile cell suspension prepared above and the P3U1 cell suspension were mixed at a cell number ratio of lung cells and P3U1 cells of 10:
After mixing at a ratio of 1:1, the mixture was centrifuged and the supernatant was removed. Next, apply about 40% PEG to the cells at the bottom of the centrifuge tube.
4000-containing PBS (-") solution 1111 was slowly added dropwise. After standing at 37°C for 4 minutes, RP
PEG was diluted by adding MI-1640 (containing 10% FC5). Then, after centrifuging and removing the supernatant, the final concentration of PsU1 cells was 1.0 x 10' cells/
Add RPMI-1640 (10% FC5) to
After diluting with (containing), it was dispensed into a 96-well plate at a rate of 100 μl/well. In addition, 100 μl of thymocytes from 961 b/c mice within 3 weeks of age were added to this plate in advance as feeder cells at a ratio of 5XIO' cells/well.
/ well.

(3) Selection of hybridomas From the day after the above fusion operation, half of the medium (100 μE) in each well was replaced with ■^T medium every day for 4 days, and the medium was further replaced with 118T medium every other day. Culture was carried out for 10 days while performing the following steps. In addition, the above) IAT medium is
RPMI-1640 medium containing 100μH Hiboki 4-nitin, 0.1μn aminopterin, 1.6μi thymidine,
10% FC5, 5X10-'M 2-mercaptoethanol, 2mM glutamine, 100 units total penicillin, and 0.1 + wg/mi streptomycin.

In addition, PBS (-) includes 8.0 g of sodium chloride, 0.2 g of potassium dihydrogen phosphate (anhydrous), 1.15 g of sodium hydrogen phosphate (anhydrous), and 0.2 g of potassium chloride.
was mixed to make 10100O1 (pH 7.4
).

The culture supernatant was then analyzed using the following procedure.

(i) Preparation of analysis plate (a) Preparation of antigen
The suspension was suspended in sugar-containing Tris-HCl buffer (pH 7.4), homogenized, and the supernatant was removed by centrifugation. The obtained precipitate was resuspended in the above buffer, and the supernatant was removed by centrifugation. Then, 1.0 g of calcium chloride and 1 g of magnesium chloride were added to the composition of PBS (-).
．． 0g] and the number of cells is 2 x 106 cells/
It was prepared so that it was al.

(b) Antigen immobilization 10 gg/ml poly-L-lysine-containing PBS (-
') was injected in 50 μl portions, and after standing at room temperature for 30 minutes, PBS was added.
(-)-Tween (T in the PBS (-)
ween200.5mj! was added. To this, add 10'' antigens prepared above at 150 ttl/
After injecting into each well (suspended in PBS (-)), centrifugation was performed, and then 50 μl of PBs (-) containing 0.5% glutaraldehyde was added to each well and left at room temperature for 15 minutes. . PBS(-)-Tey
After washing twice with PBS containing 1% bovine serum albumin (BS^) containing 100 mM glycine (-
) solution and leave it at room temperature for 30 minutes, then add PBS (
-) - Washed once with Tween. Then PBS(-)
(contains 1% BSA) and leave it at room temperature for 2 hours, then PB
Plates for analysis were prepared by washing with S(-)-Tween.

A control analysis plate on which normal gastric mucosal cells were immobilized was also prepared in the same manner as above.

(ii) Analysis of the supernatant. Take the supernatant of the well in which colonies appeared, and
50 μl/well was added to an analytical plate.

After standing for 2 hours, the wells were washed 3 times with PBS(-)-Tween, 50 μl/well of peroxidase-labeled goat anti-mouse immunoglobulin (Capel) was added, and incubated for 2 hours (37°C, 5% carbon dioxide gas). went.

After washing 5 times with PBS(-)-Tene, 0.1M citrate buffer (p
ABTS (2,2'-azinobis(3-
Ethylbenzothiazoline)-6-sulfonic acid) 2.5m
A solution of M and 5 mM of hydrogen peroxide solution was prepared immediately before use, and after injection at a rate of 100 μl/well, the reaction was carried out at room temperature for 5 to 15 minutes. Then stop the reaction (2 m
After adding M sodium azide (100 μl/well), the titer tic multiscan e (Flow Co., Ltd.) was used. . , was measured.

By this method, 10 wells containing the supernatant reacted on plates immobilized with TMK-1 cells but did not react on plates immobilized with normal gastric mucosal cells.
Selected seeds. Cells were then taken out from the colonies in these 10 wells and cloned by the limiting dilution method to establish 10 types of hybridomas.

(4) Culture of hybridomas and collection of antibodies
0 (containing 10% pcs) medium at 37° C. in the presence of 5% carbon dioxide in a carbon dioxide gas incubator, and then antibodies were recovered from the culture supernatant by ammonium sulfate fractionation. The results of characterizing these antibodies are shown in Table 1.

4 for 1gM/umbrella! 7'7 Russ is IgM ′:z:(
D IMfjA is ni (power 7ba) 1
be. Similarly, IgG+/ has a subclass of IgG1 and t
Indicates that i ijt is .

Slk Hybridoma 9 A 3.20D1 obtained by the present invention
Using monoclonal antibodies derived from 1 and 21A4,
The specificity for gastric cancer cells was investigated. The properties of the antibody were investigated using Vectastin's ABC kit according to the following procedure.

Gastric cancer tissue sections (55 cases) obtained from patients were incubated in methanol containing 0.3% hydrogen peroxide for 30 minutes to block peroxidase activity inherent in the tissue. The tissue was then washed with PBS (-) and
The cells were incubated with normal horse serum diluted approximately 20 times with water and washed with PBS(-).

This and the antibody of the present invention were then incubated at room temperature for 12020 minutes and washed three times with PBS(-). Biotinylated horse anti-mouse immunoglobulin (Vector) was added to this and incubated at room temperature for 60 minutes, followed by PBS (-
). Next, avidin and biotinylated peroxidase complex were added, and after further incubation for 60 minutes, the mixture was washed with PBS (-). A solution containing diaminobenzidine and hydrogen peroxide was added thereto, followed by incubation for 5 minutes, washing, and counterstaining with methyl green.

The results are shown in Table 2. In addition, 9A in Table 2
3.20D11 and 21A4 indicate the origin (hybridoma) of the tested antibodies.

Hereinafter, the expression "Ml woven type" in Table 2 is in accordance with the Gastric Cancer Handling Regulations, and has the following meaning. It is something that we have.

pap: papillary adenocarcinoma tublH well differentiated ductal adenocarcinoma tubzH moderately differentiated ductal adenocarcinoma por; poorly differentiated adenocarcinoma sig H signet ring cell carcinoma mucH gluoid adenocarcinoma sq; squamous cell carcinoma (mad) is medullary It means cancer, and (sci) means scirrhus (hard cancer). Furthermore, +.

The symbols ± and - have the following meanings.

+ Strongly stained.

± Partially stained.

- No staining at all.

The same operations as in Example 2 were performed to examine the reactivity between the antibody of the present invention and normal cells in each biological tissue.

The results are shown in Table 3.

1) In the same manner as in Examples 2 and 3 above, cancer cells derived from each adult tissue and neutrophils were treated in the same manner as in Examples 2 and 3 above. The reactivity with the monoclonal antibody of the invention was investigated. The results are shown in Table 4.

Below margin Kaya-2riichimote 19th Marugame 1st μλ)

[Brief explanation of drawings]

FIG. 1 is an example of a photograph of gastric cancer cells stained by the ABC method using a monoclonal antibody derived from hybridoma 9A3, and is a photograph in place of a drawing showing the morphology of the organism. The parts that appear black indicate gastric cancer cells, and the magnification is 160x. FIG. 2 is a 160x photograph of gastric cancer cells stained by the ABC method using a monoclonal antibody derived from hybridoma 9A3, and is a photograph in place of a drawing showing the morphology of the organism. The parts that appear in M color are gastric cancer cells. Figure 3 is a photograph of signet ring cells (gastric cancer cells) stained when the ABC method was performed using a monoclonal antibody derived from hybridoma 9A3, and is a photograph replacing a diagram showing the morphology of the organism. Upper part (unstained part)
indicates normal epithelium, and the lower part of the drawing (black area) indicates cancer cells. FIG. 4 is a photograph of papillary adenocarcinoma (gastric cancer cells) stained when the ABC method was performed using the hybridoma 9A3 monoclonal antibody, and is a photograph in place of a drawing showing the morphology of the organism. The parts that appear black are cancer cells, and the cell bodies and cell crests of the cancer cells are stained. FIG. 5 is a photograph of a well-differentiated ductal adenocarcinoma (stomach cancer cell) stained when the ABC method was performed using a monoclonal antibody derived from hybridoma 9A3, and is a photograph representing the morphology of the organism. Out. The parts that appear black are cancer cells. Figure 6 is a 160x photograph of a pre-poorly differentiated adenocarcinoma stained by the ABC method using a monoclonal antibody derived from hybridoma 20D11, and is a photograph in place of a drawing showing the morphology of the organism. be. The parts that appear black are cancer cells. Figure 7 is a 160x photograph of an alveolar epithelial carcinoma stained when the AflC method was performed using a monoclonal antibody derived from hybridoma 20D11, and is a photograph in place of a drawing showing the morphology of the organism. . The parts that appear black are cancer cells. Fig. 8 is a 160x photograph of a lung adenocarcinoma stained by the ABC method using the monoclonal antibody of Hypuridoma 20D11 Yamaki, and is a photograph in place of a drawing to consider the morphology of the organism. be. The parts that appear black are cancer cells.

Claims (1)

[Scope of Claims] 1. Obtained by fusion of tumor cells with B lymphoid cells of an animal immunized with human cancer cells, which substantially does not react with normal cells and is specific to cancer cells in multiple organs. A hybridoma that produces anti-human cancer monoclonal antibodies that react with cancer. 2. The scope of claim 1, wherein the cancers in multiple organs are multiple cancers selected from stomach cancer, lung cancer, breast cancer, tongue cancer, esophageal cancer, duodenal cancer, bile duct cancer, urinary organ cancer, skin cancer, colon cancer, and salivary gland cancer. 1
Hybridomas described in Section 1. 3. Hybridoma 9A3 or hybridoma 20D1
1. The hybridoma according to claim 1, which is