JPS61103837A - Human anticancer monoclonal antibody - Google Patents

Abstract

PURPOSE:The titled antibody which is specifically reacted with a cancerous cell of plural organs but not with a normal cell, useful for diagnosis, produced by a hybridoma obtained by fusion of B lymphocyte cell of an animal immunized against human cancerous cell and a tumor cell. CONSTITUTION:A hybridoma obtained by fusion of B lyophocyte of an animal immunized against human cancerous cell and tumor cell is cultivated, and a monoclonal antibody which is not reacted with a normal cell but specifically reacted with a cell of plural organ cancers (e.g., stomatch cencer, lung cancer, mammary cancer, tongue cancer, esophagus cancer, duodenum cancer, cholagiocarcinoma, urinary organ cancer, colon cancer, and salivary gland cancer) is obtained from the culture mixture. Since the antibody is reacted with a cancerous cell of the plural organs, preparation of an antibody to each organ is not required, it is further reacted with a cancerous cell independently of differentiation of the cancerous cell, namely, it is reacted with undifferentiated cancerous cell with which an existing antibody was not reacted, so it is very useful for diagnosis of cancers.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) This invention relates to monoclonal antibodies that specifically react with human cancer cells.

(Conventional technology) Cell fusion technology (Nature + main aim, 495-497,
(1975) ), 11 species of monoclonal antibodies have been produced using this technique. Various monoclonal antibodies against cancer have also been created.
Applied research is underway [GANN, 75
, 485 (1984), Proc, Natt.

Acad, Sci, USA, 75.3405 (
1978), Proe.

Natt, Acad, Sc1. USA, 76,
1438 (1979), 5clenc@, 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.

Note that there is no example in which a monoclonal antibody has been created using TMK-1 [5C-6-JCK, human pre-poorly differentiated adenocarcinoma-derived cells transplanted into nude mice] 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.

(Problems to be Solved by the Invention) This invention solves the problems of the conventional monoclonal antibodies against human cancer as described above, and has the ability to react with cancers in multiple organs without substantially reacting with normal cells. Moreover, the present invention aims to provide a monoclonal antibody that reacts regardless of the degree of differentiation of cancer cells.

(Problems and means for solving them) In order to create monoclonal antibodies having the above-mentioned characteristics, the present inventors used cell fusion technology to obtain novel hybridomas, and by screening them, all of the above-mentioned characteristics were achieved. We succeeded in selecting a hybridoma that produces seven clonal antibodies with the following characteristics, and completed this invention.

Therefore, this invention provides B+ of animals immunized with human cancer cells.
An anti-human cancer product that is produced by a hybridoma obtained by fusion of a 7-cell lineage cell and a tumor cell, and is characterized by not substantially reacting with normal cells, but specifically reacting with cancer cells in multiple organs. Regarding monoclonal antibodies.

(Specific Description) (1) Production of monoclonal antibodies The production of monoclonal antibodies according to the present invention is carried out as follows. First, ■ prepare antibody-producing cells by immunizing an animal with human cancer cells, ■ obtain a cyst/iplidoma by fusing these cells with tumor cells, and ■
Select a hybridoid that produces an anti-human cancer monoclonal antibody having the above-mentioned characteristics.
fsu -'t.

2) Obtain the desired anti-human cancer monoclonal antibody from the culture. The above-mentioned general method itself is known, for example, Japanese Patent Publication No. 58-45407, Japanese Patent Application Laid-open No. 58-45407,
9-128397 and J, Immunol,
Methods, 39285-308 (1980), etc.

■ Preparation of antibody-producing cells Antibody-producing cells are obtained by immunizing an animal with an antigen, extracting lung cells, and using a known method [for example, "Methods in
ENZYMOLOGY, 73.14 (1981) Academic Press]. It can also be prepared by sensitizing B-phosphotZ cells with the antigen in vitro ('84.10.
12 Date and notehead “Preparation method of antibody-producing cells”
reference〕.

In the present invention, the antigen TMK-1 (
After administering SC-6-JCK nude mouse transplanted human gastric poorly differentiated adenocarcinoma-derived cells (first immunization), the immunization procedure (booster immunization) was repeated twice at 10-day intervals, and 3 days after the final immunization, the animals were removed from the plate. After extracting the cells, antibody-producing cells were prepared.

The above TMK-1 is derived from human pre-poorly differentiated adenocarcinoma-derived cells 5C-6-JCK [: Cancer Clinical, 27
．． 1605-1612 (1981): ] is a tissue culture cell line.

(2) Cell fusion This is a step in which the antibody-producing cells prepared above and @cancer cells are fused in the presence of a fusion agent to obtain a hybridoma that can be subcultured. Such cell fusion operation is known as Nature (
(above), J. Immunot, Methods (above), left [monoclonal antibody-), hybridoma and ELI
This can be done with reference to SA-JP 50-60 published by Kodansha Scientific. In the case of the present invention, the above antibody-producing cells and tumor cells P3-X63 AggU, (
P3Ul) (Methods inENZYMOLO
GY, 73.3-46 (1981)) was performed in RPMI-1640 medium in the presence of about 40 polyethylene glycol (PEG).

■ Selection of hybridoma The selection of the desired hybridoma begins with P3 used in the present invention.
In case of tumor cells such as Ul, HAT medium (R containing hypoxanthine, aminobutyrin and thymidine)
PMI-1640 medium) (5science+145,
709 (1964)] [Methods i
nENZYMOLOGY173. 16-18 (198
1):).

Next, the hybridomas obtained by the above procedure are further cultured, and the culture supernatant is analyzed (confirming the presence of antibodies) to select hybridomas producing the desired antibody. Culture supernatants can be analyzed using the plaque method, agglutination reaction method, lanoimmunoassay method, etc., but the simple method is usually the ELISA method (Math, Bnzyrnot, 7).
0.419-439 (1980) etc.]
Ru.

■ Culture hybridomas in a growth medium [e.g. RP] according to the conventional method.
MI-1640 medium (10-20% fetal bovine serum (Fe
2) Culture in (Methoda inEN)
ZYMOLOGYS73.42-43 (1981)]
Alternatively, hybridomas can be intraperitoneally administered to experimental animals (mice, rats, etc.) in which these cells can proliferate and cultured in vivo (Methods in EN).
ZYMOLOGY, 73.43-44 (1981
)).

■ Recovery of antibodies - Antibodies can be collected from the culture supernatant when the hybridomas are grown in a growth medium according to a conventional method, or from the ascites of the animal when the hybridomas are grown in vivo in experimental animals. However, it can be recovered using conventional methods (for example, ammonium sulfate fractionation method). Furthermore, a purified sample with a higher antibody titer can be obtained by using r-filtration method, ion-exchange chromatography method, affinity column chromatography method, or an appropriate combination of these methods [bottom rMONOc
LONALANTIBODIES j 405 (19
80), published by Plenum Press].

(2) Confirmation of specificity Confirmation of the specificity of the created monoclonal antibody can be carried out according to the above-mentioned ELISA method using a system in which cancer cells are immobilized. It is also possible to use a commercially available kit using a tissue specimen of cancer cells. In the present invention, the ABC method [J, 1H1a, tocheffl,
Cytochem, 29. 577 (1981)] E. - C line 9 E. Kao, ABC method is good, ■AB
This can be easily done using C kit.

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

In Table g3 (Example 3), hybridoma 9A3.20D11 is a representative example of the monoclonal antibody of this invention.
As shown for the monoclonal antibodies derived from , and 21A4, the monoclonal antibodies of the present invention hardly react with normal cells, and this property is particularly remarkable in the monoclonal antibodies derived from 9A3.

The monoclonal antibodies of this invention are shown in Table 2 (Example 2)
As shown in Figure 2, it universally reacts with gastric cancer cells of a wide range of tissue types, regardless of the degree of differentiation. This property is 9
This is particularly noticeable in monoclonal antibodies derived from A3.

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, tongue cancer, esophageal cancer, duodenal cancer, bile duct cancer, and urinary organ cancer. It can react against multiple cancers such as cancer, skin cancer, colon cancer, and salivary gland @.

(3) Anti-human cancer monoclonal antibody The anti-human cancer monoclonal antibody thus obtained is prepared into a high antibody titer standard as necessary, and then used for cancer diagnosis (pathological diagnosis, radiological diagnosis). It can be used for scientific diagnosis, etc.). In addition, it may be possible to apply it to a treatment method (missile therapy) in which the chemotherapeutic agent acts only on cancer cells in combination with the chemotherapeutic agent conventionally used for the treatment of cancer (:
BRITISH MEDICAL JOURNAL 2
85.461-462 (1982), 285.1
447 (1982) ml.

(Effects of the Invention) The monoclonal antibody of the present invention specifically reacts with cancer cells and does not react with normal cells, so it can be used for cancer diagnosis. That is, when using the monoclonal antibody of the present invention bound to a labeling substance (enzyme, fluorescent substance, etc.), the antibody specifically reacts with cancer cells, so the characteristics of the labeling substance bound to the antibody are utilized. This makes it possible to detect cancer cells. Furthermore, when the monoclonal antibody of the present invention is bound to a metal matrix (ferritin, etc.) or other substances that interfere with electron beams, cancer cells can be detected using an electron microscope.

Since the monoclonal antibody of the present invention reacts with cancer cells in a plurality of different organs, there is no need to prepare antibodies for each organ.

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

The monoclonal antibody derived from hybridoma 9A3 of the present invention particularly exhibits 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.

Example 16.

(1) Cell preparation Batb/c mice were immunized by intraperitoneal administration of 106 human pre-poorly differentiated adenocarcinoma TMK-1 cells, followed by two booster immunizations at 10-day intervals after the initial immunization. went. Three days after the final immunization, the mouse tile cells were removed aseptically, loosened, and placed in RPMI-1640 medium at 97°C.
1! Thereafter, a mouse tile cell suspension (2×10 cells/d) was prepared by filtration through a nylon mesh.

On the other hand, mouse tumor cell P3Ul to be fused with the above cells
(Flow Corporation) suspension (RPMI-1640 medium) was prepared according to a conventional method.

(2) Cell fusion The mouse tile cell suspension prepared above and the P3Ul cell suspension were mixed at a cell number ratio of tile cells and P3Ul cells of 10:
After mixing at a ratio of 1:1, the mixture was centrifuged and the supernatant was removed. Next, add about 40 PEG 40 to the cells at the bottom of the centrifuge tube.
00-containing PBS←) solution ITL1 was slowly added dropwise.

After leaving this at 37℃ for 4 minutes, 1""-16"
0 (contains 0% FC3).

Toyofi PEG was diluted. After centrifugation and removing the supernatant, the final concentration of P3U1 cells was 1. 0
RPMI-1640 (10
After diluting with FC3), add 100% to a 96-well plate.
Dispense was performed at a ratio of μt/well. In addition, this plate contains Batb/c within 3 weeks of age as feeder cells in advance.
100 mouse thymocytes at a ratio of 5 x 10 cells/well
Now dispense L each into μ and leave.

(3) Selection of hybridomas From the day after the fusion procedure described above, half of the culture medium in each well is filled every day for 4 days! (100 μt) each was replaced with [T medium, and
While holding an exchange meeting for HAT medium and Shishi medium every other day,
Culture was carried out for several days. In addition, the above HAT medium is RPMI
-100 μM hypoxa/tin in 1640 medium, 0.1 μ
M aminozothelin, 16 μM thymison, 10 FC8,
5X 10-5M 2-mercagutoethanol, 2mM
Glutamine, 100 units/1nl penicillin, and UO, 1m97ml streptomycin were added.

In addition, PBS←) is a mixture of sodium chloride s, o, y, potassium dihydrogen phosphate (anhydrous) 0.2gjlC' acid-sodium hydrogen phosphate (anhydrous) 1.15g, and potassium chloride 0.2I to make 10100O. (PH7,4).

The culture supernatant was then analyzed using the following procedure.

(+) Preparation of analysis plate (a) Preparation of antigen 2 x 10 TMK-1 cells/ytl'flO, 25M, homogenized after suspension in Tris-HCl buffer (P) (7,4) The supernatant was removed by centrifugation. The obtained precipitate was resuspended in the above buffer solution, and the supernatant was removed by centrifugation. Then, calcium chloride 1. Q, 9 and magnesium chloride 1.0.9] was added until the number of cells was 2
The number was adjusted to 106 cells/rnl.

(b) Antigen immobilization Add 10 μI Anl of poly-L-lysine-containing PBS (-) to each well of a 96-well plate (7 flucon).
Inject 0 μt each, leave at room temperature for 30 minutes, and then add PBS
pween (PBS←) to Tween 2Q Q
, 5 ml]. To this, add 10 antigens prepared above at 150μt/well [PBS←]
After centrifugation, 50 μt of PBS containing 0.5% glutaraldehyde (←) was added to each well, and the mixture was left at 3 temperatures for 15 minutes. After washing twice with PBS←)-Ttvsen, l
1% bovine serum albumin (B
After adding SA)-containing PBS(-) solution and injecting it statically at room temperature for 30 minutes, it was washed once with PBS←)-Tween. Next, PBS←) (containing 1% BSA) was added, and the plate was left to stand at room temperature for 2 hours, followed by washing with PBS←)-'pveen to prepare a plate for analysis.

In addition, a control analytical grade in which normal gastric mucosal cells were immobilized was also prepared in the same manner as above.

(11) Analysis of supernatant The supernatant of the wells where colonies appeared was removed and added at 50 μt/well to the above two types of analysis plates.

After standing for 2 hours, 50 μt/well of chinonooxidase-labeled goat anti-mouse immunoglobulin (Capel), which had been washed 3 times with PBS←)-Tween, was added and incubated for 2 hours (37°C, 5% carbon dioxide). went.

After washing 5 times with PBS←)-Tween, 0.1M citrate buffer (PH4
, 2) with ABTS [: 2.2'-anonobis(3-ethylbenzothiazoline)-6-sulfonic acid 12.5 mM
A solution of 5 mM of hydrogen peroxide solution was prepared immediately before use, and after injection at a rate of 100 μt/well, a reaction was performed at room temperature for 5 to 15 minutes. Then the reaction was stopped (2mM
Sodium azide was added at 100μt/well) and then titertic multiscan■ (Flow Co., Ltd.)
D4o5 was measured.

By this method, well 10 containing the supernatant that reacts in the immobilized TMK-1 cells but not in the plates immobilized with normal gastric mucosal cells.
Selected seeds. Then, multicellular colonies in these 10 wells were taken out and cloned by the limiting dilution method to establish 10 types of hybridomas.

(4) Culture of hybridomas and recovery of antibodies
In PMI-1640 (containing 10% FC8) medium at 37°C,
The cells were cultured in a carbon dioxide incubator in the presence of carbon dioxide gas, and the antibodies were then recovered from the culture supernatant by ammonium sulfate fractionation. The results of the characterization of these antibodies are presented in the first
Shown in the table.

Table 1, Ig to L/', indicates that the subclass is IgM and its light chain is ka-ya. Similarly, Ig
G, / indicates that the subclass is IgG1 and the light chain is 2.

Example 2 Hyperidoma 9A3.20D11. obtained according to the present invention.
Using monoclonal antibodies derived from 21A4 and 21A4, the specificity for gastric cancer paper vesicles was investigated.

The specificity of the antibody was examined using the Vectastin ABC kit according to the following procedure.

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

This and the antibody of the present invention were then incubated at room temperature for 120 minutes and washed three times with PBS. Biotinylated horse anti-mouse immunoglobulin (Pecta) was added to this and incubated at room temperature for 60 minutes, followed by washing with PBS←). Next, avidin and piotinylated peroxidase complex were added, and after further incubation for 60 minutes, the mixture was washed with PBS←). After adding a solution containing diaminobenzdine and hydrogen peroxide, the mixture was incubated for 5 minutes, washed, and then counterstained with methyl green. The results are shown in Table 2. In addition, 9A3.2 in Table 2
0D11 and 21A4 indicate the origin (hybridoma) of the tested antibody.

Table 2 Table 2 (Continued) Table 2 (Continued) The expression of tissue type 1 in Table 2 is in accordance with the Gastric Cancer Handling Regulations and has the following meaning.

pap: papillary adenocarcinoma tub, S well-differentiated ductal adenocarcinoma tub2; moderately differentiated ductal adenocarcinoma por; poorly differentiated adenocarcinoma sig: punctate cell carcinoma muC; colloid adenocarcinoma 3q: squamous cell carcinoma; ) means medullary carcinoma, and (sci) means scirrhous carcinoma. Furthermore, +1 person.

The symbol - indicates the following meaning.

10. Strongly stained.

10 Partly stained.

- No staining at all.

Example 3 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.

Table 3 Reaction only with monkey neutrophils Table 3 (face down) Table 3 (continued) Table 3 (continued) Example 4 Cancer cells derived from each adult tissue in the same manner as in Examples 2 and 3 above. The reactivity of the monoclonal antibody of the present invention with the monoclonal antibody of the present invention was investigated. The results are shown in Table 4.

Below margin Table 4 Table 4 (continued) Table 4 (continued)

[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 black parts indicate gastric cancer cells. Note that the magnification is 160 times. Patent Application Agent for Patent Issuer Yusui Pharmaceutical Co., Ltd.

Claims (1)

[Claims] 1. It is produced by a hybridoma obtained by fusion of B lymphoid cells of an animal immunized with human cancer cells and tumor cells, does not substantially react with normal cells, and can be used to treat cancers in multiple organs. An anti-human cancer monoclonal antibody that specifically reacts with cells. 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
Monoclonal antibodies described in section. 3. The monoclonal antibody according to claim 2, which reacts relatively strongly with human gastric cancer cells.