JPH0356425A - Carcinostatic composition - 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 [Field of Industrial Application] The present invention provides an anticancer composition containing as an active ingredient a monoclonal antibody having specificity for N-acetylneuraminic acid having β coordination. Regarding.

[Conventional technology]

In 1975, K6hlor and M
John Illstein introduced a method for producing monoclonal antibodies using hybrid cells (hybridomas), which allows the production of unlimited quantities of antibodies with uniform specificity and properties.

Antibodies are proteins that have the ability to associate with and recognize other molecules known as antigens. Monoclonal antibodies are no different from other antibodies, but they are highly homogeneous in nature and recognize only one type of antigen or antigenic determinant. Many techniques for producing such monoclonal antibodies and hybridomas are described in Monoclonal Hybridoma Antibodies: Techniques and Applications.
onal Hybridoma Antibodies
: Techniques and Applic
aLions) J (John G.
．． Hurre, ed., 1983) is common today.

On the other hand, N-acetylneuraminic acid is present at the end of the sugar chain of complex carbohydrates and is known to play a variety of important biological functions. In recent years, the existence of a novel glycan cancer antigen containing β-coordinated N-acetylneuraminic acid in malignant tumors has been suggested, and its significance has attracted attention. Therefore, a composition containing a monoclonal antibody having specificity for β-coordinated N-acetylneuraminic acid has been expected for purposes such as cancer treatment. Among the monoclonal antibodies against these complex carbohydrates, many monoclonal antibodies whose epitope is a site containing N-acetylneuraminic acid have been obtained, but all of these are α
It reacts with N-acetylneuraminic acid, which has a β-coordination. Since they do not react with N-acetylneuraminic acid, they all convert N-acetylneuraminic acid with β coordination into F! ! Ii does not have the ability to combine,
It could not be used for the purpose of cancer treatment. [Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide an anticancer composition containing a monoclonal antibody having β-coordinated N-acetylneuraminic acid as an epitope (antigenic determinant) as an active ingredient. It's about doing. (Means for Solving the Problems) In view of the above problems, the present inventors chemically synthesized a β-coordinated sialic acid compound, created a monoclonal antibody using this as an immunogen, and produced a monoclonal antibody using the β-coordinated sialic acid compound. It was revealed that the present invention reacts specifically with cancer cells and does not react with normal cells, leading to the completion of the present invention.

That is, the present invention provides an anticancer drug containing as an effective ingredient a monoclonal antibody having specificity for N-acetylneuraminic acid having β coordination as an epitope (antigenic determinant). It is.

The present invention will be explained in more detail.

Hybridomas that produce the monoclonal antibodies contained in the composition of the present invention can be obtained by the method of Koehler et al., that is, by cell fusion of B cells (lymphocytes) obtained by immunizing an animal with an antigen and myeloma cells. Prepared by Here, as the "antigen" for producing the monoclonal antibody contained in the Mi compound of the present invention, N-
Specific examples of these compounds, which are acetylneuraminic acid derivatives, are listed below.

(B) (C) Next, an N-acetylneuraminic acid derivative having an α-coordination that does not show antigen specificity to the monoclonal antibody contained in the composition of the present invention. A specific example is shown below. (H) 0H (J) 0H The monoclonal antibody produced from the hybridoma recognizes the β-coordinated sialic acid as an epitope (antigenic determinant) and reacts specifically with it. Therefore, the monoclonal antibody produced from the hybridoma has high specificity for any N-acetylneuraminic acid having β-coordination and its derivatives. First, mice are immunized intramuscularly, subcutaneously, or intraperitoneally with the antigen described above. In this immunization, the adjuvant, i.e., the immunopotentiator, can be either an incomplete adjuvant or a complete adjuvant, such as oil, emulsifier, killed Mycobacterium tuberculosis, killed Salmonella and mixtures thereof, preferably killed Salmonella minnesota. Bacteria can be used. Next, the B cells and myeloma cells obtained above are fused.

Here, as the fusing agent, polyethylene glycol, H
VJ etc. can be used, preferably polyethylene glycol 4000. Furthermore, as the culture medium, it is preferable to use HAT medium in order to select hybridoma cells and myeloma cells. Furthermore, cloning the obtained hybridoma? Single clones are isolated using methods such as methylcellulose, soft agarose, or limiting dilution.

The antibody titers of the monoclonal antibody-producing hybridomas obtained in this way are checked in a conventional manner, and the hybridomas with high antibody titers are selected and stored. ＝Raifu′
The ridoma has been deposited with the American Thai Culture Collection (accession number HB9619AT).
CC), available to anyone.

The monoclonal antibody thus obtained can recognize cancer cells as antigens and, as a result, has anticancer activity. The reaction between cancer cells and the monoclonal antibody causes cancer cells,
For example, PC-10 (a transplantable tumor line derived from lung cancer),
EL IS using YTS-1 (human bladder cancer) etc.
A method (enzyme immunoassay), antibody dependence, cell damage test,
This was revealed by immunoadsorption tests, etc.

The anticancer compound of the present invention contains the above monoclonal antibody as an active ingredient. After the monoclonal antibody is made into a dry powder, various additives such as pH adjusters, chelating agents, stabilizers, solubilizing agents, etc. may be added to obtain the composition of the present invention. The monoclonal antibody solution may be made into a powdered composition by means such as freeze-drying. These additives are usually included in an amount of 20 to 90% by weight. In order to administer the anticancer composition of the present invention to a patient for the purpose of cancer treatment, in addition to systemic administration by intraarterial, intravenous, intramuscular, subcutaneous injection, and intravenous drip, the anticancer composition of the present invention can be administered locally into or near the cancer tissue. You may.

This composition is effective against solid cancers such as stomach cancer and lung cancer, as well as blood cancers such as leukemia. The dosage varies depending on the route of administration and the type of cancer to be treated, but the monoclonal antibody may be administered at 0.00% once. 1-500■, 0. It is 1~2000■. (Effects of the Invention) The monoclonal antibody contained in the anticancer U or substance of the present invention can specifically react with β-coordinated N-acetylneuraminic acid, so it is extremely specific to cancer cells. Collectively, they can exert anticancer effects. It is also possible to combine it with other anticancer drugs and use it for gesoting therapy.

Reference Example (11Him (A) legal 3-0-(methyl(5-acetaside-4.78,9-tetra-0-acetyl-3.5-dideoxy-β-D-glyceroD-galacto-2-non22) Lopyranosyl)onate)-1,2-di○-tetradecyl-Sn-glycerol was dissolved in methanol and then hydrolyzed with IN sodium monohydroxide at room temperature.After neutralizing the obtained reaction product with Amberlite, Compound (A) was purified by reverse-phase column chromatography and used as an immunogen. (Refer to Japanese Patent Application Laid-Open No. 164798/1983) (2) Five female 6-week-old Balb/c mice obtained from Tsutomu Oko 7 in an air-conditioned room
Those kept for one day were used in the experiment.

(3) Salmonella minnesota (Salmonella minnesota) treated with 1■ of Gomi-Gosha-4U milk fissure compound (A) and acetic acid as an adjuvant.
sota) R5954 mg was mixed with phosphate-poor physiological saline (PBS(-))1 to prepare antigen concentration.

(4) Cultivation ground Medium: Ninosui RPMI1640 was used.

At the time of use, 100 U of potassium penicillin G/tmg titer of strep-1-mycin sulfate, 1 mM of sodium hyalbate, and fetal bovine serum (Fl3S) were added in 10% increments.

HAT medium: Tidine 0. 0 3 8 8 g and hyboxanthin 0. 1 3 6 1 g and 100 g of distilled water
Melt by heating to make a 100 times concentrated stock solution -20
Stored at °C. Similarly, 0.0176 g of aminopterin was dissolved in 100 g of distilled water by adding a small amount of IN sodium hydroxide solution, and this was dissolved in 100 g of RP old 1640 medium.
It was diluted in culture medium and stored as a 100-fold concentrated storage solution at -20'C in the dark.

At the time of use, 1/100 amount of each was added to 10% FBSRPM+1640 medium to prepare a HAT medium.

In addition, as the HT medium, only thymidine and pivoxanthin preservation solution was added to l/looffi. <<5>> X63^g 8-6.5, which is a myeloma cell derived from Balb/C mouse, is used as the parent cell for Fukirich bile cell fusion.
．． 3 cells were used. The cells were RP supplemented with 10% FBS.
Subculture was carried out in MI 1640 medium, and the appearance of mutants was prevented by adding 6-thioguanine to the medium at a concentration of 3 μg/.

(Example) (a) Production of hybridomas (1) Six-week-old Balb/c broadly immune female mice were treated with Compound (A) 1■ as an immunogen and Salmonella minnesota 4■ as an adjuvant using phosphate buffered physiological conditions. Saline solution (PBS (-),
Ca. ．． After mixing with 1 ml (excluding Mg) and diluting appropriately, 5 μg was administered intraperitoneally to the mouse on the first day, 10 μg on the 12th day, 10 μg on the 16th day, 15 μg on the 26th day, and 35 μg on the 26th day.
Immunization was performed on day 1 with a 15 μg immunization schedule. After the third immunization, tile cell lymphocytes were removed from the mice and then a single cell suspension was made.

(2) Cell fusion between the tile cell lymphocytes obtained above and the parent mouse myeloma cells was carried out according to the method of Köhler and Milstein. Specifically, 3 days after the final immunization, 10s of spleen lymphocytes were incubated in the presence of 50% polyethylene glycol (PEG4000) in a medium.
It fused with 0' myeloma cells.

(3) After hybridoma and cell fusion, the obtained hybridoma cells were cultured for 15 days in RPMI-1640 medium containing HAT preservation solution and 10% FBS.

After culturing for 15 days, the supernatant of this culture was screened for antibody production against compound (A) using an enzyme antibody method.

(11) Enzyme antibody method (ELISA method) A 96-well flat-bottomed plate (manufactured by Falcon) was pretreated with ethanol and used for the experiment. J-wheat, 0.2% casein pB, dispensed onto plates and bloomed
200 μl of s (-z volume) was added and left at room temperature for 2 hours. After removing the solution by suction, 50 μl of hybridoma culture supernatant was added as a primary antibody and left at room temperature for 1 hour.

In the same way, remove the primary antibody from the plate and add PBS (-1/
After adding 150 μl of 8 solution and washing the wells 3 times, 0.
200 μl of 2% casein PBS(-) aneurysm liquid was added and left for 30 minutes. After removing this solution, 0.2% casein P
BS (-) to i! ! Secondary antibody diluted to a concentration of 50
,cl! was added and left at room temperature for 1 hour and 30 minutes. As with the primary antibody, wash the wells 3 times 7 times with PBS (=) and react.
100 μe of the solution was poured into the well and allowed to react in the dark. Cool the reaction overnight and add orthophenylenediamine and hydrogen peroxide to citric acid-phosphate buffer M (p}I=5). 75
It was prepared to have a concentration of 0.4 ./cm and 0.01% and used in the experiment. The reaction was stopped by adding 30 μe of 8N sulfuric acid, and colorimetric measurement was performed at an absorption wavelength of 490 nm. Horseradish peroxidase (HRP) as a secondary antibody
Labeled goat anti-mouse IgG, M, A antibody was used.

By this method, compounds A to F having β coordination and α
Compound H-J with coordination was investigated, and Figures 1 to 3
We obtained the results shown in the figure.

FIG. 1 and FIG. 2 each use a compound containing N-acetylneuraminic acid having β-coordination as an antigen, and examine the cross-reaction with the anti-compound (A) antibody.

This result shows that all compounds having β-coordination are positive.

Further, in FIG. 3, cross-reactivity was similarly investigated using a compound containing N-acetylneuraminic acid having α-coordination as an antigen.

This result shows that all compounds having α-coordination are negative.

(c) Preparation of anticancer composition After culturing the hybridoma obtained as above, B A
It was transplanted into the abdominal cavity of LB/C mice, and ascites fluid was collected. The monoclonal antibody was isolated and purified from the ascites by column chromatography.

The purified monoclonal antibody was dissolved in PBS at a concentration of 10 mg/s+1 to obtain the anticancer composition of the present invention.

(2) ELISA method using cancer cells as antigen Coat 1.OX10'/well of cells in a 96-sized microplate, and coat with 1.OX10'/well of cells in RPMI 1640+ECS 10% medium for 2 hours.
Cultured for 1 day. Thereafter, the wells were washed twice with PBS (-).

0.25% glutaraldehyde (in PBS (-)) was added at 100 ml/well and treated for 2 hours at room temperature.

After washing twice with PBS (-), 200 μl/well of 0.2% casein-PBS (-) was added and treated overnight at 4°C. The above antibody was diluted 10 times with io-toh, added at 50 μl/well, and incubated at 37° C. for 1 hour. Then 0.2% zein-PBS (-)
Washed 3 times with Peroxidase-labeled secondary antibody (goat anti-mouse IgG, M.A.) was diluted 2000 times,
It was added at 0 μl/well and incubated for 1 hour. Thereafter, it was washed three times with 0.2% zein-PBS (-). Substrate solution (o-phenylenediamine 400 μg
/ml, 0. 0 1% H20, in citrate buffer: p}l=5.0) was added at 100 μfl/well,
After reacting for 5 minutes, add 4N LSD4 to stop the reaction,
The OD at 95 nm was measured.

To determine the antibody titer, first set up a well without primary antibody for each well of cells (+) and (1), and then calculate this background from all wells for each cell (+) and (-). After that, the cells (
+) Well cell (1) Well value is 0. A value of 1 or more was considered to be a significant difference, and the maximum dilution factor with this significant difference was taken as the antibody titer against the cells. The results are shown in FIGS. 4 and 5. From this result, it is clear that the antibody reacts with cancer cells.

(e) Reactivity with normal cells. On a 96-well U-bottom plate, add 1 ml of the above monoclonal antibody.
It was diluted in 5 steps from 0 to 312,500 times. Normal human red blood cells 0.5% (v/v) were added to the above wells containing each diluted antibody.
(PBS solution) was added. After standing at 4℃ for 1 hour,
Cohesion was determined visually. The results are shown in Table 1.

None of the present monoclonal antibodies showed affinity with each cell when diluted 10 times. These results revealed that this monoclonal antibody specifically reacts with cancer cells and does not react with normal cells.

Table 1

[Brief explanation of drawings]

Figures 1 to 3 show cross-reactions with various derivatives of β-coordinated N-acetylneuraminic acid by enzyme antibody method, and Figure 4 shows cross-reactions between antibodies contained in the anticancer composition of the present invention and cancer cells. pc-t
Figure 5 similarly shows the reaction with cancer cell YTS-1. Figure 1 (u9) O, D Figure 2

Claims (1)

[Claims] An anticancer composition comprising, as an active ingredient, a monoclonal antibody having specificity for N-acetylneuraminic acid having β coordination as an epitope (antigenic determinant).