JPH0383597A - Monoclonal antibody specific for o-acetylated gangliosiode gm3, hybridoma capable of producing the same antibody and its production - Google Patents

Abstract

PURPOSE:To enable to obtain a cancer diagnostic agent for uterine cancer, etc., by immunizing an autoimmune mouse with an O-acetylated ganglioside GM3 and using the resultant antibody-producing cell as one of parent cells. CONSTITUTION:A ganglioside GM3 separated from a desired cell is treated with N-acetylimidazole to provide an O-acetylated ganglioside GM3 (A), which is then administered into the abdominal cavity, etc., of an autoimmune diseased mouse, such as B/WF, to afford an immunized mouse (C). Splenic cells collected from the mouse (C) are then mixed with mouse myelomatous cells, such as NS-1, at (1-10):1 ratio and fused in the presence of PEG to provide a hybridoma (D), which is subsequently cloned and then cultured in a desired culture medium to afford a culture (E). The resultant ingredient (E) is subsequently purified by precipitation with ammonium sulfate, ion exchange column chromatography, etc., to produce a monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to 0-acetylated ganglioside GM,
The present invention relates to a monoclonal antibody specific for , a hybridoma that produces the monoclonal antibody, and a method for producing the same.

[Prior art] Glycolipids, which are constituents of cell membranes, are characterized by the type, number, and number of constituent sugars.
A variety of molecular species exist due to differences in binding methods, etc., and they exhibit one-type-specific, organ-specific, and cell-specific distributions. As for its physiological functions, it has become clear that it plays an important role in interaction as a receptor for cytotoxins, hormones, etc., and as an immunological determinant such as blood group substances. In addition, as cells become cancerous, qualitative and quantitative changes occur in their composition, some of which become cancer antigens, and certain glycolipids play a role in regulating cell proliferation mechanisms mediated by growth factors and protein kinases. It has been suggested that.

In particular, regarding the relationship with cancer, a method for producing monoclonal antibodies was reported by Milstein et al. [Natur
e, 256.495 f19751] As monoclonal antibodies specific to cancer cells have been produced, it has become clear that some of these antibodies recognize sugar chains of glycolipids or glycoproteins. There is [J,
Natl, Cancer In5t,, 71.23
1i19831]. For example, Ili visceral cancer monoclonal antibody CA-19-91 (Centocor, USA)
identifies cancer-specific sugar chain monosialogangliosides.

In this way, glycolipids are being studied as clues for elucidating the canceration mechanism, as well as cancer antigens and cancer markers.

In recent years, quantitative and qualitative changes that indicate cancer-specific antigenicity have been observed in sialic acid, a component of gangliosides, and have attracted attention. For example, ganglioside GD3 is a human melanoma-related antigen that is hardly detected in normal tissues. , and several monoclonal antibodies against it have been produced (J, Exp, Wed, 155.11
33-1137 f1982), Int.

J, Cancer 29.269-275. f1
Furthermore, the monoclonal antibody Di,l prepared against the rat B49 cell line reacts specifically with human melanoma and is acetylated at the 9-position of N-acetylneuraminic acid, ganglioside G D s (9-
Recognizes O-Ac-GDil (J, Biol, C
heltl, 259.7453-745911984
11, conversely, when GD3 was acetylated using N-acetylimidazole, a reagent that acetylates the 9-position of sialic acid, it reacted with monoclonal antibody D1.1 (5science, 225.844-846 f
198411゜Thus, it has been suggested that O-acetylated sialic acid and/or O-acetylated sialic acid-containing sugar chains may be cancer-related antigens, and antibodies against them may be used to elucidate the canceration mechanism. , may be used for cancer diagnosis and cancer treatment.

Furthermore, complex carbohydrates containing sialic acid acetylated at position 9 are known to be receptors for influenza C virus (J, Biol.

Chew, 261.5947-951. f198
61. VirologL 159°102-108.
+1198711, therefore, a monoclonal antibody against this may be applicable to elucidating the infection mechanism of influenza virus and preventing infection.

On the other hand, ganglioside GM3 is represented by the following formula and is widely present in normal tissues, but monoclonal antibody M2590, which specifically reacts with human melanoma, reacts with GM (J, Biol, Cher@).

±, 13328-13333. (f19851j.

It is known that Galβl-4Glcβ1-+Cer2aNeuAc ganglioside GM can be a cancer-related antigen depending on the state of existence, especially the difference in density and amount (J, Tmmunology, 139.3171
-3176 f19. ! 17)).

[Problems to be Solved by the Invention] As mentioned above, the possibility of sialic acid-containing sugar chains as cancer-related antigens has been suggested. An object of the present invention is to provide a novel monoclonal antibody that reacts with cancer and has use as a cancer diagnostic agent.

c. Means for Solving the Problem] As a result of intensive research, the inventors of the present application succeeded in producing a monoclonal antibody that specifically recognizes O-acetylated ganglioside GMs. The present invention was completed based on the discovery that cancer diagnosis is possible using monoclonal antibodies.

That is, one aspect of the present invention provides a monoclonal antibody that reacts with O-acetylated ganglioside GM and does not react with non-O-acetylated ganglioside GM.

Furthermore, the present invention provides hybridomas that produce the monoclonal antibodies of the present invention.

Furthermore, the present invention provides a method for producing a hybridoma by immunizing an autoimmune disease mouse with O-acetylated ganglioside GM and using antibody-producing cells derived from the immunized mouse as one parent cell.

[Effect of the invention] According to the present invention, 0-acetylated ganglioside G
A novel monoclonal antibody that specifically recognizes M3, a hybridoma that produces it, and a method for producing the same have been provided. The monoclonal antibody of the present invention can be used for the diagnosis of cancer, particularly uterine cancer and ovarian cancer.

[Detailed Description of the Invention] As described above, the monoclonal antibody of the present invention specifically reacts with at least O-acetylated ganglioside GM z and does not react with ganglioside GM. As specifically shown in the Examples below, the monoclonal antibody of the present invention has O-acetylated GM s
However, O-acetylated GMs is treated with an alkali to remove the acetyl group and does not react with the original GM, so the monoclonal antibody of the present invention reacts with O-acetylated GM. It is clear that the part containing the O-acetyl group is the antigenic determinant.

Next, the method for producing the monoclonal antibody of the present invention will be explained in detail.

First, a mammal is immunized with an immunogen. The mammal to be immunized is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion, and mice and rats are preferably selected. Ganglioside GM 3 is thought to exist in mouse tissues and is thought to have extremely weak immunogenicity, so it is preferable to use animals with autoimmune diseases.As usable autoimmune disease mice, NZB
,NZW,B/WF,,MRL/1. BXSB male, S
Examples include mice with autoimmune diseases such as L/Ni.

In addition, Ba1 which increased autoantibody production ability by administering polyclonal B cell activating agents (PBA) such as Durham-negative lipid polysaccharide (LPS) and dextran sulfate.
Mice such as B/C can be used in an autoimmune disease state.

Immunogens include cell bodies containing O-acetylated ganglioside GMs, cell membrane components isolated from the cells, and O-acetylated GM isolated from the cells.
, etc. can be used - but Method in
Enzymology, 83.155-167 (1
Ganglioside GMs was separated from the cells according to the method described in 982), and this was purified by 5science, 225
, 844-846 f1984+
-O- acetylated with acetylimidazole
Preferably, acetylated ganglioside GM is used.

Immunization can be carried out by conventional methods.

That is, the above-mentioned immunogen was added to a phosphate buffer solution (hereinafter referred to as P
BS) and administered intraperitoneally or intravenously. At that time, the immunogen was bovine serum albumin (BS
A) and on a carrier such as bacterial cells. Further, an adjuvant such as Freund's adjuvant or bacterial cell adjuvant can also be injected together. In order to increase the antigenicity of the immunogen, it is preferable to adsorb the immunogen to Salmonella minnesota bacteria treated with acetic acid and administer it.The method of acetic acid treatment is described by Eur, J. Biochem.
,.

The method described in Gong, 116-122119711 was followed.

Next, the tile cells obtained from the immunized animal are fused with mouse myeloma cells. Various cells already known as myeloma cells, such as N5-1.5P-2, X 63.6.5.
3, P3-01, etc. can be used. The fusion method is
This can be carried out according to known methods. Polyethylene glycol (PEG), Sendai virus (IVJ), etc. can be used as a fusion promoter. The mixing ratio of tile cells and myeloma cells is preferably 1:1 to 10:1.

After cell fusion, hybridomas can be selected by culturing in a normal selection medium. Since the myeloma cells described above cannot grow in HAT medium (containing hypoxanthine, aminopterin, and thymidine), HAT
Cells that grow in the medium may be selected.

When the hybridoma colony becomes sufficiently large, a strain that produces the desired antibody is searched and cloned. The search for antibodies specific to O-acetylated GMs can be carried out using methods commonly used to detect antibodies, such as ELISA [Meth.

Enzysolo,, 70.419 ft9801]
, agglutination reaction method, RIA method, double immunodiffusion method, etc. Further, cloning can be performed by the limiting dilution method. That is, monocloned hybridomas can be obtained by distributing hybridomas onto a 96-well microtiter plate so that one or less hybridomas per well and growing a single colony repeatedly. At this time, it is preferable to add mouse thymocytes as feeder cells.

Hybridomas producing the monoclonal antibodies of the present invention can be stored for long periods in liquid nitrogen, and can be kept in a ready-to-distribute state.

The monoclonal antibody of the present invention can be obtained by culturing hybridomas in a medium and separating them from the culture supernatant, or by intraperitoneally administering hybridomas to mice and collecting them from the ascites fluid. Furthermore, it can be purified using general methods such as ammonium sulfate precipitation, gel filtration, and ion exchange column chromatography.

O-acetylated gangliosides that react with the monoclonal antibody of the present invention are not present in normal tissues, but only in cancer tissues, particularly uterine cancer and ovarian cancer tissues. Arita is particularly useful as a diagnostic agent for uterine cancer and ovarian cancer.

[Example 1] Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Separation and purification of glycolipids are performed using Method in Enzym.
The following procedure was carried out according to the method described in 1982, 83.155-167. Macrocytes thoroughly washed with physiological saline were added to a large amount of cold acetone, and the resulting precipitate was mixed with two bottles of chloroform and methanol at a volume ratio of 20:1) and 10:0).

Extraction operations were performed sequentially with each mixed solution of f20:10:l). The obtained crude glycolipid was treated with DEAE-Sephadex A.
-25 column chromatography (Pharmacia 1)
The mixture was separated into an acidic fraction and a neutral fraction. After treating the acidic fraction with alkali, it was purified using Iatrobeads column chromatography (manufactured by Yatron) to obtain ganglioside GM.
I got s.

Purified ganglioside GM obtained as above
After dissolving 2 mg of N-acetylimidazole in 1 ml of pyridine, a solution of 0.2 mg of N-acetylimidazole in pyridine (2 B/ml) was added.
5 ml was added and heated at 506C for 24 hours. The reaction product was qualitatively characterized by thin layer chromatography (TLC) (developing solvent: chloroform: methanol: 0.2% aqueous calcium chloride M (55:45:10 fv/v/vl), and as a result, G M 3fRf = 0. 〇-acetylated GM with a larger Rf value than 501, lRf・[
After distilling off the solvent, the reaction mixture solution in which the production of 1,58+ was confirmed was mixed with chloroform:methanol (1:1 (
It was dissolved in V/Vll and stored at -20DC. A portion of each component was isolated by Iatrobead column chromatography. The conditions for Iatrobeads column chromatography were chloroform:methanol:water (75:2).
It is a method with a polar tendency from 5:21 to 155:47:31. Transfer 300 μg of this stock solution to phosphate buffered saline (PBS).
Dissolve 1.5 ml of Salmonella minnesota cells.
The mixture was thoroughly mixed with 1.5 ml of PBS containing 2 mg and used as an immunogen. The Salmonella minnesota bacteria was washed with acetone and ether, treated with 1% acetic acid at 100°C for 2 hours, washed with water, and then dried.

Human NZB mice (female, 8 weeks old) were injected intraperitoneally with 300 µl of a PBS solution containing 5 x 10' killed B. pertussis bacteria, and at the same time, the prepared immunogen 3 (1) was injected intravenously. Similarly, intravenous injections were given three times every three weeks.

Three days after the final immunization, the mouse tile cells were removed and RPMI
Washed with 1640 medium. On the other hand, mouse myeloma cells X63.6.5.3 in the logarithmic growth phase were collected using RP&111.
Washed with 640 medium. A suspension of 1.5 x 10' tile cells and a suspension of 1 x 10' mouse myeloma 'J were mixed, and the medium was removed by centrifugation. To the mixed cells in a water bath heated to 37°C, 1 ml of 50% polyethylene glycol-RPMI 1640 medium was gradually added over a period of 1 minute, followed by gentle stirring for 1 minute to effect fusion.

Add 2 ml of RPMI 1640 medium for 2 minutes, then add 7 ml of RPMI 1640 medium for 2 minutes.
n+1 was added over 2 minutes with gentle stirring. The medium was removed by centrifugation, 40 ml of RPMI1640 medium containing 10% tallow serum was added to the cells, and 0.1 ml per well was distributed to four 96-well plates. The next day, (AT medium (4x to-' 4 aminopterin, 1.6 x 10''M thymidine, l x 10-
'M Hyboxanthin, RPM containing 10% tallow serum
11640 medium) was added to each well. The medium in each well was further replaced by half with E (AT medium) every 3 or 4 days. After 3 weeks of culture, hybridoma growth was observed in 90% of the wells.

The search for antibodies in the hybridoma culture supernatant of the hybridoma was carried out by ELISA using the 0-acetylated ganglioside GMsa compound obtained in the above immunogen preparation as the antigen.

The antigen was adsorbed onto a 500 nge ELISA microtiter plate, blocked with a 1% BSA-PBS solution, and then reacted with the culture supernatant. Furthermore, peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted with 49 using orthophenylenediamine as a substrate.
The antibody of interest was detected by measuring absorbance at 2 nm. As a result, in the hybridoma prepared by immunization and cell fusion, antibodies that reacted with O-acetylated ganglioside GM and the mixture were detected in three wells, and among these, antibodies that reacted with non-O-acetylated GM were detected. One antibody was obtained.

The obtained hybridoma was transferred from HAT medium to HT medium without aminopterin, and further added with 10% tallow serum (F).
e2) was transferred to RPM11640 medium and cultured.

Next, the obtained hybridoma was cloned by limited dilution method. That is, per hole in a 96-well plate.
Dilute cells to a density of 0.8 cells per hole 4 x 1
Cultured with 0' mouse thymocytes and EL after 2 weeks.
Antibody-producing cells were selected using the ISA method.

Repeated cloning further to create stable hybridoma M
Ac-301 was obtained.

The class of monoclonal antibody MAc-301 was determined to be IgGz by ELISA method. Hybridoma MAc-301 was deposited with the FIKEN on July 20, 1989, and its accession number is FEI No. 10869.

ELISA was performed using O-acetylated ganglioside GM obtained in Example 1 or 6M3 that was not O-acetylated as an antigen.

500 ng of each antigen was adsorbed onto a Quitar plate, and hybridoma culture supernatant was prepared using RPM1 containing 10% fetal bovine serum.
A 5-reaction mixture obtained by reacting serial dilutions with 1640 medium and further reacting with a peroxidase-labeled goat anti-mouse immunoglobulin antibody was used as a substrate and absorbance at 492 nm was measured using orthophenylenediamine. The results are shown in Figure 1.

From FIG. 1, the monoclonal antibody of the present invention is O-acetylated GM.

Although it reacts with O-a It can be seen that it does not react with non-cetylated GM.

For TLC plate A, 1cm from the bottom edge of the TLC plate.
The antigen O obtained in Example 1 with a width of 5111ffl
- Acetylated GM, compound 11 (lane 2) and 0
- 2 μg non-acetylated GM (lane 1)
Spotted with developing solvent chloroform: methanol = 0
．． 2% calcium chloride aqueous solution (55:45:to (v
After developing with /v/vll, color was developed with orcinol reagent.

On TLC plate B, 40 ng of each of the above antigens were spotted and developed in the same manner, and then reacted with a solution of MAc-301 and further reacted with a peroxidase m-specific goat anti-mouse immunoglobulin antibody. 4-chloro-1 as substrate
- A blue-purple colored spot was detected using naphthol. Figure 2 shows the results of orcinol coloring and enzyme immunostaining.
It was shown to.

From FIG. 2, it can be seen that the monoclonal antibody of the present invention reacts with GM3 that is 0-acetylated, but does not react with GM3 that is not 0-acetylated.

1 above 1 Purified ganglioside GM3 (obtained in Example 1) as biogenic antigen
Lane 1), isolated O-acetylated GM3 (
The same procedure as in Example 3 was carried out, except that lane 2) and isolated O-acetylated GM3 were treated with alkali to remove the O-acetyl group (lane 3). Note that this alkali treatment was carried out by spotting 0-acetylated GM3 on TLC and then allowing it to stand under ammonia vapor for 24 hours. The results are shown in FIG. Plate A was developed using orcinol reagent, and plate B was developed using enzyme immunostaining.

The monoclonal antibody of the present invention has O-acetylated G
GM reacts with M, but does not react with GM that is not O-acetylated, and GM3 that is O-acetylated by alkali treatment is GM that is not O-acetylated.
It can be seen that when MAc-301 is converted to MAc-3, the reactivity with the antibody MAc-301 of the present invention disappears. From this, MAc-3
It has been revealed that 01 recognizes the O-acetyl group-containing portion of O-acetylated GM3 as an antigenic determinant.

Using cells from cancer tissues from 2 cases of uterine cancer and 2 cases of ovarian cancer, the same procedure as for dog red blood cells in Example 1 was carried out to obtain acidic cells obtained by DEAE-Sephadex 8-25 column chromatography. The fraction was designated as a ganglioside fraction. The obtained ganglioside fraction was converted into sialic acid by 2 μg each.
It was spotted on LC and enzyme immunostaining was performed in the same manner as in Example 3.

Note that the developing solvent is chloroform:methanol = 2.5%
Ammonia water f55:45:10 (v/v/vll was used.

As a result, colored spots were detected in 1 case of uterine cancer and 2 cases of ovarian cancer out of 4 cases, confirming the presence of O-acetylated ganglioside as a cancer-related antigen and detection by the monoclonal antibody of the present invention.

Claims (3)

[Claims] (1) Reacts with O-acetylated ganglioside GM_3 and non-O-acetylated ganglioside G
A monoclonal antibody that does not react with M_3. (2) A hybridoma producing the monoclonal antibody according to claim 1. (3) A method for producing a hybridoma in which an autoimmune disease mouse is immunized with O-acetylated ganglioside GM_3 and antibody-producing cells derived from the immunized mouse are used as one parent cell.