JP2743015B2 - Monoclonal antibody specific to O-acetylated ganglioside GM <3>, hybridoma producing the antibody, and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an O-acetylated ganglioside GM ₃
And a hybridoma producing the monoclonal antibody and a method for producing the same.

[Prior Art] Glycolipids, which are components of cell membranes, are composed of
There are various molecular species depending on the number, binding method, etc., showing species-specific, organ-specific, and cell-specific distributions.
It has become clear that its physiological function plays an important role in the interaction as a receptor for cytotoxins and hormones, and as an immunological determinant such as blood group substances. In addition, qualitative and quantitative changes in the composition occur as cells become cancerous, some of which become cancer antigens, and certain glycolipids play a role in regulating cell growth mechanisms via growth factors and protein kinases. It has been suggested.

In particular, Milstein et al. Reported a method for producing monoclonal antibodies with regard to its association with cancer [Nature, 2
56, 495 (1975)] in which cancer cells specific monoclonal antibodies come made, some of which it has been shown to be an antibody that recognizes sugar chains of glycolipids or sugar tank cytoplasm [J. Natl. Cancer Inst., 71 , 231
(1983)]. For example, pancreatic cancer monoclonal antibody CA
-19-9 (Centocor, USA) identifies a monosialoganglioside sugar chain specific to cancer.

Thus, glycolipids have been studied as clues for elucidating the mechanism of canceration and as cancer antigens and cancer markers.

In recent years, sialic acid, a component of gangliosides, has been noted for its quantitative and qualitative changes that show cancer-specific antigenicity. For example, ganglioside GD ₃ is a human melanoma-associated antigen is hardly detected in normal tissues, monoclonal antibodies have also been produced some to this (J.Exp.Med. 155, 1133-1137 (1982 ), Int.
J. Cancer 29 , 269-275, (1982)). Monoclonal antibody D1.1 made to further rat B49 cell lines, human melanoma specifically react, N- acetylneuraminic ganglioside GD ₃ to 9 of the neuraminic acid is acetylated (9
O-Ac-GD ₃ ) (J. Biol. Chem. 259 , 7453-745).
9 (1984)). Conversely, reagents acetylated 9-position of sialic acid was acetylated GD ₃ using N- acetyl imidazole was reacted with a monoclonal antibody D1.1 (Science, 225,844-846 (1984) ).

Thus, it is suggested that O-acetylated sialic acid and / or an O-acetylated sialic acid-containing sugar chain may be a cancer-associated antigen, and an antibody against it may be used to elucidate the mechanism of canceration, It can be used for diagnosis and cancer treatment.

Also, glycoconjugates containing sialic acid acetylated at the 9-position are known to be receptors for influenza C virus (J. Biol. Chem. 261 , 5947-951,
(1986), Virology, 159 , 102-108, ((1987)). Therefore, there is a possibility that a monoclonal antibody against this can be applied to elucidation of the infection mechanism of influenza virus and protection against infection.

On the other hand, ganglioside GM ₃ is represented by the following formula is present widely in normal tissue, a monoclonal antibody M2590 which specifically reacts with human melanoma reacts multi with GM ₃ (J.
Biol. Chem. 260 , 13328-13333, ((1985)).

Ganglioside GM ₃ is present state, in particular the density, it has been found that can be a cancer-associated antigen by the difference in abundance (J.Immunology, 139, 3171-3176 (1987 )).

[Problems to be Solved by the Invention] As described above, the possibility of a sialic acid-containing sugar chain as a cancer-associated antigen has been suggested, but the object of the present invention is to
An object of the present invention is to provide a novel monoclonal antibody which specifically reacts with a sialic acid-containing sugar chain and has use as a cancer diagnostic agent.

[Means for solving the problems] The present invention intensively studied and succeeded in producing a monoclonal antibody specifically recognizing ganglioside GM ₃ which is O- acetylated, also, the monoclonal Finding that cancer can be diagnosed using antibodies,
The present invention has been completed.

That is, the present invention reacts the ganglioside GM ₃ which is O- acetylated, the ganglioside GM ₃ which is not O- acetylated provides monoclonal antibodies that do not react.

Furthermore, the present invention provides a hybridoma producing the monoclonal antibody of the present invention.

The invention further immunized with ganglioside GM ₃ which is O- acetylated autoimmune disease mice, provides a method of making hybridomas using antibody-producing cells from the immunized mice as one of the parental cell.

[Effect of the Invention] According to the present invention, O-acetylated gangliosides
GM ₃ specifically recognizing novel monoclonal antibodies, hybridomas and a manufacturing method thereof are provided for producing it. The monoclonal antibody of the present invention can be used for 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 ganglioside GM ₃ in which at least O- acetylated, but not with ganglioside GM _3. As specifically shown in the Examples below, monoclonal antibodies of the present invention will be react with O- acetylated GM _3, remove the acetyl group of O- acetylation GM ₃ and alkali treatment, the original since not react to those with GM _3, the monoclonal antibodies of the present invention is O- acetylated GM ₃
It is clear that the portion containing the O-acetyl group is used as the antigenic determinant.

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

First, a mammal is immunized with the immunogen. The mammal to be immunized is preferably selected in consideration of the compatibility with the myeloma cells used for cell fusion, and a mouse or rat is preferably used. In particular, ganglioside GM ₃ which is O- acetylated used as the immunogen in the present invention, immunogenicity is considered the presence of the mouse tissues, it is preferable to use an autoimmune disease animal because it is considered very weak . NZB, NZW,
Autoimmune disease mice such as B / WF ₁ , MRL / 1, BXSB male, SL / Ni and the like can be mentioned.

In addition, mice such as Balb / c, whose autoantibody production ability has been enhanced by administering a polyclonal B cell activator (PBA) such as gram-negative bacterial lipid polysaccharide (LPS) and dextran sulfate, are brought into an autoimmune disease state. Can also be used.

The immunogen, O- acetylated cells having ganglioside GM _3, can be used the cells from the isolated cell membranes components and isolated from the cells O- acetylated GM _3, etc., Method in Enzymology, 83 , 15
5-167 separated ganglioside GM ₃ from cells according to the method described in (1982), which Science, 225, 844-846 (1
984) O-acetylated ganglioside treated with N-acetylimidazole according to the method described in 984)
Preferably, GM ₃ is used.

Immunization can be performed by a general method. That is, the above-mentioned immunogen can be diluted with a phosphate buffer solution (hereinafter, referred to as PBS) and administered intraperitoneally or intravenously. At this time, the immunogen can be carried on a carrier such as bovine serum albumin (BSA) and cells. Also, an adjuvant such as Freund's adjuvant or bacterial adjuvant can be injected together. In order to increase the antigenicity of the immunogen, it is preferable to administer the immunogen by adsorbing it to Salmonella minnesota bacteria treated with acetic acid. Acetic acid treatment was performed according to the method described in Eur. J. Biochem., 24 , 116-122 (1971).

Next, splenocytes collected from the immunized animal are fused with mouse myeloma cells. As the myeloma cells, various known cells such as NS-1, SP-2, X63.6.5.3, and P3-U1 can be used. The fusion method can be performed according to a known method. Polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used as a fusion promoter. The mixing ratio of spleen cells to 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 cannot grow in HAT medium (including hypoxanthine, aminopterin and thymidine), cells that grow in HAT medium may be selected.

When the hybridoma colonies become sufficiently large, a strain producing the desired antibody is searched and cloned. Search antibodies specific for O- acetylated GM _3, commonly used method for the detection of antibodies, eg, ELISA method [Meth.Enzymolo., 70, 419 ( 1980)], agglutination reaction method, RIA method And the double immunodiffusion method. Cloning can be performed by the limiting dilution method. That is, a hybridoma is distributed on a 96-well microtiter plate such that the number of hybridomas is 1 or less per well, and a single colony is repeatedly grown to obtain a monocloned hybridoma. At this time, it is preferable to add mouse thymus cells as feeder cells.

The hybridoma producing the monoclonal antibody of the present invention can be stored for a long time in liquid nitrogen, and can be maintained in a state that can be distributed.

The monoclonal antibody of the present invention can be obtained by culturing the hybridoma in a medium and separating it from the culture supernatant, or by administering the hybridoma to the peritoneal cavity of a mouse and collecting the hybridoma from the ascites. In addition, common methods,
Purification can also be performed using ammonium sulfate precipitation, gel filtration, ion exchange column chromatography, or the like.

Since the O-acetylated ganglioside that reacts with the monoclonal antibody of the present invention is not present in normal tissues but is present only in cancer tissues, particularly uterine cancer and ovarian cancer tissues, the monoclonal antibody of the present invention is used for cancer, In particular, it is useful as a diagnostic agent for uterine cancer and ovarian cancer.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 Separation and Purification of Glycolipids Separation and purification of glycolipids were carried out according to Method in Enzymology, 83 , 15
It carried out as follows according to the method described in 5-167,1982. Dog erythrocytes thoroughly washed with physiological saline were added to a large amount of cold acetone, and the resulting precipitate was subjected to a chloroform: methanol: water volume ratio of (10: 20: 1), (10:10:
The extraction operation was sequentially performed with each of the mixed solutions (0) and (20: 10: 1). The obtained crude glycolipid was subjected to DEAE-Sephadex A-25.
It was subjected to column chromatography (Pharmacia) to separate into an acidic fraction and a neutral fraction. After alkali treatment acidic fractions gave the ganglioside GM ₃ was purified Ia Toro bead column chromatography (Iatron Inc.).

Preparation of immunogen Purified ganglioside GM ₃ 2 mg obtained as described above
Was dissolved in 1 ml of pyridine, 0.5 ml of a pyridine solution of N-acetylimidazole (2 mg / ml) was added, and the mixture was heated at 50 ° C. for 24 hours. The reaction product was analyzed by thin-layer chromatography (TL
C) (Developing solvent: chloroform: methanol: 0.2% calcium chloride aqueous solution (55:45:10 (v / v / v)). As a result, O-acetylation having a larger Rf value than GM ₃ (Rf = 0.50) was obtained. The formation of the GM ₃ (Rf = 0.58) was confirmed, and the solvent was distilled off from the reaction mixture solution, which was then dissolved in chloroform: methanol (1: 1 (V / V)) and stored at −20 ° C. Each component was partially isolated by Iatrobeads column chromatography under conditions of chloroform: methanol: water (75: 2).
5: 2) to (55: 47: 3). Dissolve 300 μg of this stock solution in 1.5 ml of phosphate buffer solution (PBS),
A mixture sufficiently mixed with 1.5 ml of PBS supplemented with 1.2 mg of Salmonella minnesota cells was used as an immunogen. Salmonella minnesota bacteria were washed with acetone and ether,
The mixture was treated with 1% acetic acid at 100 ° C. for 2 hours, washed with water, and dried.

Immunization method and cell fusion NZB mice (female, 8 weeks old) were intraperitoneally injected with 300 μl of PBS solution containing 5 × 10 ⁸ pertussis dead teeth, and simultaneously prepared 300 μm of immunogen
1 was injected intravenously. Thereafter, the immunogen was similarly intravenously injected three times every three weeks.

Three days after the final immunization, the spleen cells of the mouse were removed, and RPMI16
Washed with 40 media. Meanwhile, mouse myeloma cells X63.6.5.3 in the logarithmic growth phase were collected and washed with RPMI1640 medium. The suspension of spleen cells 1.5 × 10 ^{8 and} the suspension of mouse myeloma 3.1 × 10 ⁷ were mixed, and the medium was removed by centrifugation. 1 ml of 50% polyethylene glycol-RPMI1640 medium was gradually added to the mixed cells in a water bath heated to 37 ° C. over 1 minute, and gently stirred for 1 hour to perform fusion. 2 ml of RPMI1640 medium was added for 2 minutes, and 7 ml was added over 2 minutes with gentle stirring. The medium was removed by centrifugation. After adding 40 ml of RPMI1640 medium containing 10% fetal bovine serum to the cells, 0.1 ml of the medium was distributed to four 96-well plates per well. The next day, HAT medium ( ⁴ × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine, 1 × 10 ⁻⁴
0.1 ml of RPMI1640 medium (M hypoxanthine, 10% fetal calf serum) was added to each well. The medium in each well was further replaced by half the HAT medium every 3 or 4 days. After 3 weeks of culture, 90% of the wells showed hybridoma growth.

Search of antibodies selected hybridoma culture supernatant of Baiburidoma was performed by ELISA using the immunogen ganglioside GM ₃ mixture as O- acetylated obtained in the preparation of the antigen.

After 500 ng of the antigen was adsorbed on a microtiter plate for ELISA and blocked with a 1% BSA / PBS solution, the culture supernatant was reacted. Further, a peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted, and the target antibody was detected by measuring the absorbance at 492 nm using orthophenylenediamine as a substrate. as a result,
Immunization, in hybridomas prepared in cell fusion, detected antibodies three wells that reacted with ganglioside GM ₃ mixture as O- acetylated, antibodies which do not react with GM ₃ this out not O- acetylated 1 I got one.

The obtained hybridomas were transferred to HT medium from which aminopterin was removed from HAT medium, and further 10% fetal calf serum (FCS).
The cells were transferred to the containing RPMI1640 medium and cultured.

Next, the obtained hybridoma was cloned by a limited dilution method. That is to say, 0.
The cells were diluted to a density of 8 and cultured with ⁴ × 10 ⁵ mouse thymocytes per well. Two weeks later, antibody-producing cells were selected by ELISA. Cloning was further repeated to obtain stable hybridoma MAc-301.

Monoclonal antibodies MAc-301, the class was determined IgG ₃ by ELISA. The hybridoma MAc-301 was deposited on July 20, 1989 at the Japan Institute of Micro-Technology, and the deposit number is Micro-organisms No. 10869.

EXAMPLE 2 Antigen Specificity Example 1 GM ₃ have not obtained O- acetylated ganglioside GM ₃ or O- acetylated as antigen
ELISA was performed.

500 ng of each antigen was adsorbed to a titer plate, and a solution obtained by serially diluting a hybridoma culture supernatant with RPMI1640 medium containing 10% fetal bovine serum was reacted, and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. The reaction mixture was prepared using orthophenylenediamine as a substrate and 492 n
The absorbance at m was measured. The result is shown in FIG.

Than 1, the monoclonal antibody of the present invention is to react with GM ₃ which is O- acetylated, it can be seen that does not react with GM ₃ not O- acetylated.

Is lower from GM ₃ mixture as antigen O- acetylated to give a width of 5mm in Example 1 at a 1 cm (lane 2) and O- acetylation of TLC plates in Example 3 antigen specificity TLC plate A GM ₃ (lane 1) was spotted at 2 μg each and developed with a developing solvent chloroform: methanol: 0.2% calcium chloride aqueous solution (55:45:10 (v / v / v)), and then developed with an orcinol reagent.

The TLC plate B was spotted with 400 ng of each of the antigens described above, developed in the same manner, reacted with a MAc-301 solution, and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. A blue-violet spot was detected using 4-chloro-1-naphthol as a substrate. FIG. 2 shows the results of orcinol coloring and enzyme immunostaining.

From FIG. 2, the monoclonal antibodies of the present invention react with GM ₃ which is O- acetylated, but it is understood that does not react with GM ₃ not O- acetylated.

Example 4 Purified ganglioside GM obtained in Example 1 as an antigen
₃ (lane 1), isolated O-acetylated GM ₃ (lane 2) and isolated O-acetylated GM ₃ treated with alkali to remove the O-acetyl group (lane 3) The same operation as in Example 3 was performed except for using. Note that this alkali treatment, the GM ₃ which is O- acetylated After spotting on TLC, was carried out by standing under ammonia vapor for 24 hours. The result is shown in FIG. Plate A shows color development with the orcinol reagent, and plate B shows enzyme immunostaining.

The monoclonal antibody of the present invention is O-acetylated
Reacts with GM _3, but the GM ₃ not O- acetylated not react. Further, it can be seen that also eliminates reactivity with antibodies MAc-301 of the present invention and are converted to GM ₃ to the alkali treatment O- acetylated GM ₃ is not O- acetylated. Therefore, MAc-301 is a portion including the O- acetylated GM ₃ of O- acetyl groups revealed that recognized as antigenic determinants.

Example 5 Using cells of each cancer tissue of two cases of uterine cancer and two cases of ovarian cancer,
The same operation as that of the canine erythrocytes of Example 1 was performed, and the acidic fraction obtained by DEAE-Sephadex A-25 column chromatography was used as a ganglioside fraction. The obtained ganglioside fraction was spotted on TLC in an amount of 2 μg in sialic acid amount, and subjected to enzyme immunostaining as in Example 3. The developing solvent is chloroform: methanol: 2.5%
Ammonia water (55:45:10 (v / v / v)) was used.

As a result, coloring spots were detected in one case of uterine cancer and two cases of ovarian cancer out of the four cases, confirming the presence of O-acetylated ganglioside as a cancer-associated antigen and detection by the monoclonal antibody of the present invention.

[Brief description of the drawings]

1, the monoclonal antibody of the present invention, shows the results of enzyme immunoassay showing the reactivity to GM ₃ that is not GM ₃ and O- acetylated are O- acetylated, FIG. 2, the present invention shows monoclonal antibody shows reactivity to GM ₃ that is not GM ₃ and O- acetylated are O- acetylated, the orcinol color pattern and enzyme immunostaining pattern by thin-layer chromatography, 3, the monoclonal antibodies of the invention, purified ganglioside GM _3, the GM ₃ and isolated O- acetylated GM ₃ which is O- acetylated isolated by alkali treatment O-
It is a figure which shows the orcinol coloring pattern and enzyme immunostaining pattern by thin layer chromatography which show the reactivity with respect to the thing which removed the acetyl group.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (C12N 5/10 C12R 1:91)

Claims (3)

(57) [Claims] (1) O-acetylated ganglioside GM ₃
Gangliosides that are not O-acetylated
Monoclonal antibody that does not react to GM _3. 2. A hybridoma which produces the monoclonal antibody according to claim 1. 3. O-acetylated ganglioside GM ₃
Of an autoimmune disease mouse, and using an antibody-producing cell derived from the immunized mouse as one parent cell.