JP3006943B2 - Anti-glycolipid glycan monoclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to IgG3s and at least one ganglioside GD3 and 3 ', 8'-
It relates to a monoclonal antibody reactive with LD1.

[0002]

2. Description of the Related Art Ganglioside is a kind of glycolipid constituting a cell membrane and is a molecule composed of a sugar chain as a hydrophilic side chain, sphingosine and a fatty acid as hydrophobic side chains. It is known that the expression of gangliosides varies depending on cells, organs, and animal species, but it is becoming increasingly clear that gangliosides expressed during the process of canceration of cells undergo quantitative and qualitative changes. [Cancer Research (CancerRes.) 45 , 2405 (1985)].

For example, in neuroblastomas, small cell lung carcinomas, gliomas and melanomas, which are said to be highly malignant neuroectodermal tumors, gangliosides GD2, GD3, It has been reported that GM2 and the like are abnormally expressed as cancer antigens [Journal of Experimental Medicine (J. Exp. Med.) 155 , 1133 (1982), Journal of
Biological chemistry (J. Biol. Chem) 257 , 127
52 (1982), Cancer Res. 47 , 22
5 (1987), 47 , 1098 (1987), 45 , 2642 (1985), Proceeding of the National Academy
Proc. Natl. Acad.
Sci. USA) 80 , 5392 (1983)].

In order to analyze and treat these tumors, a monoclonal antibody reactive with ganglioside GD2 [Cancer Research (CancerRes.) 47 , 1098 (198)
7), ibid. 45 , 2642 (1985), Proceeding of the
National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 79 , 7629 (198
2)] and a monoclonal antibody reactive with ganglioside GM2 [Cancer Research (Cancer Res.) 46 , 41].
16 (1986), 48 , 6154 (1988), Proceeding of the National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 80 , 5392
(1983)].

[0005] Ganglioside GD3 has N-terminal at its non-reducing end.
euAcα2 → 8NeuAcα2 → 3Gal sugar chain, Ne
uAcα2 → 8NeuGcα2 → 3Gal sugar chain, Neu
Gcα2 → 8NeuAcα2 → 3Gal sugar chain or Ne
uGcα2 → 8NeuGcα2 → 3Gal sugar chain-bound ganglioside, and its expression is frequently observed in melanoma among neuroectodermal tumors in particular, and anti-ganglioside G belonging to IgM and IgG3
Five D3 monoclonal antibodies have been produced and reported to react with ganglioside GD3 [International Journal of Cancer (Int. J. Cancer) 29 , 269 (1982), Journal of Biological chemistry (J. Biol. Chem) 257 , 1275
2 (1982), Cancer Research 47 , 225
(1987), Acta Neuropatho
l.) 79 , 317 (1989), Proceeding of the National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 77 , 6114 (1980), Journal of the Experimental Medicine (JE
xp.Med.) 155 , 1133 (1982), Proceeding of
The National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 81 , 5767
(1984)].

[0006] In addition, treatment of melanoma using an anti-ganglioside GD3 monoclonal antibody belonging to IgG3 has also been attempted [European Journal of Cancer and Clinical Oncology (Eur.J.
Cancer Clin. Oncol.) 24 , suppl 2, S65 (1988), Proceeding of the National Academy of Sciences U.S.A. (Proc. Natl. Acad. Sci.
USA) 82 , 1242 (1985)], but at present sufficient therapeutic effects have not been obtained.

On the other hand, in glial cell tumor, which is one of the neuroectodermal tumors, expression of 3 ′, 8′-LD1 having the same sugar chain as GD3 at the non-reducing end,
An IgM class monoclonal antibody reacting with 8'-LD1 has been reported [Acta Neuropathol. 79 , 317 (1989)], 3 ',
8'-LD1 is also considered important as one of the cancer antigens.

[0008] Gangliosides GD3 and 3 ',
An IgM class monoclonal antibody that cross-reacts with both antigens of 8'-LD1 is known (Acta Neuropathol 79 , 317 (1989)).
No IgG class monoclonal antibody is known.

[0009]

SUMMARY OF THE INVENTION Ganglioside GD3
If a monoclonal antibody that reacts with 3 ′, 8′-LD1 and belongs to the IgG class can be obtained, it is useful for treating neuroectodermal cancer.

[0010]

Means for Solving the Problems The present inventor has proposed that monoclonal antibodies of the IgG class produced by ganglioside GD3 or a hybridoma established using GD3-expressing cells as an immunogen are ganglioside GD3 and 3 ′, 8′-.
The present inventors have found that they are useful for treating cancer because they have a cell killing activity in response to LD1, and completed the present invention.

The present invention belongs to IgG3, and has at least one
Provided are monoclonal antibodies reactive to gangliosides GD3 and 3 ', 8'-LD1 of the species. Ganglioside GD3 in the present invention is NeuAcα2 → 8Neu
Acα2 → 3Gal sugar chain, NeuAcα2 → 8NeuG
cα2 → 3Gal sugar chain, NeuGcα2 → 8NeuAc
α2 → 3Gal sugar chain or NeuGcα2 → 8NeuG
It is selected from ganglioside GD3 having cα2 → 3Gal sugar chain.

The monoclonal antibody of the present invention is prepared by fusing a spleen cell of a mouse immunized with ganglioside GD3 or a cancer cell expressing the ganglioside GD3 with a mouse myeloma cell line to prepare a hybridoma, and ganglioside GD3 and 3 ', 8. By selecting a hybridoma that produces a monoclonal antibody reactive with '-LD1, and culturing the hybridoma in a medium or administering it to a mouse to cause the mouse to undergo ascites carcinoma, and collecting from the culture or ascites. can get.

As a specific example of the hybridoma producing the monoclonal antibody of the present invention, the hybridoma KM-
641, KM-643 and KM-644 (September 1990
FERM BP-
3116, FERM BP-3117 and FERM BP-3118, respectively). The monoclonal antibodies produced by each hybridoma were identified as KM-641, KM
-643 and KM-644.

Hereinafter, the method for producing the monoclonal antibody of the present invention will be described in detail. (1) Immunization of animals and preparation of antibody-producing cells 3 to 20-week-old mice are immunized with ganglioside GD3 antigen, and antibody-producing cells in spleen, lymph nodes, and peripheral blood of the animals are prepared. The method of immunization is to subcutaneously or intravenously or intraperitoneally carry ganglioside GD3 on a carrier such as a bacterial cell such as Escherichia coli, to administer the cancer cell, or to administer cancer cells expressing ganglioside GD3 or ganglioside GD3. There is a method in which the drug is administered while being held in a liposome composed of a lipid such as phospholipid or cholesterol together with lipid A. These doses are 1
It is performed 5 to 10 times every 1 to 2 weeks after the second administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the serum reacts with ganglioside GD3. Enzyme immunoassay (enzyme immunoassay (ELISA): 1976, published by Medical Shoin)
Check with

Enzyme-linked immunosorbent assay (ELISA): 96-well EIA
Plate [Flow Laborator
ies), an ethanol solution containing ganglioside GD3, phospholipid and cholesterol was dispensed at 20 μl / well (20 pmol / well as ganglioside), air-dried, and PBS (phosphate serum containing 1% BSA (bovine serum albumin)). 1.83 g disodium, 0.21 g monopotassium phosphate,
7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2) solution (BSA-PBS) was dispensed at 100 to 200 μl / well,
The plate was allowed to stand at 4 ° C. for 1 to 2 nights to block (block) the protein-binding residues remaining on the plate. afterwards,
After discarding BSA-PBS and washing well with resin water or PBS, 100 μl / well of a sample (mouse serum, hybridoma culture supernatant, purified monoclonal antibody) diluted with BSA-PBS was added as the first antibody. 1 in ° C
Leave at night. After washing once with resin water and six times with a 2M sodium chloride solution, a 400-fold diluted solution of a rabbit anti-mouse immunoglobulin antibody-peroxidase conjugate [manufactured by DAKO] was used as the second antibody at 100 µl / well. Pour and leave at room temperature for 2 hours.

After washing well with PBS, 550 mg of ABTS substrate solution [2-ammonium 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) -2-ammonium is added.
M citrate buffer (pH 4.2)
Color solution is measured at an absorbance of OD _{415 nm} using a solution containing 1 μl / ml of hydrogen peroxide immediately before use. Before subjecting to cell fusion, spleen cells are excised from immunized mice 3 to 4 days after the final administration of the antigenic substance, and splenocytes are prepared.
The spleen was shredded in MEM (manufactured by Nissui Pharmaceutical), loosened with forceps, centrifuged (1200 rpm, 5 minutes), the supernatant was discarded, and Tris-ammonium chloride buffer (pH 7.65) was used.
For 1-2 minutes to remove erythrocytes, and wash three times with MEM to provide splenocytes for fusion.

(2) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mice (BA
LB / c derived) myeloma cell line P3-X63Ag8-U1
(P3-U1) [Current Topics in Microbiology and Immunology (Current Topics
in Microbiology and Immunology) 81 , 1-7 (1987))
[European Journal of Immunology (Eu
ropeanJ.Immunology) 6 , 511-519 (1976)], SP2
/ 0-Ag14 (SP-2) [Nature 27
6 , 269-270 (1978)), P3-X63-Ag8653 (653)
[Journal of Immunology (J. Immunology) 12
3, 1548-1550 (1979)] and P3-X63-Ag8 (X63) [Nature 256 , 495-497 (1975)]. These cell lines are 8-azaguanine medium [RPMI-1
Glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 ⁻⁵ M), gentamicin (10 μl / ml) and fetal calf serum (FCS) (CSL, 10%) added to 640 medium a further 8-azaguanine (15 [mu] g / ml) but passaging the added medium] and they are subcultured in the normal medium 3 or 4 days before cell fusion, secure 2 × 10 ⁷ or more of the number of cell fusion day I do.

(3) Cell fusion The antibody-producing cells immunized in (1) and the myeloma cells obtained in (2) are thoroughly washed with MEM medium or PBS, and
Antibody-producing cells: myeloma cells were mixed at a ratio of 5 to 10: 1, centrifuged (1,200 rpm, 5 minutes), the supernatant was discarded, and the precipitated cell group was thoroughly loosened.
At 37 ° C, polyethylene glycol-1,000 (PE
G-1,000), a mixture of 2 g of MEM, 2 ml of MEM and 0.7 ml of dimethyl sulfoxide, 0.2 to 1 ml / 10 ³ antibody-producing cells were added.
After adding 1-2 ml of MEM several times every 1-2 minutes,
To make the total volume 50 ml. Centrifugation (90
(0 rpm, 5 minutes), discard the supernatant, gently loosen the cells, and add 100% normal medium (RPMI1640 medium containing 10% FCS).
Add ml, and suspend the cells gently by sucking and blowing with a female pipette.

This suspension was added to a 96-well culture plate for 10 times.
Dispense 0 μl / well and incubate at 37 ° C. for 24 hours in a 5% CO ₂ incubator. 100 μl in culture plate
l / well of HAT medium [hypoxanthine (10
^-4 M), thymidine (1.5 × 10 ⁻⁵ M) and aminopterin (4 × 10 ⁻⁷ M)], and further culture for 24 hours. Thereafter, for every two days, the culture supernatant 100
Discard μl, add the same amount of HAT medium, and add 5% CO
₂ Incubate at 37 ° C for 10 to 14 days in an incubator.

In the well where colony-grown fused cells were observed, 100 μl of the supernatant was discarded, and the same amount of HT medium (a medium obtained by removing aminopterin from HAT medium) was added. Perform conversion to medium. After culturing in an HT medium for 3 to 4 days, a part of the culture supernatant is taken, and the above-mentioned enzyme immunoassay or immunohistological determination method (ABC method) (enzyme-antibody method;
Year), the antibody titer against ganglioside GD3 is measured. At this time, ganglioside GM3
The reactivity with other gangliosides is also measured, and those that specifically react with ganglioside GD3 are selected. Reacts strongly with ganglioside GD3, ganglioside GM
For holes that do not react with other gangliosides such as 3,
Cloning is repeated twice by the limiting dilution method, and those having a stable ganglioside GD3 antibody titer are selected as hybridoma lines producing anti-ganglioside GD3 monoclonal antibody.

(4) Preparation of Monoclonal Antibody The anti-ganglioside GD3 monoclonal antibody-producing hybridoma obtained in (3) was added to RPMI-16 supplemented with 10% FCS.
Culture in 40 medium or serum-free medium, and collect culture supernatant when cells become confluent. After adjusting the pH of the culture supernatant to 8.9 with sodium hydroxide, it is adsorbed to protein A-Sepharose 4B (Pharmacia) by a method of passing through a column or a batch method. In the former, the culture supernatant whose pH has been adjusted is passed through a column packed with protein A-Sepharose 4B, and in the case of a batch method,
A gel obtained by adding protein A-Sepharose 4B to the culture supernatant is packed in a column. The column is combined with a binding buffer (1.5 M glycine, 3 M sodium chloride, pH
8.9), and washed with elution buffer (0.1 M citric acid, p
H4.0), the IgG fraction was collected, the pH was adjusted to 7 to 8 with sodium hydroxide, and then 2M sodium chloride-PB
Dialyze against S to obtain a purified monoclonal antibody.

The subclass of the antibody is determined by using a mouse monoclonal antibody typing kit (Zymet). The protein amount was determined by the Folin method and 280
Absorbance at nm [1.0 (OD ₂₈₀ ) ≒ immunoglobulin 1 mg /
ml]. The anti-ganglioside GD3 monoclonal antibody of the present invention has high reaction specificity and is therefore useful for detection and analysis of ganglioside GD3. Further, the monoclonal antibody itself has complement-dependent cytotoxic activity (CDC activity) and antibody-dependent cell-mediated cytotoxic activity (ADCC activity), and expresses ganglioside GD3 and 3 ′, 8′-LD1. It is expected to exhibit antitumor effects such as cell killing activity on tumor cells without damaging normal cells by binding to existing tumor cells.

Hereinafter, embodiments of the present invention will be described.

[0024]

【Example】

Example 1 (1) Preparation of antigen NeuAcα2 → 8NeuAcα2 → 3G at non-reducing end
5 μg of ganglioside GD3 (manufactured by Yatron), dipalmitoyl phosphatidylcholine (manufactured by Sigma) 0.5 μmol, cholesterol (manufactured by Nacalai Tesque) 0.5 μmol, dipalmitoyl phosphatidic acid (Sigma) 0.05 μmol) and 2.5 μg of Lipid A (Funakoshi) were dissolved in 30 ml of a chloroform / methanol (2/1) solution, heated to 45 ° C. and the solvent was removed to form a uniform lipid thin film. I let it. Further, the solvent was completely removed by suction with a vacuum pump for 1 hour, and 0.5 ml
Was added and stirred at 45 ° C. to obtain an antigen solution.

(2) Preparation of antibody-producing cells 0.5 ml of the antigen solution prepared in (1) was administered to the tail vein of a mouse once a week, seven times in total, and immunized. In addition, ganglioside GD3-positive cells SK-MEL-28 (ATCC HTB 72) (1 × 10 ⁷ )
Was intraperitoneally administered once a week for a total of three times to immunize. On the third day after the final administration, spleen cells were prepared from the mice and used for cell fusion.

(3) Preparation of mouse myeloma cells The 8-azaguanine-resistant mouse myeloma cell line P3-U1 was cultured in a normal medium to obtain 2 × 10 ⁷ or more cells at the time of cell fusion and used as a parent strain for cell fusion. did.

(4) Preparation of hybridoma The spleen cells and myeloma cells obtained in (2) and (3) were 10: 1.
, And cultured in HAT medium at 37 ° C. for 14 days under 5% CO ₂ . After selecting the fused cells and further culturing by changing to HT medium, the antibody titer against ganglioside GD3 is measured, an active well is selected, the medium is further changed to normal medium, cloning is repeated twice, and enzyme immunoassay or , Immunohistological determination method (AB
C), a hybridoma that specifically reacts with ganglioside GD3 was selected. That is, a method such as Nores et al. (J. Immunol. 139 , 3171) was used from ganglioside GM3 ｛dog erythrocytes.
(1987)] and 2 ng of ganglioside GD3 (manufactured by Yatron) in 2 ml of ethanol solution containing 5 ng of phosphatidylcholine (manufactured by Sigma) and 2.5 ng of cholesterol (manufactured by Sigma). Dissolved. Of these, 20 ml was dispensed into each well of a 96-well microtiter plate (manufactured by Flow Laboratories), air-dried, and then blocked with a 1% BSA-PBS solution. The culture supernatant of the hybridoma was washed with ganglioside G.
Dispense 50 μl each to the plate on which D3 was adsorbed and the plate on which ganglioside GM3 was adsorbed, and
The reaction was performed at 4 ° C.

The reaction was carried out according to a known method (Cancer Res. 46 , 4438 (1986)), and the reaction was not carried out with ganglioside GM3 but with ganglioside GD3.
A hybridoma strain producing a mouse monoclonal antibody that specifically reacts with was selected. Here, the mouse monoclonal antibody was replaced with a mouse monoclonal antibody KM-64.
1, KM-643 and KM-644.

(5) Purification of Monoclonal Antibody (a) Preparation of Protein A-Sepharose 4B 15 g (dry powder) of bromocyan-activated Sepharose 4B (manufactured by Pharmacia) was dissolved in 100 ml of 1 mM hydrochloric acid.
After swelling for 1 minute and washing with 3 l of 1 mM hydrochloric acid, the resulting gel was washed with 75 ml of binding buffer [0.5 M sodium chloride, 0.1 M sodium bicarbonate, pH 8.3] to obtain a gel suspension.

On the other hand, 500 mg of Protein A (manufactured by Pharmacia) previously dissolved in a binding buffer having the same composition as described above was quickly mixed with the above gel suspension, and gently stirred at room temperature for 2 hours. After stirring, the gel was reacted with 200 ml of a blocking reagent (0.2 M glycine, pH 8.0) at room temperature for 2 hours to block the remaining active groups. To further remove excess adsorbed protein, 100 ml of 0.5
M sodium chloride-0.1 M acetate buffer (pH 4.0) and 10
Wash 5 times alternately with 0 ml of 0.5 M sodium chloride-binding buffer and wash with 50 ml of Protein A-Sepharose 4
B (gel) was obtained.

(B) Obtaining Cell Culture Supernatant KM-641, KM-643 and KM-644 hybridomas were each cultured in a serum-free medium [RPM-164 in 1 liter of medium.
0 (manufactured by Nissui Pharmaceutical Co., Ltd.) 10.4 g, 7.5% sodium bicarbonate 20 ml, N- (2-hydroxyethyl) piperazine-
N'-2-ethanesulfonic acid (HEPES) 10 mM,
Glutamine 0.29 g, insulin (Sigma) 3 m
g, transferrin (Sigma) 5 μl, sodium pyruvate 15 mM, sodium aselenate 125 n
M, 1 g of galactose and 0.1% of Pepol B-188 (manufactured by Toho Chiba Chemical Co., Ltd.) at 37 ° C., and when the cells become confluent, the culture supernatant is collected,
It was stored frozen at 20 ° C.

(C) Purification of antibody from cell culture supernatant The culture supernatants frozen and stored (60 l of KM-641, K
M-643 18l, KM-644 33l) were thawed, the pH was adjusted to 8.9 with sodium hydroxide, and phenylmethylsulfonyl fluoride (PMS
F, manufactured by Nacalai Tesque, Inc.) and disodium ethylenediaminetetraacetate (EDTA, manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 0.1 mM and 5 mM, mixed well, and filtered to remove cell debris. . These culture supernatants
40 ml of protein A-Sepharose 4B (gel) prepared in (a) was added, and the mixture was stirred at 4 ° C for 12 to 20 hours. The gel was recovered from the culture supernatant, packed into a column, and bound with a binding buffer (1.5 M glycine, 3 M sodium chloride,
Washed with 600 ml of pH 8.9) and the elution buffer (0.1 M
(Citric acid, pH 4.0). The IgG fraction was confirmed by measuring the absorbance (OD _{280 nm} ) of 10 ml of each eluate as one fraction. Further, the IgG fraction was collected, adjusted to pH 7 to 8 with sodium hydroxide, and dialyzed against 2M sodium chloride-PBS to obtain a purified monoclonal antibody.

(6) Determination of Subclass The mouse monoclonal antibodies KM-641, KM-643 and KM-6 were determined by ELISA using a mouse monoclonal antibody typing kit (manufactured by Zymet).
Forty-four subclasses were determined to be IgG3.

(7) KM-641, KM-643 and K
Assay for specificity of M-644 Ganglioside N-acetylgalactosamine (Ga1NAc) GM1b, GD as a standard for assaying the specificity of KM-641, KM-643 and KM-644
1a, GD1b and GT1b were manufactured by Sigma (ganglioside type V), and ganglioside GD3 and N-acetylGM2 were manufactured by Yatron. Ganglioside GM3, GM1, N-glycolyl GM2, GD
2 is a method known from dog erythrocytes, bovine brain, mouse liver, and cultured cell line IMR32 (ATCC CCL127), respectively (Journal of Biological Chemistry) 263 , 10915, 20
79 (1988)].

KM-641, KM-643 and KM-
The specificity of 644 was examined by the following TLC (thin layer chromatography) immunostaining and enzyme immunoassay. (a) TLC immunostaining TLC immunostaining is performed by a known method [Journal of Biological Chemistry (J. Biol. Chem.) 257 , 14].
365 (1982)] according to TLC Resosinol (Merck)
This was performed by a staining method. That is, ganglioside GM
3, GM2, GM1, GD3, GD1a, GalNAc
GM1b, 3 ′, 8′-LD1 and GT1b are converted to HTP
Spot on an LC plate (manufactured by Whatman), chloroform: methanol: 0.25% calcium chloride aqueous solution
(50:40:10), developed with a 0.5% polyisobutyl methacrylate (Aldrich) ethanol solution, dried, and dried with a 1% BSA-PBS solution.
Time blocked. Next, KM was used as the first antibody.
−641, KM-643 and KM-644 (10 μg
/ Ml) of a 1% BSA-PBS solution for 18 hours at room temperature, washed with a PBS solution, and added with a biotinylated anti-mouse immunoglobulin antibody (manufactured by Dako) as a second antibody, followed by reaction for 1 hour. After further washing with a PBS solution, avidin peroxidase (manufactured by Dako) was reacted for 1 hour,
HRP Color Development Reagent (Biorat)
The color was developed using.

KM-641 and KM-644 reacted with GD3 and 3 ', 8'-LD1. In addition, KM-64
3 is GD3, 3 ', 8'-LD1, GD1b and GT
Reacted with 1b.

(B) Enzyme-linked immunosorbent assay (ELISA) Ganglioside N-acetyl GM3, N-glycolyl G
M3, N-acetyl GM2, N-glycolyl GM2, G
M1, GD3, GD1a, GD2, GD1b, GT1b
96-well EIA plate [Flow Laboratorie
s) (manufactured by s) Co., Ltd., and various gangliosides and KM-641, KM-643 and K
The reactivity with M-644 was examined.

As a control, 20 μl / well of an ethanol solution containing phosphatidylcholine and cholesterol was dispensed into an EIA plate, and the absorbance was measured in the same manner as described above. The results are shown in Table 1.

[0039]

[Table 1]

KM-641 reacted strongly only with ganglioside GD3. The reactivity of KM-643 and KM-644 with various gangliosides was examined in the same manner as described above. The control was performed in the same manner as above, and the absorbance was measured. The results are shown in Tables 2 and 3.

[0041]

[Table 2]

[0042]

[Table 3]

KM-643 strongly reacted with gangliosides GD3 and GD1b. KM-644 strongly reacted with ganglioside GD3.

Example 2 KM-641, KM-643 and K by fluorescent antibody method
M-644 reactivity Human melanoma cell line SK-MEL-28 (ATC
C HTB72), WM266-4 (ATCC CRL)
1676), KHm- ／ [Journal of National Cancer Institute (J. of Natl. Can.)
cer Inst.), 59 , 775 (1977)] and 1 × 10 ⁶ human neuroblastoma cell line IMR32 (ATCC CCLK127) in a microtube (manufactured by Tref) and centrifuged with PBS (1200 rpm, 5 rpm). After washing the cells, add 50 μl (10 μl / ml) of KM-641, KM-643 or KM-644, respectively, and stir.
The reaction was performed at 30 ° C. for 30 minutes. After the reaction, the mixture was centrifuged three times with PBS (1200 rpm, 5 minutes), washed, and then anti-mouse IgG antibody and IgM antibody fluorescently labeled with fluorescein isothiocyanate (manufactured by Kappel, 20-fold diluted)
After addition of μl and stirring, the mixture was reacted at 4 ° C. for 30 minutes. After the reaction, the cells were centrifuged three times with PBS (1200 rpm, 5 minutes), washed, and then suspended in PBS.
Analysis was performed using CS-1 (manufactured by JASCO Corporation).

Further, KM-641, KM-643 and K
The analysis was performed in the same manner as described above except that the anti-ganglioside GD3 antibody R24 (MEL-1, Signet) belonging to IgG3 was used instead of M-644. KM-641, KM-643 and KM-644 as controls
The analysis was performed by the same operation as described above without any addition.

The results are shown in FIG. 1 and FIG. KM-64
1, the fluorescence intensities of KM-643 and KM-644 are shifted to the right (higher fluorescence intensity) as compared with those of the control, and the reactivity is slightly different depending on the antibody. Was shown to react directly with the glycolipid sugar chain expressed on the cell surface.
KM-641, KM-643 and KM-644
Showed a higher reactivity than the anti-ganglioside GD3 antibody R24.

Example 3 Complement-dependent cytotoxicity (Complement Dependent Cytoto
xicity: CDC) (a) Preparation of target cells Target cells suspended in RPMI-1640 medium supplemented with 10% FCS
Ganglioside GD3-positive cells G361 (JCRB)
1) and SK-MEL-28 ⁷Pieces/
Adjust to Na_Two ⁵¹CrO_FourTo 100 μCi / 1 × 10
⁷And reacted at 37 ° C for 1 hour.
Washed three times. Then, leave in the medium at 4 ° C for 30 minutes,
After spontaneous dissociation and centrifugation (1200 rpm, 5 minutes), the medium is
Plus 4 × 10⁶It adjusted to the number / ml.

(B) Absorption of complement A freeze-dried rabbit complement (manufactured by Cedar Lane Laboratory) was dissolved in water, and 2 × 10 ⁷ target cells prepared in (a) were added, and the mixture was added at 4 ° C. for 30 minutes. Reacted. This was centrifuged (1200 rpm, 5 minutes), 2 × 10 ⁷ target cells were added again, and the reaction was carried out in the same manner as described above. The cells were removed by passing through a filter to obtain a complement solution.

(C) Measurement of CDC activity KM-641, KM-643 or KM-644 was put on a 96-well U-shaped bottom plate at a final concentration of 0.05 μg / ml to 50 μm.
l / ml in each case, and 2 × 10 ⁵ target cells were added to each.
Pieces / well were added. The mixture was reacted at room temperature for 1 hour, centrifuged (1200 rpm, 5 minutes), the supernatant was removed, 50 μl of the complement solution obtained in (b) was added, and the mixture was reacted at 37 ° C. for 1 hour. After centrifugation (1200 rpm, 5 min), it was measured amount of ⁵¹ Cr in the supernatant in γ- counter. The amount of spontaneously dissociated ⁵¹ Cr was determined by adding only the medium to the target cells instead of the antibody and complement solution
And measuring the amount of ⁵¹ Cr in the supernatant in the same manner as described above. Total free ⁵¹ Cr amount was added 5 N sodium hydroxide in lieu of the complement solution, and in the same manner as described above, the supernatant ⁵¹ C
It was determined by measuring the amount of r. CDC activity was determined by the following equation.

[0050]

(Equation 1)

As a control, a medium was added in place of the complement solution, and the same procedure was carried out as above, and the amount of ⁵¹ Cr in the control was measured to determine the CDC activity. The results are shown in Table 4.

[0052]

[Table 4]

KM-641, KM-643 and KM-
644 is GD3-positive cells G361 and SK-M
It showed strong cytotoxic activity against EL-28.

Example 4 Antibody-dependent cell-mediated cytotoxicity (Antibody Dependent Cel)
l mediated Cytotoxicity: ADCC) (a) Preparation of target cells Target cells SK-MEL-28 and G361 suspended in RPMI-1640 medium supplemented with 10% FCS were each 1 × 10 ⁶
Per 0.5 ml, and add Na ₂ ⁵¹ CrO ₄ to 50 μCi / 1 × 10
Add ⁶ cells and incubate at 37 ° C for 1 hour.
Washed twice. Then, the mixture was allowed to stand in a medium at 4 ° C. for 30 minutes, spontaneously dissociated, centrifuged (1200 rpm, 5 minutes), and adjusted to 2 × 10 ⁵ cells / ml by adding the medium.

(B) Preparation of effector cells 50 ml of human venous blood is collected, and 0.5 ml of sodium heparin (Takeda Pharmaceutical, 1000 units / ml) is added and mixed gently. This was centrifuged (1500 to 1800 g, 15 minutes) using Leuko-PREP (manufactured by Nippon Becton Dickinson) to collect the cell layer, and then centrifuged three times in an RPMI-1640 medium (1500 to 1800 g).
g, 15 minutes), washed, and suspended (5 × 10 ⁶ cells / ml) in RPMI-1640 medium supplemented with 10% FCS to obtain effector cells (lymphocytes).

(C) Measurement of ADCC activity KM-641, KM-643 or KM-644 was added to a 96-well U-shaped bottom plate at a final concentration of 0.05 μg / ml to 50 μm.
g / ml in the range of 50 μl each, 1 × 10 ⁴ target cells / well (50 μl) and 5 effector cells
× 10 ⁵ cells / well (100 μl) were added. After reacting at 37 ° C. for 4 hours, the mixture was centrifuged (1200 rpm, 5 minutes), and the amount of ⁵¹ Cr in the supernatant was measured with a γ-counter. Spontaneous dissociation ⁵¹
The Cr content was determined by adding only the medium to the target cells instead of the antibody and effector cells, and measuring the ⁵¹ Cr content of the supernatant in the same manner as described above. The total amount of free ⁵¹ Cr was determined by adding 5 N sodium hydroxide instead of the antibody and effector cells, performing the same procedure as above, and measuring the amount of ⁵¹ Cr in the supernatant. ADCC activity was determined by the following equation.

[0057]

(Equation 2)

[0058] As a control, was added to the medium instead of the effector cells were performed in the same manner as described above to determine the measured ADCC activity ⁵¹ Cr-amount control. The results are shown in Table 5.

[0059]

[Table 5]

KM-641, KM-643 and KM-
644 is a ganglioside GD3-positive cell, SK-
The antibody exhibited antibody-dependent cell-mediated cytotoxic activity against MEL-28 and G361.

Example 5 Therapeutic Effect on Transplanted Tumors 2 × 10 ⁷ human melanoma cells C24.22 were transplanted into the abdominal skin of 7 Balb / c nu / nu mice per group. After transplantation, K
200 μg of M-641 from the day of tumor implantation, or
It was intravenously administered 5 times from 7 days after tumor implantation. Further, as a control group, anti-Sialyl Le ^a monoclonal antibody AMC
-462 (ECACC86050801) was administered in the same manner. The therapeutic effect on the transplanted tumor was represented by the tumor volume and the number of days of survival calculated by the following formula.

[0062]

(Equation 3)

In addition, comparison of the effect by the antibody dose was performed using AMC-462 at 200 μg / animal or KM from the day of tumor implantation.
This was carried out by administering -641 at 200 µg / mouse or 100 µg / mouse, respectively. The results are shown in FIGS. A
In the control group to which MC-462 was administered, remarkable tumor growth was observed.
By administering 0 μg / animal, tumor growth was significantly suppressed (FIG. 3), and prolongation of survival days was also observed (FIG. 4). Further, in the group to which KM-641 was administered from the 7th day after transplantation, the survival days were not prolonged (FIG. 4), but the tumor growth was significantly suppressed compared to the control group (FIG. 3). ). 100 days after transplantation, the number of engrafted tumors was 7/7 in the control group, 200 μg / mouse of KM-641 / 200 μg / mouse on day 7 after tumor transplantation, and 200 μg / mouse of KM-641 was co-administered with tumor. Group 4/7 showed the efficacy of KM-641 administration. FIG. 5 shows the results of the examination of the dose of KM-641.
It was shown to. As shown in FIG. 5, not only 200 μg / animal,
Even at 0 μg / animal, a significant tumor growth inhibitory effect was observed.

[0064]

According to the present invention, gangliosides GD3 and 3 ', 8' useful for treating cancers derived from the neuroectodermal system.
-IgG class monoclonal antibodies reactive to LD1 are provided.

[Brief description of the drawings]

FIG. 1 shows ganglioside G obtained by the fluorescent antibody method of Example 2.
It is a figure showing the reactivity to D3-positive cells KHm- お よ び and IMR32. The vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity. Row (a) is KM-641, row (b) is KM-643, row (c) is KM-644, row (d) is R24.
The reactivity of each is shown. The dotted line shows the reactivity without addition of the antibody.

FIG. 2 shows ganglioside G obtained by the fluorescent antibody method of Example 2.
D3-positive cells SK-MEL-28 and WM266-4
FIG. 4 is a diagram showing the reactivity with respect to. The vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity. (a) Column is KM-641,
Column (b) shows the reactivity of KM-643, column (c) shows the reactivity of KM-644, and column (d) shows the reactivity of R24. The dotted line shows the reactivity without addition of the antibody.

FIG. 3 is a graph showing the effect of KM-641 on the transplanted tumor of Example 5 by tumor volume. The vertical axis indicates tumor volume, and the horizontal axis indicates days after tumor implantation. A white circle is AMC-4
62: 200 μg / animal simultaneously administered with tumor transplantation, black circle: 200 μg / animal of KM-641 / administered simultaneously with tumor implantation, open square: 200 μg / animal of KM-641 / day 7 days after tumor implantation The groups administered from the eyes are shown.

FIG. 4 is a graph showing the effect of KM-641 on the transplanted tumor of Example 5 in terms of the number of days of survival. The vertical axis indicates the survival rate, and the horizontal axis indicates the number of days after tumor implantation. The white circle is AMC-46
2, a group administered 200 μg / animal simultaneously with tumor transplantation, a black circle represents a group administered 200 μg / animal of KM-641 simultaneously with tumor transplantation, and a white square represents a group administered 200 μg / animal of KM-641 7 days after tumor implantation. The groups administered from the day are shown.

FIG. 5 shows KM- against the tumor volume of the transplanted tumor of Example 5.
FIG. 7 shows the effect of the dose of 641. The white circle is AMC
-462: 200 μg / animal was administered at the same time as tumor transplantation, black circles: 200 μg / animal of KM-641 were administered simultaneously with tumor implantation, open squares: 100 μg / animal of KM-641 was implanted with tumor. The groups administered simultaneously are shown.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:91)

Claims (7)

(57) [Claims] 1. A monoclonal antibody belonging to IgG3 and reacting with at least one ganglioside GD3 and 3 ′, 8′-LD1. 2. Ganglioside GD3 is NeuAcα.
2 → 8 NeuAcα2 → 3Gal sugar chain, NeuAcα2
→ 8 NeuGcα2 → 3Gal sugar chain, NeuGcα2 →
8 NeuAcα2 → 3Gal sugar chain or NeuGcα2
The monoclonal antibody according to claim 1, which is selected from ganglioside GD3 having → 8 NeuGcα2 → 3Gal sugar chain. 3. The hybridoma KM-641 (FERM BP-
3116), hybridoma KM-643 (FERM BP-3117)
Or the monoclonal antibody according to claim 1, which is produced by hybridoma KM-644 (FERM BP-3118). 4. A hybridoma belonging to IgG3 and capable of producing a monoclonal antibody reactive to at least one ganglioside GD3 and 3 ′, 8′-LD1. 5. The hybridoma KM-641 (FERM BP-
3116), Hybridoma KM-643 (FERM BP-3117)
The hybridoma according to claim 4, wherein the hybridoma is selected from the group consisting of: and hybridoma KM-644 (FERM BP-3118). 6. An antitumor agent, which comprises the monoclonal antibody according to claim 1 as an active ingredient and is effective against cancers derived from the neuroectodermal system. 7. The antitumor agent according to claim 6, wherein the cancer derived from the neuroectodermal system is malignant melanoma.