JPH0530991A - Ganglioside-resistant monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide the subject antibody widely recognizing ganglioside molecule species and useful for treating melanoma, etc., by immunizing a mouse with a lactone type GD3 ganglioside, fusing the splenic cells of the immunized mouse with myeloma cells, and culturing the fused cell strain. CONSTITUTION:A lactone type GD3 ganglioside obtained by treating with glacial acetic acid a GD3 ganglioside extracted from a bovine brain is subjected together with a Salmonella minnesota stain to an ultrasonic treatment to produce an antigen solution. The antigen solution is administered into the abdominal cavity of a Balb/c mouse to immunize the mouse, and the splenic cells of the mouse are collected and fused with mouse myeloma cells. The fused cells are cultured in a HAT medium to produce hybridoma cells. The hybridoma cells are screened by an enzymatic immunoassay method, and the positive clones are selected. The positive clones are cloned by a limiting dilution analysis method, etc., and the clone is cultured to produce the objective ganglioside- resistant monoclonal antibody widely recognizing various ganglioside molecule species.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-ganglioside monoclonal antibody which widely recognizes various gangliosides distributed in melanoma, neuroblastoma, pancreatic cancer and the like.
More specifically, the present invention relates to ganglioside GM4, GM
3, GM2, GM1a, GM1b, SPG (α2-
3), SPG (α2-6), GD3, GD2, GD1
a, a GD1b, and an anti-ganglioside monoclonal antibody that reacts widely with GT3, which are melanoma, neuroblastoma,
The present invention relates to an anti-ganglioside monoclonal antibody that can be used as a therapeutic agent for pancreatic cancer and the like.

[0002]

2. Description of the Related Art Monoclonal antibodies that recognize gangliosides as cancer-related antigens have been the anti-GM3 antibody M2590 (Taniguchi, M., et .
al. , Jpn. J. Cancer Res. , 75 ,
418-426, 1984) and DH2 (Doi,
T. , Et al . , Cancer Res. , 48 ,
5680-5685, 1988), or anti-GD3 antibody R-24 (Houghton, AN, et a .
l . Proc. Nalt. Acad. Sci. USA,
82 , 1242-1264, 1985), anti-GD2 antibody L72 (Irie RF, et al . Proc.
Nalt. Acad. Sci. USA, 83 , 8694
-8698, 1986) and the like have been reported. These monoclonal antibodies are under development as therapeutic agents for melanoma.

[0003]

Melanoma, neuroblastoma, and pancreatic cancer are malignant and easy to metastasize, and their treatment is difficult as compared with other cancers, and the development of a therapeutic method thereof is desired by clinicians. By the way. Previously reported, anti-ganglioside monoclonal antibodies as anti-cancer antibodies each have high antigen-recognition specificity and show remarkable effects on a specific population of a specific tumor, but are effective on all tumors of the same type. It is hard to say that you have. Furthermore, it has little effect on many types of tumors.

[0004]

In order to solve these problems, various tumor cells require antibodies that recognize a wide range of gangliosides that are present in relatively large amounts. Thus, the present invention provides anti-ganglioside monoclonal antibodies that recognize a wide variety of ganglioside molecular species. Use of the anti-ganglioside monoclonal antibody of the present invention solves the above-mentioned conventional problems.
That is, it becomes possible to treat various cancers by using the antibody of the present invention in a form bound to a toxin or an anti-cancer agent or in an unmodified form. Furthermore, administration of a radioisotope-labeled monoclonal antibody of the present invention or a (Fab ′) ₂ fragment of a radioisotope-labeled monoclonal antibody of the present invention to a patient enables imaging of various cancers.

The anti-ganglioside monoclonal antibody of the present invention reacts with various gangliosides. That is, it strongly reacts with ganglioside GM4 and GM3, and further, SPG
(Α2-3), GD3, GD2 and GT3, and also GM2, GM1a, GM1b, SPG (α2-6),
It also reacts with GD1a and GD1b. These names for gangliosides refer to Svennerholm nomenclature (S
vennerholm, L .; J. Lipid Res.
5 , 145-155 (1964)). The anti-ganglioside monoclonal antibody of the present invention belongs to immunoglobulin class IgG3.

The anti-ganglioside monoclonal antibody of the present invention can be prepared as follows. Immunization of Animals with Ganglioside Antigens Immunization of, for example, mice with ganglioside antigens has already been clarified (Higgins, TJ et .
al . Molec. Immun. , 22 , 1265
-1271, 1985). That is, for example, lactone type GD
Dry 5 ug of 3 gangliosides to a tube. To this was added 5 mM phosphate buffered saline (pH 7.2,500 μl) containing cells of Salmonella minnesota strain (1 g) that had been previously treated with 1N hydrochloric acid, and added.
The ganglioside is adsorbed on the cells by ultrasonic treatment for a minute to form an antigen solution. This antigen solution is intraperitoneally administered to Balb / c mice. In addition to the method described above, other commonly used methods can be adopted. For example, killed tuberculosis bacteria, emulsifiers, etc. are used as adjuvants,
A method of subcutaneously or intravenously administering to mice or rats can also be adopted.

[0007]Preparation of splenocytes and myeloma fusion cells Creating a cell line that produces a monoclonal antibody
For example, splenocytes and mau of mice immunized with gangliosides
It is carried out by fusion with Smyeloma cells, and the method is
It has been revealed in various places. Mouse / mouse
Cell fusion is "experimental medicine"6(10) 909 (1988)
Year)). First, cancer
The spleen is excised from a mouse immunized with glioside and minced
A mesh of appropriate size, eg 200 mesh
Suspension in a suitable medium, for example RPMI1640
It Meanwhile, RPMI 1640 culture containing 10% fetal bovine serum
10 in the ground^FiveMyeloma cells cultured at 1 / ml, eg
For example, serum-free P3-X63-Ag8-U1 (P3U1)
5 × 10 with RPMI1640 medium^FivePieces / ml
To suspend. Mouse splenocyte suspension and mouse myelo
20ml of suspension is mixed and salted at 1000rpm for 5 minutes
Pellet to form pellet and decant supernatant to complete
Excluding. This pellet was heated to 37 ° C with 50% poly
RPMI1 containing ethylene glycol (PEG 1500)
Add 1 ml of 640 medium gradually with stirring for about 1 minute
Continue stirring. Then, 9 ml of RPMI1640 medium
Add slowly with stirring for 4 or 5 minutes, then add 100
Spin down at 0 rpm for about 5 minutes. After removing the supernatant, remove the cells
RPMI1640 medium containing 20% fetal bovine serum 50 ml
Suspend in 100 μl in each well of a 96-well cell culture plate.
Dispense by l. 5% CO ₂37 ° C in culture device, 24
After a while, HAT medium (hypoxanthine, aminopte
RPMI16 containing phosphorus, thymidine, 10% fetal bovine serum
40 medium; Litterfield, Science1
45  709-710, 1964) and culturing
By, only fused cells are grown. The above fusion details
In addition to cell preparation methods, other conventional fusion methods may be used.
I can do it. For example, polyethylene glycol as a fusing agent
Other myeloma cell lines using 6000, HVJ, etc.
Can also be used to prepare fused cells. Thus profitable
Various gangliosides using the culture supernatant of fused cells
The antibody that widely recognizes is screened.

Antibody screening In order to obtain antibodies that widely recognize gangliosides,
ELISA using multiple types of gangliosides as antigens
The method is performed to screen for antibodies that react with many types of gangliosides. It has already been clarified by an ELISA method using ganglioside as an antigen (Higash
i, H. , Et al . J. Biochem. 95 ,
785-794, 1984). For example, ganglioside is dissolved in ethanol to a concentration of 200 pmol / ml, and 50 μl thereof is dispensed into each well of a 96-well polystyrene plate. The mixture is left at 37 ° C. for about 3 hours, and ethanol is evaporated to adsorb ganglioside to each hole of the polystyrene plate. 100 μl of 5 mM phosphate buffered saline (PBS) (pH 7.2) containing 5% bovine serum albumin (BSA) was dispensed into each well,
Leave at room temperature for 3 hours. This is sucked out, 50 μl of the culture supernatant of the fused cells is dispensed into each well and left at room temperature for 2 hours. The culture supernatant was sucked out and each well was washed 3 times with 100 μl of PBS, and then 5% PBS containing 0.5% BSA was added to each well.
Dispense 0 μl each and leave at room temperature for 15 minutes. This was sucked out, and 0.2% goat anti-mouse immunoglobulin (Kappel) with 0.1% horseradish peroxidase was added to each hole.
Dispense 50 μl of PBS containing 5% BSA and leave at room temperature for 2 hours. This was sucked out, and each well was washed 5 times with 200 μl of PBS, and then 50 μl of citrate phosphate buffer (pH 5.0) containing 0.1% o-phenylenediamine and 0.01% hydrogen peroxide was added to each well. Each of them is added and the color reaction is performed at room temperature for 10 minutes. Add 1N hydrochloric acid to each hole to stop the reaction,
The absorbance at 510 nm is measured. By repeating such screening, a fused cell producing a monoclonal antibody that reacts with various gangliosides is cloned. The desired fused cells thus obtained are cultured, and the anti-ganglioside monoclonal antibody of the present invention can be obtained from the culture supernatant. Alternatively, the fused cells can be intraperitoneally administered to mice, for example, and anti-ganglioside monoclonal antibodies can be obtained from the serum or ascites fluid.

The anti-ganglioside monoclonal antibody of the present invention can be used as a therapeutic agent for melanoma, neuroblastoma, pancreatic cancer and the like. For example, human melanoma cell KHM
It exerts a remarkable growth-suppressing action on the −1 / w strain and is particularly useful as a therapeutic agent for melanoma. A known method may be applied to the preparation of an antitumor agent containing an anti-ganglioside monoclonal antibody as an active ingredient, and the administration method is preferably intravenous injection. The injection can be prepared by a conventional method using a physiological saline, a water-soluble solvent such as sterile water Ringer's solution, a known carrier such as a stabilizer, an additive and the like. Alternatively, the anti-ganglioside monoclonal antibody can be directly linked and dried to give an injection. In this case, the drug is dissolved in physiological saline or the like before use. Further, the anti-ganglioside monoclonal antibody of the present invention can be used as a cancer missile therapeutic agent by binding to a toxin or another antitumor agent. The dose of the anti-ganglioside monoclonal antibody of the present invention may vary depending on symptoms, age, sex and the like, but is usually 50 to 500.
mg / day.

The anti-ganglioside monoclonal antibody of the present invention can be used for in-vivo diagnosis (imaging) of various cancers. That is, anti-ganglioside monoclonal antibody or its (Fab ′) ₂ fragment was ¹³¹ I,
It is possible to visualize the cancer site where the antibody is accumulated by labeling with a radioisotope such as ¹¹¹ I and administering it to a living body and scanning with a gamma camera.

[0011]

As is apparent from the above detailed description,
The anti-ganglioside monoclonal antibody of the present invention has the property of reacting broadly with various gangliosides, and is useful as a treatment for various cancers such as melanoma, neuroblastoma, and pancreatic cancer, and in vivo diagnosis of various cancers. Can also be used for

[0012]

[Examples] Next, the present invention will be described in more detail with reference to Examples. Example 1 Production of Monoclonal Antibody 1-1 Lactone type GD3 Bal with ganglioside
Immunization of b / C mouse 10 μ of GD3 ganglioside extracted from bovine brain and purified by Q-Sepharose (Pharmacia) column
g was incubated in the presence of glacial acetic acid at 25 ° C. for 8 hours (Yu. RK, et al ., J. Bioche.
m. , 98 1367-1373, 1985). The lactone type GD3 ganglioside was obtained by dialysis of the reaction solution and freeze-drying. Dry 5 μg of this in a test tube,
To this was added 500 μl of a 5 mM phosphate buffered saline containing salmonella Minnesota strain cells (1 g) that had been treated with 1N hydrochloric acid in advance. The lactone type GD3 ganglioside was adsorbed to the cells by ultrasonic treatment for 5 minutes to obtain an antigen solution. 500 μl of this antigen solution was added to Balb /
c mice (female, 5 weeks old) were intraperitoneally administered.

1-2 Preparation of Mouse Spleen Cells Spleens excised from Balb / c mice immunized with the lactone type GD3 ganglioside by the method of 1-1 were cut into small pieces and passed through a stainless mesh (200 mesh, manufactured by Abe Kagaku). The cells were suspended in 10 ml of RPMI1640 medium. This was collected in a 50 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes to remove the supernatant. Next, the cells were resuspended in 10 ml of 50 mM ammonium chloride aqueous solution, left at 4 ° C. for 10 minutes and then pipetted to lyse the red blood cells. This was centrifuged at 1000 rpm for 5 minutes and the supernatant was removed. 10 ml of RPMI1640 medium was added to the pellet and the number of lymphocytes was counted, followed by centrifugation at 1000 rpm for 5 minutes. RPMI1640 medium was added to the pellet to prepare 10 ⁷ cells / ml. 1-3 Preparation of mouse myeloma cells 10 ^{5 in} RPMI1640 medium containing 10% fetal bovine serum
Mouse myeloma cells P3-X6 cultured in cells / ml
RPMI1 without 3-Ag8-U1 (P3U1) in serum
The cells were washed with 640 medium and suspended in serum-free RPMI1640 medium such that the number of cells was 5 × 10 ⁵ cells / ml.

1-4 mouse lymphocytes and mouse myelo
Fusion of Ma strains The method of preparing mouse / mouse fused cells has been clarified in various places, but mainly "Experimental Medicine" 6 (10) 909.
(1988). 10 ml of spleen cell suspension of immunized Balb / C mouse prepared in 1-2,
10 ml of the mouse myeloma (P3U1) cell suspension prepared in 3 was mixed and centrifuged at 1000 rpm for 5 minutes to form a pellet, and the supernatant was completely removed. 1 ml of RPMI1640 medium containing 50% polyethylene glycol (PEG1500) heated to 37 ° C. was gradually added to this pellet while stirring, and stirring was continued for 1 minute. Next, 9 ml of RPMI1640 medium was gradually added over 4 or 5 minutes, followed by centrifugation at 1000 rpm for 5 minutes. After removing the supernatant, the cells were suspended in 50 ml of RPMI1640 medium containing 20% fetal bovine serum and dispensed in 100 μl portions.

1-5 The cell suspension prepared in HAT selection 1-4 was dispensed into each well of a 96-well cell culture plate in an amount of 100 μl and cultured at 37 ° C. in a 5% CO ₂ incubator. . After 24 hours, 100 μl of HAT medium was dispensed into each well of the culture plate. Furthermore,
From the start of culture, 2, 3, 5, 8, 11, 14, 17,
On day 21, the operation of replacing half of the culture medium with fresh HAT medium was repeated. Due to this HAT section, myeloma and spleen cells that have not fused are killed, and only fused cells grow. 1 hybridoma that has been raised
Cultured in RPMI1640 medium containing 0% fetal bovine serum,
The culture solution was used as an analysis sample for the enzyme immunoassay method (ELISA method).

1-6 Enzyme- Linked Immunosorbent Assay (ELISA) Appropriate raw materials, for example, various gangliosides purified from bovine brain were dissolved in ethanol to prepare 1 nmol / ml solutions. 50 μl of this ethanol solution was dispensed into each well of a 96-well polystyrene plate (Flow Laboratories), left at 37 ° C. for about 3 hours, and ethanol was evaporated to adsorb ganglioside to each well of the polystyrene plate. 100μ of PBS containing 5% BSA in each hole
It was dispensed in increments of 1 and left at room temperature for 3 hours. This was sucked out, 50 μl of the culture supernatant of the fused cells was dispensed into each well, and left at room temperature for 2 hours. Suck out the culture supernatant and fill each well with 10
After washing 3 times with 0 μl PBS, add 0.5% BSA to each well.
50 μl of the contained PBS was dispensed and left at room temperature for 15 minutes. This was sucked out, and 50 μl of PBS containing 0.1% horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Cappel) and 0.25% BSA was dispensed into each well and left at room temperature for 2 hours. Suck this out and put 2 in each hole
After washing 5 times with 00 μl of PBS, 0.1% o-
50 μl each of phenylenediamine and 0.01% hydrogen peroxide-containing citrate phosphate buffer (pH 5.0) were added, and a color reaction was performed at room temperature for 10 minutes. 50 μl of 1N in each hole
Hydrochloric acid was added to stop the reaction, and the absorbance at 510 nm was measured. As a result, antibodies that react with various types of gangliosides were selected. Thus, the hybridoma cell line LD3 producing anti-ganglioside monoclonal antibody was cloned. This mouse hybridoma LD3 has 1
It was deposited with the National Institute of Micro-Industrial Research on May 22, 991, and the deposit number is Micro-organism Research Institute No. 12267 (FERM P-1226).
7) is given.

1-7 Specificity of Monoclonal Antibody The reaction specificity of the anti-ganglioside monoclonal antibody LD3 was examined by the enzyme-linked immunosorbent assay described in 1-6.
LD3 is GM4, GM3, GM2, GM1a, GM1
b, SPG (α2-3), SPG (α2-6), GD
3, reacted with GD2, GD1a, GD1b, GT3.
Figure 1 shows LD3, GM4, and GM3 in the ELISA system.
The reaction curve with is shown. Table 1 shows the reactivity of LD3 with various gangliosides. When 50 pmol of various ganglioside antigens are coated on each plate and reacted with anti-ganglioside monoclonal antibody LD3, the absorbance is 0.2 or more and less than 0.5 +, 0.5
It was set as ++ when it was 0.75 or more and less than 0.75, and as ++ when it was 0.75 or more.

[Table 1] Table 1 Reactivity between LD3 antibody and various gangliosides ──────────────────── Ganglioside reactivity ───────────── ──────── GM4 +++ GM3 ++++ GM2 + GM1a + GM1b + SPG (α2-3) ++ SPG (α2-6) + GD3 ++ GD2 ─ ─ ────────────────── ─────────────

1-8 Purification of antibody LD3 LD3 cells were cultured in serum-free ITES-eRDF (Kyokuto Pharmaceutical) medium and the culture supernatant was collected to obtain a protein fraction (80%).
An ammonium sulfate precipitated fraction) was prepared. This is Se
Phacryl S-200 (Pharmacia) column chromatography was fractionated to obtain a purified LD3 antibody.

Example 2 Anti-proliferative effect of purified antibody LD3 on human melanoma cell KHM-1 / w strain Human melanoma cell KHM-1 / w strain was treated with RPMI1640 medium containing 30% human serum at 2 × 10 ⁵ cells / ml. Cultured at a density. 25, 50 of purified antibody LD3,
The cells were added at a concentration of 100 μg / ml and the number of viable cells was counted after 24 hours (FIG. 2). 25, 50 of purified antibody LD3,
The number of viable cells in the system added at a concentration of 100 μg / ml is 3 when cultured in 30% human serum medium.
1.8%, 12.8%, 3.7%, purified antibody LD
3 showed a remarkable growth inhibitory effect on the melanoma cell line KHM-1 / W.

Example 3 Preparation of Injectable Solution Hybridoma LD3 was added to RPMI1 containing 10% calf serum.
The cells are cultured in 640 medium or serum-free e-RDF medium (Kyokuto Pharmaceutical). Alternatively, the ascites is obtained by proliferating in the mouse abdominal cavity.
Ammonium sulfate is added to the culture supernatant or ascites fluid at 4 ° C. to 80% saturation to precipitate the protein fraction. This is fractionated by a gel filtration method using Sephacryl S-200 (Pharmacia) to obtain purified LD3 as an IgG fraction. This is connected and dried as it is to obtain an injection. This injection is used by dissolving it in physiological saline at the time of use.

[Brief description of drawings]

FIG. 1 is a graph showing the results of examining the reactivity of antibody LD3 with gangliosides GM4 and GM3 by the enzyme immunoassay method of Example 1-6.

FIG. 2 is a human melanoma cell KH of antibody LD3.
5 is a graph showing the results of examining the complement-dependent cytotoxic activity against the M-1 / W strain by the method described in Example 2.

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Claims (7)

[Claims] 1. An anti-ganglioside monoclonal antibody which recognizes a wide variety of ganglioside molecular species. 2. Ganglioside GM4, GM3, SPG
The anti-ganglioside monoclonal antibody according to claim 1, which reacts with (α2-3), GD3, GD2 and GT3. 3. Ganglioside GM4, GM3, GM
2, GM1a, GM1b, SPG (α2-3), SPG
The anti-ganglioside monoclonal antibody according to claim 1, which reacts with (α2-6), GD3, GD2, GD1a, GD1b, and GT3. 4. The anti-ganglioside monoclonal antibody according to claim 1, which is produced by a mouse / mouse fusion cell line of spleen cells of Balb / c mouse immunized with lactone type GD3 ganglioside and mouse myeloma cells. 5. The anti-ganglioside monoclonal antibody according to claim 1, wherein the immunoglobulin class is IgG3. 6. An antitumor agent containing the anti-ganglioside monoclonal antibody according to claim 1 as an active ingredient. 7. The antitumor agent according to claim 6 for treating melanoma.