JPS63270699A - Monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:An antibody which specifically recognizes saccharides containing N-glycolylneuraminic acid, is reacted with N-glycolyl GM2 ganglioside, glycolyl GM3 ganglioside and IV<3> NeuGc-nLcOSe4 Cer but not with VI<3> NeuGc-nLcOSe6 Cer. USE:A diagnosticum for human cancer. A drug is bonded to the antibody and is applicable to missile therapy. A reagent for detecting H-D antibody. A diagnosticum and remedy for Marek's disease of chicken. PREPARATION:A mouse is immunized against a cell containing a glycoliid containing N-glycolylneuraminic acid as an immune source and the splenocyte of the immunized animal is fused with a mouse myeloma cell. Then the prepared hybridoma is cultivated, a strain to produce the aimed antibody is searched and cloned, a monocloned hybridoma is obtained and cultivated to produce an antibody.

Description

[Detailed Description of the Invention] [Object of the Invention] (Industrial Application Field) The present invention relates to a monoclonal antibody, and more particularly, the present invention relates to a monoclonal antibody that specifically recognizes a certain type of N-glycolylneuraminic acid-containing sugar chain. It relates to monoclonal antibodies that

(Prior art and its problems) Glycolipids are constituents of cell membranes.
Due to differences in binding methods, a variety of molecular species ζ exist, and they exhibit species-specific, organ-specific, and cell-specific distributions. Their functions include acting as receptors for bacterial toxins, hormones, etc.
It is becoming clear that in addition to acting as immunological legal bases such as blood group substances, they play important roles in controlling proliferation or culture and in interactions between cells. Furthermore, it has been shown that qualitative and quantitative compositional changes occur as cells become cancerous, and that some of them can become cancer antigens. It has also been suggested that changes in its composition may be directly involved in the canceration mechanism, such as by acting as a regulator of the mechanism.

On the other hand, Milstein et al. reported a method for establishing a cell line that produces uniform antibodies with specificity for one type of antigenic determinant [Nature, 52
56, pp. 495-497 (1975)], it became possible to qualitatively and quantitatively quantify trace substances. In order to search for cancer antigens, this technology has been used to create many monoclonal antibodies specific to cancer cells, but some of these antibodies recognize sugar chains of glycolipids or glycoproteins. was revealed [Journal Fist of National Cancer Institute (J, Natl.
, Cancer In5t, ), No. 71a, No. 231
~251 pages, (1983)].

For example, antibodies that react with glycolipids such as GD2 ganglioside or GD3 ganglioside have been obtained as monoclonal antibodies against human melanoma. Moreover, monoclonal antibody N519-9 specific for pancreatic cancer reacts with a glycolipid having a sialosyl Lewis type A vJ button. These antibodies are useful for diagnosing cancer and observing the progress of treatment, and attempts are being made to use them for treatment. Qualitative and quantitative changes in glycolipids associated with canceration are caused by changes in the activities of various glycosyltransferases in the sugar chain biosynthesis mechanism due to abnormal gene expression. A sugar chain structure that does not occur is created. Its sugar chain structure can be used as a gamma marker.

In this way, the importance and usefulness of glycolipids as clues to the elucidation of the canceration mechanism and as cancer antigens and cancer markers has been highlighted, and their applications in critical fields such as diagnosis and treatment have been highlighted. Be expected.

Among glycolipids, those containing sialic acid, which is an acidic sugar, in their sugar chains are collectively called gangliosides. The main types of sialic acid are N-acetylneuraminic acid and N-glycolylneuraminic acid. Sialic acid is widely detected in various organs, cells, and body fluids of animal species, but N-glycolylneuraminic acid has so far not been found in normal humans or chickens.

Detected in serum sickness patients, sheep, horses, pigs, rabbits,
The anthropophilic antibody that agglutinates guinea pig red blood cells is Hanganatz 1u-Deich.
er, hereinafter referred to as rH-DJ) antibody. The antigen recognized by this antibody is called HD antigen. N
- Glycolylneuraminic acid-containing ganglioside is HD
It has been reported to have antigenic activity [Biochemical and Biophysical Research Communications (Biochem, Biophys, Res,
Commun, ), Vol. 79, pp. 388-395 (
(1977)], the sugar chain structure of NeuGc α2-3 Gal was identified as the main antigenic determinant.

In recent years, N, a ganglioside with HD antigen activity, has been
-Glycolyl GM 3 gang 1 nooside (113Nf
! By immunizing chickens with uGc-LacCer), antibodies that react with various gangliosides having HD antigen activity were prepared from the serum, and using these antibodies, N-glycolylneuraminic acid was found to be characteristic of human cancer tissues. [Biken 9 Journal (Biken J,
), Vol. 25, pp. 47-50 (1982)]. In addition, several types of H
-D antigen-active N-glycolylneuraminic acid-containing gangliosides were detected, and glycoproteins with HD-antigen active sugar chains were detected in tissues of teratomorphic species [Gann, No. 75]. Vol., pp. 1025-1029 (1984)], when glycolipids with HD antigen activity detected from human colon cancer tissues were analyzed using antibodies prepared in chicken, N-glycolyl GM2 ganglioside, N-glycolyl GM2 ganglioside, N-glycolyl 0M3 ganglioside, 0-
Acyl-N-glycolyl 0M3 ganglioside, IV3
NeuGc-nLcOse4 Cer, which were not detected in normal tissues, Cancer Res, Vol. 45, No. 379.
6-3802 (1985)].

Gangliosides with HD antigen activity were also detected in cell lines of Marek's disease, a type of chicken lymphoma (Journal of φ Biochemistry).
Tokyo) [J.

Biochem, (Tokyo)], Volume 95, No. 7
pp. 85-794 (1984)].

N-glycolylneuraminic acid and N-glycolylneuraminic acid-containing sugar chains are considered cancer-related antigens, and detecting them with high sensitivity and accuracy is extremely important for cancer diagnosis.

Contains N-glycolylneuraminic acid or N-glycolylneuraminic acid 8! In order to efficiently detect i-chain, immunoassay methods are considered to be superior in terms of detection sensitivity and accuracy.

N-glycolylneuraminic acid-containing a! with HD antigen activity! Antibodies that react with J chain have conventionally been used to inject H-D into chickens.
It was obtained by immunizing with a purified glycolipid antigen having antigenic activity and separating it from the serum. However, this method has some drawbacks. That is, (1) in order to obtain antiserum, a large amount of purified antigen is required each time. (2) Mainly due to individual differences in immunized animals;
There are variations in affinity and potency. (3) Since antibodies other than the target antibody are also present, complicated operations are required to purify the target antibody.

(4) There are disadvantages such as there is a limit to the amount that can be produced at one time. Therefore, in order to perform immunological assays accurately and with maximum effectiveness, it has been desired to be able to supply large quantities of antibodies of stable and uniform quality that are free from contamination with other antibodies. Production of such antibodies has already been reported as a monoclonal antibody production technique.

The present inventor has already invented a monoclonal antibody that specifically recognizes N-glycolylneuraminic acid-containing sugar chains that have HD antigen activity.

The application has been filed as Special Patent Publication No. 61-305248.

Among the monoclonal antibodies specifically obtained in this application,
YHD-02 and YHD-05 are N-glycolyl 0M
2 ganglioside, N-glycolyl GM3 ganglioside, I'V3NeuGc-nLcose4 Cer and VI3NeuGc-nLcOse6 Cer.

Therefore, as a result of further research, the present inventors succeeded in obtaining a monoclonal antibody exhibiting antigen specificity different from that of the monoclonal antibodies described above, thereby completing the present invention.

[Structure of the Invention] (Means for Solving the Problems) The present invention provides a monoclonal antibody that specifically recognizes N-glycolylneuraminic acid-containing sugar chains, :I IJ le G
M3 ganglioside and IV3NeuGc-nLcO
Reacts with sea Cer, VI3NeuGc-nLc
This invention relates to a monoclonal antibody characterized by not reacting with Ose6 Cer.

Here, specifically recognizing N-glycolylneuraminic acid-containing sugar chains means N-7 cetylneuraminic acid-containing sugar chains,
This means that it does not recognize any sugar chain that does not contain sialic acid.

The structures of the glycolipids described herein are shown below.

N-glycolyl 0M2 ganglioside (II3NeuG
c-GgOse3 Cer)GalNAcβ1 +4G
alβ1-4Glcβl −+ICer↑ 2 aNeuGc N-glycolyl GM3 ganglioside (II” Neu
Gc-Lac Cer)Galβ1-4Glcβ1 +
ICer↑ 2 aNeuGc IV3NeuGc-nLcose4 CerGalβI
→4GIcNAcβl→3Galβ1 →4Glcβ1
-+ICer↑ 2 aNeuGc V13NeuGc-nLcOse6 CerGalβ1
→4GIcNAcβI+3Galβ1-+4GIcNA
cβ1+3ca1↑ 2 aNeuGc β1-4GIcβ1-+ICer N-aceti JL/GM2 ganglioside (II3Neu
Ac-GgQse3 Cer)GalNAcβ1 +4
Galβ1 →4Glcβ1−+IGet)↑ 2 aNeuGc Asialo GM2 (GgOse3 Cer) CalNA
cβ1 →4GalβI →4Glcβ1 ”IC:e
rN-acetiJl/GM3 ganglioside (IT3Ne
uAc-Laccer)Galβ1+4Glcβ1+
1cer↑ 2aNeuAc CDH (Lac Cer) Galβ1-4GIcβ1-+IC:er [wherein, Ga
l is galactose, Glc is glucose, Ga1NAc
stands for N-acetylgalactosamine, GlcNAc stands for N-acetylglucosamine, Neu Gc stands for N-glycolylneuraminic acid, Neu Ac stands for N-acetylneuraminic acid, and Car stands for ceramide. ] The monoclonal antibody of the present invention has reactivity with several types of gangliosides having HD antigen activity, which are considered to be major cancer-related antigens, and has reactivity with gangliosides containing N-acetylneuraminic acid and glycolipids that do not contain sialic acid. It doesn't react.

Sugar chains having HD antigen activity that are recognized by the monoclonal antibody of the present invention exist not only in glycolipids but also in glycoproteins. Therefore, the monoclonal antibodies of the present invention that recognize these infant buttons, which are considered to be human cancer-related antigens,
It is extremely useful for cancer diagnosis.

The monoclonal antibody of the present invention is extremely useful for human cancer diagnosis by tissue, cell diagnosis, blood, urine diagnosis, image diagnosis, etc., and is also useful for missile therapy that binds drugs to antibodies and treatments that utilize cytotoxicity. Application is possible. Furthermore, the monoclonal antibody of the present invention can also be used to detect HD antibodies. It is also applicable to the diagnosis and treatment of Marek's disease in chickens.

The monoclonal antibody of the present invention can be used as a useful tool in basic research on the relationship between sugar chains and carcinogenic mechanisms, the role of sugar chains in vivo, and the like.

The monoclonal antibody of the present invention can be obtained by the following method.

First, a proforaminal animal is immunized with an immunogen. At this time, I will be immunized! The mammalian animal is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion, and it is more preferable to use mice and rats. In the case of glycolipids, it is more preferable to use animals with autoimmune diseases, and it is especially preferable to use autoimmune mice. Examples of usable autoimmune disease mice include NZB, NZW, B/WF, M
RL/1. Examples include BXSB male, SL/Ni, etc.

In addition, polyclonal B cell activators (PBA) such as Durham-negative bacterial lipid polysaccharide (LPS) and dextran sulfate
), the ability to produce autoantibodies was increased.

Normal mice, such as Ba1b/c, may be brought into an autoimmune disease state and used as immunized animals.

N-glycolylneuraminic acid is present in many animals including mice.

It is known that N-glycolylneuraminic acid-containing glycolipids containing gangliosides having HD antigen activity, which are the object of the present invention, are widely present in mouse tissues.
For mice, these glycolipids are self-antigens;
Immunogenicity is considered to be extremely weak. In conventional methods using normal mice such as Ba1b/c mice as immunized animals, it is extremely difficult to obtain hybridomas producing monoclonal antibodies against N-glycolylneuraminic acid-containing glycolipids. On the other hand, it is known that mice with autoimmune diseases produce antibodies against self-antigens, such as antinuclear antibodies and antierythrocyte antibodies.

The present inventor attempted to produce a hybridoma producing a monoclonal antibody against a glycolipid having HD antigen activity, and found that the desired hybridoma could be produced very easily by immunizing mice with an autoimmune disease. completed.

As an immunogen, any of the cells themselves containing N-glycolylneuraminic acid-containing glycolipids, cell membrane components separated from the cells, and N-glycolylneuraminic acid-containing glycolipids separated from the cells can be used. It is. It is also possible to use glycolipids together with phospholipids and cholesterol as liposomes. Immunization is carried out by a general method, and the above immunogen may be diluted with phosphate buffered saline (hereinafter referred to as rPBSJ) and administered intraperitoneally or intravenously. At that time, the immunogen was bovine serum albumin (BSA).
It may be carried on a carrier such as a bacterial cell or a bacterial cell, or an adjuvant such as Freund's adjuvant or a bacterial cell adjuvant may be injected together.

Lung cells collected from immunized animals are fused with mouse bone marrow hepatocytes. As myeloma cells, various known cells,
For example, N5-1.5P-2, X63. El, 5.3
, P3-U 1, etc. are used.

The fusion method is performed according to a known method. Examples of fusion promoters include polyethylene glycol D-Az (PEG),
Sendai virus (HvJ) h9 is used. The ratio of tile cells and myeloma cells used is the same as in the general method,
A ratio of 1 to 1 to 10 pairs is preferred.

After completion of the fusion, hybridomas are selected by culturing in a normal selection medium. The myeloma cells described above are HA
Since they cannot grow in T medium (a medium containing hypoxanthine, aminopterin, and thymidine), HArj; cells that grow underground may be selected.

When the hybridoma colony becomes sufficiently large, a strain producing the desired antibody is searched for and cloned.

The antibody-producing strain can be searched for using methods generally used to detect antibodies, such as the EL ISA method [Method in Enzymology (Meth, Enzya+o1.), Vol. 70, pp. 419-439 (1980). ], agglutination reaction method, RIA method, double immunodiffusion method, etc.

Specifically, a plate to which a purified glycolipid antigen has been attached is blocked with BSA, then mixed with the culture supernatant of a test hybridoma, and an enzyme-labeled antibody against a mouse antibody is further spread. The presence of antibodies bound to the antigen is confirmed by measuring enzyme activity, and the desired antibody-producing strain is selected.

Further, cloning is performed by the limiting dilution method. That is, on a 96-well microtiter plate, hybridomas are distributed so that one or less per well is grown, and a single colony is grown. At this time, it is preferable to add mouse thymocytes as feeder cells.

The above cloning is repeated to obtain monocloned hybridomas.

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

The monoclonal antibodies 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 is also possible to purify using common methods such as ammonium sulfate precipitation, gel filtration, ion exchange column chromatography, etc.

(Examples of the Invention) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples do not limit the scope of the present invention in any way.

Example 1 ■) Isolation and purification of antigens and various glycolipids ■ Rabbit thymus cell membrane components Thymus tissues obtained from two 6-week-old rabbits were homogenized in a phosphate buffer solution and centrifuged at 800 rpm for 5 minutes. did. The supernatant was centrifuged at IO, 000g for 1 hour, and the resulting pellet was fractionated into PB310d and used as an immunogen.

■ Glycolipid Bovine kidney was homogenized in cold acetone, and a large amount of cold acetone was added.
:10:0) (20:10:1) The extraction operation was performed sequentially using each mixed solution.

The obtained crude glycolipid was treated with DEAE-Sephadex A-2.
5 column chromatography to separate it into an acidic fraction and a neutral fraction, and the acidic fraction was subjected to Iatrobead column chromatography to obtain N-glycolyl 0M2 ganglioside. Similarly, from horse racing ticket blood cells, N-glycolyl GM3
Ganglioside was extracted from raw blood cells by IT3NeuGc-n.
LcOse4 Cer and VI3NeuGc-nLc
Ose@Car, N-acetyl 0M2 ganglioside and N-acetyl GM from the cerebrum.

Gangliosides were obtained from CDH from macrocytes.

N-acetyl 0M2 ganglioside in formic acid IN
Asialo GM2 was obtained by performing rtn anti-rserttt at 00° C. for 1 hour, DEAE-Sephadex A-25 column chromatography, and then Iatrobead column chromatography.

2) Immunization method and cell fusion Emulsify the rabbit thymus cell membrane component PBS solution and the same amount of Freund's complete adjuvant, and add 300 ml to NZB.
It was injected intraperitoneally into mice (male, 6 weeks old). Thereafter, 20 gg of N-glycolyl GM3 ganglioside was adsorbed to 80 gg of Salmonella minnesota bacteria treated with acetic acid.
200 l of PBS solution was injected intravenously 5 times every 2 weeks.

Three days after the final immunization, the lungs were removed from the mice, dissociated into single cells, and then the lung cells were washed with RPM11640 medium. On the other hand, mouse myeloma cells X63.
e, 5.3 were collected and washed with RPM11640 medium. A suspension of 4.7 x 10' lung cells and a suspension of 9.2 x 10' mouse myeloma were mixed, and the medium was removed by centrifugation. The mixed cells were treated with 50% polyethylene glycol-RPM11 in a water bath heated to 37°C.
640 medium 2- was gradually added over 1 minute, and fusion was performed by gently stirring for 1 minute.

RPMI 1640 medium 4- for 2 minutes, then 14-
was added over 2 minutes with gentle stirring. Remove the medium by centrifugation and add RP containing 10% tallow serum to the cells.
Add 120ml of M11640 medium to 96-well plate 12.
0.14 per hole was distributed on each sheet. The next day, HAT medium (4X10-7M aminopterin, 1.6X10-5
M Thymidine, I X 10'4M Hyboxanthin, 10
RPM11640 medium containing % tallow serum) 0.1r&
t was added to each well. Further, half of the medium in each well was replaced with HAT medium every 3 or 4 days. After 3 weeks, hybridoma growth was observed in 90% of the wells.

3) Selection of hybridomas Antibodies in hybridoma culture supernatants were searched for by ELISA method.

As antigens, N-glycolyl 0M2 ganglioside, N
-Glycolyl GM, gangliosides and rV3NeuG
c-nLcOse4 Cer was used. Antigen 500ng
was adsorbed onto a microtiter plate for ELISA, and blocked with a 1% BSAΦPBS solution.
The culture supernatant was reacted. Furthermore, the antibody of interest was detected by reacting with a peroxidase-labeled goat anti-mouse immunoglobulin antibody and measuring the absorbance at 492 nm using orthophenylenediamine as a substrate. As a result, in hybridomas produced by immunization and cell fusion, N
-Glycolyl 0M2 ganglioside, N-glycolyl G
M3 ganglioside and IV3NeuGc-nL
Antibodies reacting with cOse4Cer were detected in one well.

Hybridomas in which antibody activity was detected were transferred to HT medium from which aminopterin was removed from HAT medium, and further transferred to RPM11640 medium containing 10% tallow serum (Fe2) and cultured.

This hybridoma was cloned by the limited dilution method. That is, cells were diluted to a density of 0.8 cells per hole in a 96-well plate and cultured with 4×10 5 mouse thymocytes per hole, and 2 weeks later, antibody-producing cells were selected by ELISA. Cloning was further repeated to obtain a stable hybridoma, YHD-03.

The class of monoclonal antibody YHD-03 was determined to be gG2a by ELISA method.

The hybridoma, YHD-03, is EuropeanC
It has been deposited in the collection of Animal Ce1l and has an accession number (Provisional Acc.
ession Nu+5ber) 87042208 is attached.

Example 2 1) Measurement of antigen specificity of YHD-03 by ELISA method Using 2 nmol of various glycolipids fi O as the antigen, ELISA
practiced law. The antigen adsorbed on the plate was reacted with the hybridoma culture supernatant, and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using orthophenylenediamine as a substrate, the absorbance of the 492n layer was measured to examine the reactivity of monoclonal antibodies to various antigens. The results are shown in the table.

YHD-03 contains N-glycolyl 0M2 ganglioside, N-glycolyl 0M3 ganglioside and IV3 N
reacts with euGc-nLcOse4 Cer, especially N
- Reacts strongly with glycolyl 0M3 ganglioside, I
It reacted weakly with V 3 NeuGc-nLcOse4 Cer, but not with VI 3NeuGc-nLcOge6 C
It did not react with er.

Also, N-acetylno-yramic acid-containing glycolipid N
-Acetyl 0M2 ganglioside and N-acetyl GM
It was found that it did not react with gangliosides, and did not react with sialic acid-free Asia r:2GM2 and CDH.

2) Thin layer chromatography (hereinafter referred to as "rTLc")
Measurement of reactivity of YHD-03 on Plate 2 Various glycolipids were spotted at a width of 6 m+a at a location 1 cm from the bottom edge of the TLC plate, and developed with an appropriate solvent system. Of the two plates that underwent the same operation, one
The plate was colored with orcinol reagent. The other piece was subjected to enzyme immunostaining. That is, the antibody of the present invention was reacted, and then a peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted. 4-chloro-1-naphthol was used as a substance, and a bluish-purple colored spot was detected.

The figure shows the results using 6 types of gangliosides as antigens. Chloroform: methanol: 2.5 as developing solvent.
N ammonia water (55:45:10 volume ratio) was used.

A is color development using orcinol reagent, B is YHD-03
This is a plate on which enzyme immunostaining was performed using. 1 is N-acetyl 0M2 ganglioside, 2 is N-acetyl GM3 ganglioside, 3 is N-glycolyl 0M2
ganglioside, N-glycolyl 0M3 ganglioside for 4, IV 3NeuGc-nLcose4C for 5
er, 6 is VI” NeuGc-nLcOse6 C
er was expanded.

The monoclonal antibody YHD-03 of the present invention comprises N-glycolyl 0M2 ganglioside, N-glycolyl 0M3 ganglioside and IV3NeuGc-r+LcO8e4
Reacts with Cer, but Vl 3NeuGc-nLcO
It was confirmed that it did not react with se6Cer and N-acetylneuraminic acid-containing glycolipids.

[Effects of the Invention] According to the present invention, a novel monoclonal antibody that specifically reacts with an N-glycolylneuraminic acid-containing sugar chain can be provided. This antibody is very effective in elucidating the mechanism of cancer development, diagnosing it, and treating it.

[Brief explanation of the drawing]

The figure shows the reactivity of the monoclonal antibody YHD-03 of the present invention with various glycolipids.

Claims (1)

[Claims] A monoclonal antibody that specifically recognizes N-glycolylneuraminic acid-containing sugar chains, comprising N-glycolyl GM_
A monoclonal antibody characterized in that it reacts with 2 ganglioside, N-glycolyl GM_3 ganglioside, and IV^3NeuGc-nLcOse_4Cer, but does not react with VI^3NeuGc-nLcOse_6Cer.