JPH06253887A - Heparitinase monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain a heparitinase (heparan sulfate specific endoglucuronidase) monoclonal antibody substantially participating in the metastasis of cancer cell. CONSTITUTION:A monoclonal antibody specifically recognizing the heparitinase having a molecular weight of about 95,000 and having immunoglobulin G subclass. A monoclonal antibody specifically recognizing the antigen of the former antibody in its manifestation and having immunoglobulin M subclass. A fused cell of mouse spleen cell and mouse myeloma cell capable of producing the monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention aims to provide a heparitinase (heparan sulfate-specific endoglucuronidase) monoclonal antibody which is importantly involved in the metastasis of cancer cells.

Heparitinase is described by Oosta et al. (J. B.
iol. Chem. , 257, 11249-1125
5, 1982), it is an enzyme that specifically hydrolyzes the sugar chain of heparan sulfate proteoglycan, which was first found in human platelets, in endotype. The enzyme has been identified.

This enzyme is generally considered to be involved in the metabolism of normal cell membrane heparan sulfate. However, malignant cancer cells and activated immune system cells are interstitial and vascular to tissue-dependent. It is also suggested that when it moves from tissue to blood vessel, it decomposes the sugar chain of heparan sulfate proteoglycan, which is a major component of extracellular matrix, and contributes to the destruction of extracellular matrix.

Further, cancer cells that have acquired metastatic potential have extremely high heparitinase activity (Takashi Yokota, Kazuo Oishi, Miya Miya, Hiroshi Otsuna, Proc. Of the 79th Annual Meeting of the Japanese Society of Pathology). 139, 1990), Kramer et al. (Kr
amer. R.K. H. Vogel. K. G. , Nic
olson, G.M. L. J. Bicl. Chem. , 2
57, 2678-2686, 1982) confirmed that this enzyme degrades basement membrane heparan sulfate proteoglycans.

Furthermore, the correlation between this enzyme activity and the frequency of metastasis of cancer cells is very high (Nakajima, M., Iri).
mura, T .; , DiFerrante, D .; T. , D
iFrrante, N.M. , Nicolson, G .;
I. , Science, 220, 611-613, 19
83, Vlodansky, V.I. , Cancer Re
s. , 43, 2704-2711, 1983), it has been clarified that the metastasis of cancer cells can be suppressed by the inhibitor of this enzyme (Irimura, T., Nakaji).
ma, M .; , Nicolson, G .; L. , Ann.
N. Y. Acad. Sci. , 556, 496-49
8, 1988).

From the above, it is clear that there is a very close relationship between heparitinase and the metastatic potential of cancer cells. A wide variety of methods have been developed and are being effectively implemented for the early diagnosis of cancer.However, even for the presence or absence of metastatic ability of cancer cells, no such diagnostic method has been developed. The development of law is urgently needed.

Therefore, in order to develop this early diagnostic method, development of a monoclonal antibody having extremely high specificity for the above-mentioned enzyme is urgently needed. Moreover, it is considered that the use of such an antibody leads to the most accurate diagnostic method.

Conventionally used antibodies against heparitinase are polyclonal and have many specificities such as insufficient purification of the enzyme as an antigen and reactivity with substances other than this enzyme. However, there was nothing to satisfy.

[0009]

SUMMARY OF THE INVENTION Here, the present inventors overcome the above-mentioned drawbacks observed in conventional heparitinase polyclonal antibodies, and can stably produce a monoclonal antibody against this enzyme. It is intended to obtain a hybridoma.

[0010]

That is, the present invention proposes an antibody which specifically recognizes heparitinase having a molecular weight of about 95,000 and a subclass of which is immunoglobulin G. A monoclonal antibody characterized by a subclass of immunoglobulin M, which is an antibody that specifically recognizes an antigen in carrying out the method.
The present invention proposes a fusion cell of mouse spleen cells and mouse myeloma cells, which is characterized by producing a monoclonal antibody and the monoclonal antibody.

According to the present invention, a hybridoma can be obtained which does not have the drawbacks found in the conventional heparitinase antibody and which can stably produce a monoclonal antibody against the present enzyme.

[0012]

EXAMPLES The present invention will now be specifically described with reference to Examples. (1) Method for Purifying Antigen The method for purifying antigen is the method of Yokota et al. (Takashi Yokota, Kazuo Oishi, Miya Koyama, Hironori Katayama, Hiroshi Otsuna, Proc. According to.

Sublines selected by the organ-specific metastatic ability of Mouth Luis lung cancer cells were collected, suspended in a citrate buffer solution (pH 4.0) containing a surfactant, and then suspended at a low temperature (4) by a cell disruptor. The cells were destroyed at (° C). An extract was obtained by centrifugation and dialyzed against deionized water, followed by ion exchange (Wako Co.) chromatography, heparin-
It was purified about 14,000 times using a cephalos-chromatography (manufactured by Falmatia), a hydrophobic chromatography (manufactured by Tosoh) and a gel filtration chromatography (manufactured by Falmatia). The purity of this antigen is SD
It was confirmed by S-electrophoresis and gel filtration chromatography that the product was a single product and had a molecular weight of about 95,000.

(2) Test method Measurement of heparitinase activity and inhibition of activity by antibody Bovine kidney-derived heparan sulfate (manufactured by Seikagaku Corporation) was used as CN.
-Immobilized on Sepharose (manufactured by Pharmacia),
0.1 M Tris-hydrochloric acid buffer solution (pH of 15 M NaCl)
It was suspended in 7.2) and used as a substrate.

The enzyme obtained by the above method (1), the substrate and the mouse-derived immunoglobulin (1 g) G or IgM are mixed, the above buffer is added, and the mixture is reacted at 37 ° C. with gentle shaking. Let

The mixture was placed in ice water to stop the reaction, and the amount of uronic acid in the liberated reaction product was measured by the sulfuric acid-carbazole reaction method (B
itter, T .; Muir, H .; M. , Ann. Bi
ochem. , 4, 330, 1962). Inhibition of the present enzyme activity by the present antibody is achieved by
Instead of antibody against other substances (IgG or IgM)
The same procedure was used, except that The inhibitory activity was represented by the residual activity of this enzyme.

Preparation of Monoclonal Antibodies Monoclonal antibodies are commonly used in
Vivo stimulation method (Kohler,
C. Milstein, C .; , Nature, 25
8, 495, 1975). (1)
500 μl with the antigen (500 μg / ml.PBS) obtained in
Of the incomplete Friend Ajyu band were mixed and administered intraperitoneally to mice. Blood was collected from the second week after immunization, and the serum antibody titer was measured by the ELISA method. After confirming the increase in serum antibody titer, 100 μg of the antigen was finally intraperitoneally administered as a booster as described above.

Three days after the final immunization, the spleen was aseptically removed, and the spleen cells and mouse myeloma cells (P3NSI / 1
-Agl strain, polyethylene glycol (molecular weight 1,
500, B.I. D. (Manufactured by Company H) was used for fusion to prepare a hybridoma.

This hybridoma was treated with HA containing 10% FCS.
It was suspended in a T medium (manufactured by Kibco), dispensed into a 96-well microplate by the limiting dilution method, and cultured under the conditions of 37 ° C. and 5% CO ₂ . Screening was carried out by the ELISA method and the indirect fluorescent antibody method using a flow cytometer using the antigen and mouse Louis lung cancer cells.

The antibody-producing cells against the present antigen were cloned three times by the limiting dilution method, and cloned to obtain cells producing highly specific antibodies. These cells were statically cultured under the medium and conditions described above. After collecting the culture medium and determining the isotype of Ig, IgG was purified by protein G or protein A-Sepharose (manufactured by Pharmacia), and IgM was purified by ammonium sulfate precipitation method.

The titer of the antibody obtained by this method was determined by ELISA (di-antibody Sangerichi method), and the specificity to the antigen obtained in (1) above was determined by the Western blotting method (To
wbin, H .; , Staehlin, T .; , Gordo
n, J. , Proc. Natl. Acad. Sci.
U. S. A, 76, 4350, 1979).

(3) Test Results As shown in Table 1, the antibodies obtained in (2) above are
Heparitinase activity was inhibited by about 50% at 6.25 ng. The titer of this antibody by ELISA is IgG, Ig
M was 1 to 2 × 10 ³ .

[Table 1]

The specificity of this antibody for heparitinase is that it reacts only with heparitinase (immunized antigen) as shown in FIG. 1 by Western blotting method, and no reaction with miscellaneous proteins is observed. Not shown, showing very high specificity. In this figure, a is a molecular weight marker.
Cars and b indicate IgG antibody use, and C indicates IgM antibody use. Properties of heparitinase antibody IgG IgG1-κ igM IgM1-κ

When the Ruis lung cancer cells transplanted into mice using this antibody were stained with the enzyme antibody method, as shown in FIG. 2, the tumor tissues of the transplanted site were scarcely stained, but the metastatic site (lung). The tumor tissue stained very clearly. Further, even when the tumor tissue of mouse sarcoma 180 having no metastatic ability used as a control was similarly stained, it was scarcely stained as shown in FIG.

[0025]

As is clear from the above, the heparitinase monoclonal antibody obtained by the present invention has very high titer and specificity and is used for the examination of the metastaticity of cancer cells. And is considered to be effective.

[Brief description of drawings]

FIG. 1 is a diagram showing the specificity of an antibody of the present invention for heparitinase.

FIG. 2 is a diagram showing a routine transplanted into a mouse using the antibody of the present invention.
It is a micrograph of histologically stained lung cancer cells in the chair.

FIG. 3 is a photomicrograph of a stained mouse sarcoma 180 tumor tissue having no metastatic potential.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical display location G01N 33/573 A 8310-2J (C12P 21/08 C12R 1:91) (72) Inventor Oami Hiroshi 1-1-36-1116 Seishincho, Edogawa-ku, Tokyo (72) Inventor Takashi Yokota 1-1-3-2-201 Shimotakaido, Suginami-ku, Tokyo

Claims (3)

[Claims] 1. A monoclonal antibody, which is an antibody that specifically recognizes heparitinase having a molecular weight of about 95,000, wherein the subclass is immunoglobulin G. 2. A monoclonal antibody, which is an antibody that specifically recognizes the antigen according to claim 1, wherein the subclass is immunoglobulin M. 3. A fused cell of a mouse spleen cell and a mouse myeloma cell, which produces the monoclonal antibody according to claim 1 or 2.