JPH0325408B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel anticancer agent, and more particularly to an anticancer agent containing a cancer cell-derived sugar chain-related antigen (hereinafter referred to as "GRA") as an active ingredient. Immune-competent cells, especially T, which plays a major role in cell-mediated immunity
Although lymphocytes carry out a rejection reaction based on foreign cell antigens during transplantation immunization, this immunosuppression is not observed or is weak against cancer cells.
Therefore, cancer cells proliferate in the body without being destroyed,
Eventually, it causes the death of the cancer-bearing host. The present inventor was conducting intensive research on the host's immune response to cancer cells and its application to cancer treatment, and discovered that cancer cell-specific antigens, which are not found in differentiated normal cells, act as immunogens on the host. death,
We have discovered that there is a GRA with extremely high immunogenicity that establishes a specific immune response with cancer cells.
We discovered that when this GRA is administered to a host, it induces antigen-specific cell-mediated immunity and destroys cancer cells. Completed the invention. Therefore, the present invention provides an anticancer agent containing GRA as an active ingredient. The GRA of the present invention comprises human or animal cultured cancer cells,
It can be obtained from cancer cells having GRA, such as transplanted cancer cells, naturally occurring cancer cells, cancer cells generated by chemical substances or viruses, and cancer cells derived from surgical tissues, as follows.
That is, first, cell membrane components are separated from the cancer cells, and then a lectin that specifically binds to terminal galactose [JBC 250 , 8518-8523 (1975);
Biochem.Biophys Res.Comm. 62 , 144 (1975);
Z.Immunitaetsforch, 138 , 423-433 (1969);
Br.J.Exp. Pathol. 27 , 228-236 (1946); Proc.
Nath.Acad.Sci.USA, 75 , No.5, 2215−2219
(1978); Biochemistry 13 , 196-204 (1974);
Carbohydrate Research, 51 , 107-118 (1976)],
For example, peanut lectin, castor bean (Ricinus
Communis) lectin, etc., and binds to the lectin and separates it. Cancer cell membrane components can be separated by known methods such as a homogenate method and a solubilization method using a solubilizing agent. More advantageously, for example, after homogenizing cancer cells in physiological saline or an appropriate buffer, the precipitated portion is collected by centrifugation or the like, and then dissolved in physiological saline or a buffer using a solubilizing agent. This can be carried out by removing the supernatant by centrifugation or the like. As the solubilizer used, there are various surfactants that are generally known to be able to solubilize cell membranes, such as "Triton X-100".
(manufactured by Wako Pure Chemical Industries, Ltd.), "NP-40" (manufactured by Ciel Corporation), nonionic surfactants such as dichitonin and urea, and anionic surfactants such as sodium dodecyl sulfate (SDS). Furthermore, the separation of GRA that binds to lectin from the cell membrane components obtained above can be carried out by conventional physicochemical or biochemical means that utilize the properties of GRA. Examples of such methods include affinity chromatography using a column carrier containing lectin, immunoprecipitation using GRA antibodies, etc.
Examples include dialysis, gel filtration, electrophoresis, physical precipitation using a glycoprotein precipitant such as polyethylene glycol or acetone, or a combination of these as appropriate. More advantageously, affinity chromatography using a column carrier containing lectin is preferred, and the column carrier can be easily obtained, for example, by immobilizing lectin on an insolubilized support. Here, the lectin can be immobilized on the insolubilized support according to a conventionally known biological material immobilization method. Among these, immobilization methods using cyanogen bromide activated polysaccharide method, N-hydroxysuccimide ester method, etc. are preferred. Among these, the cyanogen bromide activated polysaccharide method is a method in which an insoluble support is treated with cyanogen bromide, and then the obtained activated product is coupled with a lectin under mild conditions to immobilize the lectin. When treating an insoluble support with cyanogen bromide, for example, use a basic compound such as sodium hydroxide or sodium hydrogen carbonate to maintain the pH at 7.5 to 12, and then treat the insoluble support with water, acetonitrile, or 0.1M sodium hydrogen carbonate at room temperature. The treatment may be carried out for about 1 to 12 minutes in a solvent such as a buffer solution (PH≈8.7) or a buffer solution with a pH of 7.5 to 1.2 such as 0.01M phosphate buffer (PH≈7.7). The amount of cyanogen bromide to be used relative to the insoluble support is usually approximately equal in weight. In this case, the insoluble support has low nonspecific adsorption to biological substances in general, high porosity, functional groups that can immobilize biological substances under mild conditions, and sufficient chemical and physical properties. Any stable, conventionally known insoluble support can be used. For example, aminoethyl cellulose,
Cellulose-based supports such as carboxymethylcellulose, bromoacetylcellulose, p-anilinocellulose, cross-linked dextran-based supports such as Cephadex, CM-Sephadex (manufactured by Pharmacia), Cepharose 2B, Cepharose 4B,
Examples include agarose-based supports such as Sepharose 6B (manufactured by Pharmacia). When coupling the cyanogen bromide-activated support thus obtained with a lectin, the cyanogen bromide-activated support is used 30 to 80 times the weight of the lectin, and an appropriate solvent such as 0.1 mol sodium hydrogen carbonate ( Containing 0.5M sodium chloride, PH8.4) in aqueous solution,
Usually about 0 to 40℃, preferably about 10℃ at 2 to 8℃
It is enough to react for ~20 hours. In this way, a carrier for affinity chromatography containing a lectin is produced. According to chromatography using the carrier for affinity chromatography containing the above lectin, the target GRA binds to the lectin in the carrier and is collected on the column. Then, an exchange reaction is carried out through the column through a substance that binds to lectin, such as galactose, a disaccharide having a terminal galactose, or an oligosaccharide, or a highly concentrated salt, an aqueous solution of potassium thiocyanate,
Pass through an adsorption/separation agent (eluent) such as boric acid buffer
Obtain by dissociating GRA. The GRA thus obtained contains glycoproteins, glycopeptides, glycolipids and/or saccharides having galactose ends. This can also be lyophilized and stored if necessary. This GRA can be used alone as an active ingredient,
It can also be used in combination with other antibacterial agents and anticancer agents. The anticancer agent containing GRA of the present invention as an active ingredient is
It can be in any form as long as it effectively contains GRA, the main drug, but usually it is a liquid solution,
It is administered intravenously, subcutaneously, or intramuscularly as a suspension or emulsion. They can also be provided as dry products which can be made into liquid form by addition of suitable carriers before use. Such liquid preparations may contain suspending agents such as methylcellulose, emulsifying agents such as lecithin, preservatives such as methyl-p-hydroxybenzoate, or stabilizers that do not by themselves adversely affect the immune function of humans or animals. , a buffering agent, etc. As the aqueous carrier, physiological saline can be used, and as the non-aqueous carrier, vegetable oils such as sesame oil, mineral oils such as paraffin, animal and vegetable oils such as squalene, or propylene glycol can be used. Furthermore, such a solution can also contain a suitable adjuvant for immune promotion. Examples of the adjuvant include Freund's complete adjuvant, saponin for animals, and aluminum hydroxide for humans. The anticancer agent of the present invention can be administered to cancer patients once or multiple times over a long period of time to treat the cancer, or can be administered to those at risk of developing cancer to protect them. GRA's LD ₅₀ (mouse intraperitoneal) is 500 as sugar content
Since the toxicity is low at more than mg/Kg, it can be administered in a wide range of doses. Therefore, GRA in the anticancer agent of the present invention
There are no particular restrictions on the concentration, but in general the amount of sugar
0.001 to 100 μg/ml is preferred. Although the dosage varies depending on the severity of the disease, age, and sex, it is usually preferable to administer the amount of sugar in 0.001 to 1000 μg/Kg/day in one to several doses. Reference example 1 (localization of GRA) Production of FITC-labeled lectin (PNA-FITC); Peanut lectin (PNA, manufactured by EY) 10 mg
0.85% NaCl 0.01M-phosphate buffer (PH
7.2) Dissolve in 2ml. FITC (manufactured by Sigma) 2
mg is dissolved in 1 ml of 0.5M bicarbonate buffer (PH=9.0) and 0.5 ml thereof is added to the above PNA buffer. After stirring for 2 hours at room temperature, Sephadex G25
(10 mm x 300 mm, manufactured by Pharmacia) and collect the first peak. E/P ratio = 1.0 GRA localization of various cancer cells; 1 x ¹⁰⁶ various human cultured cancer cells were treated with 0.85% NaCl
After washing with 0.05M Tris-HCl buffer (PH = 7.2) by centrifugation three times, the PNA-
Add 100μ of FITC (200μg/ml) and allow to react at room temperature for 30 minutes. 0.85% after reaction completion
After washing three times with 0.01M NaCl phosphate buffer (PH=7.2), the cells are mounted on a glass slide and examined under a fluorescence microscope. The results are shown in Table 1. The cancer cells tested were all known and were obtained from Daiichi Pathology, Niigata University School of Medicine.

[Table] Reference Example 2 (Preparation of GRA) Production of insolubilized lectin (PNA-Sepharose); After thoroughly washing 3 g of CNBr-activated Sepharose 4B (manufactured by Pharmacia) with 1 mM HCl.
0.1M-sodium hydrogen carbonate (PH=8.5) 200ml
Add 5 ml of 0.01M phosphate buffer (PH=7.7) containing 20 mg of PNA, and react at 25° C. with occasional stirring for 2 hours to obtain PNA-Sepharose. Preparation of GRA; (a) BT-1 (Burkitt lymphoma) cells 1.3×
10 Wash ⁸ pieces three times with physiological saline, and add 2% "Triton X-100" (manufactured by Wako Pure Chemical Industries, Ltd.), 0.85%
NaCl, 2mM- _CaCl2 , 2mM- _MgCl2
0.01M-Tris-HCl buffer (PH=7.4) 30ml
and stir at 4°C for 15 minutes. after that
Ultracentrifugation was performed at 100,000×g for 2 hours. Ultracentrifugation supernatant
Of the 28ml, 14ml is 0.1% Triton X-100,
0.85% NaCl, 2mM- _CaCl2 , 2mM-
_Affinity chromatography (φ0.5 x 1
cm). After washing with the same buffer, 0.1M-lactose, 0.85% NaCl, 2mM-CaCl2, ₂
mM _MgCl2 , 0.1% Triton X-100
Elute with 0.01M Tris-HCl buffer (PH=7.4), and add the eluate to 0.85% NaCl and 2mM
MgCl ₂ , 2mM CaCl ₂ in 0.01M Tris
Dialyze with hydrochloric acid buffer for 48 hours to obtain 17 ml of GRA solution.
obtain. The protein and sugar content of this
As a result of measurement using the Folin-Lowry method and the phenol sulfuric acid method, the protein content was 644 μg, and the sugar content was 644 μg.
It was 120μg. Hereafter, this will be referred to as "GRA-1"
It is called. (b) After washing 1 x ¹⁰ _C3H mouse breast cancer cells three times with physiological saline, 2% Triton X-100,
0.85% NaCl, 2mM- _CaCl2 , 2mM-
MgCl2 in _0.01M Tris-HCl buffer (PH=
7.4) Add 30ml and stir at 4℃ for 30 minutes.
Thereafter, ultracentrifugation _was performed at 100,000
MgCl2 in _0.01M Tris-HCl buffer (PH=
7.4) overnight. This dialysis fluid
Concentrate to 3 ml with Immersible-CX Ulra-filters (manufactured by Millipore), and 1 ml of this
0.005% Triton X100, 0.85% NaCl, 2mM
-CaCl ₂ , 2mM-MgCl ₂ equilibrated with Tris-HCl buffer (PH=7.4) Reference Example 2
-PNA-Sepharose Affinity chromatography (φ0.5 x 2 cm). After thorough washing with the same buffer, 0.1M-lactose, 0.85%
NaCl, 2mM- _CaCl2 , 2mM- _MgCl2 ,
0.01M Tris in 0.005% Triton X-100
Elute with hydrochloric acid buffer (PH=7.4) and remove the eluate.
0.85% NaCl, 2mM- _CaCl2 , 2mM-
MgCl2 in _0.01M Tris-HCl buffer (PH=
7.4) for 48 hours to obtain 2 ml of TSGA solution. The protein content of this product was 156 μg and the sugar content was 94 μg. This is referred to as “GRA-M” below.
-1". Example 1 (i) GRA-M-1 obtained in Reference Example 2 (b) was diluted with physiological saline to adjust the sugar content to 1.0 μg/ml and protein content to 1.6 μg/ml. , anti-cancer drugs
It was ranked No.1. (ii) C ₃ H/He spontaneous breast cancer tumor masses were aseptically cut into 5 mm squares and implanted subcutaneously on the backs of 10 syngeneic C ₃ H/He mice (7 weeks old). Tumor colonization and proliferation were confirmed on the 7th day. These five animals were given anticancer drug No. 1 prepared in (i) for one day.
A dose of 300μ was administered subcutaneously at two-day intervals. remaining 5
The animals served as untreated controls. from the first dose
Ten days later, the tumor was removed by surgery, the average volume was determined, and histopathological observation was performed. Tumor volume: Administration group 22.3 mm ³ (Fig. 1) Control 162.7 mm ³ (Fig. 2) This means that there was a tumor reduction of 86.3%. Histopathological observation: Control group (Fig. 3) formed cancer nests;
The histology appears to be medullary ductal carcinoma, with proliferation of tumor cells seen throughout the tissue. In contrast, in the drug-administered group (Figure 4), the cancer cells had melted and died, necrotized, calcified and fibrosed in the areas where cancer cells had once formed, and there were only a few cancer cells in some areas. The antitumor properties of the anticancer agent of the present invention were confirmed.

[Brief explanation of the drawing]

Figure 1 is a photograph showing the state of cancer in the GRA-M-1 administered group of tumor-bearing mice, Figure 2 is a photograph showing the state of cancer in the non-administered group of tumor-bearing mice, and Figure 3 is a photograph showing the same non-administered group. Figure 4 is a photograph of the cancer cell tissue of the same administration group.

Claims (1)

[Scope of Claims] 1. An anticancer agent containing as an active ingredient a cancer cell-derived sugar chain-related antigen consisting of a cancer cell membrane component that binds to a lectin that specifically binds to terminal galactose. 2. The anticancer agent according to claim 1, wherein the lectin that specifically binds to terminal galactose is peanut lectin or castor bean lectin.