JPH0366320B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to Substance D having tuberculin activity, a method for producing the same, and an antitumor agent containing it as an active ingredient. It is widely known that Mycobacterium tuberculosis has antitumor activity, and attempts have been made to treat cancer patients by administering Mycobacterium tuberculosis, especially BCG. However, direct administration of BCG cells to patients also brings about various undesirable side effects. Therefore, in order to reduce side effects, components with antitumor activity are separated from the cells. As examples, cell walls, cell wall skeletons, water-soluble adjuvants, wax D, and polysaccharides have been reported.However, many of these substances have strong effects and also have relatively strong side effects.
On the other hand, drugs with weak side effects also have weak main effects, so it cannot be said that these drawbacks have been fully overcome. Therefore, the present inventors focused on the delayed allergic reaction, which is a characteristic of the immune response to Mycobacterium tuberculosis. We have been conducting research on treating tumors by administering them to living organisms and eliciting reactions. In research on applying delayed allergic reactions to tumor treatment, there have been some attempts to use other bacterial species or dinitrochlorobenzene (DNCB) as antigens, but currently most humans are infected with natural infections or with BCG vaccination. Taking into consideration that the patient has the ability to have a delayed allergic reaction to Mycobacterium tuberculosis through forced inoculation, it is considered easier and safer to utilize the reaction against Mycobacterium tuberculosis. The present inventors have already revealed that a Mycobacterium tuberculosis body extract that has the activity of inducing a tuberculin reaction in Mycobacterium tuberculosis sensitized animals exhibits a strong antitumor effect in Mycobacterium tuberculosis sensitized tumor-bearing animals, and has patented (JP-A-55-13204, JP-A-55-50894, JP-A-55-
50895) was filed. However, subsequent studies revealed that these extracts contained impurities unrelated to the original active substance and were not necessarily desirable as therapeutic agents. As a result of intensive studies based on the above points, the present inventors succeeded in isolating a novel and powerful tuberculin-active substance derived from Mycobacterium tuberculosis and clarifying its properties. The tuberculin active substance of the present invention is isolated by subjecting a water-soluble extract derived from Mycobacterium tuberculosis to a nucleic acid removal operation, a carbohydrate removal operation, and a low activity protein removal operation, and this substance is a novel substance.
It was also confirmed that it had an extremely strong delayed allergic reaction-inducing activity, and it was named Substance D. The tuberculin active substance of the present invention is hereinafter referred to as substance D, and its manufacturing method and properties will be explained in more detail. (A) Cultivation of raw material bacteria and production of water-soluble extract The raw material of the present invention is Mycobacterium tuberculosis (Mycobacterium sp.)
Bacteria belonging to Mycobacterium bovis strain BCG are used, especially Mycobacterium bovis strain BCG.
ATCC19015), Mycobacterium tuberculosis
tuberculosis H37Ra, ATCC25177,
Mycobacterium tuberculosis H37Rv,
ATCC25618) etc. are suitable. The culture may be carried out in accordance with a commonly used method suitable for this purpose. For example, a good culture can be obtained by statically culturing at 37° C. for 3 to 8 weeks in Sauton's medium or meat extract/glycerin medium, and this can be filtered to obtain bacterial cells. In some cases, the obtained viable cells may be heat sterilized by the receiving method and used. Further, the method for producing an extract from bacterial cells may be any conventional method. For example, by thoroughly mixing bacterial cells with an appropriate buffer solution,
A suspension is obtained by crushing the bacterial cells using a bacterial cell crusher, and if necessary, a buffer is added to sufficiently extract the substances inside the bacteria, and then this is filtered or centrifuged to obtain an extract. I can do it. The operation is preferably carried out at low temperature in the presence of a buffer solution with a pH near the neutral range. (B) Purification operation Substance D is obtained by purifying the above-mentioned extract by the treatment described below to remove impurities such as fats, nucleic acids, carbohydrates, and low-activity proteins, and then making it into an intermediate purified product, which is then subjected to further purification operations. It can be manufactured by adding Any method for removing impurities may be used as long as it does not reduce the activity of the target substance D. Some methods can remove multiple types of impurities at the same time, and it is not necessarily necessary to remove each impurity individually. Furthermore, there is no need to particularly limit the order of the removal process, but when using the removal method described below, it is recommended that the order described is easier to operate. Of the removal methods, the removal of lipids will not be particularly described, but most of them are removed as a secondary effect by the operations described below. (1) Production of intermediate purified product A method for removing impurities suitable for producing intermediate purified product will be explained below. Nucleic acid removal operation As a method for nucleic acid removal operation, conventional methods such as a method using a precipitant, a method using a nucleolytic enzyme, and an affinity chromatography method can be used, but a method using a precipitant is the simplest and preferable. It is. As a precipitant, polyvalent metal salts, basic antibiotics or their salts, basic polymer substances or their salts, etc. can be used, but among these, the method using streptomycin sulfate is suitable and is easy to operate. . The amount used is usually 0.1 to 1% of the liquid amount.
That's about it. As for the operation method, if necessary, a nucleic acid precipitant is dissolved in an appropriate amount of water in advance and added to the liquid to be treated, stirred thoroughly and left to stand to form a precipitate, and the precipitate is removed by filtration or centrifugation. Obtain the supernatant. Streptomycin remaining in the supernatant can be easily removed by dialysis or the like. The pH during treatment is preferably in the neutral range or near it, and the temperature is suitably 0 to 25°C. It is preferable to generate the precipitate over a sufficient period of time, such as by allowing it to stand overnight. Low activity glycoprotein removal operation As an operation for removing proteins with low delayed allergic reaction activity, it is possible to apply various fractionation methods focusing on the difference in properties between low activity proteins and substance D. The present inventors have conducted repeated studies, taking into consideration the fact that many of the proteins in tuberculosis bacteria precipitate at around PH4.5.
It was found that substance D was recovered in the supernatant. The method of applying the difference in isoelectric points is simple and suitable for mass processing, and is suitable for this operation. A recommended operating method is, for example, to fill a dialysis tube with the liquid to be treated and dialyze it against an appropriate buffer. More specifically, it is preferable to use a buffer such as acetic acid or phosphoric acid, adjust the pH to 4.0 to 4.5, and generate a precipitate over a sufficient period of time at a low temperature. The generated precipitate can be removed by centrifugation or the like to obtain a supernatant from which low activity proteins have been removed. Carbohydrate Removal Operation As a method of carbohydrate removal operation, since most of the carbohydrates contained in the material to be treated have a high molecular weight, it is also possible to separate and remove them using a method using a molecular sieve, such as a gel filtration method. Furthermore, since many carbohydrates are precipitated by organic solvents, this can also be used. Other methods can also be used as long as they do not reduce the activity of Substance D. Among these, methods using organic solvents are simple and suitable for large-scale processing. The organic solvent is not particularly limited as long as it does not reduce the activity of the target product, but alcohols such as ethanol are particularly suitable. The amount of organic solvent to be used can be determined empirically, but is usually 10 to 40.
It is appropriate to use it as a percentage. The treatment involves adding an organic solvent to the liquid to be treated little by little while stirring.
The treatment temperature is below room temperature, preferably 0 to 15°C. After addition of the organic solvent, the mixture is allowed to stand still to sufficiently form a precipitate, and then the precipitate mainly consisting of carbohydrates is removed by centrifugation or the like. Fractionation treatment using an ion exchanger By performing the purification operations described in ~ above, it is possible to obtain a substance whose delayed allergic reaction activity per protein is more than twice that of purified tuberculin. By processing the substance, it is possible to obtain an intermediate purified product suitable for purification of Substance D. When the purified product obtained is brought into contact with a cation exchanger under selected conditions, mainly substances with low activity are collected by the ion exchanger, and substance D remains in the liquid. A method is conventionally known in which the substances that are not captured by the cation exchanger are further fractionated using an anion exchanger, and the substances that are not captured are separated. (Japanese Patent Application No. 50,894/1989) However, it is also possible to adsorb the target substance on an anion exchanger, wash away carbohydrates as contaminants, and then elute it using an appropriate eluent and recover it. As the intermediate purified product to obtain Substance D, it is possible to use either a product that is not adsorbed to an anion exchanger and is eluted as is, or a product that is adsorbed and eluted, but the latter method is suitable for removing contaminants. It is suitable for subsequent processing because of its small amount of oxidation. The ion exchanger used in this treatment may be one normally used for protein purification, and for example, one based on cellulose or crosslinked dextran may be used. Ion exchange groups include, for example, cation exchange types such as CM (carboxymethyl) group and P (phospho) group.
groups, SE (sulfoethyl) groups, SP (sulfopropyl) groups, and anion exchange types include DEAE (diethylaminoethyl) groups, TEAE (triethylaminoethyl) groups, and QAE (diethyl-(2-hydroxypropyl)-aminoethyl) groups. The basic one is sufficient to achieve the purpose. As for the operation method, a normal ion exchange treatment method may be used. The operation can also be performed using the batch method, but
An effective method is to fill a column with an ion exchanger. The treatment with a cation exchanger is preferably carried out in an acidic range, and particularly good results can be obtained when carried out in a weakly acidic range with a pH of 5 or less. The optimum pH value can be selected through experiments. Treatment with an anion exchanger can be carried out in a medium to acidic range. Preferably, it is carried out in a neutral range. The ion exchanger and the solution to be treated must be sufficiently equilibrated in advance using a buffer solution with a desired pH value according to a conventional method. Buffer formulations may be of the conventional type, such as phosphoric acid, acetic acid, citric acid, trishydroxymethylaminomethane (Tris), and the like. Regarding the order of treatment, either cation exchange or anion exchange treatment may be performed first. By performing the above fractionation treatment, an intermediate purified product can be obtained. (2) Purification of Substance D Regarding the purification of Substance D, any method may be used as long as it does not reduce the activity of the target Substance D. The components contained in the intermediate purified product used as the material are mainly proteins and carbohydrates, and various mild methods for fractionating these are known, and typical methods will be described below. There is no need to specifically limit the order of these fractionation treatments, but when using the fractionation treatments in the description, it is recommended that the order described is easier to operate. Fractionation using Molecular Sieve As will be described later, Substance D has a narrow apparent molecular weight distribution when using a molecular sieve, so it can be purified from an intermediate purified product by, for example, gel filtration. Molecular sieves have a molecular weight cutoff of 2
It is appropriate to use one that can fractionate between 30,000 and 30,000 proteins. For example, Sephadex G200, G150, G100, G75, Sephacryl S200, S300 (all manufactured by SQUEDEN, Fulmacin), or Biogel P100, P60 (manufactured by Biorad, USA) are suitable. . The buffer solution is commonly used, and its pH should be medium to slightly acidic. The processing temperature is room temperature or lower, and the flow rate and separation method depend on the specifications such as the type of molecular sieve used, the height of the column, and the small diameter. It is necessary to fractionate a range of 30,000 to 30,000. If necessary, this operation can be repeated multiple times to further increase the degree of purification. Fractionation using an ion exchanger As mentioned above, Substance D can be adsorbed to an anion exchanger by selecting conditions, so it can be fractionated using this. The anion exchanger to be used can be the one used in the production of the intermediate purified product, and the operating method is also similar to the method for producing the intermediate purified product, but the elution method is D.
It is possible to determine the optimal method depending on the adsorption power of the substance to the ion exchanger through experiments. For example, a concentration gradient elution method using sodium chloride or the like is suitable, as it easily removes low activity proteins, etc.
The desired substance D can be obtained. The optimal salt concentration gradient is 0 to 0.12M.
In this case, appropriate operating conditions can be selected depending on the specifications such as the type of ion exchanger used, the height of the column, and the size of the diameter. This operation can be performed multiple times if necessary to further increase the degree of purification. By performing the above fractionation processing, a fraction containing substance D can be obtained. The freeze-drying method is most suitable for obtaining solid substance D from these fractions, and long-term storage is possible. (C) Properties of Substance D The lyophilized substance D is a white powder that is soluble in water but insoluble in methanol, ethanol, ether, and acetone. D substance by Davis method (Ann.NYAcad.Sci.,
121, 404 (1964), a single stained band is observed (however, the mixing ratio of acrylamide and N,N'-methylenebisacrylamide was 20:1, and the staining
Proteins were stained with 0.1% Coomassie brilliant blue dissolved in %TCA, or sugars were stained using the PAS staining method. ) Also, the mobilities of the protein staining band and the sugar staining band matched, indicating that Substance D was a highly purified glycoprotein (Figure 5). When the molecular weight of Substance D was measured using a Cephadex G75 Superfine column in a neutral phosphate buffer containing 0.15M sodium chloride, it was found that bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 43,000), and chymotoribus were standard proteins. Nogen A
(molecular weight 25000), ribonuclease A (molecular weight)
13700), it is calculated as 26000 (3rd
figure). The ultraviolet absorption spectrum of substance D is shown in FIG. 1, and the infrared absorption spectrum is shown in FIG. When substance D is subjected to a sedimentation reaction with concanavalin A using a 1% agar plate gel according to the double immunodiffusion method, a single sedimentation line is observed (fourth line).
figure). This is another proof that substance D is a glycoprotein. The composition of substance D is as shown below. Composition (G / 100g) Asparagic acid (G / 100g) 11.4 proline 36.4 glycine 36.4 glycine 3.0 Alanine 4.0 osoleysin 2.4 leucine 1.5 mannose 19.0 Gluro trout 1.2 amino acid 1.2 amino acids 1.2 amino acids 1.2 amino acids 1.2 amino acid ℃, 20 hours hydrolyzed Hitachi KLA It was analyzed using a -5 type amino acid analyzer.
Sugars were hydrolyzed in 1N hydrochloric acid at 100°C for 2 hours, followed by dehydrochlorination, reduction, and acetylation, and analyzed using a Shimadzu GC4BM gas chromatography analyzer. The numerical values indicate the content (g) of each composition in 100 g of lyophilized substance D. The biological properties of Substance D include strong delayed allergic reaction activity and antitumor properties. The strength of the delayed allergic reaction activity (that is, the strength of the tuberculin reaction activity) of Substance D is more than 8 times as active per dry weight in BCG vaccine-sensitized guinea pigs compared to the commonly used purified tuberculin (PPDs). shows. On the other hand, tumor engraftment and growth can be suppressed by mixing substance D with tumor cells and implanting the mixture in animals that have been sensitized with BCG vaccine in advance. Similarly, if tumor cells are previously transplanted into a sensitized animal and Substance D is administered into the engrafted tumor, an effect of inhibiting tumor growth or an effect of prolonging survival is observed. The antitumor effect of Substance D is observed not only in mouse syngeneic tumor lines but also in guinea pig syngeneic tumor lines. (D) Uses of Substance D Based on its strong delayed allergic reaction activity and tumor activity, Substance D is used in delayed allergic reaction tests (tuberculin test) and tumor treatment or prevention. When used in delayed allergic reaction tests,
An intradermal reaction test is appropriate, and it is used in the same manner as purified tuberculin. That is, substance D is preferably prepared using phosphate buffered sodium chloride solution (hereinafter abbreviated as PBS), and the concentration is
Inject 0.1 ml of the preparation prepared at 0.1 to 1000 ng/ml intradermally into animals or humans, and administer 24 or 48
After some time, local redness and induration are measured to determine the degree of delayed allergic sensitization. When used to treat tumors, intratumoral administration is appropriate. That is, a preparation prepared using D substance, preferably PBS, at a concentration of 0.1 to 1000 μs/ml as protein is injected into a human or animal tumor at 0.1 μs/ml.
One to several weekly injections at a dose of ~1 ml/dose are recommended. It is also conceivable to mix substance D with tumor cells that have been previously inactivated by X-ray irradiation, chemotherapeutic agents, etc., and use them for treatment. The dose and interval of administration should be appropriately selected depending on the type of animal and tumor. The toxicity of Substance D is extremely weak. For example, when Substance D obtained from BCG is injected subcutaneously into mice,
Acute toxicity shows an LD ₅₀ value of 100mg/Kg or more,
It is sufficiently safe compared to the dose. When using Substance D, the above-mentioned fraction containing Substance D may be used as it is depending on the purpose, but it is generally better to formulate it according to the manufacturing method of biological preparations. An example of a method for manufacturing Substance D injections is to dissolve Substance D in distilled water for injection, add additives and excipients, and filter the mixture for sterilization to produce a stock solution. This predetermined amount is dispensed and freeze-dried to produce an injection drug. This injection is dissolved and administered using distilled water for injection before use. As an antitumor agent, an injection drug may also be used which is made into an emulsion by mixing the undiluted solution with a suitable surfactant and oil (for example, mineral oil such as normal paraffin, animal oil such as stawalane or squalene, or vegetable oil such as sesame oil). The present invention will be further explained below with reference to Examples and Test Examples. Example 1 Production of intermediate purified product from BCG bacteria Mycobacterium bovis BCG (ATCC19015) was used as a meat extract. The cells were statically cultured at 37° C. for 6 weeks using a glycerin medium. Wet bacterial cells 2 obtained by filtering the culture through cheesecloth
After adding and suspending 10 kg of 50 mM phosphate buffer (PH7.0), the bacterial cells were crushed using a Dyno-Mill while cooling on ice. The obtained microbial cell fragments were centrifuged under cooling to remove solid matter, and a cell-free extract 8.2 was obtained. This is combined with streptomycin sulfate 24.6 as a nucleic acid precipitant.
After adding g and stirring thoroughly, the mixture was allowed to stand overnight at 4°C to form a precipitate, and the mixture was centrifuged to obtain a supernatant 7.8. 6 kg of solid ammonium sulfate is added to the above nucleic acid removed supernatant.
was added, stirred to saturation, allowed to stand overnight to form a precipitate, and centrifuged to obtain a precipitate. This precipitate is suspended in a small amount of distilled water, packed into a dialysis tube, and distilled water
Dialyzed against 10 for 2 days. Then add extra dialysis fluid
The mixture was made into 10mM acetate buffer (PH4.2) and further dialyzed at 4°C for 3 days. The dialysis fluid was replaced with fresh fluid twice daily. After completion of dialysis, centrifugation was performed to obtain supernatant 2.2. To this supernatant, cold ethanol 1.0 was added little by little while stirring, and after standing at 4°C overnight, centrifugation was performed to obtain a supernatant, which was concentrated under reduced pressure to about 100 ml. CM cellulose (CM52, Watmann) equilibrated in advance with 70mM acetate buffer (PH4.1)
was packed into a column to create a bed of 15 x 200 mm. The supernatant was dialyzed against the same buffer and passed through this column at a flow rate of 30 ml/h, followed by elution with the same buffer. In eluate, absorbance 0.05 (280nm)
The portion showing the above was fractionated. This fraction contained 238 mg of protein. This fraction was then concentrated under reduced pressure and then dialyzed against 5mM Tris-HCl buffer (PH8.0). This dialysate was equilibrated with the same buffer using a QAE Sehadex A-25 (manufactured by Pharmacia) column (15 x 160).
mm) at a flow rate of 30 ml/h, and then washed with 700 ml of the same buffer. Next, the intermediate purified product was eluted using the same buffer solution to which 0.15M sodium chloride was added. Absorbance in eluate 0.01
(280 nm) or higher was collected and used as an intermediate purified product. The protein content of the obtained intermediate purified product was 106 mg. The intermediate purified product was lyophilized after dialysis against distilled water. Example 2 Production of substance D from intermediate purified product Sephadex equilibrated in advance with 10mM phosphate buffer to which 0.15M salt was added
Using a G100 column (26.4 x 960 mm), the intermediate purified product of Example 1 dissolved in 5 ml of the same buffer was developed with the same buffer at a rate of 11 ml/h according to a conventional method.
Collect the amount of effluent from 330 to 375 ml and collect approximately 2 ml.
It was concentrated under reduced pressure. Next, using a Sephadex G75 Superfine column (16 x 1480 mm) that had been equilibrated with the above buffer solution in advance, the above concentrate was developed with the same buffer solution at a rate of 6 ml/h. Effluent volume: 130~
A portion of up to 160 ml was collected. This fraction contained 11 mg of protein. Furthermore, this fraction was added to 5mM Tris-HCl buffer (PH
8.0) and passed through a QAE Sephadex A-25 column (10 x 450 mm) that had been equilibrated with the same buffer at a rate of 10 ml/h.
Linear concentration gradient elution was performed using 150 ml each of the same buffer and 0.12M saline-added buffer. The protein content and carbohydrate content of the eluate are measured, and the salt concentration is approximately
The glycoprotein peak eluted at 0.07 to 0.09M [ ] was fractionated. This fraction was put into a dialysis tube, and after dialysis against the above buffer solution 1, chromatography was again performed under the same conditions using a QAE Sephadex A-25 column. The obtained fraction containing the target product was dialyzed against distilled water and then lyophilized to obtain 5.4 mg (weight of lyophilized product) of Substance D. The protein content of the obtained substance D is 0.74mg
It was hot. Substance D of the present invention derived from BCG bacteria is hereinafter referred to as BD substance. In addition, the protein amount and sugar amount were measured by the following method. (i) Protein: Lowry method using bovine serum albumin as standard (Stauffer, CE, Analtical Biochemistry,
69, p. 646, 1975). (ii) Carbohydrates: Phenol sulfate method using glucose as standard (Dubois, M.etal, Analytical
Biochemistry, vol. 28, p. 350, 1956). Example 3 Production of substance D derived from Mycobacterium tuberculosis Mycobacterium tuberculosis
H37Ra (ATCC25177) using Sauton medium,
The cells were statically cultured at 37°C for 8 weeks. After heat sterilizing the culture at 100° C. for 60 minutes, 240 g of wet bacterial cells were filtered through cheese cloth, and 6.4 mg (protein amount) of an intermediate purified product was obtained according to the method of Example 1. Then, according to the method of Example 2 from the intermediate purified product,
0.15 mg (protein amount) of Substance D was obtained. The following substance D of the present invention derived from Mycobacterium tuberculosis is RD
It is called a substance. Example 4 Freeze-dried preparation 10 mg of BD substance was dissolved in 100 ml of distilled water for injection,
Next, 5 g of mannitol was added and dissolved, followed by sterilization and filtration using a Nuclepore filter (0.2 μm, manufactured by Nucleipore Corporation). The obtained filtrate was aseptically dispensed into vials in 1 ml portions and lyophilized to obtain a lyophilized preparation of the substance of the present invention. Example 5 Diagnostic drug preparation 1 mg of BD substance was dissolved in 2 distilled water for injection,
Next, 10 g of lactose was added and dissolved, followed by sterilization and filtration using a Nuclepore filter (0.2 μm; manufactured by Nucleipore Corporation). The resulting filtrate was aseptically dispensed into vials in 2 ml portions and lyophilized to obtain a diagnostic preparation of the substance of the present invention. Test Example 1 Delayed allergic reaction activity test for BD substances.
Using 6 female Hartley guinea pigs inoculated with the following products (manufactured by Co., Ltd.) to achieve delayed sensitization, the BD substance of Example 2 was expressed as the weight of the lyophilized product.
1, 5, 25 ng (= 10 ^-9 g), and PPDs (purified tuberculin, manufactured by Nippon BCG Manufacturing Co., Ltd.) were injected in titer units of 50, 100, and 200 ng into the flanks of guinea pigs at six intradermal locations. , Local redness diameter after 24 hours (arithmetic mean value of vertical and horizontal diameters of redness, unit: mm)
was measured. The average value of the redness diameter at each dose of active substance is shown in Table 1. The activity ratio based on PPDs was calculated from the relationship between the dose of the active substance and the redness diameter, and was approximately 50 to 80 times higher. Substance D was injected intradermally into the flanks of three non-sensitized guinea pigs at a dose of 1 μg of the freeze-dried product.
When the local area was observed after a period of time, redness was hardly detectable, and the skin reactogenicity was specific to sensitized animals.

[Table] Test Example 2 Delayed Allergic Reaction Activity Test for RD Substance Hartley female guinea pigs 8 were inoculated with a paraffin suspension of heat-killed Mycobacterium tuberculosis Aoyama B strain 10 weeks ago to achieve delayed sensitization. Using a head, 0.5, 2 ng and 0.5 ng of the RD substance of Example 3 as protein amount and
A test was conducted in the same manner as in Test Example 1 using PPDs at doses of 50 and 200 ng in titer units. The results are shown in Table 2.

[Table] Test Example 3 Antitumor test on P815 mastocytoma 6-week-old female DBA/2 with 500 μg of BCG vaccine
Sensitized mice. Example 2 at 3 weeks after sensitization.
50 μg of BD substance and 5 P815 mastocytoma cells
0.1 ml of a cell suspension containing ×10 ³ cells was inoculated intradermally into the flank of the mouse. As a control, only tumor cells were inoculated into the other group, and the average survival days after transplantation were compared. The results are shown in Table 3. Substance D of the present invention showed a significant (P<0.005) survival effect.

[Table] Test Example 4 Anti-tumor test on IMC carcinoma 5 × 10 ⁵ IMC carcinoma cells were transplanted intradermally into the flank of female CDF ₁ mice sensitized in the same manner as in Test Example 3, and administered twice a week from the day after transplantation. 6 times in total at the rate of
0.7 μg of substance B of Example 2 dissolved in PBS was administered into the tumor. The control group received PBS only.
The tumor was excised on the 35th day after transplantation, and its weight was measured. The results are shown in Table 4. Substance D of the present invention showed a significant (P<0.02) tumor growth inhibiting effect.

[Table] Test Example 5 Antitumor test on Line 10 hepatoma 1 x 10 ⁶ Line 10 hepatoma cells were intradermally transplanted into the flank skin of male Strain 2 guinea pigs that had been sensitized with 500 μg of BCG vaccine for 8 weeks. 36 μg of the BD substance of Example 2 dissolved in PBS was administered into the tumor twice, for a total of 10 times. The control group received PBS only. Antitumor effects were compared based on survival days and the presence or absence of lymph node metastasis. Table 5
As shown in Figure 2, Substance D of the present invention exhibited strong survival prolonging and metastasis inhibiting effects.

[Table] Test Example 6 Acute Toxicity BD substance was dissolved in physiological saline and administered subcutaneously to 10 female ddY mice (group 1) at a dose of 100 mg/kg body weight. The substance of the present invention did not inhibit body weight gain during the observation period of one week after administration, and there were no cases of death. This result shows that the LD ₅₀ of Substance D of the present invention when administered subcutaneously is 100 mg/Kg or more.

[Brief explanation of drawings]

Figure 1 is an ultraviolet absorption spectrum diagram of substance D (1 mg/ml distilled water), Figure 2 is an infrared absorption spectrum diagram of substance D, Figure 3 is a diagram showing the correlation between molecular weight and eluate volume, and Figure 4 is substance D and concanavalin A
(con A). FIG. 5 is a diagram showing a polyacrylamide electrophoresis image of substance D. In FIG. 3, a...bovine serum albumin, b...ovalbumin, c...chymotrypsinogen A, and d...ribonuclease A are shown. In Figure 4, P is the center hole, concanavalin A (5 mg/ml), and A is substance D (0.5 mg/ml).
mg/ml), and hereafter B, C, D, E, and F indicate substance D that was diluted 2 times in sequence. In Figure 5,
... Protein staining, Y... Sugar staining.

Claims (1)

[Claims] 1. Substance D, which is derived from Mycobacterium tuberculosis and has the following properties and has tuberculin activity. (1) It is a glycoprotein and appears as a white powder. (2) Solubility in solvents: Soluble in water, insoluble in methanol, ethanol, ether and acetone. (3) Molecular weight: 26,000 according to the gel filtration method using Cephadex G75 Superfine (4) Ultraviolet absorption spectrum: 240~240 for aqueous solution
It has no absorption peak at 300nm. (5) Infrared absorption spectrum; around 1650 cm ^-1 and
Has an absorption band around 1530cm ^-1 (6) Amino acid composition (g/D substance 100g) Aspartic acid 0.5, Threonine 9.1, Serine
1.4, glutamic acid 11.4 proline 36.4, glycine
3.1, alanine 4.0, valine 7.4, isoleucine 2.4,
Leucine 1.5 (7) Sugar composition (g/D substance 100g) Mannose 19.0, Glucose 1.2 (8) A single sedimentation line is formed between concanavalin A and concanavalin A by double diffusion method using an agar plate. (9) Delayed allergic reaction activity in BCG vaccine-sensitized guinea pigs was 8
Indicates more than double. 2. Using Mycobacterium bovis or Mycobacterium human tuberculosis, which can produce substance D by separating water-soluble substances from the bacterial cells and then performing operations for removing nucleic acids, carbohydrates, and low-activity proteins, A method for producing substance D, which comprises performing these operations. 3 D derived from Mycobacterium tuberculosis and has tuberculin activity
An antitumor agent containing a substance as an active ingredient. 4 D derived from Mycobacterium tuberculosis and has tuberculin activity
A tuberculin diagnostic agent that contains the substance as an active ingredient.