JPS6363559B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying a proteinaceous physiologically active substance having anticancer activity. Mr. Carswell et al.
-tte-Gue′rin (BCG) administered CD-1
The serum of Swiss mice obtained by intravenous injection of endotoxin after 2 weeks was cultured in L.
Having cytocidal ability against cells and Meth A
Tumor was loaded with sarcoma (BALB/c×
We discovered that tumors in C57BL/6) F ₁ mice developed into hemorrhagic necrosis.
Factor) [Proc.Nat.Acad.Sci.
USA Vol. 72 (No. 9) pp. 3666-3670 (1975)]. They then partially purified TNF from the serum and obtained an active fraction that was 20 to 30 times more purified than the original serum, and the active substance was separated into α-globulin fractions by cellulose acetate membrane electrophoresis. [Proc.Nat.Acad.Sci.USA Vol. 73 (No. 2) 381-385]
Page (1976)]. Ma¨nnel et al. investigated the properties of antitumor factors (cytotoxic factors) using mouse serum obtained by the method of Carswell et al. It was discovered that the active volume fraction fluctuates, and the molecular weight is 55,000-60,000 in high-salt buffers, and 125,000 in low-salt buffers or serum due to association.
~150,000 [Infec.
Immun. vol. 28 (No. 1) pp. 204-211 (1980)]. However, evaluation of this factor in animals (e.g. Meth A
Carswell
It cannot be determined whether it is the same as the TNF reported by et al.
Furthermore, although they investigated various properties by measuring activity using Lcell, they did not describe the purification method. In addition, Matthews et al. induced TNF in rabbits, investigated the properties of rabbit TNF using the serum, and determined that the molecular weight of rabbit TNF was
It was reported that the gel filtration method using 200 was in the range of 40,000 to 50,000 [Br.J.Cancer Vol. 38, pp. 302-309 (1978)]. Based on the above-mentioned state of the art, the present inventor administered a substance having a reticuloendothelial system activating effect to a mammal,
Then by injecting endotoxin,
We also conducted intensive research to find a highly practical method for purifying physiologically active substances with anticancer effects induced by adding endotoxin to a tissue culture system containing activated macrophages derived from mammals. As a result, we have found a simple purification method that allows the physiologically active substance to be obtained with relatively high purity in good yield and is suitable for large-scale purification. The present invention provides one of the substances having reticuloendothelial system activation effect.
endotoxin from Gram-negative bacteria by administering the species or species to a mammal and then injecting endotoxin from Gram-negative bacteria or into a tissue culture system containing activated macrophages from mammals. A crude solution of a proteinaceous physiologically active substance that has an anticancer effect induced by adding
(1) Adsorb the physiologically active substance by contacting it with a basic anion exchanger using a buffer solution with a pH of 6.0 to 9.0 and a salt concentration of 0.2M or less, and (2) a salt concentration higher than that at the time of adsorption. The adsorbed physiologically active substance is eluted from the basic anion exchanger using a buffer solution with a concentration of PH6.0~9.0 as eluent
The physiologically active substance fraction is subjected to gel filtration using a buffer solution of (4) as required. The present invention relates to a method for purifying a proteinaceous physiologically active substance having an anticancer effect, which is characterized by subjecting it to affinity chromatography using an immobilized dye having an anticancer effect. In order to induce the physiologically active substance according to the present invention (hereinafter referred to as the present physiologically active substance), first,
Carswell et al.'s method [Proc.Nat.Acad.Sci・
USA Vol. 72 (No. 9) pp. 3666-3670 (1975)], mammals (e.g., mice, rabbits, guinea pigs, etc.) are given one or more substances that activate the reticuloendothelial system intravenously. Inject intravenously or intraperitoneally. Gram-positive bacteria, protozoa, or yeast are usually used as the substance having a reticuloendothelial system activation effect, and are administered in a live state, a dead state (for example, after heat treatment with formalin), or as a bacterial cell extract component. Here, as Gram-positive bacteria, for example,
Propionibacterium acnes (Corynebacterium
parvum), Propioniba-cterium granulosum
(Corynebacterium granulo−sum)
Propionibacteria.bacillus Calmette−Gu−e′rin
(BCG), Mycobacterium smegmatis-like
Mycobacteria.Nocardiaerythropolis,
Examples include Noca-rdias such as Nocardia gardneri. As a protozoan. Examples include malaria parasites and Toxoplasma gondii. In the case of yeast,
Zymosan extracted from Saccharomyces cerevisiae etc. is usually used. Moreover, synthetic polymer compounds such as pyran copolymers can also be used. Endotoxin obtained from Gram-negative bacteria 7 to 14 days after administration, such as Escherichia coli, Pseudomonas aeruginosa,
Typhoid-derived lipopolysaccharide is injected intravenously into the mammal. 1.5 to 2 hours after injection,
Body fluids (eg, ascites, lymph fluid, etc.) and/or serum or plasma of the mammal are obtained, and organs such as the liver and spleen of the animal are homogeneously crushed and extracted with physiological saline. These body fluids, serum, plasma, and/or organ extracts are used as crude solutions of the physiologically active substance, and usually serum or plasma is used. Another method of inducing this physiologically active substance is by adding endotoxin to a tissue culture system containing activated macrophages of mammalian origin. For example, one of the substances that has the effect of activating the reticuloendothelial system.
Activated macrophages derived from peritoneal exudate, glenoid, or liver are collected from mammals to which the species or two or more species have been administered, and endotoxin is added to the tissue culture system. The active substance is liberated. This supernatant is also used as a crude solution of the physiologically active substance. The physiological activity of this physiologically active substance is evaluated by the following method. (a) Evaluation using L cell: Carswell et al.'s method [Proc.Nat.Acad.Sci.
USA Vol. 72 (No. 9) pp. 3666-3670 (1975)]. A plate made by Linbro (USA) was used as a culture vessel, and non-essential amino acids, 10% heat-inactivated fetal bovine serum, and 100 units of penicillin were added.
ml, Eagle's minimum essential medium (MEM medium) containing streptomycin 100 μg/ml and Lcell
(S). The cell suspension (1×10 ⁵ cells) and the same volume of diluted sample are cultured at 37° C. for 48 hours in air containing 5% carbon dioxide. The titer is calculated by plotting the dilution of the sample and the viable cells of L cells in grams, and then calculating the dilution corresponding to 50% cell-killing ability of L cells. One unit is the amount of physiological activity required to kill 50% of L cells. (b) Evaluation using Meth A sarcoma tumor-bearing mice: According to the method of Carswell et al. (ibid.)
(BALB/c×C57BL/6) 2×10 ⁵ Meth A sarcoma cells were implanted intradermally in the axillary region of F ₁ mice, and after 7 days, the size of the implanted tumor was 7 in diameter.
0.5 ml of the sample diluted with physiological saline was injected into the tail vein of a mouse with a size of ~8 mm and good blood circulation without hemorrhagic necrosis, and after 24 hours it was judged according to the following criteria. (-): No change (+): Faint hemorrhagic necrosis (): Moderate hemorrhagic necrosis (necrosis extending from the center of the transplanted cancer surface to more than 50% (): Significant hemorrhagic necrosis (necrosis of the transplanted cancer) The central part is severely necrotic, with only a small amount of surrounding cancer tissue remaining) The details of the purification method of the present invention will be explained below.
Prior to the first step, contacting with the basic anion exchanger, the crude solution may be dialyzed against the buffer used during contact with the anion exchanger or diluted with a low salt buffer. You may. The contact of the crude solution with the basic anion exchanger is
Either a column method or a batch method may be used.
In this step, the crude solution is brought into contact with a basic anion exchanger using a buffer solution with a pH of 6.0 to 9.0 and a salt concentration of 0.2M or less to adsorb the physiologically active substance, and then the ion exchanger is used with the same buffer solution. This is done by washing the body to remove unadsorbed proteins, and then eluting the physiologically active substance using a buffer with a high salt concentration. Specific examples of basic anion exchangers include:
Diethylamino group-containing anion exchangers such as DEAE-Sephadex A-50, DEAE-Sepharose CL-6B, DEAE-Sephacel (manufactured by Pharmacia, Sweden), AIEC DE52 (manufactured by Watmann, UK), Servacel AE (manufactured by Serva, Sweden), Made by
Aminoethyl group-containing anion exchangers such as (West Germany), QAE-Sephadex A-50 (manufactured by Pharmacia), CellexQAE (manufactured by Biorad, USA)
Examples include quaternized aminoethyl group-containing anion exchangers such as. As a buffer solution. Examples include dilute Tris-HCl buffer and phosphate buffer. The salt added to adjust the salt concentration is preferably sodium chloride or potassium chloride.
The protein concentration in the eluate is measured by absorbance at 280 nm, and the concentration of the physiologically active substance is measured as the L cell killing titer described above. Although one contact with the anion exchanger is sufficient, rechromatography may be preferred in column methods. The eluate containing the physiologically active substance obtained in the above step is concentrated by a conventional method such as ultrafiltration or freeze drying, and then subjected to gel filtration using a carrier suitable for separating substances with a molecular weight of 30,000 to 70,000. A buffer is used as the eluent, and its pH is usually 6.0 to 9.0. The salt concentration in the buffer is 0.5M or more, preferably 0.7 to 2.0M when mouse serum or plasma is used as the crude solution. When using serum or plasma of other animal species, the concentration is preferably 0.15 to 2.0M, although it is not particularly limited. Specific examples of carriers for gel filtration include Cephadex G-75, 100, 150,
200 (manufactured by Pharmacia), Sefacrylic S-200,
300 (manufactured by Pharmacia), Bio-GelP-30, 60,
100, 150, 200 (manufactured by Biorad), CPG-
100 (350 Å, 240 Å, 170 Å, 120 Å) (manufactured by Electronucleonics, USA), and the like.
As the buffer solution and the salt to be added, those listed in the above steps can be used in the same manner. The fractions containing this physiologically active substance obtained by gel filtration are collected, concentrated using conventional methods such as ultrafiltration and freeze-drying, and dialyzed against physiological saline. Approximately 200% compared to serum or original plasma
A ~10,000-fold purified solution is obtained, and the activity recovery rate evaluated using L cell through two steps is approximately 50-100%. Furthermore, if purification is required, use Cibacron Blue as the concentrate obtained earlier.
It is subjected to affinity chromatography using a carrier fixed with F3G-A (manufactured by Ciba Geigy). Cibacron Blue F3G-A (dye) is immobilized on a carrier and insolubilized using known methods [for example, the method of Bo¨hme et al., J. Chromatogr. 69, pp. 209-214 (1972
)], or commercially available immobilization carriers [for example, Blue Sepharose CL-6B (manufactured by Pharmacia), Afui Gel Blue (manufactured by Biorad)] may be used. After dialyzing the concentrated solution of this physiologically active substance against a dilute buffer solution with a pH of 7.0 to 8.0 (e.g., phosphate buffer, Tris-HCl buffer), it is applied to the Cibacron Blue F3G-A immobilization carrier. . Through this operation, contaminant proteins such as albumin are adsorbed, and the physiologically active substance is eluted in the non-adsorbed fraction. The degree of purification of this eluate is approximately 1,000 to 30,000 times higher than that of original serum or plasma, and the activity recovery rate through the three steps is approximately 43 to 95%. The purified solution of the physiologically active substance obtained in this way is subjected to dialysis or gel filtration to adjust the pH and salt concentration, is over-sterilized, and if necessary is heated and then freeze-dried to produce the physiologically active substance. A purified product can be obtained. Approximately 3000 units of refined products here are
In the evaluation using Meth Asarcoma tumor-bearing mice described above, it showed (-) activity. The purified product of this physiologically active substance also showed cytocidal activity against various cultured human cancer cells. Table 1 shows the cell killing rate of 800 units of the purified product after 48 hours.

[Table] On the other hand, for normal cells such as human and mouse cultured fibroblasts, the purified product of this physiologically active substance
Even at 2×10 ⁶ units, no cell killing effect was shown. Furthermore, BALB/
In a test in which the purified product of this physiologically active substance was administered to C mice and A/Jax mice bearing tumors with neuroblastoma Neuro-2a, there was a significant difference compared to the control group (physiological saline administration group). Suppression and regression of cancer growth were observed. In addition, in some individuals, established cancers regressed and disappeared without causing hemorrhagic necrosis. The purified product of this physiologically active substance has an extremely excellent anticancer effect and has little species specificity, so it can be expected to be an extremely useful antitumor agent. The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to these Examples. Example 1 A female rabbit (weight 2.5-3 kg) was treated with formalin-killed Propionibacterium acnes (Coryne
−bacterium parvum; Wellcome, UK) 50
mg was injected via the ear vein. Eight days later, 100 μg of endotoxin (lipopolysaccharide derived from water coli 026:B6, manufactured by Difco, USA) was injected into the rabbit via the ear vein, and 2 hours later, whole blood was collected from the heart. The collected blood was centrifuged at 5000 rpm for 30 minutes to remove blood cells and insoluble solids. 1200 ml of serum with a titer of 12800 units/ml was obtained from 20 rabbits. After diluting the serum with 600 ml of 0.02M Tris-HCl buffer (PH7.8), DEAE-Sepharose CL- which had been equilibrated in advance with 0.02M Tris-HCl buffer (PH7.8) containing 0.1M sodium chloride was added. 6B
(manufactured by Pharmacia) column (6 x 36 cm),
It was added gradually. Then add 1000ml of column equilibration buffer (0.02M Tris containing 0.1M sodium chloride).
After washing with hydrochloric acid buffer, PH7.8), 0.1M of 1.5
0.02M Tris-HCl buffer (PH7.8) containing sodium chloride and 1.5% containing 0.3M sodium chloride
Using 0.02M Tris-HCl buffer (PH7.8),
Elution was performed by a linear sodium chloride salt gradient with a gradienter. The active fraction was collected by fractionating into 18 ml portions at a flow rate of 60 ml/hour and concentrated. The activity recovery rate at this stage was 92%, and the degree of purification was 150 times that of the original serum. Next, the concentrated solution was dialyzed into a 0.005M phosphate buffer (PH7.4) containing 0.15M sodium chloride, and then
Gel filtration was performed. Using a Sephacryl S-200 (manufactured by Pharmacia) column (5 x 80 cm), the column was sufficiently equilibrated with the same buffer solution, and then the concentrated solution was applied thereto, and gel permeation elution was performed with the same buffer solution. Flow rate 60ml/
The active fraction was collected by fractionating 10 ml at a time. The active fraction obtained immediately after the albumin fraction was concentrated by ultrafiltration to obtain a purified product of the physiologically active substance. At this stage, the activity recovery rate was 92% and the degree of purification was 52 times, and throughout all stages the activity recovery rate was 85% and the degree of purification was 7800 times. The specific activity of the purified product of this physiologically active substance was approximately 1.4 x 10 ⁶ units/mg protein. Figures 1 and 2 show the patterns of chromatography on a DEAE-Sepharose CL-6B column (first step) and gel filtration on a Sephacryl S-200 column (second step), respectively. Example 2 2200 ml of rabbit serum containing this physiologically active substance was
After diluting with 1200 ml of 0.02 Tris-HCl buffer (PH7.2), add a DEAE-Sepharose CL-6B column ( 8
x 26 cm). It was then washed with 1000 ml of 0.02 M Tris-HCl buffer (PH 7.2) containing 0.13 M sodium chloride, and then washed with 1000 ml of 0.02 M Tris-HCl buffer (PH 7.2) containing 0.13 M sodium chloride.
7.2) and 0.02M containing 0.3M sodium chloride in 2.0
Elution was performed using a tris-hydrochloric acid buffer (PH 7.2) with a sodium chloride linear salt concentration gradient using a gradienter. At a flow rate of 90ml/hour
The active fraction was collected by fractionating 18 ml each. Active fraction containing 0.1M sodium chloride
After dialyzing against 0.02M Tris-HCl buffer (PH7.2), it was equilibrated in advance with the same buffer.
DEAE-Sepharose CL-6B column (2.5×
3.0 cm). The adsorbed biologically active substance was transferred to a column containing 350 ml of column equilibration buffer and 350 ml of 0.3M sodium chloride.
Using 0.02M Tris-HCl buffer (PH7.2),
Elution was performed by a linear sodium chloride salt gradient with a gradienter. The active fraction was collected by fractionating into 7 ml portions at a flow rate of 25 ml/hour and concentrated. Next, the concentrated solution was applied to a Sephacryl S-200 column equilibrated with 0.01M Tris-HCl buffer (PH7.8) containing 1.0M sodium chloride, and eluted with the same buffer. The active fraction concentrate obtained by gel filtration was dialyzed overnight against 0.02M phosphate buffer (PH7.1), and then Bluecepharose CL-6B (Pharmacia) equilibrated with the same buffer was used. The sample was applied to a column (1.0×10 cm) of a commercial manufacturer (Japan). After thorough washing with the same buffer at a flow rate of 2.5 ml/hour and 3 ml/fraction, the solution containing 1.5 M sodium chloride was used.
The adsorbed substances were eluted with 50 ml of 0.02M phosphate buffer (PH7.1). No activity was observed in the adsorbed fraction, and all activity was recovered in the non-adsorbed fraction. This was collected and lyophilized to obtain a purified product of this physiologically active substance that was 30,000 times more pure than the original serum. Activity recovery throughout all stages was 77%. Example 3 Propionibacterium acnes (Corynebacterium acnes) killed with formalin was administered to 6-week-old ddY female mice.
-rium parvum; Wellcome Ltd., UK) 1 mg was administered intraperitoneally, and 10 days later, endotoxin (E. coli
026: B6-derived lipopolysaccharide) 10 μg was intravenously injected, and 2 hours later, blood was collected from the orbital venous plexus using a capillary pipette. The collected blood is
Blood cell components were removed by centrifugation at 3000 rpm for 15 minutes. In this way, mouse serum with a titer of 10240-51200 units/ml was obtained. 500ml of the serum containing 0.2M sodium chloride
Dialysis was performed overnight against 0.05M Tris-HCl buffer (PH7.8). The dialyzed serum was placed on a Buchner funnel,
This physiologically active substance was adsorbed by gradually applying DEAE-Sephadex A-50 (500 g dry weight) equilibrated with the same buffer solution in advance. The ion exchanger was then washed with the same buffer in an amount three times the volume of the ion exchanger to remove non-adsorbed substances. Next, the physiologically active substance was eluted with 0.02M Tris-HCl buffer (PH7.8) containing 1.0M sodium chloride in the same volume as used for washing. At this stage, the activity recovery rate was 100%, and the degree of purification was 36 times that of the original serum. The eluate was concentrated by ultrafiltration and then subjected to gel filtration. Sephadex G-200 column (3.5 x 95 cm) contains 0.7M sodium chloride.
Equilibration was performed with 0.02M Tris-HCl buffer (PH7.8), and elution was performed using the same buffer at a flow rate of 40 ml/hour. This physiologically active substance was fractionated as a single peak from the latter half of the albumin fraction. The active fraction was concentrated by ultrafiltration. At this stage, the activity recovery rate was 80% and the degree of purification was 6.5 times, and throughout all stages the activity recovery rate was 80% and the degree of purification was 240 times. The concentrated solution was dialyzed against 0.02M phosphate buffer (PH7.1) and then applied to a column (1.0 x 10 cm) of Afuigel Blue (manufactured by Biorad) equilibrated with the same buffer. It was eluted with a buffer solution. All flow rates were 2.5 ml/hr. Activity has been eluted in the non-adsorbed fraction. The fractions were collected and lyophilized to obtain a purified product of the physiologically active substance. At this stage, the activity recovery rate was 81% and the degree of purification was 5 times higher, and throughout all stages the activity recovery rate was 65% and the degree of purification was 1200 times higher. The specific activity of the purified product of this physiologically active substance was approximately 2.4×10 ⁵ units/mg. Example 4 600 ml of mouse serum containing this physiologically active substance was
DEAE-Sepharose CL-6B column diluted with 300 ml of 0.02M Tris-HCl buffer (PH7.8) and pre-equilibrated with 0.02M Tris-HCl buffer (PH7.8) containing 0.1M sodium chloride. (6
×36cm). Then, after washing with 500 ml of column equilibration buffer, 0.02 M Tris-HCl buffer containing 0.3 M sodium chloride (PH
7.8), elution was performed using a linear sodium chloride concentration gradient using a gradienter. The active fraction was collected by fractionating into 18 ml portions at a flow rate of 75 ml/hour and concentrated. Next, the concentrated solution was transferred to a Sephacryl S-200 column (5 x 80
cm) and eluted with the same buffer. The active fraction was collected in 10 ml fractions at a flow rate of 63 ml/hour and concentrated. The concentrated solution was subjected to affinity chromatography using an Affigel blue column under the same conditions as in Example 3, and compared to the original serum,
A purified product of this physiologically active substance with a purity of 1100 times was obtained. Activity recovery throughout all stages was 49%.

[Brief explanation of drawings]

FIGS. 1 and 2 respectively show the chromatography pattern using DEAE-Sepharose CL-6B and the gel filtration pattern using Sephacryl S-200 in Example 1.

Claims (1)

[Scope of Claims] 1. One or two substances having a reticuloendothelial system activating effect
By administering a species or more to a mammal and then injecting an endotoxin derived from a gram-negative bacterium,
Alternatively, a crude solution of a proteinaceous physiologically active substance with anticancer activity induced by adding endotoxin derived from Gram-negative bacteria to a tissue culture system containing activated macrophages derived from mammals (1) PH6.0 ~
9.0, adsorb the physiologically active substance by contacting it with a basic anion exchanger using a buffer solution with a salt concentration of 0.2M or less, and (2) using a buffer solution with a salt concentration higher than the salt concentration during adsorption. The adsorbed physiologically active substance is eluted from the basic anion exchanger, and then (3) the eluate containing the physiologically active substance is used as a carrier and eluate suitable for separating substances with a molecular weight of 30,000 to 70,000 at a pH of 6.0 to 70,000. Gel filtration using 9.0 buffer, and if necessary (4)
The physiologically active substance fraction has the following structural formula: 1. A method for purifying a proteinaceous substance having anticancer activity, the method comprising subjecting it to affinity chromatography using an immobilized dye having an anticancer effect. 2. The purification method according to claim 1, wherein the mammal is a mouse. 3. The purification method according to claim 1, wherein the mammal is a rabbit.