JPS60139624A - Protein having antitumor activity - Google Patents

Abstract

NEW MATERIAL:A protein composed of the subunit of a polypeptide having the amino acid sequence of formula at the C-terminal, and having the following characteristics. Molecular weight, 40,000+ or -5,000 (by gel-filtration) and 17,500+ or - 2,000 (by SDS-polyacrylamide gel electrophoresis); isoelectric point, 5.0+ or -0.3 (electrophoresis at isoelectric point); specific activity, 5X10<6>-10X10<7> unit/mg-protein by the estimation using the L-M cell defined in the text. USE:Antitumor agent having low species-specificity, wide effective range, and low toxicity. PREPARATION:A rabbit is administered with one or more material having reticuloendothelial system activation activity (e.g. Gram-positive bacteria, yeast, etc.), and after 7-14 days, injected with endotoxin obtained from Gram-negative bacteria. A crude solution of the protein of the present invention can be obtained 1.5-2hr after injection. The crude solution is purified to the titled protein by basic anion-exchange chromatography, heat-treatment, gel-filtration, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance having anti-ajXI activity.

In this specification, amino acids and peptides are referred to as IUPAC-
Indicated by the abbreviations adopted by the IUB Committee on Biochemical Nomenclature (CIIN), for example, the following abbreviations are used.

In addition, when an amino acid or the like can have optical isomers, 5 isomers are shown unless otherwise specified.

Ala: Arani/Gin: Glutamine Glu: Glutamate Gly: Glycine 11e: Isoleucine Leu: Leucine Phe: Phenylalanine Set: Serine Tyr: Tyrosine Val: Valine □ Various substances that have a reticuloendothelial system activation effect, such as various types of Duram There have been many reports on the existence of substances that are induced by positive bacteria and endotoxin and have physiological activities such as tumor cytotoxicity. For example, Carswell et al.
Bacillus Calmet in 5w1ss mouse
The serum of the mice obtained by administering te-Guerin (BCG) and intravenously injecting endotoxin two weeks later showed cytotoxic effects on cultured cells, and meth I was given cancer. (IIALB/c
XC57BL10)F+T
We named it F (tumor Necrcis!5Factor) [Proc, Nat, ^cad, sci, υS
8, 72°3 (16G to 1975)']. After that, Ru
f f et al. [J, 1mmuno1. ．． 125゜1B71(
1980)] and Matthews et al. [[lr, J, C
ancer, 42.4113 (1980)] is the C
Attempts were made to purify TNF from rabbit serum prepared according to the method of Kamersweil et al., and results were obtained that were about 2000 times and about 1000 times more purified than the original serum, respectively. However, in any case, the purified product has not been confirmed to have an anti-inflammatory effect in animal experiments.

JP-A No. 57-140725 discloses that one or more substances having a reticuloendothelial system activating effect are administered to mammals (mice, etc.).

(rabbits, guinea pigs, etc.) and then by injecting gram-negative bacterium entodoxy/, or by adding gram-negative bacterium entodoxy/ to a 1 m culture system containing activated macrophages of mammalian origin. The authors disclose that they have isolated and purified a proteinaceous physiologically active substance that has anticancer effects induced by the above-mentioned cancer.Furthermore, the molecular weight of the substance was determined by gel filtration method and 5DS-polyacrylamide gel electrophoresis method. 39,000 ± 5,000, the isoelectric point is pH 3,9 ± 0.3 (isoelectric focusing method),
It has been disclosed.

The anticancer activity of the above substance is extremely excellent, but in order to isolate and purify it, the crude solution must be subjected to a complicated purification process, and the recovery rate of its activity is low.
It has the following drawback.

The present inventors have been diligently researching the purification method for obtaining the above-mentioned substance in good yield from rabbit serum prepared by method α described in this specification, and in the process,
By adopting a purification method different from the purification method of No. 140725, a new protein with excellent antitumor activity, which is different from the above-mentioned substances, was surprisingly produced in good yield (i.e., the recovery rate throughout the entire process was about 50 to 50%). 70%)
However, as a result of further research, the present invention was completed.

In the present invention, the C-terminal portion =・5er-Gly-Gln-Val-Tyr-Ph
The present invention relates to a protein having a structure consisting of subunits of a polypeptide having an amino acid sequence structure represented by e-Gly-11e-11e-Ala-Leu, and having the following properties.

a) Molecular weight to, ooo ± 5,000 (gel filtration method) 17.500 ± 2,000 (5DS-polyacrylamide gel electrophoresis method) b) Equal weight 5.0 ± 0.3 (isoelectric focusing method) )C)
In addition, the physiologically active substance of the present invention has a specific activity of SX 106 to 107 units/■ protein in the evaluation using LM cells described in Experimental Example 3.
A sarcoma-bearing tumor BA1. In the activity 1□ evaluation using B/c mice, 3,000 to 5,000 per mouse.
The activity when the unit is administered intravenously is (+) or higher.

In addition, in electrophoresis using cellulose acetate membrane, α-
The globulin region was electrophoresed, and various lectin columns [Co
nA-Sepha0-S (Pharmacia), RCA-1
- Gel (E, Y, Laboratory Co., Ltd.).

□ WGA-Seph 10-S 6MB (Pharmacia), UE
A-1-ge/lz, LPA-1-gel (hereinafter E, Y, u; j! la) 'J-Company, )].

The physical properties of the novel physiologically active substance of the present invention were measured by each method of Experimental Examples 1 to 8 described below.

Experimental Example 1 Molecular weight measurement □ A) Using a column (1.5 x 100 cm) of Cef 1 Kryl S-200 (Pharmacia), 0. IM Na
C1/ 50mM heptacid buffer (pl+7・4) V'1
・5) Standard protein for molecular weight measurement (Pharmacia; ribonuclease A, chymotrypsinogen A)
A molecular weight calibration curve was created using the following: The physiologically active substance of the present invention was eluted between ovalpdin and chymotrypsinogen A, and had a molecular weight of 40,000±5,000.

1) Methods of Segrest et al.
Enzymology.

2B-B, 54 (1B72), ^cadem+c
Press Inc., New York, T.
10 μg of the sample was applied to an SDS/polyacrylamide gel using ris/glycine/SDS (sodium dodecyl sulfate) (pH 8,3), and electrophoresis was performed. A molecular m standard curve was created using a standard molecular weight kit (Pharmacia), and the molecular weight was determined by activity evaluation in LM cells and staining (Coomassie Brillant Tatami Blue R-250). The physiologically active substance of the present invention has 17,500±2
, 000 showed a single staining band and activity.

Experimental Example 2 Isoelectric Pointing Measurement Isoelectric Focusing H Place (5J-107 manufactured by At-Co., Ltd.)
1ECI! ), Fal! 5% polyacrylamide flat gel containing light (Ffurmacia, pH 4-6.5) and glycerol (thickness 1■■, length foes).

A wide range of Ocs> was created. 0.04M D on the lig pole side
Pre-phoresis was performed at 700V for 50 minutes using L-glutamic acid and 0.2M L-histidine on the cathode side. Next, apply 50ugik to the sample and apply 700V for 1 hour, 5
Electrophoresis was performed at 00V for 10 hours. After the electrophoresis is complete, remove the gel
．． Cut out 5 pieces of gel width, then each gel piece is 0.15M N
Extraction was performed with 18.2>0.2 ml of 0.02M Tri 5-IIcI buffer (p) containing aCl, and each extract was evaluated for activity using LM cells. The isocenter of the physiologically active substance of the present invention was 5.0±0.3.

Experimental Example 3 Activity evaluation using LM cells Activity evaluation using LM cells was performed using the method of Ruff et al. [edited by E. Pick, Lymphokines, 2, 235 (1981);
^cadem 1cPress Inc,] or K
The method of Ull et al. [J, Immunol, +120.12
79 (1981)], which the present inventors have improved, and the physiologically active substance of the present invention is derived from L-M cells (ATCC
The objective was to evaluate the effect of killing CCLl, 2).

That is, 0.11 and 105 samples/■ sequentially diluted with medium.
A suspension of LM cells in a medium having a concentration of 0.11 was added to a 96-well tissue culture microplate (Flow Laboratory). The medium is Eagle's Minimum Essential Medium containing 1% (Vha) fetal bovine serum (its composition can be found, for example, in "Tissue Culture" edited by Junnosuke Nakai et al., Asakai Shoten,
(described in 1907) was used. The microplate was incubated at 37° C. for 48 hours in air containing 5% carbon dioxide. After culturing, add 20 μl of glutaraldehyde.
was added to fix the cells. After fixation, the microplate was washed, dried, and diluted with 0.11% methylene blue solution.
In addition, surviving cells were stained. After washing away excess methylene blue and drying, remove remaining methylene blue to 0.
．． Extract with 30 N hydrochloric acid solution, and measure the absorbance at 665 n
Laboratory Co., Ltd.). ``This absorbance is proportional to the number of surviving cells. The amount of physiological activity required to kill 50% of LM cells is defined as 1 unit/-1, and the dilution rate of the sample corresponding to 50% of the absorbance of the control without the sample is shown in the graph. □ Calculate the reciprocal of the dilution rate to determine the physiological activity of the sample (expressed in units/i+1)
And so.

'On the other hand, the protein amount was determined by the method of l1ranford et al.' [
^nal.

11iochem, 72, 248 (1970)] by a dye binding method using Coomassie Brilliant Blue G-250.

The specific activity of the physiologically active substance of the present invention is %5X106~1X1
07 units 1-g 1 m' of white matter.

Experimental Example 4 Activity evaluation using Meth^sarcoma-bearing mice 2 x 105 Meth cells were transplanted into the abdominal ridge of IIAL II/C mice, and 7 days later, the size of the transplanted anal ulcer was 7 in diameter. A mouse with a yi tumor of ~8-1 and no hemorrhagic necrosis and good blood circulation was selected, and a sample of 0.211 diluted with physiological saline was injected into the tail vein, and 24 hours later, the next injection was performed. Judgment of necrotic reaction according to the criteria
- 0 □ (-): No change - □゛ (+): Faint hemorrhagic necrosis (4)): ' Moderate hemorrhagic necrosis (more than 50% of the transplanted cancer surface Wat□te necrosis) (4++):! ! Severe hemorrhagic necrosis (severe necrosis in the central part of the transplanted cancer, with only a small amount of surrounding cancer tissue remaining).Also, 20 days after sample administration, whether the cancer had completely regressed was determined. After observation, we found a complete cure rate.

The activity of the physiologically active substance of the present invention measured by the above method is shown in the table below.

5.000 0 2 1 3 0 2/(ilo, 00
0 (i 0 1 2 3 3/820.000 0
0 0 156113 control (physiological saline) ti6000
0/G0 complete cure rate: number of mice with complete regression of cancer/number of experimental mice Experimental Example 5 Determination of amino acid sequence structure The following study was conducted using the physiologically active substance of the present invention.

0.1% SDS according to the method of Segrest et al.
Electrophoresis of the physiologically active substance of the present invention was carried out using a 12.5 Vo polyacrylamide plate gel containing the following. Approximately 10 ug of sample was added to SDS-containing Tris/glycine buffer (pH
8,3) as the electrophoresis solution at 120V for 5 hours,
Protein staining was performed with Coomassie Brilliant Blue R-250. The physiologically active substance of the present invention has a molecular weight of approximately 17,500.
It was stained as a single band at a mobility of ±2,000. Next, the same sample was treated with 2-mercaptoethanol, a disulfide bond reducing agent, and then subjected to electrophoresis under the same conditions, with almost no change in molecular weight.

(1) Identification of C-terminal amino acid The C-terminal amino acid of the peptide chain of the physiologically active substance of the present invention was identified by a hydrazine decomposition method and an enzymatic method using carboxypeptidase.

8) Hydrazine decomposition method Red mud method [Bull, Chem, Soc, Jap,
25, 214 (1952)]. Anhydrous hydrazine 0.0.
11 was added, and L'IQ- =
3 disassembled. After lyophilizing the reaction product, 50 μl of the residue
of distilled water, extract and remove amino acid hydrazide derivatives with 50t11 of benzaldehyde, and remove 25μ of the aqueous layer.
1 was subjected to amino acid analysis.

Amino acid analysis was carried out using a fluorescent amino acid analysis system (Shima γ1 (Seisakusho)) using orthophthalaldehyde. As a result, leucine was mainly detected.

B) Carboxypeptidase method 15 μg of the physiologically active substance of the present invention was added to 0.05M 177@buffer (pH 8,0) containing 0.05M SDS 10
g/di lysed L, carboxypeptidase A (diimpropyl full 4 sips, sulfate-treated carboxypeptidase A.

Sigma) was added at an enzyme/substrate molar ratio of 1:25, and the reaction was carried out at 25°C. After the reaction, 40% trichloroacetic acid @ 5 μl was added and stirred.
Centrifuged for minutes. The supernatant was subjected to amino acid analysis, and the amino acids liberated from the C-terminus of the physiologically active substance of the present invention were analyzed at 8 hours.6 As a result, leucine was mainly present until 12 hours after the start of the reaction. was detected, and almost no other amino acids were released.

Next, carboxypeptidase 8 (Sigma) was allowed to act in the same manner, but no basic or amino acid release was observed from the physiologically active substance of the present invention.

These results revealed that the C-terminal amino acid of the physiologically active substance of the present invention is leucine.

(2) Determination of the amino acid sequence of the C-terminal portion The amino acid sequence of the C-terminal portion of the physiologically active substance of the present invention was determined by two methods. The first method involves direct action of carboxypeptidase on the physiologically active substance of the present invention, sequentially releasing amino acids from the C-terminus, and determining the sequence.
In this method, the physiologically active substance of the present invention is fragmented, a peptide fragment containing the C-terminal portion is isolated, and the sequence is determined by the Edman degradation method.

A) Carboxypeptidase method 15μg of the physiologically active substance of the present invention was added to 0.05M SDS.
0.05M including! 77 Wli buffer (p+I6.
0) After reacting dilytic ML and carboxypeptidase A at 25°C for 10 minutes, 5 μl of carboxypeptidase Y (Oriental yeast) was added to the enzyme/substrate molar ratio i:
Add at a ratio of 2.10, 30% at 25°C.
, and reacted for 180 minutes. After adding 1.5 μl of 40% trichloroacetic acid to each reaction solution and stirring, the mixture was centrifuged at 5,000 rpm for 2 minutes, and the supernatant was subjected to amino acid analysis. From the order of released amino acids, the amino acid sequence of the C-terminal portion of the physiologically active substance of the present invention is = (Phe, Gly,
Ile)-11e-Ala-Leu (The amino acids in parentheses indicate the amino acids that may be sequenced from C; lfla to positions 4 to 6).

B) Amino acid sequence of C-terminal peptide fragment Physiologically active substance of the present invention 5! t 3.0■ was dissolved in 50 mM ammonium acetate buffer (pH 4,0>1.0et) and heated in a boiling water bath for 5 minutes. Staphylococcus aureus (strain V8) protease (Staphylococcus aureus (strain V8) was added to this denatured protein solution.
Add 50 μg of US protease (Waushi 7Ton Co., Ltd.) and react at 25°C for 24 hours to fragment the bioactive substance of the present invention. The reaction solution is freeze-dried, and the residue is reduced to 0.1
% trifluoro vinegar 3110-dissolved in 3 ml, 5,0
After centrifugation at 00 rpm for 10 II), the supernatant was subjected to reversed phase high performance liquid chromatography to isolate and purify the peptide fragments. Chromatography carbon chain CIl
4.OX 250 +n column of silica,
The elution was carried out using TSKgel 0DS-120T (Toyo Soda) using a linear concentration gradient elution method (0 to 80%) of acetonitrile containing 0.1% trifluoroacetic acid. Flow velocity is 0
．． 7ml/win, elution peptide wavelength 23On+
It was detected by absorbance of z. As a result, 811 peptide fragments were isolated as homogeneous peptides. After concentrating both parts of each peptide fragment to dryness under reduced pressure, 0.2 et
was dissolved in 10% acetic acid and a portion thereof was used to analyze the amino acid sequence of the C-terminal portion with carboxypeptidase A and carboxypeptidase Y in the same manner as above. As a result, one of these peptide fragments was found to be It was a C-terminally derived peptide having l1e-Ala-Leu as a partial structure. The peptide fragment was subjected to amino acid analysis according to a conventional method. That is, the peptide fragment was dissolved in 0.2 ml of 6N hydrochloric acid.
Hydrolyzed by heating in vacuum at 110°C for 24 hours,
The decomposition products were concentrated to dryness under reduced pressure and analyzed.

As a result, this peptide has 1 mole of Set + 1 mole of A1a, 0172 mole, 1 mole of Val, and 2 moles of 11e2.

Leu 1 mol, Tyrl mo/l<, Phe 1 mol,
It was revealed that the peptide consisted of 1 mole of Glu (or Gln), a total of 11 amino acids.

Next, the amino acid sequence of the peptide derived from the C-terminus of
277 (1950)] from the N-terminus.

By dissolving the peptide in dimethylallylamine/buffer, coupling reaction with phenyl thiocyanate, and cleaving with trifluoroacetic acid, the resulting thiazolinone derivative of the N-terminal amino acid was heated with IN hydrochloric acid at 80°C for 10 minutes. It was converted into a 3-phenyl-2-thiohydantoin derivative (PTH derivative). This PTH amino acid derivative was identified by high performance liquid chromatography.

High performance liquid chromatography was performed using the method of Zimmerman et al.

77.569 (Seal 977)], Zorbax O
It was carried out using a DS column (4,6x, 25011. DuPont). As a result, the amino acid sequence from the N-terminus of the peptide is 5et-Gly-Gln-Val'-Tyr
-Phe-, Gly-11e-11e-Ala-, Le
It became clear that u.

Experimental Example 6 Adsorption to Lectin Column Various commercially available lectin solids and chemotactic resins were packed into a Sepacol Mini column (Bio-Rad, Inc.), and 150mM NaC
After thorough washing with 50 mM phosphate buffer (pH 7,5) containing 1, 100 μg of a sample of the physiologically active substance of the present invention dissolved in the same buffer was given as (), and then eluted with the eluent shown below. After that, the activity of the cable passing fraction and the elution fraction was measured by L-M.
It was measured by an activity evaluation method using cells.

In all columns, all activity was recovered in both flow-throughs.

Column used: Eluent Con^-Sepharose 0. IMα-methyl-d-ma/noside WG^-Sepharose 0.1M N-acetyl-D-glucosamine ζ RC^-■-gel 0.1M lactose υE^-1-gel 0.05M α-fucose LP8-■
Hegel 0.1M sialic acid Experimental Example 7゛ Electrophoretic mobility Separax S (Fuji Photo Film) was used as the cellulose acetate membrane, pl+ 8. fi, ionic strength 0
．． Electrophoresis was performed at 0.06 to 0.07. After the electrophoresis was completed, sections with a width of 1■■ were prepared. The obtained sections were extracted with physiological saline, and the activity in LM cells was evaluated to determine the mobility. At the same time, a sample for staining was run and stained with Bonn-3R (Half J1 Chemicals).

The physiologically active substance of the present invention was electrophoresed in the a-globulin region and showed activity at the position of a single stained band.

Experimental Example 8 Influence of disulfide bond reduction Dithiothreitol (0.1mM
, 1'mM) or 2-mercaptoethanol (0,1mM)
M, 1 mM), 20 mg of the physiologically active substance of the present invention
/ml was reacted for 2 hours at 25°C in 0.15M NaC1150mM phosphate buffer (pH 7.4). After the reaction, residual activity was measured using LM cells, and the remaining activity was measured using LM cells without using a disulfide bond reducing agent. The ratio of the remaining activity to that of the control was calculated.The results are shown in the table below.

As is clear from the above explanation, the novel physiologically active substance of the present invention has a molecular weight in solution, that is, a molecular weight at the time of gel filtration, of to, ooo ± 5,000, and 5DS
- Subunit resolution of polyacrylamide gel electrophoresis [
The molecular weight after finishing is 17.5'00±2,000.

Moreover, when the extracted sample after 5DS-polyacrylamide gel electrophoresis is subjected to gel filtration, the active fraction has a molecular weight of 40.
,000±5,000, the physiologically active substance of the present invention is considered to be a protein having a subunit structure.

Next, the novel physiologically active substance ccu of the present invention will be explained. ' One or more substances having reticuloendothelial system activating effects are injected intravenously or intraperitoneally into rabbits. As the substance having reticuloendothelial system activating effect, Durham-positive bacteria, 'B live animals or yeast are usually used, and they can be used in live bacterial state, dead bacterial state (e.g. after heat treatment or formalin treatment) or as a bacterial cell extract component. Administered □. Here, Durham-positive bacteria, e.g.
i'''Pro'pionibacter'iumac
nes (Corynebacterium parv
u'm), Propionibac-teriul
granulosum (Corynebacterium
Propioni such as M granulosum)
bacteria, bacillus Calmet
te-Gu5rin (13CG), Mycobact
Mycob like er gous smegmat'is
acter ia, Nocardia erythro
polis.

Nocardia gar, Noc like dneri
ard! As is an example. Examples of protozoa include malaria parasites and toxoplasma. For yeast, usually Saccharomyces cerev+5
Zy■osan extracted from iae etc. is used.

Also, synthetic polymeric compounds such as pira/copolymers can be used.

7-14r-+ after administration of a substance that activates the reticuloendothelial system
84. t+ 7/2J Seizanko) h However, a lipopolysaccharide derived from Escherichia coli, Pseudomonas aeruginosa, or Salmonella typhi is injected intravenously or intraperitoneally into the rabbit. 1.5 to 2 hours after the injection, a crude solution of the physiologically active substance of the present invention (hereinafter simply referred to as crude solution) is obtained. That is, the rabbit's body fluids (eg, abdominal fluid).

lymph fluid, etc.) and/or serum or plasma.

Or, evenly destroy the liver, **, etc. organs of the rabbit □
Crush and extract with physiological saline.

Or, in advance, one of the substances that has a reticuloendothelial system activation effect.
When endotoxin is added to a tissue culture system containing macrophages (e.g., alveolar macrophages, peripheral blood macrophages, etc.) of rabbits or normal rabbits that have been injected intravenously or intraperitoneally with one or more species, endotoxin is present in the supernatant. The physiologically active substance of the invention is liberated.

The crude solution thus obtained is purified by the means described below to obtain the physiologically active substance of the present invention. ” The gist of a preferred purification method for obtaining the physiologically active substance of the present invention is to subject a crude solution, especially serum or plasma, to the following step.

(1) Pretreatment of crude solution Basic anion exchange chromatography (3) Heat treatment (4) Gel filtration (5) Zn2++ rate affinity chromatography (6) Gel filtration Below, these steps are explained below. Explain details.

The protein concentration in the crude serum and eluate and the concentration of the physiologically active substance of the present invention are measured by the protein plaster measurement method and activity evaluation using LM cells described in Experimental Example 3, respectively.

Purification step 1 The crude solution is treated with a chelating agent and diatomaceous earth powder, and then filtered with a filter. As the chelating agent, ethylenediazinetetraacetic acid disodium salt is preferred. Examples of diatomaceous earth powder include Celite and Supercell. The filtration operation is carried out using one type of filter or a combination of several types of filters having a pore size of 0.2 to 3 μm. The recovery rate Lt of activity in this step is about 95 to 100%.

Purification step 2 The filtrate obtained in the above step may be brought into contact with the basic anion exchanger using either the column method or the NO(FUTHI method).Prior to the contact with the basic anion exchanger. The filtrate may be dialyzed against the buffer used during contact with the anion exchanger, or diluted with a low salt buffer.

In this step, pHe is 0 to 8.0. Using a buffer solution with a salt concentration of 0.2 M or less, the filtrate is brought into contact with a basic anion exchanger to adsorb the physiologically active substance of the present invention, and then the ion exchanger is washed with the same warm buffer solution at 0. After removing non-adsorbed proteins, the physiologically active substance of the present invention is eluted using a buffer solution with a high salt concentration.

Specific examples of salt-based anion exchangers include DEAE-Sephadex A-50, DEAE-Sephadex A-50, DEAE-Sephadex CL-
6B, DEAE-Cef7 Cell (Pharmacia)
Anion exchangers containing diethylaminoethyl groups are preferred. As a buffer solution, for example, dilute Tris-H
CI buffer and yWt buffer are mentioned. The salt added to adjust the salt concentration is preferably sodium chloride or potassium chloride.

In addition, if the degree of purification has not reached 600 times or more compared to the original serum or raw plasma after the completion of purification step 2, purification step 2
Repeat.

Purification Step 3 The eluate obtained in the above step is concentrated using ultrafiltration, lyophilization, or a basic anion exchange column of an appropriate size, and then subjected to heat treatment. Heat treatment is usually 60 to 70
The treatment is carried out at 60°C for 30 minutes and 2 hours, preferably at 60°C for 30 minutes. As a buffer solution for heat treatment,
For example, TriS-11CI buffer or phosphorus@buffer may be used, and the pl+ thereof is 6.0 to 9.0.

The degree of purification through purification steps 1 to 3 is more than 600 times, and the recovery rate of activity is about 78-100%.

Purification step 4 The eluate containing the physiologically active substance of the present invention is purified by ultrafiltration, lyophilization, or by using an i-based anion exchange column of an appropriate size, and then reduced to a molecular ffl 3'0,000. ~7
It is subjected to gel filtration using a carrier suitable for the separation of 0,000 substances. A buffer is used as the eluent, and its pH
is usually 6.0 to 9.0. The salt concentration in the buffer solution is 1.
It is 0M or less. Specific examples of carriers for gel filtration include Sephadex G-150, G-200, and Sepharyl S.
-200 (Pharmacia), Biogel I'=1
50. P-200 (Bio-Rad), Toyo Pearl H
Examples include W-55 and HW-65 (Toyo Soda).

As the buffer solution and the salt to be added, those listed in purification step 2 are used in the same manner. The physiologically active substance-containing fractions of the present invention obtained by gel filtration are collected, ultrafiltrated, and freeze-dried using a small basic anion exchanger! Concentrate with ・etc. Purification degree through purification steps 1 to 4 is approximately fx 10' to 4X
10' times, the recovery of activity is about 70-95%.

Purification step 5 The solution obtained in purification step 4 was applied to a Zn2+ chelate Sepharose column prepared according to the method of J. Porath et al. [Nature, 258, 598 (Wataru 975)]. In addition, since Sepharose CL-61 (Pharmacia) coupled with iminodiacetic acid is commercially available, this may also be used.

7710-s CL-E coupled with iminodiacetic acid
Flow zinc chloride at a concentration of 1 g/ml into the IB column, and
Chelate adsorption of 2+. Subsequently, after washing the column with the buffer used for elution, the solution obtained in purification step 4 is used as it is or after being concentrated.

The buffer used in this step and the salt concentration and type used therein are the same as those used in purification step 2. Most of the contaminant proteins are adsorbed by this operation,
The physiologically active substance of the present invention is eluted in the non-adsorbed fraction. Purification degree through purification steps 1 to 5 is approximately 2X 10' to 7X
104 times, the recovery of activity is about 50-76%.

Purification Step 6 The eluate obtained in Purification Step 5 is subjected to gel filtration using the same carrier, buffer, and salt as in Purification Step 4.

However, the column used is one that is smaller in diameter and longer than the one used in purification step 4. The active fractions are collected and subjected to concentration, dialysis, fi over sterilization and, if necessary, freeze-drying, etc., to obtain the novel physiologically active substance of the present invention. Purification steps 1-6
The degree of purification is about 4X104-1.5X105 times, and the activity recovery rate is about 50-70%.

The physiologically active substance of the present invention thus obtained showed excellent anticancer effects against various syngeneic mouse cancers and human H transplanted into nude mice. That is, mouse colon cancer Co1
On 20-transplanted B^LD/c mice or malignant black M111 (i)-transplanted C5711L/(i-series mice and neurobud @ Neuro 2a-transplanted A-strain mice were treated with the physiologically active substance of the present invention 5,000 ~10,0
In tests in which 00 units were administered intravenously once to three times, greater inhibition of cancer growth and regression was observed compared to the control group (physiological saline administration group). Also, human-made lung ship 1'
c-10. Malignant black MIIMV-2 or neural 1'B
The physiologically active substance of the present invention 10,000-30,
In tests in which 000 units were administered intravenously once to seven times, significant cancer growth inhibition and regression were observed compared to the control group (physiological saline administration group).

As explained above, the physiologically active substance of the present invention can be obtained from a crude solution at a good yield of about 50 to 70%.

This substance has an extremely excellent anti-malignant tumor effect, has little species-specificity, has a wide range of effects, and has almost no toxicity. It can be used to treat M tumors. It is usually administered parenterally or locally, and the clinical dosage varies depending on the administration method, patient's symptoms, body weight, etc., but is usually about 10 3 to 10 8 units per dose for adults.

The present invention will be described in more detail by referring to Reference Examples and Examples below, but the present invention is not limited to these Examples.・Reference example Preparation of iminodiacetic acid immobilized resin 15 g of commercially available Epoxy Activation Co., Ltd. Phallose CL-61 was mixed with 100 g of distilled water.
■I was added to the mixture and allowed to swell for 2 hours. The mixture was then suction-filtered on a glass filter and thoroughly washed with distilled water. After thorough draining, the gel was suspended in a 2M aqueous solution of thorium carbonate containing 1 g of thorium iminodiacetate, and reacted at 65° C. for 24 hours with gentle shaking. After the reaction was completed, the reaction solution was suction-filtered using a glass filter and thoroughly washed with distilled water to obtain iminodiacetic acid-immobilized resin 451.

Resin is 0.05M IJ containing 0.15M NaCl
y acid buffer (pH 7.4) and used in purification step 5 of the example. Resin regeneration was carried out in Q, 15M NaC
0 containing l. ．． 05M ethylenediaminetetraacetate solution.

Example Propionibacter (U.S.A.C.) us ac, which was treated with formalin to kill bacteria in female rabbits (body weight 2.5-3.0 kg)
nes (Corynebac-terium par
Vum) 50 mg was injected through the ear vein. After 8 days, 100 μg of endotoxin (E. coli 020:n (Ideki Boboli Zaccharide, Difco) was injected into the ear vein, and 2 hours later, whole blood was collected from the heart. The collected blood was treated with 100 units per 100 cells. After adding sutrium, refrigerated centrifugation was performed at 5,000 rpm for 30 minutes to remove blood cells and undissolved solids.

Plasma 247 with a titer of 3,000 units was obtained from 400 rabbits.

(Purification step 1) After adding 24 g of disodium ethylenediamide/tetraacetate and 240 g of Celite to this plasma 241 and stirring for 1 hour,
It was sequentially filtered through filters with pore sizes of 3μ, 1u, and 0.2μ.

(Purification step 2) After adding 0.04M Tris-11CIl'a buffer to the filtrate 2i1 (pH 7,8>121, 0.1M
A DEAE-Sephas CL-61 (Pharmacia) column (27
×4501) gradually. Then add column equilibration buffer 1 751 and 0.04M containing 0.15M NaCl.
Tris-HC1 buffer (pH 7,8>50
0.04 containing 0.28 M NaC]
MTris-1-ICI buffer (pl+7.
2> was used for elution. The flow rate was 51/hr, and the eluate was fractionated into 81 fractions to collect active fractions. This active fraction was diluted by adding the same volume of 0.04M Tris-11C1 buffer (pl+7.8), and then applied to a DEAE-Sepha0-S CL-613 column (
IOX 13cm>. Then 0, IM Na
0.04M Tris-HCl containing CI
lt Wi liquid (P117.8) Flow rate 20 using 1 /
After washing at 0ml/hr, 0.04M Tris-11CI buffer containing 0.18M NaCl (P
117.2)5/ at a flow rate of 200 ml/hr. The eluate was fractionated into 2501 fractions and active fractions were collected. At this stage, the recovery rate of activity is 90% and the purity is 66%.
It was 0 times.

('b'i manufacturing process 3) Next, this eluate was transferred to a container and immersed in a 70°C water bath.
The eluate was heated while stirring until the temperature of the eluate reached 6θ°C. Thereafter, it was transferred to another hot water bath at 60°C, heated for 30 minutes, and then rapidly cooled to 4°C. The heat-treated solution is
By ultrafiltration! Shrunk. The recovery rate of activity in the purification steps up to this point was 88%.

(Purification step 4) 0.005M phosphate buffer containing 0.1M NaCl (
Cef7cryl S-20 sufficiently equilibrated at pH 7.4)
The concentrated solution was applied to a column (5×80 cm ) of 0 (Pharmacia) and eluted with the same buffer. The flow velocity is 40m
The active fraction was collected by fractionating 40 x 1/hr, and the active fraction was concentrated by ultrafiltration. The activity recovery rate through purification steps 1 to 4 was 82%, and the degree of purification was 2.0×10 4 times.

(f/l manufacturing step 5) A concentrated solution of the active fraction obtained by gel filtration was applied to a 2+ Zn chelate Sepha 0-scolumn. A column (+, OX 20c) packed with chelateceph 10-su (iminodiacetic acid immobilized resin) prepared by the method described in the reference example.
m ) k:, ll1g/m1 (D zinc chloride aqueous solution 120
ml was flowed at a flow rate of 20 ml/hr. Next item O, 1M
0.05 M S/rj1 buffer (plζ7) containing NaCl
．． After sufficient equilibration in step 4), the 0-condensate solution obtained in the previous step was run at a flow rate of 20 e+I/hr, and the non-adsorbed fraction eluted with the same buffer solution at +20 I/hr was collected.The activity was Most of the activity was recovered in both portions.The activity recovery rate through purification steps 1 to 5 was 66%, and the degree of purification was 5.0×10′.

(Purification step 6) The active fraction obtained in the pre-purification step was concentrated and
Contains NaC1 b O,005M') y (It m
New solution (pH 7,4>) Thoroughly equilibrated Toyovar H
W-55 (Toyo Soda) Power 5 A (1,5X90 c
m) k: Attached. The same buffer was used at a flow rate of 4 ml/hr to collect the active fraction. The recovery of activity throughout the entire purification process was 60%, and the degree of purification was 7.5×30 times. The specific activity of the physiologically active substance of the present invention thus obtained was 106 units of O, OX/protein.

Patent applicant: Dainippon Pharmaceutical Co., Ltd. Asahi Kasei Industries, Ltd. Representative Kojima - Akira

Claims (1)

[Claims] (1) At the C-terminal part...--8er-Gly-Gln-Val-
Tyr-Phc-Gly-11e-11e-Ala
A protein having a structure consisting of subunits of a polypeptide having an amino acid sequence structure represented by -Leu, and having the following properties. a) Molecular weight 40,000±5,000 (gel filtration method) 17.500±2.000 (5DS-polyacrylamide gel electrophoresis method) b) Equal weight 5.0±0.3 (isoelectric focusing method) ) C)
The specific activity in the evaluation using LM cells as defined in the text is 5.
X 106 to IX 107 units/g protein (2) By administering one or more substances having a reticuloendothelial system activating effect to rabbits, and then injecting endotoxin derived from Gram-negative bacteria, or The protein according to claim 1, which is induced by adding endotoxin ( ) derived from Gram-negative bacteria to a tissue culture system containing macrophages derived from the protein. □