RU2082430C1 - Method of antitumor agent preparing - Google Patents

Abstract

FIELD: biotechnology, medicine, oncology. SUBSTANCE: invention relates to method of preparing insoluble form of recombinant human alpha-interferon. Method involves preparing a mixture of mono-, di- and oligomers of human recombinant alpha-2-interferon at molecular-weight distribution from 17 to 5000 kDa by reduction of disulfide bonds in recombinant protein and oxidation of the formed thiols. Insoluble form can be obtained from water-soluble recombinant human alpha-2-interferon or directly from body inclusion forming in cells of the strain-superproducer. EFFECT: simplified method, increased stability of the end product. 3 cl, 5 tbl

Description

 The invention relates to biotechnology and can be used to obtain preparations of recombinant human alpha-2-interferon (RhIFN-α2) as a substance in the manufacture of antitumor agents based on the insoluble form of the recombinant human alpha-2-interferon, as well as for research purposes.

 Alpha-2-interferon is a representative of an extensive class of cytokines. Along with well-studied antiviral activity, the protein exhibits antitumor and immunomodulatory effects. For widespread use of drugs based on alpha-2-interferon, its large-scale production is necessary. The latter can be achieved by constructing producers using the recombinant DNA methodology (DVGoedell et al. Nature, 1980, vol. 287, p. 411a: V. Kravchenko et al. RF application N 5023641/13 of 01.22.92) RhIFN -α2 isolated from E. coli cells exhibits all the basic biological properties of the natural protein.

 One of the problems associated with obtaining drugs based on recombinant alpha-2-interferon is to ensure high purity of the drug. In the preparation of highly purified protein, much attention is paid to the removal of partially degraded, improperly folded, as well as oligomeric forms of RhIFN-alpha-2 (SJ Talnovski et al. Methods Enzymol. 1986, vol. 119, p. 153-165; US patent N 4534906; patent USA N 4432895) for two reasons: 1) oligomeric forms exhibit lower specific activity; 2) the possibility of side effects of these components is not ruled out.

 In contrast to alpha-2-interferon and its soluble oligomeric forms, insoluble protein oligomers do not exhibit antiviral activity in vitro (J. A. Langer, S. Pestka, Methods anzymol. 1986, vol. 119, p. 248-255). Therefore, the use of an insoluble form of recombinant human alpha-2 interferon, which is based on oligomers, as an antitumor agent is an unobvious solution.

 No information on antitumor agents based on the insoluble form of recombinant alpha-2-interferon obtained without special addition of precipitating agents was found in the literature.

 As a prototype can be considered a method of obtaining an insoluble complex of alpha-interferon and copper ions, intended for injection drugs (US patent N 4871538). The drug is prepared by mixing alpha-interferon at a concentration of 0.1-1 mg / ml, chloride, acetate, sulfate or copper citrate (0.05-0.09 mg / ml) in a buffer with a pH of 7.5-3.2. Although the preparation is simple in terms of the composition of the components, the optimum values for the concentrations of protein and copper salts and pH values are within narrow limits and deviations from these values lead to a sharp decrease in the yield of the insoluble form of alpha interferon. In addition, the presence in the environment of other proteins capable of forming complexes with copper ions prevents the complex formed and causes the dissolution of the already formed.

 The aim of the invention is to simplify the method of obtaining an insoluble form of recombinant human alpha-2-interferon and increase the stability of the target product.

 To achieve the goal, an insoluble oligomeric form of recombinant human alpha-2-interferon is obtained, which is a mixture of mono-, di- and oligomers with a molecular weight distribution of 17 to 5000 kD, or from insoluble inclusion bodies (TB) formed in the cells of the strains super-producers, or from highly purified water-soluble RhIFN-α-222.

 A method of obtaining an insoluble form of a2-2-interferon from inclusion bodies.

As a source of recombinant human alpha-2-interferon, transformed with plasmid pIF1 ⁶ encoding the synthesis of RhIFN-alpha-2, E. coli strain (RF application N 5023641/13) was used. Superproduction of a recombinant protein is accompanied by the formation of insoluble alpha-2-interferon inclusion bodies.

 Insoluble inclusion bodies after lysis or ultrasonic disintegration of E. coli SG 20050 cells (pIF 16) are precipitated by centrifugation together with cell wall debris. Removal of a significant part of the impurity proteins is achieved by washing the precipitate with buffer solutions, solutions of a nonionic detergent, for example, Triton X-100 at a concentration of up to 1%) and denaturing agents (for example, urea at a concentration of 3-8 M). The losses of alpha-2-interferon are negligible. The complete dissolution of interferon is carried out in solutions of 5-7 M guanidinium chloride (Gdm.HCl) and alpha-2-interferon is again precipitated, removing the denaturing agent by dialysis of the resulting solution against water or buffer. As a result of solubilization and reprecipitation, additional removal of impurity proteins is achieved.

As a result of the studies, the following method was chosen to obtain an insoluble, partially purified form of recombinant alpha-2-interferon:
1) destruction of cells, removal of soluble fraction;
2) washing insoluble material (TB) with solutions with increasing urea concentration to remove ballast proteins;
3) dissolution of the precipitate 7 M Gdm.HCl;
4) precipitation of RhIFN-2 with dialysis against water, washing the precipitate with water.

 The precipitated protein is collected by centrifugation, washed with distilled water and stored as a suspension in water. If necessary, the protein can be freeze-dried or stored as a solution in 7 M Gdm.HCL.

 The insoluble form has the following characteristics. The content of alpha-2-interferon, estimated by gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions, is 78-80%. The bulk of the protein is oligomers with unnatural intermolecular disulfide bridges. The molecular weight of SS oligomers is very high, because they practically do not fractionate in a 15% polyacrylamide gel in the presence of sodium dodecyl sulfate, but without preliminary treatment with 2-mercaptoethanol, and are retained at the interface between the concentrating and separating gels.

 A method of obtaining an insoluble form from soluble RhIFN-α.

In unfractionated inclusion bodies, depending on the producer cell growth phase, various amounts of cellular components are found: membrane proteins, ribosomal proteins, host and plasmid DNA, RNA, etc. Given the high requirements for the purity of recombinant proteins, which are drug substances, the insoluble form of alpha-2 interferon can be obtained from a highly purified water-soluble protein. To standardize the purification procedure and the renaturation process, the insoluble form, the preparation of which is described above, is destroyed by reducing disulfide bonds and reversible sulfitolysis of the formed thiols. After sulfitolysis in a polyacrylamide gel in the presence of sodium dodecyl sulfate without treatment with 2-mercaptoethanol, only the monomeric form of interferon was detected. The process of renaturation of the sulfitated monomeric form is carried out under specially selected conditions in a mixture of oxidized and reduced glutathione. The chosen renaturation regimen promoted not only the removal of modifying groups, but also mainly the correct closure of disulfide bonds. Final purification is carried out by ion exchange chromatography. Thus obtained alpha-2-interferon had a high degree of purity and good solubility in water. In general, this scheme for producing water-soluble alpha-2-interferon includes the following main stages:
1) the destruction of biomass and obtaining a partially purified insoluble form of RhIFN-a2 as described above;
2) the destruction of SS-oligomers and the translation of all protein molecules into one form by sulfitolysis;
3) renaturation of the sulfated protein in the presence of a mixture of oxidized and reduced glutathians;
4) additional purification by chromatography on cation exchange resin.

Purified according to the described scheme, RhIFN-α2 has the following characteristics:
1) the concentration of water-soluble alpha-2-interferon, determined by the Lowry method is 0.3-1.25 mg / ml;
2) under completely denaturing conditions, the protein has a high degree of purity, more than 95%. Depending on the batch, 1-4% of the protein corresponding to the molecular weight of the alpha-2-interferon dimer can be detected in the preparation;
3) in the absence of pretreatment with 2-mercaptoethanol during electrophoresis under native conditions, the main band of the alpha-2-interferon monomer and 1 or 2 minor bands of its conformational isomers are found;
4) a high degree of purity is confirmed by the results of HPLC;
5) an admixture of proteins of E. coli SG 200 50, determined by enzyme-linked immunosorbent assay, less than 100 ng / mg alpha-2-interferon;
6) an admixture of bacterial DNA determined by hybridization with labeled probes, less than 6 ng / mg;
7) an admixture of lipopolysaccharides, determined using the TAL test, 0.3-0.4 ng / mg protein;
8) the specific activity of the drug, determined by the suppression of the cytopathic effect of the virus of vesicular stomatitis on the cell culture of human skin-muscle tissue L68 is (1-6) • 10 ⁸ IU / mg;
9) with intraperitoneal administration to outbred mice at a dose of 10 ⁶ units / ml / mouse, no deaths were observed (group of 5 animals);
10) intravenous administration of 10 ⁶ units / ml / kg of weight did not cause a pyrogenic reaction in rabbits for 3 hours

 Obtaining an insoluble fraction from highly purified alpha-2-interferon is carried out by restoring natural disulfide bonds with subsequent oxidation of the formed thiols in a neutral or slightly alkaline environment in the presence of atmospheric oxygen. The resulting oligomers precipitate; upon gel electrophoresis without preliminary processing of the material with 2-mercaptoethanol, they are detected as a set of discrete bands. Along with the oligomeric one, the monomeric form of RhIFN-α2 is found in the precipitate. The ability to form insoluble oligomeric forms is a protein property that is independent of the nature of the host strain, and can be used to produce an insoluble fraction of recombinant alpha-2 interferon isolated from various sources.

Example 1. 5 g of biomass of E. coli SG 200 50 (pIF 16), superproducing human alpha-2-interferon, suspended in 100 ml of buffer A (50 mm Tris HCl, pH 7.0-8.0) containing 2 mm EDTA. The suspension is voiced at maximum power in five series for 1 minute at intervals of 1-2 minutes, then centrifuged at 12,000 rpm for 30 minutes. The collected precipitate was suspended in 100 ml of buffer A containing 2 mM EDTA and 1% X-100 newt, stirred for 30 minutes, and the precipitate was collected by centrifugation as described above. The collected precipitate is sequentially treated, as described above, with solutions of 3 and 6 M urea in buffer A. The collected precipitate after washing is dissolved in buffer A containing 7M Gdm.HCL for 1 h, the resulting solution is centrifuged at 12000 rpm for 30 min and collect the supernatant. The solution is transferred to a dialysis bag and dialyzed against distilled water during the day. The precipitate of the insoluble form of RhIFN-α2 was collected by centrifugation at 12000 rpm, 2 ^° C, for 10 min, suspended in 100 ml of water and centrifuged again. The precipitate was suspended in 20 ml of water and stored as a suspension at 4 ^° C. Electrophoretic purity of alpha-2-interferon under completely denaturing conditions of about 80% Yield 30 mg / g biomass. (Drug N 1).

Example 2. To a 3.4 ml solution of a partially purified insoluble form of alpha-2-interferon (11.75 mg / ml) add 23 ml of buffer A and 2.7 ml of a sulfitation solution containing 200 mg / ml Na ₂ S ₄ O ₆ and 100 mg / ml Na ₂ S ₄ O ₆ in water, and left for 20 hours at room temperature.

All subsequent procedures are carried out at a temperature of 4-6 ^o C. A solution of sulfated protein is dialyzed for 16 hours against 10 l of a 5 mm solution of ammonium bicarbonate, pH 9.0. The precipitate formed during dialysis is separated by centrifugation at 12,000 rpm for 20 minutes at 2 ^° C. 160 ml of 0.1 M Tris HCL, pH 8.0, containing 2 mM EDTA are added to the supernatant. To the resulting solution were added 49 mg of reduced glutathione and 9.8 mg of oxidized glutathione, the mixture was stirred on a magnetic stirrer and left for 16 hours to renature the protein.

 The renatured protein solution was dialyzed against 10 L of a 50 mM sodium acetate solution, pH 4.5, for 16 hours. The dialyzed solution was applied at a rate of 15 ml / h to a 10 ml column filled with carboxymethyl cellulose and equilibrated with 0.1 M sodium acetate, pH 5.0. After applying the solution, the column is washed with 50 ml of 0.1 M sodium acetate, pH 5.0 and the protein is eluted with a linear gradient of sodium acetate (from 0.1 to 0.5 M), pH 5.0. The total volume of the gradient is 100 ml, the elution rate is 20 ml / h, the volume of fractions is 3 ml. The yield of alpha-2-interferon is determined by the absorption peak at 280 nm (usually eluted with 0.3 M salt), confirmed by the results of electrophoretic analysis in a 15% polyacrylamide gel under completely denaturing conditions when 30 μl of the analyzed fraction is added to the well. Fractions containing alpha-2-interface combine, measure the volume, protein concentration and biological activity.

Example 3. 35 ml of alpha-2-interferon-containing solution (concentration 0.34 mg / ml) obtained after chromatography was transferred to a dialysis bag and dialyzed against 4 L of 50 mM Tris HCI, pH 9.0 for 16 hours at 4 ^o C. 2-mercaptoethanol is added to the solution to a final concentration of 0.1 M, the mixture is kept at 37 ^o C for 3 hours and again dialyzed against two shifts of 2 L each with 50 mM Tris HCI, pH 7.0 over 20 hours The precipitate of the insoluble form of alpha-2-interferon is collected by centrifugation at 10,000 rpm for 20 minutes The precipitate was suspended in 2.6 ml of Tris HCI, 7.0, and stored as a suspension until further use. The yield of recombinant alpha-2-interferon is 10.6 mg. (Example N 2)
Example 4. The study of the antitumor activity of the insoluble form of RhIFN-α2 (Preparation No. 1).

 Testing of the antitumor activity of an insoluble form of recombinant human alpha-2-interferon is carried out by the method (Subrenal capsule assay, A. Bogden et al. Proc. Sympos. 1970, p.231-250) using primary breast tumors, skin melanoma and human breast cancer metastases. For this, xenografts (pieces of tissue) of the tumor are implanted under the renal capsule of mice and the tumor growth rate is determined on the sixth day by measuring their mass. As a control, physiological saline and highly purified water-soluble recombinant human alpha-2-interferon are used (commercial preparation Reaferon). The amount of insoluble form of recombinant alpha-2-interferon introduced is determined based on the specific activity of the drug.

Tumor tissue removed from patients during surgery is placed in a nutrient medium with antibiotics and, after a short wash (20-30 minutes), cut into small fragments. The latter are carefully separated from necrotic or bloody areas, from macroscopically visible adipose tissue and only after that they are cut into small pieces. Samples with a weight of 1 mg were taken and implanted into mice (C 57 BI / 6X TWA / F ₁ ) under a kidney capsule (2 xenografts per animal), the operation was performed under hexanal anesthesia, and all stages of the operation were performed under sterile conditions. Animals are slaughtered on the 6th day under ether anesthesia, xenografts are removed and weighed.

A suspension of the insoluble fraction of RhIFN-α2 (preparation No. 1), the commercial preparation RhIFN-2 (Reaferon, NPO Vector) was administered to mice intraperitoneally (ip) in a volume of 0.5 ml daily for 5 days. A single dose of reaferon is 2 • 10 ⁴ IU per animal. The insoluble fraction is introduced in an equivalent weight (protein) amount (0.1 μg, assuming the specific activity of soluble interferon equal to 2 • 10 ⁸ IU / mg). Each group includes 10 experimental and 10 control animals. From the results of the experiment, are presented in table. 1, it is seen that the insoluble fraction of RhIFN-α2 with an interferon content of 80% has a pronounced antitumor effect.

 Example 5. Comparative evaluation of the drug N 1 from TV and Reaferon on the antitumor effect on the metastatic tissue of breast cancer in the lymph node.

 For heterotransplantation, the tumor tissue is treated and injected under the capsule of the kidneys of mice, and after treatment, they are removed and weighed according to the method described in example 4.

For treatment of experimental mice, the preparation of the insoluble fraction of RhIFN-α2 (Example 1) and alpha-2-interferon are administered intramuscularly (IM) daily for 5 days, 0.2 ml each, a single dose is 2 • 10 ⁴ IU per mouse. In the experiment, no 10 experimental and 10 control animals were used. Each specimen is implanted with 2 xenografts. The results are presented in table. 2. It is seen that the insoluble fraction of RhIFN-α22 exhibits a higher anti-cancer activity than the soluble alpha-2-interferon against human breast cancer metastases.

 Example 6. The effect of the insoluble fraction of RhIFN-2, homogeneous by a22-IFN, from TB N 2 and reaferon on the primary cancer of the human breast.

 The process of heterotransplantation of tumor tissue under the capsule of the kidney of mice and the selection of xenografts after treatment is carried out according to the method described in example 4.

For treatment, each drug is administered intraperitoneally (i / b) and intramuscularly (i / m) daily for 5 days. A single (per injection) dose is 2 • 10 ⁴ ME per mouse. The volume of input material is 0.5 ml with a / m introduction or 0.2 ml with a / m introduction. Each group includes 10 experimental and 10 control animals. The results of the experiments are presented in table. 3.

 The data obtained indicate that the insoluble fraction of RhIFN-a2 homogeneous with respect to α2-interferon has a more pronounced antitumor effect on the tissue of the primary cancer of the human breast.

 Example 7. The effect of the drug N 2 on the tissue metastases (in regional lymph nodes) tumors of the human mammary gland.

 Pieces of metastatic tissue of a human breast tumor are treated, cut into small pieces weighing 1 mg and transferred under the capsule of the kidney of mice according to the method described in example 4. 2 xenografts are implanted into each mouse. Experimental mice are treated, the control drug is not administered. The treatment is carried out according to the scheme described in example 4. The animals are killed on the 6th day, xenografts are removed and weighed. In experiments and control using 10 individuals. The results are shown in table. 4.

 From the table. Figure 4 shows that alpha-2-interferon and drug N 2 have a pronounced antitumor effect. The drug N 2 causes regression of the tumor.

 Example 8. The study of the effect of the drug RhIFN-α2 from tissue melanoma of the skin (primary tumor).

 To study the antitumor and / or growth-inhibiting effect of the preparations, tissue pieces of the primary tumor of skin melanoma are treated, cut, small pieces weighing 1 mg are taken and implanted under the kidney capsule into mice according to the method described in Example 4. 2 xenografts are implanted into each mouse. In the experiment and control took 10 mice. Experimental mice are treated with preparations as in Example 4. After 5 days, the mice are sacrificed, grafts are removed and weighed on a torsion balance. The observation results are presented in table. 5.

 As a result of the experiments, a significant inhibitory effect of the preparations on the skin melanoma tissue was shown. Moreover, there is even a regression, a decrease in the mass of xenografts.

 Thus, a method for producing an insoluble form of RhIFN-α2 having antitumor activity when tested in the SRCA test, using a breast tumor and human melanoma as a model, is proposed. The sensitivity of melanoma to the action of the drug is higher than the sensitivity of breast cancer. The sensitivity of metastatic tissue to the action of the insoluble form of RhIFN-α2 is higher than the sensitivity of primary tumors.

Claims (3)

 1. A method of obtaining an antitumor agent based on an insoluble form of alpha-2-interferon, characterized in that an insoluble mixture of mono-, di- and oligomers of recombinant human alpha-2-interferon is obtained with a molecular weight distribution of from 17 to 5000 kDa by restoring disulfite bonds of the recombinant protein and oxidation of the formed thiols.  2. The method according to p. 1, characterized in that the insoluble form of alpha-2-interferon is obtained by restoring a water-soluble recombinant human alpha-2-interferon in the presence of a disulfide bond reducing agent at pH 7 10, dialyzed against water or a buffer solution in the absence of reducing agent during the day before the formation of a precipitate of the target product.  3. The method according to p. 1, characterized in that an insoluble form of alpha-2-interferon is obtained directly from the inclusion bodies formed in the cells of the superproducer strain, for which the bacterial cells are destroyed, the water-soluble components are separated by centrifugation, the impurity components are sequentially extracted with 0.1 solutions 1.0% Triton X100, 3 M and 6 M urea, dissolve the precipitate in 7 M Gaunidine chloride and precipitate the protein by dialysis against water or a buffer solution containing no guanidine salts.

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