RU2730657C1 - Anti-tumour agent based on recombinant vaccinia virus strain and method for production thereof - Google Patents

Abstract

FIELD: biotechnology; medicine.SUBSTANCE: invention relates to biotechnology and medicine. Antineoplastic agent based on recombinant vaccinia virus strain contains recombinant strain VV-GMCSF-Lact of vaccinia virus in amount of (1-2)×10PFU capable to suppress the growth of solid malignant tumour, deposited in the Federal Budget Institution of Science the State Scientific Centre of Virology and Biotechnology VECTOR under number V-688, and pharmaceutically acceptable solvent to 1 ml. Initial components are specified at content per 1 ml. Method for preparing the anti-tumour agent is also provided.EFFECT: invention provides higher anti-tumour efficacy of the agent and wider range of its functional activity in relation to glioblastoma, lymphosarcoma and breast carcinoma (solid tumours of humans and animals).7 cl, 3 dwg, 5 ex

Description

The group of inventions relates to the field of medicine and relates to an antitumor agent containing a recombinant vaccinia virus strain W-GMCSF-Lact and a method for its production. The agent can be used to treat malignant neoplasms (solid tumors) of mammals.

In connection with the large and growing problem of fighting cancer, the colossal variety of types of tumors, instability or lack of an immune response to tumor cells and the emergence of immunodeficiency states in cancer, work on the development and production of new antineoplastic agents with a multifunctional mechanism of action becomes especially urgent. It is very important that these drugs have highly selective oncotoxic properties, which would facilitate the recognition and destruction of only tumor and metastatic cells, as well as the release of tumor-associated antigens and their presentation to the host's immune system. Non-pathogenic or attenuated natural and recombinant viruses - oncolytic viruses that have mechanisms for recognizing cancer cells, have selective cytolytic potential and, possibly, express genes of immunologically active and other proteins that specifically enhance their antitumor activity - are almost ideally suited for the role of such agents. In addition to direct lysis of cancer cells, oncolytic viruses are able to induce the body's immune response by stimulating adaptive and innate immunity (1-3).

There are known agents based on bacterial oncolytic strains of Clostridium ghonii with the ability to suppress the growth of solid malignant tumors (4). As oncolytic strains, the strains Clostridium ghonii MW-DCG_HNCv18, Clostridium ghonii MW-DCG_CCv17 and Clostridium ghonii MW-DCG_LCv26 deposited at the National Measurement Institute, 1/153 Bertie Street, Victoria, Port Australia, Melbourne correspond. registration numbers V12 / 001485, V12 / 001487 and V12 / 001486.

There is a known agent based on the oncolytic strain ZhEV-15 ^L of the Coxsackie B6 virus, which selectively infects and lyses human tumor cells and has a fragment of the genomic sequence, which is its marker feature (5). This strain was deposited in the FBSI SSC VB "Vector" under registration number V-576.

A known composition for the treatment of oncological diseases, containing a recombinant strain of the Sendai virus belonging to the genus Respirovirus, the Paramyxoviridae family, obtained on the basis of viral material deposited in the American collection of cell cultures ATCC under the number PTA-13024 and a pharmaceutically acceptable carrier (6). Pharmaceutically acceptable carriers include fresh chicken embryonic allantoic fluid, lyophilized allantoic fluid, saline or aqueous buffers such as PBS and the like. In addition, the composition of the present invention may contain other additives such as adjuvants, stabilizers, antioxidants and the like.

The disadvantages of the known composition are the multicomponent nature, laboriousness of its preparation, as well as the use of non-standardized components.

Currently, a wide range of viruses have been described that are used for virotherapy of malignant neoplasms. Currently in the USA, Canada, China and European countries (Germany, Hungary) a number of vaccinia virus (VACV) strains have been tested in clinical trials for their antitumor potential (7). GL-ONC1 and Pexa-Vec / TG6006 are the leaders in the number of clinical trials among recombinant vaccinia viruses.

Known antitumor agent GL-ONC1 based on a recombinant vaccinia virus strain, including an attenuated version of L-IVP, containing three insertion cassettes encoding a fusion protein of luciferase (Renilla) and green fluorescent protein (Aequorea), β-galactosidase and β-glucuronidase, and pharmaceutical suitable carrier (8). The transgenes are inserted at loci F14.5L, J2R (encodes viral thymidine kinase) and A56R (encodes viral hemagglutinin), which determines the attenuation of the recombinant virus. The antitumor agent GL-ONC1 specifically infects tumor cells and destroys them by oncolysis, which further triggers an antitumor immune response.

The closest to the claimed agent, the prototype, is the Pexa-Vec antitumor agent containing the recombinant vaccinia virus JX-594 strain and a pharmaceutically acceptable excipient (9). Vaccine virus strain JX-594 was obtained on the basis of the American vaccine strain Wyeth by inserting two transgenes into the region of the viral thymidine kinase (virulence factor) gene deletion: human granulocyte-macrophage colony-stimulating factor (GM-CSF) and E. coli-galactosidase. Pexa-Vec is in phase 3 clinical trials for human hepatocellular carcinoma.

The method for preparing the Pexa-Vec antitumor agent consists of three main stages, including obtaining a vaccinated material for the antitumor agent, purifying it and diluting the purified vaccinated concentrate with a pharmaceutically acceptable solvent to a virus concentration of 1 × 10 ⁹ pfu. More specifically, the known method includes the following steps:

1. Obtaining vaccinated material for a drug, including the production of virus in HeLa cells (culture of producer cells), lysis of producer cells with a hypotonic lysis buffer (obtaining a crude lysate containing virus and cell debris).

2. Purification of vaccinated material for a drug, including the treatment of lysate of producer cells with enzymes: nuclease (benzonase) to remove DNA from producer cells (HeLa) and protease (TrypLE ™ Select) to remove proteins from producer cells (HeLa), tangential filtration of vaccinated material.

3. Dilution of the purified vaccinated concentrate with a suitable solvent to a virus concentration of 1 × 10 ⁹ pfu of JX-594 (one dose).

The known method makes it possible to obtain the target product with the DNA content of the producer cells - 3 μg / dose, the protein content of the producer cells - 4 mg protein / dose (10).

The disadvantages of the Pexa-Vec anticancer agent (prototype) are:

- insufficient oncolytic activity of Pexa-Vec, associated with the use of a less virulent vector strain of vaccinia virus (Wyeth against L-IVP) and due to the lack of insertion of the gene of the oncotoxic protein lactaptin;

- low level of expression of GM-CSF Pexa-Vec (40 pg / ml);

- insufficient level of Pexa-Vec attenuation, associated with the preservation of an additional virulence gene - the viral growth factor;

- unsatisfactory onkoselectivity of Pexa-Vec due to the removal of only one gene - the thymidine kinase gene;

- the presence in the Pexa-Vec genome of an extended insertion (3000 bp) of a non-sense reporter gene β-galactosidase of E. coli.

The disadvantages of the known method of obtaining the antitumor agent Pexa-Vec are:

- the use of a culture of producer cells that does not provide the maximum specific concentration of the virus (the number of viral particles per cell of the producer culture);

- insufficient (not maximum) degree of drug purification, which can be explained by the absence of a stage for removing cell debris (clarification with low-speed centrifugation) after treatment of the lysate of producer cells with enzymes (benzonase + trypsin), as well as insufficient use of only tangential filtration to remove impurities.

The task of a group of inventions is to create a highly effective antitumor agent with a higher oncolytic activity and increased levels of attenuation, oncoselectivity and expression of GM-CSF.

EFFECT: increasing the antitumor effectiveness of the agent and expanding the range of its functional activity.

The task is achieved by the proposed antitumor agent, which contains a recombinant vaccinia virus (strain VV-GMCSF-Lact FBSI GNTs VB "Vector" - V-688), obtained in a culture of kidney cells of the African green monkey 4647 and producing secreted granulocyte-macrophage factor colony stimulating -CSF) human and non-secreted oncotoxic protein lactaptin, and a pharmaceutically acceptable solvent, with the following content of components (per 1 ml):

Vaccine virus strain VV-GMCSF-Lact (1-2) × 10 ⁷ PFU pharmaceutically acceptable solvent up to 1 ml

The proposed tool is a frozen solution - a dense solidified white mass. After thawing - a colorless liquid, transparent or slightly opalescent, without impurities, pH 6.2-6.8. The inventive agent is sterile, non-toxic, does not contain mycoplasms and foreign viral agents. The product does not contain preservatives or antibiotics. The content of endotoxins in the medicinal product does not exceed 0.5 EU / ml, the content of bovine serum albumin is not more than 50 ng / ml, the content of cellular DNA is not more than 10 ng / ml, and the protein content is not more than 100 μg / ml.

The specific activity of the proposed agent is measured as the dose of infection causing a 50% lytic effect of MDA-MB-231 human breast cancer cells, and is not more than 0.1 pfu / cell.

As the pharmaceutically acceptable solvent, physiological saline or physiologically acceptable buffered saline can be used.

Characteristics of the active component. The double recombinant strain VV-GMCSF-Lact is designed on the basis of the moderately pathogenic Russian L-IVP strain of the vaccinia virus, contains deletions of fragments of the viral thymidine kinase and growth factor genes, in whose regions the gene of granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-structure corresponds to the cDNA of messenger RNA of human GM-CSF (GenBank Ace. M11220.1), in the central part of the viral thymidine kinase gene between positions 81277 and 81308 bp. (GenBank Ace. KP233807.1); the human GM-CSF gene is expressed under the control of the natural vaccinia virus P7.5k promoter and produces a secreted form of biologically active human GM-CSF in mammalian cells; the lactaptin gene encoding a fragment of human kappa-casein 23-134 amino acid residues in the left terminal region of the viral genome between positions 7770 and 8071 bp. (GenBank Ace. KP233807.1); The lactaptin gene is expressed under the control of the synthetic vaccinia virus P7.5synth promoter and produces the oncotoxic recombinant protein lactaptin, which specifically induces apoptotic death of human cancer cells in vitro and in vivo (11). The insertion of the lactaptin gene as a transgene into the deletion region of the viral growth factor gene contributes to additional attenuation (weakening) of the virus in relation to normal cells and enhances its lytic (cytotoxic) activity in relation to cancer cells. The VV-GMCSF-Lact strain was deposited in the State collection of viral pathogens and rickettsioses of the FBSI SSC VB "Vector" under the number V-688 (12).

As a rule, oncolytic viruses are able to activate more than one cell death pathway in infected cells; however, it is known that the main pathway of death induced by adenoviruses is autophagy, while for vaccinia virus programmed necrosis prevails (13). The target of the recombinant vaccinia virus VV-GMCSF-Lact is EGFR-positive cells. Increased expression of the EGFR (epidermal growth factor receptor) gene is characteristic of many human cancer cells. The combination of deletions of VGF and tk genes (double recombinants) provides the virus with additional targeting against cancer cells and dramatically reduces its virulence against healthy cells. The main mechanism of death of EGFR-positive breast cancer cells under the influence of the proposed antitumor agent based on the recombinant VV-GMCSF-Lact strain of vaccinia virus is RIPK-1-independent EGFR-dependent necrosis. In addition, tumor cells infected with the VV-GMCSF-Lact virus are actively captured by peritoneal macrophages.

The proposed antitumor agent is intended for parenteral (intratumoral) administration into a solid malignant tumor at an effective dose (1-2) × 10 ⁷ PFU / ml × 10 ⁷ PFU / ml once a week in a course of 4 injections. A solid malignant tumor can be glioblastoma, lymphosarcoma, breast carcinoma.

The task is also achieved by the proposed method of obtaining the claimed agent, which includes the following main stages:

Stage 1. Obtaining a vaccinated material for an antitumor agent, including accumulating a culture of producing cells 4647, infecting a culture of producing cells 4647 with a vaccinia virus, cultivating an infected producer culture, collecting vaccinating material for a drug.

Stage 2. Purification of vaccinated material for the agent, including the following stages: precipitation of vaccinated material by ultracentrifugation, homogenization of vaccinated material with ultrasound, processing of the homogenate with enzymes (benzonase and trypsin), clarification of vaccinated material at low-speed centrifugation (removal of cell debris by ultra-purified double-purified vaccinated material) ultracentrifugation in a layer of 36% sucrose (removal of ballast high molecular weight cellular components).

Stage 3. Obtaining an antitumor agent by diluting the purified vaccinated concentrate with 0.9% sodium chloride solution to a virus concentration of at least 1.0 × 10 ⁷ pfu / ml and not more than 2.0 × 10 ⁷ pfu / ml.

The proposed method is as follows.

Stage 1. Obtaining vaccinated material is carried out in a standard way. First, the accumulation of the producer culture is carried out by serial passages of the cell culture of the 4647 line (African green monkey kidney cells, certified for the production of vaccines at the FBSI SSC VB "Vector"), recovered from the seed bank. After accumulation of the required number of cells, the resulting culture-producer is used to obtain vaccinated material for an antitumor agent. Virus production is carried out in roller bottles with a monolayer of producer cells 4647. Growth medium is removed from roller bottles with a monolayer of producer cells and 10 ml of a virus suspension is added to each bottle (strain VV-GMCSF-Lact FBUN SSC VB "Vector" - V- 688) with a titer of at least 10 ⁷ pfu / ml to infect cells and incubate in a roller installation at a temperature of (37 ± 1) ° С for 1 hour to adsorb the virus on the cells. Then, an additional supporting medium (DMEM medium, kanamycin 0.06 mg / ml) is added to each roller bottle. Infected containers are incubated in a roller installation at a temperature of 36.5-37.5 ° C for 2 days until a 100% CPP is formed (cytopathic effect).

Stage 2 Purification of vaccinated material for antitumor agent, obtaining a viral concentrate

Obtained in stage 1 vaccinated material is transferred into sterile centrifuge beakers and concentrated by ultracentrifugation at 4-8 ° C and 15300 rpm for 1 hour. The supernatant is removed and the pellet is resuspended in 1 mM Tris-HCl, pH 9.0.

The resulting suspension of vaccinated material is sonicated (homogenized) on an ultrasonic disintegrator. The sonication is carried out three times for 20 seconds, with cooling after each treatment for 10 seconds on ice.

To the obtained homogenate of vaccinated material, benzonase enzyme is added to a final concentration of 40-42 activity units / ml and MgCl ₂ and incubated for 30-40 minutes at room temperature. A trypsin solution is added to the homogenate with benzonase to a final concentration of 0.10-0.12 mg / ml and incubated for another 30-40 minutes at 37 ° C. Enzyme treatment allows the destruction of nucleic acids and proteins of producer cells.

The homogenate of vaccinated material treated with enzymes is clarified by centrifugation at 3000 rpm for 10-15 minutes at a temperature of 4-8 ° C (removal of cell debris).

The clarified concentrated vaccinated material is subjected to additional purification from ballast high molecular weight cellular components in the mode of double ultracentrifugation in a layer of 36% sucrose. Ultracentrifugation is carried out at 12000 rpm for 60-80 minutes at a temperature of 4-8 ° C

объема, наносимого на слой 36%-ной сахарозы при первом ультрацентрифугировании), обрабатывают ультразвуком на ультразвуковом дезинтеграторе (как указано выше) и проводят повторное ультрацентрифугирование в слое 36%-ной сахарозы.After the first ultracentrifugation in a layer of 36% sucrose, the precipitates of vaccinated material are reduced in 0.001 M Tris-HCl, pH 9.0 (in

volume applied to the layer of 36% sucrose during the first ultracentrifugation), sonicated on an ultrasonic disintegrator (as indicated above) and repeated ultracentrifugation in a layer of 36% sucrose.

After the second ultracentrifugation in a layer of 36% sucrose, the precipitates of vaccinated material are resuspended in 0.9% sodium chloride solution (saline) and treated with ultrasound as described above. As a result, a purified concentrate of an antitumor agent based on a recombinant vaccinia virus with a virus titer of 5.8 × 10 ⁸ - 1.6 × 10 ⁹ PFU / ml is obtained.

Stage 3 Obtaining an antineoplastic agent.

Purified vaccinated material is diluted with 0.9% sodium chloride solution to a calculated virus concentration of at least 1.0 × 10 ⁷ pfu / ml and not more than 2.0 × 10 ⁷ pfu / ml, packaged and stored at minus 40-42 ° C.

The defining differences between the proposed agent and the prototype are:

- the product contains a lytically active (virulent) VV-GMCSF-Lact strain with a double deletion of the thymidine kinase and growth factor genes, which ensures high attenuation of the virus and the safety of its use for immunodeficient cancer patients. To enhance the antitumor activity, the strain contains, as transgenes, the GM-CSF gene and the cytotoxic protein gene lactaptin, which provide specific death of cancer cells.

- the strain W-GMCSF-Lact of the vaccinia virus is used in a drug with an experimentally selected, optimal virus titer of (1-2) × 10 ⁷ PFU / ml, which allows for selective infection and active lysis of a wide range of human tumor cells with a small amount of viral particles ...

The defining differences between the proposed method and the prototype are:

- the cells of the kidney of the African green monkey line 4647 are used as a producer culture for the production of the virus, which makes it possible to obtain the maximum yield of viral particles (1-2) × 10 ⁷ PFU / ml) compared to the kidney cells of the African green monkey line Vero (6, 4 × 10 ⁶ pfu / ml), since 4647 cells are larger than Vero cells, they have a larger volume of cytoplasm, which allows an increase in the concentration of viral particles in one cell.

- the homogenate of vaccinated material treated with enzymes is clarified by centrifugation at 3000 rpm for 10-15 minutes, which allows to effectively remove cell debris and ensure the maximum degree of purification of the antitumor agent;

- the clarified concentrated vaccinated material is subjected to additional purification from ballast high-molecular cellular components by two-time ultracentrifugation at 12000 rpm in a layer of 36% sucrose for 60-80 minutes, which makes it possible to obtain a product with a minimum amount of ballast high-molecular cell components that can cause side toxic effects.

The proposed agent has increased antitumor efficacy due to higher oncolytic activity, as well as high levels of attenuation, oncoselectivity and expression of GM-CSF. The inventive agent has a wider range of functional activity (in comparison with the prototype), since it exhibits oncolytic activity against such solid malignant tumors as glioblastoma, lymphosarcoma and breast carcinoma.

The invention is illustrated by the following figures:

FIG. 1. Antitumor activity of VV-GMCSF-Lact against human glioblastoma U87-MG in vivo. Growth dynamics of the U87-MG tumor in SCID mice treated with the claimed agent.

FIG. 2. Antitumor activity of VV-GMCSF-Lact against drug-resistant lymphosarcoma in RLS mice in vivo. Dynamics of changes in the paw size of CBA mice with intramuscularly transplanted RLS tumor cells during treatment with the claimed agent.

fig. 3. Antitumor activity of VV-GMCSF-Lact in relation to the developed human breast adenocarcinoma tumor MDA-MB-231. Tumor growth dynamics in SCID mice treated with the claimed agent.

For a better understanding of the essence of the invention, examples of its implementation follow.

Example 1. Obtaining the claimed antineoplastic agent based on the recombinant strain VV-GMCSF-Lact of the vaccinia virus.

Construction of recombinant plasmid DNA pVGF-FR2-PE / L-Pat, transfection of CV-1 cells with the recombinant plasmid pVGF-FR2-PE / L-Pat, and production of the recombinant vaccinia virus VV-GMCSF-Lact strain are carried out according to a known method (12). The purified recombinant vaccinia virus strain VV-GMCSF-Lact with a titer of 10 ⁹ PFU / ml is stored in packaged form at -80 ° C (industrial strain).

Stage 1. Obtaining vaccinated material for an antitumor agent

The accumulation of the producer culture is carried out by serial passages of the cell culture of the 4647 line, certified for the production of vaccines at the FBSI SSC VB "Vector", restored from the seed bank. A cell culture that has formed a dense monolayer without signs of degeneration and contamination by microbial flora is used for subculturing. The technique of the passage is as follows: the growth medium is removed from the culture mattresses with a capacity of 50 ml, 10 ml of phosphate-buffered saline (PBS) heated in a thermostat to a temperature of (32 ± 0.5) ° C is added to each mattress. The cell monolayer is washed with gentle shaking, then removed with a PBS pipette. The procedure for washing the PBS monolayer is repeated twice, then 1 ml of a dispersing solution heated in a thermostat to a temperature of 32 ± 0.5 ° C (100 ml of PBS / EDTA solution + 50 mg of trypsin (Trypsin, for cell culture, Gerbu) is added to the mattresses) ), incubate the mattresses at (37 ± 0.5) ° C until the cells are detached, then 9 ml of the growth medium is added. The vial with cells is shaken 2-3 times, avoiding the formation of abundant foam, carefully pipet the contents to obtain a homogeneous suspension.

Next, the resulting cell suspension is diluted with growth medium (culture medium DMEM, 10% fetal calf serum, 0.06 mg / ml kanamycin) to a concentration of 0.10 million viable cells in 1 ml of growth medium. In a culture mattress with a capacity of 650 ml, 40 ml of a diluted cell suspension is poured, the mattress is placed in a thermostat at a temperature of (37 ± 0.5) ° C and incubated for 4-5 days until a monolayer is formed.

After the formation of a monolayer, the procedure for removing cells from a mattress with a capacity of 650 ml is repeated as described above for mattresses with a capacity of 50 ml, but with the only difference that 50 ml of washing solution of PBS, 3 ml of trypsin solution are introduced into a mattress with a capacity of 650 ml with a cell monolayer , 27 ml of growth medium for cell counting and 190 ml of growth medium.

After accumulation of the required number of cells, the resulting culture-producer is used to obtain vaccinated material for the drug. The production of the virus is carried out in 8 roller bottles with a monolayer of cell-producers of the 4647 line (roller installation INCUDRIVE DI (Schuett, Germany) or similar). The surface area of each roller bottle is 850 cm ² .

Growth medium is removed from roller bottles with a monolayer of producer cells, and 10 ml of virus suspension (VV-GMCSF-Lact strain VV-GMCSF-Lact FBUN SSC VB "Vector" - V-688) with a titer of at least 10 ⁷ pfu / ml is added to each bottle for infection cells (multiplicity of infection 1 pfu / cell). Containers with infected cells are placed in a roller installation at a temperature of (37 ± 0.5) ° C for 1 hour to adsorb the virus on the cells. Then, an additional 90 ml of support medium (DMEM medium, kanamycin 0.06 mg / ml) is added to each roller bottle.

Infected containers are incubated in a roller installation at a temperature of (37 ± 0.5) ° C for 2 days until a 100% CPP is formed (cytopathic effect). After 2 days, roller bottles with a virus-infected culture of producing cells are mechanically shaken to discard non-exfoliated cells into the culture medium. The resulting vaccinated material is subjected to a further preparation procedure.

Stage 2. Purification of vaccinated material for anticancer agent.

All stages of cleaning are carried out at a temperature of 6 ° C. The vaccinated material obtained in stage 1 is transferred into sterile centrifuge beakers with a capacity of 250 ml (200 ml per beaker) and concentrated by ultracentrifugation at 15300 rpm for 1 hour (Avanti J-E centrifuge, JA-14 rotor). The supernatant is removed, and the sediments are reduced in a buffer solution of 0.001 M Tris-HCl at the rate of 10% sediment to 90% buffer solution (15 ml of buffer per sediment from one roller bottle).

The resulting suspension of vaccinated material is sonicated (homogenized) on an ultrasonic disintegrator (UZD2-0.1 / 22, Russia). The oscillation frequency of the tip is 22 kHz, the amplitude is 14-16 μm, the disintegration power is 300 W. The sonication is carried out three times for 20 seconds, with cooling after each treatment for 10 seconds on ice.

To the resulting homogenate of vaccinated material, add the benzonase enzyme (Benzonase, ≥250 units / mkl; Sigma-Aldrich # E1014-25KU) to a final concentration of 40 activity units / ml and 60 μl of a 1 M MgCl ₂ solution per 30 ml of homogenate and incubate 30 minutes, shaking occasionally. Trypsin solution (360 mg / ml) is added to the homogenate with benzonase to a final concentration of 0.12 mg / ml and incubated for another 30 minutes at 37 ° C.

The enzyme-treated homogenate of vaccinated material is transferred into sterile centrifuge beakers and clarified by centrifugation (Avanti J-E centrifuge, JS-13.1 rotor) at 3000 rpm for 10 minutes (removal of cell debris).

The clarified concentrated vaccinated material is subjected to additional purification from ballast high molecular weight cellular components in the mode of double ultracentrifugation in a layer of 36% sucrose. For this, 5 ml of a 36% sucrose solution are added to centrifuge tubes with a capacity of 50 ml for the JS-13.1 rotor of the Avanti J-E centrifuge (Beckman Coulter, USA). Then the clarified concentrated vaccinated material in a volume of 30 ml is carefully layered along the wall of centrifuge tubes using a glass pipette onto a layer of 36% sucrose. Centrifugation mode - 80 min at 12000 rpm.

The precipitates after the first ultracentrifugation in a layer of 36% sucrose are reduced in 15 ml of 0.001 M Tris-HCl, pH 9.0, sonicated three times for 20 seconds, with cooling after each treatment for 10 seconds on ice. The oscillation frequency of the tip is 22 kHz, the amplitude is 14-16 μm, the disintegration power is 300 W. The resulting suspensions are combined in pairs and in a volume of 30 ml is carefully layered on the wall of centrifuge tubes using a glass pipette on a 5 ml layer of 36% sucrose. Ultracentrifugation is carried out in an Avanti J-E centrifuge (Beckman Coulter, USA), using a JS-13.1 rotor, centrifugation mode is 80 min at 12000 rpm.

After the second ultracentrifugation in a layer of 36% sucrose, the precipitates are resuspended in 8 ml of 0.9% sodium chloride solution (saline) and sonicated three times for 20 seconds, with cooling after each treatment for 10 seconds on ice. The oscillation frequency of the tip is 22 kHz, the amplitude is 14-16 μm, the disintegration power is 300 W.

The purified concentrated vaccinated material for the preparation is stored in a low-temperature freezer at a temperature of minus (40 ± 2) ° C.

Stage 3 Obtaining an antineoplastic agent.

The purified concentrated vaccinated material obtained in step 2 is diluted with 0.9% sodium chloride solution to a virus concentration of 1.0 × 10 ⁷ pfu / ml. After thorough mixing, control the pH value, which should be 6.5 ± 0.3 (if necessary, the pH is adjusted with diluted 1: 1 hydrochloric acid). The result is an antitumor agent containing, in 1 ml:

strain W-GMCSF-Lact of vaccinia virus - 1 × 10 ⁷ pfu

saline solution - up to 1 ml.

The antitumor drug is controlled according to the following parameters: transparency, color, pH, mechanical inclusions, sterility, abnormal toxicity, mycoplasmas, foreign agents, bacterial endotoxins, bovine serum albumin, cellular DNA, protein, specific activity.

Example 2.

The proposed antitumor agent is obtained analogously to example 1, except that the enzyme benzonase (42 units of activity / ml) and MgCl ₂ are added to the resulting homogenate of vaccinated material and incubated for 40 minutes, then a trypsin solution is added to the homogenate with benzonase to a final concentration of 0.10 -mg / ml and the mixture is incubated for another 40 minutes at a temperature of 37 ° C, cell debris is removed from the homogenate of vaccinated material treated with enzymes by centrifugation at 3000 rpm for 15 minutes, ultracentrifugation in a layer of 36% sucrose is carried out for 60 minutes, and the purified vaccinated material is diluted with phosphate buffered saline (PBS) to a virus concentration of 2.0 × 10 ⁷ pfu / ml and an antitumor drug is obtained containing, in 1 mm:

strain W-GMCSF-Lact of vaccinia virus - 2 × 10 ⁷ pfu

phosphate-buffered saline (PBS) - up to 1 ml.

Example 3. Evaluation of the antitumor efficacy of the proposed agent based on the recombinant strain VV-GMCSF-Lact of vaccinia virus against human glioblastoma U87-MG in vivo

The studies were carried out using SCID mice, 10-12 weeks old, diluted with an SPF vivarium of the Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia).

Human glioblastoma cells U-87-MG were cultured in αMEM medium (GIBCO, USA) supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin sulfate, 0.25 μg / ml amphotericin in the presence of 10% fetal bovine serum. The cells were cultured at 37 ° C in an atmosphere of 5% CO ₂ . U87-MG cells were transplanted subcutaneously into the back of SCID mice at 2 x 10 ⁶ cells per mouse in 0.1 ml of saline.

SCID mice with transplanted human glioblastoma U87-MG tumor cells upon reaching tumor nodes with an average size of 100 mm ³ were randomly divided into experimental and control groups of 10 animals each. The mice of the experimental group were injected intratumorally with the drug at a dose of 1 × 10 ⁷ pfu / mouse, 4 injections, once a week, and the control animals were injected with physiological saline according to a similar scheme. The effectiveness of the antitumor action of the drug was assessed by the index of tumor growth inhibition (TPO) after a course of 4 intratumoral injections.

FIG. 1 shows the growth curves of U87-MG tumors in SCID mice in the experimental and control groups. The arrows mark the days on which the injections were performed. (*) - p <0.05. From the data of FIG. 1 shows that a drug based on the recombinant vaccinia virus strain VV-GMCSF-Lact has high antitumor efficacy against EGFR-positive human glioblastoma U87-MG. The TPO index was 87%.

Example 4. Evaluation of the antitumor efficacy of the claimed agent based on the recombinant VV-GMCSF-Lact vaccinia virus against drug-resistant lymphosarcoma in RLS mice in vivo

The studies were carried out using mice of the CBA line, 10-12 weeks old, breeding the vivarium of the ICBFM SB RAS (Novosibirsk, Russia).

RLS mouse lymphosarcoma cells were cultured in IMDM medium (SIGMA, UK) supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin sulfate, 0.25 μg / ml amphotericin in the presence of 10% fetal bovine serum. The cells were cultured at 37 ° C in an atmosphere of 5% CO ₂ .

RLS cells were transplanted into CBA mice intramuscularly into the hind paw in an amount of 1500 cells per mouse in 0.1 ml of saline. RLS mouse lymphosarcoma is a drug-resistant tumor model and is characterized by resistance to cyclophosphamide (14). RLS tumor growth is induced by intraperitoneal or intramuscular (in the thigh) tumor transplantation. Intramuscular injection of tumor cells leads to the development of a tumor between muscle fibers, which makes it possible to simulate intratumoral administration of the drug when the drug is injected into the thigh of experimental animals.

Mice transplanted into the leg intramuscularly with cells of cyclophosphamide-resistant lymphosarcoma RLS were randomly divided into experimental and control groups, 10 animals each. 7 days after transplantation of tumor cells, the mice of the experimental group were intratumorally (in the thigh muscle) injected with the proposed agent at a dose of 1 × 10 ⁷ pfu / mouse, in a course of 3 injections, once a week, and the control animals were similarly injected with saline. The course of drug administration was limited to 3 injections, since when the experiment was extended for another week, the size of the tumors of the animals in the control group did not allow the animals to approach the feeding trough. The continuation of the experiment in this case contradicts the norms of a humane attitude towards animals.

The effectiveness of the antitumor effect of the drug was assessed by the tumor growth inhibition index (TPO). To calculate the TPO index, the volume of a paw with a tumor was measured in animals of the experimental and control groups.

FIG. 2 shows curves of changes in the volume of a paw with a tumor in animals of the experimental and control groups. The arrows mark the days on which the injections were performed. (*) - p <0.05. From the data of FIG. 2 shows that the drug based on the recombinant VV-GMCSF-Lact vaccinia virus has high antitumor efficacy against the drug-resistant RLS tumor (lymphosarcoma) of the mouse. The TPO index was 84%.

Example 5. Evaluation of the antitumor efficacy of the proposed agent based on the recombinant strain VV-GMCSF-Lact of vaccinia virus against the developed tumor of adenocarcinoma of the human breast MDA-MB-231 in vivo

The studies were carried out using SCID mice, 10-12 weeks old, diluted with an SPF vivarium of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences (Novosibirsk, Russia).

Human breast adenocarcinoma cells MDA-MB-231 were cultured in Leibovitz L-15 medium (SIGMA, UK) supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin sulfate, 0.25 μg / ml amphotericin in the presence of 10% fetal serum cattle (GIBCO, USA). MDA-MB-231 cells were transplanted subcutaneously in the back region of SCID mice in 0.1 ml of saline supplemented with Matrigel (Matrigel ™, BD Bioscience, USA) in a volume ratio of cell suspension: Matrigel 1: 1.

Since the aim of the study was to study the effect of a drug on a developed tumor (≥500 mm ³ ), SCID mice were preliminarily divided into experimental and control groups; The animals of the experimental group were subcutaneously transplanted 3 times more tumor cells (4.5 × 10 ⁶ cells) than the animals of the control group (1.5 × 10 ⁶ ). Such an experimental setup is necessary, since after transplantation of the same number of tumor cells and in the absence of treatment, the animals of the control group do not survive until the end of the experiment.

When studying the antitumor efficacy of the proposed agent against a developed tumor, it is advisable to increase the total dose of the drug. The course of therapy consisted of 6 daily injections of the virus at a dose of 1 × 10 ⁷ pfu / mouse. Treatment was started when the tumor volume in the experimental group was> 500 mm ³ .

The effectiveness of anticancer drug therapy was assessed by the tumor growth inhibition index (TPO) after the end of the course of treatment.

FIG. 3 (A) shows curves of tumor growth in animals of the experimental and control groups. The brackets with arrows indicate the period of drug administration. From the data of FIG. 3 (A) shows that the proposed antitumor agent provides an effective and reliable reduction in the size of the developed tumor MDA-MB-231 in comparison with the initial tumor size (by 42%).

Since the difference in the initial sizes of tumors in the control and experimental groups does not allow to directly correctly calculate the TPO index, the obtained data were transformed into the values of the change in tumor growth relative to the initial tumor size in (%).

FIG. 3 (B) shows curves of tumor growth in animals of the experimental and control groups. The brackets with arrows indicate the period of drug administration. Data are presented as a percentage in relation to the size of tumors at the time of initiation of treatment. From the data of FIG. 3 (B) shows that an antitumor agent based on the recombinant strain VV-GMCSF-Lact of vaccinia virus has high antitumor efficacy against the developed tumor of adenocarcinoma of the human breast MDA-MB-231.

For a developed tumor, the TPO index was calculated using the formula:

TPO = ((% V _control -% V _experiment ) /% V _control ) × 100%, where

% V _control - the percentage of change in tumor size at the final point of the experiment relative to the initial point of the experiment (animals of the control group);

% V _experiment - percentage of change in tumor size at the final point of the experiment relative to the initial point of the experiment (animals of the experimental group);

According to the data obtained, the TPO index for the developed tumor was 77.6%.

The experimental data described in examples 3-5 indicate that the claimed antitumor agent based on the recombinant vaccinia virus VV-GMCSF-Lact strain has high antitumor efficacy against solid tumors in humans and animals.

The proposed agent based on the recombinant VV-GMCSF-Lact strain can be used as a new viral drug with antitumor and antimetastatic activity and low toxicity for the treatment of solid tumors in humans and animals.

Sources of information

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Claims (9)

1. An antitumor agent based on a recombinant vaccinia virus strain, containing a recombinant vaccinia virus strain and a pharmaceutically acceptable solvent, characterized in that, as a recombinant strain, the agent contains a recombinant vaccinia virus strain VV-GMCSF-Lact, which is capable of suppressing malignant tumor growth in solid FBUN SSC VB "Vector" under the number V-688, and a pharmaceutically acceptable solvent with the following content of components (per 1 ml): vaccinia virus VV-GMCSF-Lact strain - (1-2) × 10 ⁷ PFU, pharmaceutically acceptable solvent - up to 1 ml. 2. An antineoplastic agent according to claim 1, characterized in that it contains saline or a physiologically acceptable buffer solution as a pharmaceutically acceptable solvent. 3. The antitumor agent according to claim 1, characterized in that it is intended for parenteral administration by intratumoral injections into a solid malignant tumor selected from the group consisting of glioblastoma, lymphosarcoma, breast carcinoma. 4. A method of producing an antitumor agent based on a recombinant vaccinia virus strain according to claim 1, including the production of a virus in a culture of producer cells followed by collection of vaccinated material, treatment with nuclease to destroy nucleic acid of producer cells and protease to destroy proteins of producer cells, release from cellular debris, purification of vaccinated material by removing ballast high-molecular cell components with subsequent dilution of the purified vaccinated concentrate with a pharmaceutically acceptable solvent to the required concentration of the virus, characterized in that the production of the VV-GMCSF-Lact vaccinia virus strain is carried out in a culture of cells-producers of line 4647 vaccinated material is concentrated, the resulting suspension is homogenized by sonication, benzonase enzyme is added to the resulting vaccinated material homogenate to a final concentration of 40-42 activity units / ml and MgC l ₂ and incubated for 30-40 minutes at room temperature, then trypsin solution is added to the homogenate with benzonase to a final concentration of 0.10-0.12 mg / ml and the mixture is incubated for another 30-40 minutes at 37 ° C from the enzyme-treated homogenate, cell debris is removed by centrifugation at 3000 rpm for 10-15 minutes, and the clarified concentrated vaccinated material is subjected to additional purification from ballast high-molecular cellular components in the mode of double ultracentrifugation at 12000 rpm in a layer of 36% sucrose for 60-80 min. 5. The method of claim 4, characterized in that the collected vaccinated material is concentrated by ultracentrifugation, the supernatant is removed, and the precipitate is resuspended in 1 mM Tris-HCl, pH 9.0 6. The method of claim 4, characterized in that the treatment of the suspension of vaccinated material with ultrasound is carried out three times for 20 s, with cooling after each treatment for 10 s in ice. 7. The method of claim 4, characterized in that after the first ultracentrifugation in a layer of 36% sucrose, the precipitates of vaccinated material are reduced in 0.001 M Tris-HCl, pH 9.0, and after the second ultracentrifugation, the precipitates are resuspended in 0.9% sodium chloride solution and treated with ultrasound.

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