RU2482129C2 - Newcastle disease virus strain for creating based anticancer candidate preparation and studying of oncolysis mechanisms - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to virology, and concerns the Newcastle disease virus strain. The characterised strain NDV/Mallard/Adigeya/8/2008 is recovered from a dead duck, and deposited in the Collection of Cultures of the State Research Centre for Virology and Biotechnology "Vector" under registration number No. V-512.

EFFECT: presented strain may be used to study the oncolytic properties and mechanisms, and to develop the anticancer candidate preparations based thereon.

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Description

The invention relates to strains of the Newcastle disease virus for studying the oncolytic properties and mechanisms of oncolysis, as well as for developing candidate anticancer drugs based on viruses that can selectively lyse neoplasms of mammals and can be used in virology, microbiology, medicine and veterinary medicine.

According to the World Health Organization, more than five million people die from malignant tumors every year in the world, and more than three million cases of new diseases are recorded. To date, the cure rate of cancer is only 26%. Oncology mortality rates in the Novosibirsk region are among the highest in Siberia and exceed the average level in Russia. In the dynamics of the detectability and mortality of the population from cancer in Novosibirsk, there has been a steady increase.

The development of highly effective cancer treatment methods is one of the most urgent tasks of modern medicine. Despite some progress in this area, our ability to detect tumors in the early stages and selectively affect them remains extremely limited.

The main disadvantage of the currently used methods of chemotherapy and radiotherapy is their low therapeutic index (TI). This indicator characterizes the difference in the degree of effect of the corresponding therapeutic agent on tumor and normal cells of the body (in a simplified form, the ratio of the number of killed cancer cells to the number of killed normal cells). The therapeutic index of modern chemotherapeutic agents does not exceed 6: 1. According to the opinion accepted in the international scientific community, TIs of drugs targeted by bacterial vectors and oncolytic viruses can reach 100,000: 1, which is due to their high specificity for tumor cells.

The idea of the possibility of using viruses for the treatment of tumors arose in the first half of the 20th century. However, the date of birth of modern "virotherapy" is considered to be 1991, when the journal "Science" published the results of a study of the oncolytic potential of genetically modified herpes simplex virus type 1 (Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. (1991) / Experimental therapy of human glioma by means of a genetically engineered virus mutant. // Science. 1991 May 10; 252 (5007): 854-6). To date, in the world, as a result of large-scale studies, a number of promising drugs based on various oncolytic viruses have been obtained. However, to date, the receipt of such drugs is associated with complex manipulations at the genetic level. We should not forget that to create existing anticancer drugs based on viruses, mammalian viruses were used, which are conditionally pathogenic for humans and can significantly increase their pathogenicity during evolution or due to spontaneous mutagenesis.

Currently, evidence has been obtained that the Newcastle disease virus (hereinafter - VBI) has oncolytic activity and is able to selectively lyse cancer cells, while remaining safe for normal mammalian tissue cells. Several studies have demonstrated the ability of this virus to specific oncolysis. A significant difference was found in the rate of virus replication in tumor and normal mammalian cells (Zhan X, Slobod KS, Krishnamurthy S, Luque LE, Takimoto T, Jones B, Surman S, Russell CJ, Portner A, Hurwitz JL. (2008) / Sendai virus recombinant vaccine expressing hPIV-3 HN or Federal protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections. // Vaccine. 2008 Jun 25; 26 (27-28): 3480-8. Epub 2008 May 1). There is an assumption that the high tropism of VBI to tumor cells is due to impaired synthesis of proteins responsible for the elimination of VBI (Elankumaran S, Rockemann D, Samal SK. (2006) / Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death . // J Virol. 2006 Aug; 80 (15): 7522-34).

Known antitumor drugs based on VBI P line V1 703 and Cassell73, which undergo the first stage of clinical trials (Ravindra PV, Tiwari AK, Sharma B, Chauhan RS. / Newcastle disease virus as an oncolytic agent. // Indian J Med Res. 2009 Nov ; 130 (5): 507-13).

However, information on these strains, their properties, and potential uses are not currently published.

Known recombinant viruses of Newcastle disease with oncolytic activity (US application No. 2010178684, IPC C12N 7/01, publ. 07/15/2010 and US application No. 200800206201, IPC C12N 7/00, publ. 08/28/2008).

However, when using these genetic engineering constructs, there is a high probability of reverse mutations, low viability, and complex production technology. In addition, there is a possibility of initiation of mutations in the host DNA due to the presence of vector structures in the genome of these viruses.

According to applicants, the closest oncolytic strain (prototype) is the Newcastle virus strain NDV HUJ, deposited in 2006 in the UK virus collection under number 06072401, which has been adapted to chicken embryo cells and has gone through several passages. The prototype strain NDV HUJ is published and described in US patent No. 7223389, IPC A61K 48/00, publ. May 29, 2007. "Composition and method for the treatment of tumors."

However, the prototype strain when used in the treatment of tumors requires prior adaptation to the culture of tumor cells, which reduces its therapeutic potential when used in oncology.

The technical result of the claimed technical solution is the creation of an oncolytic strain of the Newcastle disease virus, capable of specifically destroying mammalian neoplasms in the in vivo system without preliminary adaptation to the tumor cell culture.

The specified technical result is achieved by obtaining a new strain NDV / Mallard / Adigeya / 8/2008 of the Newcastle disease virus, capable of selectively lysing mammalian cancer cells, without causing an infectious disease and the death of mammals.

The strain of the Newcastle disease virus NDV / Mallard / Adigeya / 8/2008 for the creation on its basis of a candidate anticancer drug and the study and mechanisms of oncolysis, deposited in the Collection of microorganism cultures of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Rospotrebnadzor under registration number No. V-512.

The obtained data reveal the presence of strain NDV / Mallard / Adigeya / 8/2008 the ability to specifically destroy mammalian neoplasms in the in vivo system without prior adaptation to the culture of tumor cells, which is not currently described in the literature. This makes this strain a promising agent for creating anticancer drugs based on it through preliminary adaptation with a wide spectrum of interaction and a high therapeutic index.

Thus, obtaining a domestic producer strain of VBN to create a drug with oncolytic activity on its basis is promising. The relevance of this is confirmed by the absence in Russia of commercial analogues of such strains.

The recommended strain is effectively cultivated in the system of developing chicken embryos (RKE) in titers of 5.2 ± 0.2 lg EID ₅₀ / ml. The strain does not cause lethal disease in mice with intranasal and any of the varieties of parenteral infection. Moreover, the virus is highly pathogenic for chickens (ICPI = 2), causing the death of all infected chickens within 24 hours of infection. The ability to specific lysis of mammalian tumor cells was found in this strain.

To identify the oncolytic properties of strain NDV / Mallard / Adigeya / 8/2008, experiments were carried out to infect mice with tumor cells previously incubated with strain NDV / Mallard / Adigeya / 8/2008. The results of this experiment revealed the presence of the proposed VBI strain the ability to neutralize tumor cells, preventing the formation of the tumor. This can occur under the influence of the immune system induced by the presence of the virus, or, more likely, by the direct lysis mechanism of the tumor cell strain NDV / Mallard / Adigeya / 8/2008 proposed by us. The second hypothesis is also supported by the fact that with the introduction of strain NDV / Mallard / Adigeya / 8/2008 into an already formed tumor, a decrease in the dynamics of tumor growth was observed. Morphological examination revealed the destruction of the tumor tissue by the studied strain, and at the ultrastructural level, viral particles were found inside the cancer cells. When the killed virus was introduced to the animals, no changes in the growth dynamics of the neoplasms were observed. Thus, the presence of live viral particles is necessary for the manifestation of oncolytic activity, and one of the probable markers of oncolytic activity is the structure of the fusion protein cleavage site corresponding to highly pathogenic VBI variants and the ability to lyse cells. The proposed strain has these markers.

The obtained data reveal the presence of strain NDV / Mallard / Adigeya / 8/2008 the ability to specifically destroy mammalian neoplasms in the in vivo system without prior adaptation to the culture of tumor cells, which is not currently described in the literature. This makes this strain a promising agent for creating anticancer drugs based on it through preliminary adaptation with a wide spectrum of interaction and a high therapeutic index.

Characterization of the claimed strain. Strain NDV / Mallard / Adigeya / 8/2008 is a typical representative of the Newcastle disease virus of the genus Avulavirus family Paromyxoviridae. The inventive strain isolated from dead duck during an outbreak in the Republic of Adygea. The strain was deposited in the collection of cultures of microorganisms of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "(FGUN SSC WB" Vector ") Rospotrebnadzor under registration number No. V-512.

Source of receipt. From pathological material obtained from a bird that died during an outbreak in the Republic of Adygea (spleen, lung, small intestine, brain), an isolate of the Newcastle disease virus, which was called NDV / Mallard / Adigeya / 8/2008, was isolated. The virus was isolated at RCE at the first passage; the death of RSC was observed 1.5-2 days after infection. The virus was indicated in the RGA with rooster erythrocytes, but the horse erythrocytes did not agglutinate: the hemagglutinating activity was 1: 64-1: 128 GAU / 0.2 ml. The infectious activity of the isolated virus was 5.2 lg EID ₅₀ / ml.

Virus identification. The primary identification of isolated influenza A virus was carried out by RT-PCR with the detection of the results in a closed tube in real time using primers and a probe specific for the conserved regions of the M gene of Newcastle disease virus, according to (Miller PJ, Decanini EL, Afonso CL. / Newcastle disease: evolution of genotypes and the related diagnostic challenges. / Nfect Genet Evol. 2010 Jan; 10 (1): 26-35. Epub 2009 Sep 30).

Genetic and phylogenetic analysis: RNA was extracted from the virus suspension using the Ribosorb reagent kit (FGUN TsNIIE), the reverse transcription reaction was performed from the terminal, universal primer Random dN6 using the M-MuLV enzyme reverse transcriptase (FGUN TsNIIE). For sequencing, a section of the Newcastle disease virus genome was selected containing the C-terminal segment of the M gene, the N-terminal segment of the F gene, and the intergenic region. Using the nucleotide sequence of this site, the amino acid sequence of the site of proteolytic cleavage of the fusion protein, which is the main marker of pathogenicity for the Newcastle disease virus, can be recreated. For sequencing, primers based on SEPRL were used (Kim LM, King DJ, Curry PE, Suarez DL, Swayne DE, Stallknecht DE, Slemons RD, Pedersen JC, Senne DA, Winker K, Afonso CL. / Phylogenetic diversity among low- virulence newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates. // J Virol. 2007 Nov; 81 (22): 12641-53. Epub 2007 Sep 12).

PCR was performed on DNA-Engine devices (Bio Rad, USA) using reagents manufactured by the Federal State Institution of Research and Development. Amplification fragments were sequenced using the cycle sequence method with the ABI PRISM Big Dye ™ v.1.1 kit (Applied Biosystems, USA) according to the manufacturer's instructions using the ABI-3100 PRISM ™ Genetic Analyzer capillary automatic sequencer (Applied Biosystems, USA). Alignment of nucleotide sequences was performed using the DNASTAR program block according to the Clustal algorithm.

Phylogenetic analysis was performed using the MEGA 3.1 program using the Neighbor Joining method using the Kimura 2-parameter algorithm with the Bootstrap Test of Phylogeny (1000 repetitions) [Kumar S.].

Phylogenetic analysis of the nucleotide sequence of the genes showed that the strain NDV / Mallard / Adigeya / 8/2008 belongs to class 2, genotype 7. The strain is closest to the strains previously described in Southeast Asia.

Cultural properties. Strain NDV / Mallard / Adigeya / 8/2008 during cultivation of a primary culture of chicken embryo fibroblast cells (FEC) on a monolayer causes a cytopathic effect (CPD) on days 2-4 (cultivation temperature 37 ° C, in an atmosphere of 5% CO ₂ ).

Pathogenicity for humans. Newcastle disease virus strains are not pathogenic for humans.

Pathogenicity for poultry. Strain NDV / Mallard / Adigeya / 8/2008 refers to highly pathogenic variants of the Newcastle disease virus: intracerebral pathogenicity index = 2.

Pathogenicity for mammals. The strain is not pathogenic for mammals.

For long-term storage, the strain must be lyophilized using a solution of gelatin and sucrose (SAG) as a protective medium. Lyophilization with the addition of SAGE in a ratio of 1: 4 allows you to maintain stable infectious activity of the virus for at least 2 years when stored at -70 ° C.

The following culture media are used to cultivate the strain in cell culture: MEM Needle, MEM Needle × 2AVK, Med 199, DMEM containing 2 μg / ml trypsin, 2 mmol / L glutamine, 100 μg / ml Penicillin and 100 IU / ml Streptomycin.

The invention is illustrated by the following drawings. Figure 1 shows the dynamics of the growth of KREBS tumors nubbed with highly and low pathogenic VBI strains in BALB / C mice. Figure 2 shows a graph of the dynamics of growth of KREBS tumors in BALB / C mice infected with low and highly pathogenic VBI strains two weeks after inoculation of tumor cells. Figure 3 shows the central part of the tumor - the field of necrosis and tumor "islets". Figure 4 shows activated macrophages in a tumor. Figure 5 shows myelinated structures and viral particles in the intercellular space. Figure 6 shows the formation of viral particles in the EPR region. 7 shows a tumor cell infected with VBI. On Fig presents viroplasm in the perinuclear space. Figure 9 shows a destroyed tumor cell filled with viroplasm and virus-like particles.

Example 1. Identification of the oncolytic properties of strain NDV / Mallard / Adigeya / 8/2008 upon infection of mice with cells of the transplanted line of tumors KREBS 2, previously incubated with strain NDV / Mallard / Adigeya / 8/2008.

To detect neutralizing activity, three groups of 10 mice were injected with tumor cells incubated with NDV / Mallard / Adigeya / 8/2008 (highly pathogenic), tumor cells incubated with NDV / Chukotka / goose / 7/2008 (low pathogenic, obtained from the collection of the department zoonotic infections and influenza FGUN SSC VB "Vector"), tumor cells incubated with physical. solution (control group). The measurement of tumors began from the 15th day of the experiment, when the tumor in the control group was clearly determined visually and tactilely. Tumor growth was observed for 28 days. There were no significant differences in the growth dynamics of the neoplasms in the control group and in the group infected with the NDV / goose / Chukotka / 7/2008 strain. In the group infected with NDV / Mallard / Adigeya / 8/2008, tumor formation was not observed.

The results of this experiment show the presence of a VBN strain pathogenic for birds that can neutralize mammalian tumor cells and prevent tumor formation. This process can occur under the influence of the immune system induced by the presence of the virus, or by the mechanism of direct lysis of tumor cells by viral particles. If the second assumption is considered true, the presence of oncolytic activity in the pathogenic strain for birds and the absence of it in the non-pathogenic one is explained by the fact that apatogenic strains of VBI are not able to lyse cells, while the studied strain, on the contrary, has this ability, which allows it to lyse cancerous mammalian cells, preventing tumor formation. Figure 1 shows the dynamics of the growth of KREBS tumors nubbed with highly and low pathogenic VBI strains in BALB / C mice. The abscissa axis shows the time of measuring the size of the tumor (days), the ordinate axis represents the tumor volume (mm ³ ).

Example 2. The study of the oncolytic properties of the strain NDV / Mallard / Adigeya / 8/2008 on a model of a solid tumor of KREBS 2 mice with the introduction of the virus into the formed tumor in vivo.

To evaluate the effect of VBI on an already formed tumor, the following experiment was performed: three groups of 13 mice were intramuscularly infected with a tumor. After 2 weeks, 0.2 ml of live NDV / Mallard / Adigeya / 8/2008 virus, killed NDV / Mallard / Adigeya / 8/2008 virus and nat. solution accordingly. Tumor growth was observed for 28 days. There were no significant differences in growth dynamics between the group infected with the killed NDV / Mallard / Adigeya / 8/2008 and the control group. In the group infected with live NDV / Mallard / Adigeya / 8/2008, a decrease in the tumor growth rate was observed. In figure 2. a graph of changes in the dynamics of growth of KREBS tumors in BALB / C mice infected with low and highly pathogenic VBI strains two weeks after inoculation of tumor cells is shown. The abscissa axis shows the time of measuring the size of the tumor (days), the ordinate axis represents the tumor volume

(mm ³ ).

12 hours after infection, 3 mice from each group were opened. Visual analysis showed the presence of numerous necrosis in the tumor body in mice infected with live NDV / Mallard / Adigeya / 8/2008. Structural analysis at the light-optical level revealed an increase in macrophage activity in the tumor area and the presence of active lysis of tumor cells in the area of virus injection (Figs. 3, 4), cells with signs of initiation of appoptosis were also detected. Figure 3 shows the central part of the tumor - the field of necrosis and tumor "islets". Figure 4 shows activated macrophages in a tumor. At the ultrastructural level using electron microscopy, myelinated structures and viral particles in the intercellular space were detected (Fig. 5). Figure 5 shows myelinated structures and viral particles in the intercellular space.

A morphological study of the material selected on the 11th day after infection at the light-optical level revealed almost complete destruction of the tumor tissue, in fact, active mitoses were preserved only in the peripheral region of the tumor, macrophage activity was also noted.

At the ultrastructural level, viral particles were detected inside the tumor cells, the formation of viral particles on the EPR and budding of freshly formed virions, viroplasma in the perinuclear space, destroyed tumor cells completely filled with viroplasma and virus-like particles were also detected (6, 7, 8, 9) . Figure 6 shows the formation of viral particles in the EPR region. 7 shows a tumor cell infected with VBI. On Fig presents viroplasm in the perinuclear space. Figure 9 shows a destroyed tumor cell filled with viroplasm and virus-like particles.

The data obtained indicate the unique ability of strain NDV / Mallard / Adigeya / 8/2008 to exhibit selective lytic activity against mammalian tumor cells, as well as actively replicate and persist in these cells for a long time without preliminary adaptation, destroying the neoplasm.

Thus, the unique properties of the proposed strain allow it to be used to study the mechanisms of destruction of mammalian cancer cells at the ultrastructural level using light-optical and electron microscopy methods. These properties are promising for basic and applied medical research.

These properties make the strain NDV / Mallard / Adigeya / 8/2008 very promising for further studies of oncolytic activity and the development of candidate antitumor drugs.

Claims (1)

The strain of the Newcastle disease virus to create on its basis a candidate anticancer drug and study the mechanisms of oncolysis, deposited in the Collection of microorganism cultures of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "of the Federal Service for Supervision of Human Rights under registration number No. V-512.

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