Poxviral oncolytic vectors

FIELD: biotechnologies.

SUBSTANCE: pox virus of variolovaccine is proposed, which includes a defect F2L gene and a suicide gene. Pox virus has oncolytic activity. Besides, a reproduction method of such pox virus and its use for treatment of proliferative diseases or diseases with increased activity of osteoclasts is proposed.

EFFECT: improving compound application efficiency.

31 cl, 12 dwg, 3 tbl

 

The technical field

Oncolytic viruses represent a new class of therapeutic agents used to treat cancer, with a unique ability to tumor-dependent self-sufficiency (HERMISTON. A demand for next-generation oncolytic adeno viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.322-30.). Oncolytic viruses are capable of selective replication in cancer cells and therefore offer the power levels of action and specificity, which are potentially much larger than the standard anti-cancer therapy (FISHER. Striking out at disseminated metastases: the systemic delivery of oncolytic viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.301-13.). The advantage of using these viruses is that when its replication they symbolise their host cells. Cancer cells are the ideal hosts for many viruses, because they inactivated virus interferon way or motivovany genes tumor suppressor, which allow smooth flow of viral replication (CHERNAJOVSKY et al. Fighting cancer with oncolytic viruses. The British medical journal. 2006, vol.332, no.7534, p.170-2.).

Some viruses are by nature capable of selectively replicate in tumor cells, but oncolytic viruses can also be obtained by modification of natural viruses. To this end the main strategies currently used for modification of viruses, include: functional deletions in the substantial viral genes; tumor - or tone-specific promoters used to control the expression of these viral genes; and modifying the tropism to redirect adenovirus on the surface of a cancer cell. In the near future it is necessary to optimize oncolytic adenoviruses, in order to fully realize their potential as critical anti-cancer tools and, thus, to improve the prognosis for patients with malignant gliomas (JIANG, et al. Oncolytic adenoviruses as antiglioma agents. Expert review of anticancer therapy. 2006, vol.6, no.5, p.697-708.).

For example, ONYX-015, a selectively modified adenovirus to replicate and destroy cells that have p53 mutations, developed by the company "Swollen Pharmaceuticals for potential treatment of various solid tumors, including tumors of the head and neck, gastrointestinal and pancreatic tumors. He is a recombinant adenovirus that carries the mutation "loss of function" in the locus EV, the product of which is a 55 kDa protein that binds to the p53 tumor protein suppressor gene and inactivate it. Thus, adenovirus ONYX-015, assumed not affects normal cells. Mutations in the p53 gene is a tumor suppressor gene are the most common type of genetic abnormalities in cancer, which occurs in more than half of all major types of cancer. So these are the entrances are susceptible to the virus, which will be easy to replicate and cause cell death. Research continues in phase III application of ONYX-015 regarding the treatment of recurrent head and neck cancer, studies phase II relative to the treatment of colorectal tumors, tumors of the ovary, pancreas and oral cavity, and research phase I is a relatively diseases of the digestive system, tumors of the esophagus and liver (COHEN, et al. ONYX-015. Onyx Pharmaceuticals. Current opinion in investigational drugs. 2001, vol.2, no.12, p.1770-5.).

Natural oncolytic viruses are replication-competent viruses possessing an innate ability to selectively infect and destroy tumor cells. Despite the fact that they were used in the original attempts to treat cancer live viruses five decades ago, the interest in natural oncolytic viruses behind support established genetic engineering of adenoviruses and herpes viruses as cancer therapy. However, recently there was a renewed interest in high potency and selectivity of these natural agents (ROBERTS, et al. Naturally oncolytic viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.314-21.).

Among the natural oncolytic viruses, viruses ospowiki (from the poxviridae) possess many of the key features required for an ideal viral cage for use in oncolytic virotherapy. They include a short period of Polow is doing, with the rapid intercellular spread, strong analytical ability, a greater ability to clone and clear molecular biology. In addition, although they are capable of replication in human cells, they do not consider natural health issue and is particularly well characterized by the fact that they were introduced to millions of people during the campaign to eradicate smallpox. Early clinical results using or vaccine strains or genetically modified strains ospowiki demonstrated antitumor effects (THORNE, et al. Vaccinia virus and oncolytic virotherapy of cancer. Current opinion in molecular therapeutics. 2005, vol.7, no.4, p.359-65.).

On the contrary, poxvirus of myxoma is a new oncolytic candidate who has no history of direct use in humans because it has a distinctive and absolute tropism for mind-owner in relation to Lagomorpha (rabbits). Virus myxoma, as recently shown, may also be selectively infect and destroy human tumor cells, a unique tropism, which is associated with deregulirovannym intracellular signalling pathways that are found in most human cancers. This review outlines the existing data on the tropism of the virus myxoma to human cancer cells, as well as preclinical data, pokazivaushee is its ability to infect and destroy tumors in animal models of cancer (STANFORD, et al. Myxoma virus and oncolytic virotherapy: a new biologic weapon in the war against cancer. Expert opinion on biological therapy. 2007, vol.7, no.9, p.1415-25.).

Technical problem

Injection of high doses of poxviruses necessary to achieve antitumor effect caused problems associated with toxicity. Most adverse events are minor adverse reactions that are typically associated with vaccinia virus are limited and include fever, headache, fatigue, myalgia, chills, local skin reactions, nonspecific rash, erythema multiforme, swollen lymph nodes and pain in the site of inoculation. Other reactions might require additional treatment methods (for example, VIG, first-line therapy and cidofovir, second-line therapy). Adverse reactions that might require further evaluation or treatment include inadvertent inoculation, generalized vaccinia (smallpox) (GV), eczema after vaccination (EV), progressive vaccinia (smallpox (PV), vaccine-induced Central nervous system disease, and fetal vaccinia (smallpox) (CONO, et al. Smallpox vaccination and adverse reactions. Guidance for clinicians. MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports / Centers for Disease Control. 2003, vol.52, no.RR-4, p.1-28.).

Thus, there is a need for safer poxviruses with the same oncolytic activity as their natures who's copies.

Background of invention

In the US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15/11/1994 describe a modified recombinant poxvirus, particularly a vaccinia virus having inactivated insignificant virus encoded genetic functions so that the recombinant poxvirus had reduced toxicity and enhanced security. In particular, genetic functions inactivated by deletion of the open reading frame that encodes a virulence factor, or insertional inactivation open reading frame that encodes a virulence factor. In more detail, this patent describes the vaccinia virus in which the open reading frame for J2R, B13R+B14R, A26L, A56R, C7L - K1L, and I4L have been inactivated. This virus (NYVAC) can be designed as a vector for alien nucleic acid and used as a vaccine to induce an immunological response in an animal host. However, N YVAC unable to effectively replicate in most mammalian cells and can not be used as oncolytic virus (XIANGZHI, et al. Vaccinia virus K1L protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for ACAP2. Journal of virology. 2006, vol.353, no.1, p.220-233.).

WO 2004/014314 (DAVID KIRN (US)) 19/02/2004 describes a modified vaccinia virus, which includes one or more m the grants in its viral genome. Described mutations are located in one or more of the following classes polyp of aptidon: 1) interferon-modulating polypeptide; 2) the presence of complement control polypeptide; 3) TNF or chemokine - modulating polypeptide; 4) the inhibitor of serine protease; 5) IL-lp-modulating polypeptide; 6) non-infectious EEV form of the polypeptide; and (7) the viral polypeptide that acts to inhibit the release of infectious virus from cells (anti-infective viral form of the polypeptide). In addition, also disclosed mutations in vaccinia virus A41L or C11R.

Areas of the genome ospowiki, such as A34R, A41L, A53R, B5R, B7R, B8R, B13R, B15R, B18R, B22R, B28R, B29R, CUR, E3L, K2L, NIL, vC12L, vCKBP described in more detail in this application. The methods of the invention involve the use of any of poxviruses, discussed by the authors. The inventors also disclose methods for treating cancer by introducing into a cancer cell or patient an effective amount of the modified vaccinia virus.

Disclosure of inventions

The inventors surprisingly found that poxviruses, including defective F2L gene, have an improved safety profile, but retain the equivalent oncolytic activity (compared with their natural copy).

The present invention relates to a poxvirus, including defective F2L gene.

As used throughout the application the terms in the sole is the number used in the sense what they mean "at least one", "at least one", "one or several" or "many" components or steps referenced, unless the context clearly indicates otherwise. For example, the term "cell" includes a variety of cells, including mixtures thereof.

The term "and/or"used by the authors, includes the meaning "and", "or" and "or any other combination of elements that are related to the specified term.

The term "about" or "approximately"used by the authors, means within 20%, preferably within 10%, and more preferably within 5% of the given value or range.

Authors use the terms "including" and "include" mean that the products, compositions and methods include the components or steps, which are referred to, but exclude others. "Consisting essentially of"is used to define the products, compositions and methods, shall mean excluding other components or stages of any significant value. Thus, the composition consisting essentially of the following components, does not eliminate the pollutants in trace quantities and pharmaceutically acceptable carriers. "Consisting of" shall mean excluding more than trace elements in quantities of other components or stages.

Authors use the term "poxvirus, including defective gene"refers to a poxvirus, including deletion, substitution or addition of one or more nucleic acids of the defective gene, or any combination of these features, and these modifications lead to the inability of the virus to produce a protein having the activity of a protein produced by the unmodified gene. In a preferred embodiment of the invention poxvirus, including the defective gene, concerns poxvirus, which was deleted a gene sequence. The mutation can be implemented in many ways known to the qualified specialists in the field of technology, using recombinant methods. Methods for modifying the genome of the poxvirus is available in this area. For example, methods disclosed in MCCART, et al. Systemic cancer therapy with a tumor selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.. Cancer res.. 2001, no.61, p.8751-57., KIM, et al. Systemic armed oncolytic ans immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF. Molecular Therapeutic. 2006, no.14, p.361-70., WO 2004/014314 (DAVID KIRN (US)) 19/02/2004 and US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15/11/1994 can be used to produce a poxvirus of the invention. The methods disclosed in the example of this application, especially related to receiving poxvirus according to the invention. Genome sequences of various poxviruses are available in this area, for example, the genome of vaccinia virus, cowpox virus, virus Saraguro, virus Ectomelia, virus myxoma available in Genbank (inventory number NC_006998, NC_003663, NC_005309, NC_004105, NC_001132, respectively).

Authors use the term "poxvirus" refers to a virus belonging to the family from the poxviridae. According to a preferred variant implementation, poxvirus according to the invention belongs to the subfamily Chordopoxvirinae, more preferably classified in the genus orthopoxvirus genus and even more preferably of a type Vaccinia virus.

For example, can be used strains of vaccinia virus Dairen I, IHD-J, L-IPV, LC16M8, LC16MO, Lister, LIVP, Tashkent, WR 65-16, Wyeth, Ankara, Copenhagen, Tian Tan and WR. According to a particularly preferred variant implementation, poxvirus according to the invention represents the strains of Copenhagen vaccinia virus,

Poxvirus ospowiki contains a large double genome DNA (187 pairs of cilaisnowbuy) and is a member of the only known family of DNA viruses that replicate in the cytoplasm of infected cells. Because infected cell must supply a large number of precursors of DNA in the cytoplasmic sites of replication, the virus encodes and expresses a lot of the enzymatic activities required for the metabolism and synthesis of DNA, including deoxyuridine 5'-triphosphate-nucleotidases (dUTPase).

Deoxyuridine 5'-triphosphorylated (dUTPase, EU 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate in the Pris is according ions of Mg (2+). dUTPaa, deleting dUTP pool of dNTP and production dUMP, involved in maintaining the fidelity of DNA replication and in providing a precursor for the production of TSR by timidilatsintazei. dUTPaa ospowiki is a 15-kDa protein, encoded by a gene F2L (MCGEOGH.. Nucleic Acids Research. 1990, no.18, p.4105-10; BROYLES.. Virology. 1993, no.195, p.863-5.). The sequence F2L gene of vaccinia virus available in the gene Bank by stock number M, sequence and location F2L gene in different genomes of poxviruses also available in gene Bank, for example, by stock number NC_006998, DQ121394, NC_001611, AY689436, AY689437, NC_008291, DQ437594, DQ437593, AY313847, AY313848, NC_006966, NC_005309, NC_003391, NC_003389, NC_001132, NC_003310, NC_002188, M35027, AY243312, AF170726, DQ011157, DQ011156, DQ011155, DQ011154, DQ011153, X94355, Y16780, AY318871, U94848, AF198100 and M34368.

Gene nomenclature used in this description is the range of strain Copenhagen ospowiki, and is also used for homologous genes from the poxviridae other, unless otherwise specified. However, gene nomenclature may differ depending on the strain of smallpox. For more information, correspondence between genes Copenhagen and MVA, see Table I, ANTOINE. Virology. 1998, no.244, p.365-396.

According to a preferred variant implementation, the poxvirus of the present invention further includes a defective gene J2R. J2R gene encodes timedancing (TC), which are part of the "reutilization" way for the synthesis is deoxyribonucleotide pyrimidine. The reaction catalyzed by TC, involves the transfer of the γ-phosphorylated from APR 2 desoxy-thymidine (dThd)to produce thymidine 5'-monophosphate (dTMP). TC of vaccinia virus is of type 2. TC type 2 have a shorter polypeptide chain compared to type 1, ~25 kDa, but the form homotetrameric. They are sensitive to inhibitors feedback dTDP or dTTP, which are generated at the end of the metabolic pathway. The TC type 2 is more narrow specificity to the substrate compared to the TC type 1 and only phosphorylate 2 deoxyuridine (dU) and/or dThd (EL OMARI et al. Structure of vaccinia virus thymidine kinase in complex with dTTP: insights for drug design. BMC structural biology. 2006, no.6, p.22.).

Poxviruses, defective at the site J2R, and methods for their preparation are available in the art. For example, the guide MCCART, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes, cancer research. 2001, vol.61, no.24, p.8751-7., PUHLMANN, et al. Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant. Cancer gene therapy. 2000, vol.7, no.1, p.66-73., GNANT, et al. Systemic administration of a recombinant vaccinia virus expressing the cytosine deaminase gene and subsequent treatment with 5-fluorocytosine leads to tumor-specific gene expression and prolongation of survival in mice. Cancer Research. 1999, vol.59, no.14, p.3396-403 can be used to obtain a poxvirus with a deletion of the plot J2R.

According to a preferred variant embodiment, the poxvirus according to the invention further includes a target nucleic acid.

In a preferred embodiment, realized the value of the target nucleic acid contains at least one target sequence, encoding a gene product, which is a therapeutic molecule (that is, a therapeutic gene). "Therapeutic molecule" is a molecule that has a pharmacological or protective activity when properly administered to a patient, especially a patient suffering from a painful condition or disease or the person who should be protected from the disease or condition. Such pharmaceutical or protective activity is an activity that is expected, will be associated with a favorable effect on the course or a symptom of a specified disease or specified state. When a qualified technician selects during an existing invention the gene encoding a therapeutic molecule, it actually connects your choice with the previously obtained results, and can reasonably be expected, without undue experiment, except for carrying out the invention according to the formula, to get this pharmacological property. According to the invention, the target sequence may be homologous or heterologous Milenium cells into which it is introduced. Preferably the specified target sequence encodes all or part of the polypeptide, particularly a therapeutic or prophylactic polypeptide, providing therapeutic or preventive property. Polypeptide, as I understand, is lubiteleiam product polynucleotide regardless of size, and regardless of the glycosylation, and includes peptides and proteins. Therapeutic polypeptides include as a primary example, those polypeptides that can compensate for defective or imperfect proteins in the animal or human body, or those that act through toxic effects to limit or remove harmful cells from the body. They may also be giving immunity polypeptides that act as endogenous antigen to induce a humoral or cellular response, or both.

Examples of polypeptides encoded therapeutic gene include genes encoding cytokines (alpha, beta or gamma interferon, interleukin, especially IL-2, IL-6, IL-10 or IL-12, tumor necrosis factor (TNF), kolonie-stimulating factor GM-CSF, C-CSF, M-CSF...), immunostimulatory polypeptide (V, W and the like), a coagulation factor (FVIII, FIX...), growth factor (transforming growth factor TGF, fibroblast growth factor FGF and the like), the enzyme (urease, renin, thrombin, metalloproteinase, synthase nitric oxide NOS, SOD, catalase...), enzyme inhibitor (alpha 1-antitripsin, antithrombin III, a viral protease inhibitor, an inhibitor of plasminogen activator PAI-1), CFTR (transmembrane conductance regulator cystic fibrosis) protein, insulin, dystrophy, antigen MHC class I or II, polypeptide, which can modulate/regulation is ü the expression of cellular genes, polypeptide capable of inhibiting a bacterial, parasitic or viral infection or its development (antigenic polypeptides, antigenic epitopes, transdominant options, inhibiting the action of a native protein by competition...), inducer or inhibitor of apoptosis (Bax, Bcl2, BclX...), the cytotoxic agent (P21, P16, Rb,...), apolipoprotein (ApoAI, ApoAIV, ApoE...), an inhibitor of angiogenesis (angiostatin, endostatin...), angiogenic polypeptide (family of vascular endothelial growth factors VEGF, FGF family, the CCN family, including CTGF, Cyr61 and Nov), the scavenger of oxygen radicals, a polypeptide having an antitumor effect, an antibody, a toxin, an immunotoxin and a marker (beta-galactosidase, luciferase, etc.) or any other target genes recognized in the art as useful for the treatment or prevention of a clinical condition.

Suitable antitumor genes include, among others, the genes that encode genes tumor suppressor (e.g., Rb, p53, DCC, NF-1, tumor Wilm, NM23, BRUSH-1, p16, P21, R, R, and their respective mutants), products of suicide gene, antibodies, polypeptide, inhibiting cell division or signal transduction.

According to a particularly preferred variant embodiment, the poxvirus of the present invention further includes a suicide gene.

Suicide gene refers to a gene that code is the dominant protein capable of converting the precursor of a drug into a cytotoxic compound.

Suicide genes include, among others, the genes encoding the protein having the cytosine-desaminase activity, thymidine kinase activity, uracil-phosphoribosyl transfersno activity, polynucleotide-phosphorylase activity and/or thymidylate kinase activity.

Examples of suicide genes and the corresponding precursors of the medicinal product, including the one remaining nucleobase, are disclosed in the following table:

Table 1
Suicide geneThe prodrug
Thymidine kinaseGanciclovir;
ester of ganciclovir, Sadovoy acids;
penciclovir;

Suicide geneThe prodrug
acyclovir;
valacyclovir;
(E)-5-(2-bromovinyl)-2'-deoxyuridine;
idowu is in;
2'-Exo-metanarrative
Sitoindosides5-Fertilizin
Polynucleotides6-methylpyridoxine; fludarabine
Uralspetstransmash5-Fertilizin; 5-fluorouracil inside the body
TimetrackingAzidothymidine

According to a preferred variant embodiment of the invention, the suicide gene encodes a protein that has at least the activity D. D involved in the pyrimidine metabolic pathway by which exogenous cytosine is converted to uracil by hydrolytic deamination. While activity D been demonstrated in prokaryotes and lower eukaryotes (JUND, et al.. Journal of Bacteriology. 1970, no.102, p.607-15.; BECK, et al.. Journal of Bacteriology. 1972, no.110, p.219-28.; NOURISH, et al.. Journal of Infectious Diseases. 1974, no.130, p.112-18.; ESDERS, et al., J. biol. chem.. 1985, no.260, p.3915-22.), they are not present in mammals (KOECHLIN et al. Biochemical pharmacology. 1966, no.15, p.435-46.; POLAK, et al.. Chemotherapy. 1976, no.22, p.137-53.),

CDaa will also desaminase analogue of cytosine, 5-fertilizin (5-FC), thus forming a 5-fluorouracil inside the body (5-FU), which is a connection, which is what I highly cytotoxic when it is converted to 5-fluoro-UMP (5-FUMP). Cells that do not have activity D, or due to mutation, which inactivates the gene encoding the enzyme, or because they do not have this natural enzyme, as in mammalian cells, are resistant to 5-FC (JUND, et al. Journal of Bacteriology. 1970, no.102, p.607-15.; KILLSTRUP, et al.. Journal of Bacteriology. 1989, no.171, p.2124-2127.). In contrast, mammalian cells, which have migrated sequence encoding activity D, were sensitive to 5-FC (HUBER, et al. Cancer Research. 1993, no.53, p.4619-4626.; MULLEN, et al.. Proceedings of the National Academy of Sciences of the United States of America. 1992, no.89, p.33-37.; WO 93/01281 (US HEALTH)). In addition, neighboring, untransformed cells become sensitive to 5-FC (HUBER, et al.. Proceedings of the National Academy of Sciences of the United States of America. 1994, no.91, p.8302-6.). This phenomenon, which is called the effect "witness", is due to cells that Express the activity CD, secreting 5-FU, which intoxicated neighboring cells by direct diffusion through the plasma membrane. This property is 5-FU is relatively passive diffusion is the advantage compared to tk/GCV reference system in which the effect of the "witness" requires contact with cells that Express tk (MESNIL et al. Proceedings of the National Academy of Sciences of the United States of America. 1996, no.93, p.1831-35.). All of the benefits that D offers within the context of gene therapy, in particular anti-cancer gene Ter the FDI can therefore be easily understood.

Genes codA Saccharomyces cerevisiae (S. cerevisiae) FCY1, Candida Albicans FCA1 and E. coli, which respectively encode D these two organisms known, and their sequences have been published (SEQ ID No:4; SEQ ID No:5; SEQ ID no:6, respectively).

In this respect, according to a more preferred variant of the embodiment of the present invention, a gene encoding a protein having the activity CD is FCY1, FCA1 or CodA or their equivalent. Analogues of these genes affect gene having the sequence of nucleic acid, which has a degree of identity of at least more than 70%, mostly more than 80%, preferably more than 90%, and most preferably more than 95% with the nucleic acid sequence of the parent gene.

Patent WO 2005/007857 describes a gene that encodes a protein having improved activity CD. These polypeptides obtained from native CD by supplementing amino acid sequence. According to another preferred variant of the embodiment of the present invention, a protein having the activity CD, is a polypeptide, disclosed in WO 2005/007857, and more preferably the polypeptide FCU1-8, presented in the sequence identifier SEQ ID No:2 and their equivalents.

In prokaryotes and lower eukaryotes, uracil is converted to UMP under the action of ur is certstoreparameters (UR). This enzyme converts 5-FU to 5-FUMP. According to another preferred variant of the embodiment of the present invention, the suicide gene encodes a protein having the activity of UPR-Basins.

Consider UPR-Pelvis may be of any origin, in particular prokaryotic origin, fungal or yeast origin. By way of illustration, a nucleic acid sequence encoding UR from E. coli (ANDERSEN, et al. Characterization of the upp gene encoding uracil phosphoribosyltransferase ofEscherichia coli K12. European Journal of Biochemistry. 1992, no.204, p.51-56.), from Lactococcus laclis (MARTINUSSEN et al. Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis. Journal of Bacteriology. 1994, vol.176, no.21, p.6457-63.), from Mycobacterium bovis (KIM, et al. Complete sequence of the UPP gene encoding uracil phosphoribosyltransferase from Mycobacterium bovis BCG. Biochemistry and molecular biology international. 1997, vol.41, no.6, p.1117-24.) and from Bacillus subtilis (MARTINUSSEN et al. Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis. Journal of Bacteriology. 1995, vol.177, no.1, p.271-4.) can be used in the context of the invention. However, most especially preferable to use the yeast UPR-Pelvis and in particular S. cerevisiae encoded FUR1 gene, the sequence of which is disclosed in KERN, et al. The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene. 1990, vol.88, no.2, p.149-57, which is introduced by the authors by reference. As a guide sequence genes and sequences corresponding UPRTa can be the Aiden in the literature and databases of specialists (SWISSPROT, EMBL, Genbank, Medline etc).

Application EP 0998568 AND describes the FUR1 gene, no 105 nucleotides in the 5' coding portion, allowing the synthesis UPRT from which have been removed 35 of the first residue in the N-terminal position, and beginning with methionine at position 36 in the native protein. The expression product of the gene-mutant called FUR1Δ105 capable complementarian fur1 mutant of S. cerevisiae. In addition, the truncated mutant shows higher activity UR than that of native enzyme. Thus, according to a particularly preferred variant of the embodiment of the present invention, the suicide gene encodes a mutant deletions native ectasy. The deletion is preferably located in the N-terminal region of the original UPR-Basins. It can be full (affecting all remnants of the specified N-terminal region) or partial (affecting one or more continuous or discontinuous residues in the primary structure). In General, the polypeptide consists of the N-terminal, Central and C-terminal parts, each of which represents approximately one third of the molecule. For example, since UPR-Pelvis S. cerevisiae has a 251 amino acid, its N-terminal part consists of the first 83 residues, starting with the so-called initiator methionine, which is located in the first position of the native form. As for UR E. coli, its N-terminal is part covers the provisions 1-69.

Preferred protein having the activity of UPR-Basins, includes the amino acid sequence of almost such as presented in the sequence identifier SEQ ID No:1 of EP 0998568 AND starting with a Met residue in position 1 and ending with the Val residue at position 216. The term "almost" refers to the degree of identity with the specified sequence SEQ ID No:1 EP 0998568 more than 70%, mostly more than 80%, preferably more than 90%, and most preferably more than 95%. Still more preferably it comprises the amino acid sequence presented in the sequence identifier SEQ ID No: 1 EP 0998568 A. As mentioned above, it may contain additional mutations. In particular, there may be mentioned the replacement of the serine residue in position 2 (position 37 in native UR) alanine residue.

According to another preferred variant of the embodiment of the present invention, the suicide gene encodes a protein that has at least one activity CD and one activity UR. Patent application WO 96/16183 and EP 0998568 AND describe the use of the fused protein, encoding an enzyme with two domains, with activity CD and UR, and demonstrate that the transfer of the hybrid gene codA::upp or FCY1::FUR1 or FCY1::FUR1A105 (i.e. FCU1), which carries a plasmid expression, HC which increases the sensitivity of the transfected Into 16 cells to 5-FC. According to a more preferred variant of the embodiment of the present invention, the suicide gene encodes a polypeptide comprising the amino acid sequence, almost like presents in the sequence identifier SEQ ID No:3 (coda:: upp), SEQ ID No:1 (FCU1) or FCY1:: FURL the Term "almost" refers to the degree of identity with the specified sequence, more than 70%, mostly more than 80%, preferably more than 90%, and most preferably more than 95%. Still more preferably, it comprises the amino acid sequence, almost like presents in the sequence identifier SEQ ID No:3 (coda::upp), SEQ ID No:1 (FCU1) or FCY1::FUR1. As mentioned above, it may contain additional mutations.

Nukleinovokisly sequence can be easily obtained by cloning, PCR, or by chemical synthesis according to conventional techniques. They can be native genes or genes derived from them by mutations, deletions, substitutions and/or additions of one or several nucleotides, in Addition, their sequences are described in the literature, and can be accessed specialists, skilled in the art.

Experts, skilled in the art, capable of clone sequence is eljnosti D or UR on the basis of published data and make possible mutations, to test the enzymatic activity of the mutant forms in a cell-free or cell system according to the technology of the prior art or based on the Protocol indicated in the application EP 0998568 and fusing, in particular in the phase, the polypeptides with the activity D and UR, and therefore with all or part of the corresponding genes.

According to a more preferred variant embodiment, the poxvirus of the invention further includes nukleinovokisly sequence comprising the gene encoding permease.

Permease concerns transmembrane protein involved in the transfer of a medicinal product, including the one remaining nucleobase, or its predecessor across the cell membrane.

Permease includes, but restrictions listed, perinparasa, cytokinemia and nucleoside transporters. According to a preferred variant embodiment of the present invention, permease is a purine or setosipennis S. Cerevisiae. The conveyors nucleobase S. cerevisiae consist of the purine-cytosine, permease known as FCY2, and uretilirmis known as FUR4. The purine - cytosine permease, FCY2 mediates import protons and adenine, guanine, gipoksantina and cytosine through the plasma membrane of yeast (Grenson 1969, Jund and Lacroute 1970, Polak and Grenson 1973, Chcvallier et al. 1975, Hopkins et al. 1988). Protein FCY2 Opera is the duty to regulate the transport of 5-fertilizin, analogue of cytosine (Grenson 1969, Jund and Lacroute 1970). FCY2 gene encodes a protein of 533 amino acids (58 kDa), as originally intended, has 10-12 transmembrane rotating domains (Weber et al. 1990), nine of which currently approved (Ferreira ct al. 1999). FCY2 shows similar affinity for purine nucleobase and cytosine (Brethes et al. 1992). The capture of uracil in S. cerevisiae is mediated by precipitously, FUR4 (Jund and Lacroute 1970, Jund et al. 1977). FUR4 is a uracil-proton symporter (Hopkins et al. 1988), which apparently is a protein of 633 amino acids (71,7 kDa) with 10 transmembrane domains and a long cytoplasmic hydrophilic N - and C-terminal tails (Jund et al. 1988, Gamier et al. 1996). Protein FUR4 may also mediate the transport of 5-fluorouracil inside the body, analogue of uracil (Jund and Lacroute 1970).

Amino acid sequence FCY2 and Fur4 especially available in swissprot database (inventory number R 17064 and R, respectively). Preferably, permease has an amino acid sequence selected from the group comprising amino acid sequence SEQ ID N0:1 and SEQ ID N0:2, as disclosed in patent application WO 2006/048768.

In this respect, according to a preferred variant embodiment of the present invention, permease is selected from the group comprising FCY2 and Fur4 and their analogues. Analogues Fur4 and FCY2 related polypeptide having the amino acid consistently is th which has a degree of identity of at least more than 70%, mostly more than 80%, preferably more than 90%, and most preferably more than 95% with the amino acid sequence of the parent protein as described by the authors above and which retain the ability to transport the drug, including one remaining nucleobase through the cell membrane.

Specialist, skilled in the art can choose permeate to be associated with drug or a precursor of the drug, including one remaining nucleobase. For example, FCY2 and Fur4 preferably contacted with 5-fertilizing (5-FC).

In accordance with a more preferred embodiment, the poxvirus of the invention may optionally include the elements necessary for the expression of a target nucleic acid.

In accordance with a more preferred embodiment, the poxvirus of the invention may optionally include elements necessary for expression nukleinovokisly sequence comprising the gene encoding permease. These elements necessary for the expression of a target nucleic acid and/or nukleinovokisly sequence comprising the gene encoding permease included the elements required for transcription of the indicated DNA into mRNA is, if necessary, for translation of mRNA into a polypeptide. Transcription promoters suitable for use in various systems of vertebrates, is widely described in the literature. For example, additional suitable promoters viral promoters, such as RSV, MPSV, SV40, CMV or 7,5k, promoter ospowiki, the inducible promoters, etc. Preferred promoters are isolated from poxviruses, for example, C, H5R, TK, P28, P11 or K1L of vaccinia virus. Alternatively, you can use synthetic promoter, such as described in CHAKRABARTI.. Biotechniques. 1997, no.23, p.1094-97., HAMMOND, et al.. Journal of Virological Methods. 1997, no.66, p.135-38. and KUMAR.. Virology. 1990, no.179, p.151-8, as well as the chimeric promoters between early and late the poxvirus promoters.

Target nukleinovokisly sequence and nukleinovokisly sequence comprising the gene encoding permease can optionally include additional functional elements, such as the sequence of the intron, the target sequence, the transport sequence, a secretion signal, a nuclear localization signal, IRES, poly And sequence termination of transcription, tripartite leader sequence, sequences involved in replication or integration. Of these sequences has been reported in the literature, and they can be easily derived guests are welcomed the specialists in this field of technology.

The invention also relates to a method for obtaining poxvirus according to the invention, in which method:

(i) poxvirus according to the invention is introduced into a cell

(ii) the specified cell is cultivated under conditions which are relevant in order to allow the products specified poxvirus, and

(iii) specified poxvirus isolated from the cell culture.

While poxvirus may, of course, be isolated from the supernatant of the culture, it can also be isolated from the cells. One of the commonly used methods is the decomposition of cells through successive cycles of freezing/thawing to assemble virions in lisany supernatant. Then these virions can be amplified and purified using the methods of the art (chromatographic method, ultracentrifugation, in particular through the gradient of cesium chloride, and so on).

The present invention also relates to compositions that include poxvirus according to the invention in combination with a pharmaceutically acceptable auxiliary substance.

The composition in accordance with the invention is more particularly intended for the preventive or healing therapy of diseases through gene therapy and more specific focus on proliferative diseases (cancers is e education tumors, restenosis, etc) or focused on diseases associated with increased activity of osteoclasts (e.g., rheumatoid arthritis, osteo porous).

The composition in accordance with the invention can be made traditionally for its introduction locally, parenterally or by digestive. In particular, a therapeutically effective amount of the recombinant vector or the poxvirus of the present invention is combined with a pharmaceutically acceptable auxiliary substance. It is possible to provide a large number of routes of administration. Examples that may be mentioned include intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumoral, intranasal, intra-lungs and intratracheal route. In the case of these three latter embodiment it is preferable that the introduction took place with the help of an aerosol, or by instillation. The introduction may occur as a single dose or dose, which is repeated in one or more cases, after a certain time interval. The appropriate route of administration and dosage varies depending on many parameters, such as the patient, the disease to be treated, or the target gene(s) (s) is(are) transferred(s). Drugs based on viral particles is according to the invention, can be formulated in the form of doses comprising from 104up to 1014pfu (plaque-forming unit), mainly from 105up to 1013pfu, preferably from 106up to 1012pfu, more preferably from 106up to 107.

The composition can also include a solvent, adjuvant or excipient which is acceptable from a pharmaceutical point of view, as well as solubilizers, a stabilizing agent and a preservative. In the case of injecting preference is given to compositions in aqueous, nonaqueous, or isotonic. It can be represented as a single dose or multidose, in liquid or dry form (powder, lyophilized, and so on), which can be prepared while using using the appropriate solvent.

The present invention also relates to the use of poxvirus or compositions according to the invention for the preparation of a medicinal product which is intended for treatment of the human or animal body gene therapy. This drug can be administered in vivo (for example, intravenous injection, in an accessible tumor, light spray, into the vascular system, using the appropriate catheter, etc). The preferred use is in the treatment or prevention R is the same formations, tumors and diseases that result from undesirable cell proliferation. Conceivable applications that may be mentioned include cancers of the breast, uterus (especially caused by papilloma viruses), prostate, lung, bladder, liver, intestines, pancreas, stomach, esophagus, larynx, Central nervous system (e.g., glioblastoma) and blood (lymphoma, leukemia, etc). Another preferred use is in the treatment or prevention of rheumatoid arthritis, osteoporosis and other diseases associated with increased activity of osteoclasts. It can also be used in the context of cardiovascular disease, for example, to inhibit or delay cell proliferation of smooth muscle walls of the blood vessel (restenosis). Finally, in the case of infectious diseases, it is possible to apply the drug in AIDS.

When poxvirus, composition or method of the invention are used to treat cancer, the preferred route of administration is a system path, so as poxvirus according to the invention in a condition specific to target tumor cells.

The invention also encompasses a method for treating a disease characterized by the fact that poxvirus, a composition in accordance with the invention are introduced into the organism is ozaena or cell, desperately in need of such treatment.

According to an advantageous variant embodiment, a therapeutic use or a method of treatment also includes additional Studio, in which the pharmaceutically acceptable amount of a prodrug, mainly analogue of cytosine, in particular 5-FC, is introduced into the host organism or cell. By way of illustration it is possible to use a dose of from 50 to 500 mg/kg/day, with a preferred dose of 200 mg/kg/ day or 100 mg/kg/day. Within the context of the present invention, the prodrug is administered in accordance with standard practice (e.g., oral, systematically).

Preferably, the introduction, occurring after the introduction of the poxvirus or compositions in accordance with the invention, preferably at least after 3 days, more preferably at least 4 days and even more preferably at least 5 days after the introduction of the poxvirus or compositions in accordance with the invention. According to even more preferred embodiment of the present invention, the introduction of the prodrug occurs after 7 days after administration of therapeutic agent. Preferred oral route. It is possible to enter a single dose of prodrug or doses that are repeated for some time, which is sufficient to allow the production of toxic metal which lits within the host organism or cell.

In addition, the composition or method according to the invention can be combined with one or more substances that patentiert the cytotoxic effect of 5-FU. In particular, one can mention the drugs, which inhibit the enzymes of the pathway for de novo biosynthesis of pyrimidines (for example, mentioned below), drugs such as leucovorin (Waxman et al., 1982, Eur. J. Cancer Clin. Oncol. 18, 685-692), which, in the presence of the product of the metabolism of 5-FU (5-FdUMP), increases the inhibition timedilation, reducing the pool of dTMP, which is required for replication, and finally, drugs such as methotrexate (Cadman et al., 1979, Science 250, 1135-1137), which, by inhibiting dihydrotetrazolo and increasing the pool of PRPP (phosphoribosylpyrophosphate)causes an increase in the introduction of 5-FU in cellular RNA.

According to the present invention, drugs that inhibit the enzymes of the pathway for de novo biosynthesis of pyrimidines, preferably selected from the group consisting of PALA (N-(phosphonacetyl)-L-aspartate; Moore et al., 1982, Biochem. Pharmacol. 31, 3317-3321), Leflunomide, A (the active metabolite of Leflunomide; Davis et al., 1996, Biochem. 35, 1270-1273) and brequinar (Chen et al., 1992, Cancer Res. 52, 3251-3257).

The composition or method in accordance with the invention can be combined with one or more substances that are effective in cancer treatment. Environments of the pharmaceutical substances effective in anti-cancer therapy that can be used in Association or in combination with compositions according to the invention, mention can be made of alkylating agents, such as, for example, mitomycin C, cyclophosphamide, busulfan, ifosfamide, isospeed, melphalan, hexamethylmelamine, thiotepa, chlorambucil, or dacarbazine; antimetabolites such as gemcitabine, capecitabine, 5-fluorouracil inside the body, cytarabine, 2-fordetection, methotrexate, edatrexate, tomudex or trimetrexate; topoisomerase II inhibitors such as, for example, doxorubicin, epirubicin, etoposide, teniposide, or mitoxantrone; inhibitors of topoisomerase I such as, for example, irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan; antimitoticescoe drugs, such as, for example, paclitaxel, docetaxel, vinblastine, vincristine or vinorelbine; and platinum derivatives, such as, for example, cisplatin, oxaliplatin, spiroplatin or carboplatin.

The compositions or methods according to the invention can also be used in combination with radiation therapy.

The compositions or methods according to the invention can also be used in combination with one or more other agents, including, among others, immunomodulatory agents, such as, for example, alpha, beta or gamma interferon, interleukin (about what bennoti, IL-2, IL-6, IL-10 or IL-12) or tumor necrosis factor; agents that affect the regulation of cell surface receptors, such as, for example, inhibitors of the receptor for epidermal growth factor (in particular, cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib, erlotinib or lapatinib) or inhibitors of the receptor for human epidermal growth factor 2 (in particular, transtuzumab); and agents that affect angiogenesis, such as, for example, an inhibitor of vascular endothelial growth factor (especially bevacizumab or ranibizumab).

A brief description of the Figures in the drawings

Figure 1. Sensitivity in vitro to 5-FC from infected with vaccinia virus human colorectal tumor cells (LoVo). The LoVo cells infected at MOI of 0.0001 these viruses (example (•) VVTK-/FCU1 (■) or VVTK-F2L-/FCU1 (◊), is subjected to the action of various concentrations of 5-FC. The survival of cells determined after 5 days after infection. The results expressed in percentage of cell viability in the presence or in the absence of drugs. Values represented as mean ±SD of three individual determinations without the death of cells due to viral replication.

Figure 2. The efficiency of viral replication in vitro in LoVo infected at MOI of 0.0001 indicated virus on day 5 after infec the simulation. Values represented as mean ±SD of three individual determinations.

Figure 3. The mean tumor volume ±SEM p/LoVo the Swiss bare mice after the injection of the virus. After 7 days after inoculation of the tumor (palpable tumor), mice treated with 107pfu buffer+salt solution (◊), buffer + 5-FC (♦), VVTK-F2L-/FCU1 + saline (□) or VVTK-F2L-/FCU1 + 5-FC (■). Animals treated with saline or 5-FC at 100 mg/kg/j twice daily gavage feeding, after 7 days after viral injection for 3 weeks. Twice per week to determine the volume of the tumor.

Figure 4. The mean tumor volume ±SEM p/HepG2 tumors in Swiss bare mice after the injection of the virus. After 14 days after inoculation of tumor (palpable tumor), mice treated with 107pfu buffer + water (◊), or buffer + 5-FC (♦), or 106pfu VVTK-/FCU1 + water (o), or 106pfu VVTK-/FCU1+5-FC (•) (A); or buffer + water (◊), or buffer + 5-FC (♦) or 106pfu VVTK-F2L-/FCU1 + water (0), or 106pfu VVTK-F2L-/FCU1+5-FC (♦) (In). Animals treated with 5-FC at 100 mg/kg twice daily gavage feeding, after 7 days after viral injection, and within 3 weeks. Twice per week to determine the volume of the tumor.

Figure 5. The ratio of the output of the virus in dividing cells compared to confluent cells. PANC1 cells (tumor of the pancreas of a person), H1299 (human lung tumor or U118MG (tumor gliomas person) infect with 100 pfu (∎) VVTK-/FCU1 or (□) VVTK-F2L-/PCU1. After 48 hours after infection to determine viral titers. Values represent the ratio between the outputs of the virus in dividing cells compared to confluent cells.

Figure 6. Viral titers (pfu/mg tissue) in the organs or tumors at day 6 and day 21 after the/in infection in Swiss Nude mice bearing subcutaneous human tumor with 1×106PFU VVTK-/FCU1 (∎) or VVTK-F2L-/FCU1 (□).

Figure 7. The survival of the Swiss bare mice after treatment with 1×108pfu VVTK-/FCU1 (∎) or VVTK-F2L-/FCU1 (◊) by/injection.

Figure 8. Survival of immunocompetent B6D2 mice after treatment with 1×107pfu (A) or 1×108pfu (IN) VVTK-/FCU1 (■) or VVTK-F2L-/FCU 1 (◊) by/injection.

Figure 9. The average number of pock marks on the tails after the/in the injection of 1×106pfu VVTK-/FCU1 or VVTK-F2L-/FCU 1 the Swiss bare mice at day 13 after infection and at day 34 post-infection.

Figure 10. The average number of pock marks on the tails after the/in the injection of 1×107pfu VVTK-/FCU1 or VVTK-F2L-/FCU 1 the Swiss bare mice at day 15 after infection and at day 31 after infection.

Method(s) for carrying out the invention

Examples

Design vector plasmids

Build a Shuttle plasmid for deletion F2L using DNA strain Copenhagen vaccinia virus (inventory number M). Flanking DNA regions F2L amplified with what omashu PCR. The primers of the rising flank area F2L represent 5' - CGC GGA TCC GAA AGC GAT GAA HUNDRED AAT GTT C - 3' (SEQ ID no:7; the BamHI site is underlined), and

5' - TCC CCC GGG GTT AGT TTC CTT AAC AAA TST AAC - 3' (SEQ ID No:8; SmaI site underlined), Primers downstream of the site represent a 5' - GCC TGG CCA ACA AAT AGA GGA GAT CAA GGG T - 3' (SEQ ID No:9; plot MscI underlined)and 5' - GCC CAG CTG ACC ACT ACA TCA ATT TTA CAA AAG - 3' (SEQ ID No:10; PvuII site is underlined). Amplificatory DNA fragment cleaved by the restriction enzyme SmaI/BamHI or MscI/PvuII and are ligated into the corresponding sites in the plasmid PpolyIII. Re-plot the downward flank area F2L amplified by PCR using primers 5' - GCC GCA TGC TCC AGA ATT GAT CAT AGT GGA TA - 3' (SEQ ID No:11; SphI site underlined)and 5' - GCT HUNDRED GAG TTA GTT TCC TTA ACA AAT HUNDRED AC - 3' (SEQ ID No:12; XbaI site underlined) and inserted into plasmid PpolyIII. Re-plot is used to remove the cassette selection during production deleted viruses. The cassette selection corresponding to the slit gene GFP/GPT when the control pH5R promoter ospowiki, is inserted into the plot SmaI/SphI plasmid PpolyIII. The resulting plasmid is a recombinant Shuttle plasmid called pAF2L to remove F2L gene.

The preparation of recombinant viruses ospowiki.

Cells infect CEF strain Copenhagen VVTK-FCU1 (Vaccinia virus, defect is in the gene J2R, expressing FCU1 gene under the control of a synthetic promoter p11k7.5) at MOI of 0.1 and incubated at 37°C for 2 hours, then transferout with coprecipitation CaCl2recombinant Shuttle plasmid (0.2 mkg). Cells are incubated for 48 hours at 37°C. the resulting dilution of the virus used to infect cells in SERIES in selective medium containing gipoksantin at a final concentration of 15 μg/ml xanthine at a final concentration of 250 µg/ml and mycophenolate acid at final concentration of 250 μg/ml Fluorescent (GFP) and positive (GPT selection) plaques are isolated and selected by several rounds of selection in CEF cells in the presence of selective medium GPT. The presence or absence of VVTk-FCU1 account for 40 cycles of PCR with primers in the region deletions. After elimination of the parent virus use dual remote virus to infect CEF without selective environment GPT to fix the cassette selection. Afluorescent plaques are isolated and selected for 2 cycles in CEF. The final recombinant viruses W amplified in CEF, clean and viral source solutions titrated on CEF using the test plaques.

Cellular sensitivity in vitro to 5-FC.

Human tumor cells convert the corresponding recombinant W at MOI of 0.0001. In total, 3×105cells/well was placed on 6-hole Cup for the culture in 2 ml of medium, containing various concentrations of 5-FC. The cells are then cultured at 37°C for 5 days, and viable cells counted using exception Trianon blue. The results, shown in figures 1, 2 show that the activity FCU1 equivalent viruses, defective in the gene J2R than virus defective in the gene J2R and F2L.

In vitro replication in cultured cells.

Dividing cells or confluent cells infect, 6-of linkovich disks, with 100 PFU of virus (almost MOI of 0.0005), Add 2 ml of medium with 10% FCS for dividing cells and without additives for merged cells. Cells are harvested 48 hours after infection. Cells stored at -20°C and treated with ultrasound for release of the virus, the virus is also determined quantitatively by titration drives on the cells of SER. The relationship between replication in dividing cells and confluent cells, like all cells. Both virus VVTK-/FCU1 and WTK-F2L-/FCU1 replicated more in dividing cells than in confluent cells.

As an indirect value for the quantitative determination of viral specificity to replicate, determine the yield of virus obtained in dividing cells compared with confluent tumor cells (human pancreatic cancer PANC1; human lung tumor N; human glioma tumor U118MG), Confluent cells are placed on the square is nsity at 1×10 6cells/well and cultured in complete media for 7 days, then 1 day before infection, the cells are washed and cultured in medium without serum. Dividing cells are placed on the plate at 3×105cells/well one day before infection. To assess the level of cell division, the number of titrated thymidine incorporated in the nucleic acid, determined after 5 hours, 24 hours and 48 hours after culturing cells on the tablet. During this period, the incorporation of thymidine is relatively constant in confluent cells, whereas in dividing cells the increase in the inclusion of notice over time. Then infect cells with 100 pfu of virus, and after 48 hours after infection, the ratio between the output of the virus produced in dividing tumor cells and confluent tumor cells, determined by titration disks on CEF. The results, depicted in figure 5, show that both virus VVTK-/FCU1 and WTK-F2L-/FCU1 more replicate in dividing cells than in confluent cells.

Subcutaneous tumor model.

Female Swiss Nude mice obtained from Charles River Laboratories. Animals used in research, are uniform in age (6 weeks), and body mass ranging from 20 to 23 shveycarskim naked mice injected subcutaneously (s/C) in the side 5×106of LoVo cells/100 μl. When is the tumor reached a diameter of 50-70 mm 3mice randomisiert arbitrary manner and treated with the indicated vectors for in vivo experiments.

The biodistribution of the virus.

The presence of VV-FCU1 and WTK-F2L-/FCU 1 assess viral titration in samples of organs and tumors 1×106viruses injected intravenous (IV) injection into the tail vein of naked mice with set p/LoVo tumors. Mice wortlaut on day 14 after infection and tumors and other organs are harvested and weighed. Tumors and organs homogenized in PBS, and the titers determined on CEF, as described previously. Viral titers to standardise milligram of tissue. Viral titers to standardise milligram of tissue. The results, shown in Table 2 and 3 (range of viral titers are presented in pfu/mg tissue), show that after 14 days the virus according to the invention is mainly detected in the tumor. In the second set of experiments, in accordance with the same conditions as described above, mice wortlaut on day 6 and day 21 after infection. The results, depicted in figure 6, show that both virus VVTK-/FCU1 and VVTK-F2L-/FCU1 target tumor approximately 1000-10000 times more virus in the tumor than in the other organs analyzed except for the tails in the case of VVTK-/FCU1. A small amount of VVTK-/FCU1 detected in lung, spleen, kidney and lymph nodes (less than 10 pfu/mg) and more in the skin, x is the OST and the bone marrow at day 6, and the skin and the tail at day 21. On the contrary, VVTK-F2L-/FCU1 has a higher specificity to the tumor with only a small amount in the lungs, spleen, kidney, lymph nodes and skin at day 6, and in the skin and the tail at day 21.

Table 2
TumorLightSpleenKidneyHeartLymph nodes
VVTK-/FCU1(from 0.2 to 3.3)×1050.1 to 20-2,20-1,80-0,30-61
VVTK-F2L/FCU10-8,1×l040-5,70-9,30.2 to 30,20-83

Muscles
Table 3
OvariesLeatherTailBone marrowThe brainHeart
VVTK-/FCU12,2-740,1-2413,5-7·1040-8000-1,80-220-0,3
VVTK-F2L/FCU10-1080-880,8-16,3n/R0-0,20-1,80,2

Antitumor activity of a poxvirus of the invention to p/C model of the tumor.

Naked mice with established p/LoVo tumors (50-70 mm) treated once nutripure or twice intravenously (tail vein) these vectors at a dose of 1.104PFU, 1.106PFU ABO 1.107PFU, respectively. Starting from day 7 after virus injection, 5-FC is administered orally through a feeding tube at 100 mg/kg (0.5 ml 5-FC 0.5% in water) twice a day for 3 weeks. Tumor size was determined twice a week using calipers. Tumor volume is calculated in mm3using the formula (p/6) (length × width2). The results, depicted in figure 3, show that 2 viruses have similar efficacy with oncolytic activity (p<0.05), and able in order to control tumor growth and joint activity (oncolytic virus and therapeutic gene FCU1) with the introduction of 5-FC, which could further improve the control of tumor growth (p<0,01).

Naked mice with established p/tumors HepG2 cells hepatocellular liver carcinoma, human) treated intravenously (tail vein) these vectors at a dose of 1.106PFU in accordance with the following scheme: after 14 days after inoculation tumors (palpable tumor) mice treated: buffer + water, or buffer + 5-FC, or 106pfu VVTK-/FCU1 + water, or 106pfu VVTK-/FCU1+5-FC, or 106pfu VVTK-F2L-/FCU1 + water, or 106pfu VVTK-F2L-/FCU1+5-FC. Animals treated with 5-FC at 100 mg/kg twice a day orally through a feeding tube, after 7 days after viral injection, and within 3 weeks. Tumor size was determined twice a week using calipers. Tumor volume is calculated in mm3using the formula (π/6) (length × width2). The results, depicted in figure 4, showing the control of tumor development after injection of VVTK-/FCU1 and VVTK-F2L-/FCU1 (p<0,0001) relative to the buffer. The activity does not increase after administration of 5-FC. Self oncolytic activity of the virus is very strong even during the injection of the viruses at a dose of 1×106PFU.

Viral pathogenicity.

Viral pathogenicity assess the survival studies, conducted on Swiss bare mice (figure 7) and immunocompetent mice B6D2 ((6 nedelec Charles Rivers) (figure 8)). Mice/enter 1.107or 1.108PFU all viruses in 100 μl of buffer on the mouse. The mice were observed daily during the experiment. The Swiss bare mice (figure 7) injection of 1×108PFU VVTK-/FCU1 leads to death in 40% of animals after 3 days after infection. The remaining mice die between day 50 and day 80 after infection. Introduction VVTK-F2L-/FCU1 is less pathogenic, most animals die between day 65 and 115 (p<0,05). There is no evidence of toxicity with both viruses at 107pfu. All mice die after/in the injection of 108pfu VVTK-/FCU1. Group treatment VVTK-F2L-/FCU1 has a significantly longer survival up to 92% (p<0,00005) compared with VVTK-/FCU1 infected mice. Therefore, this result demonstrates reduced toxicity with dual remote virus VVTK-F2L-/FCU1.

Model lesion tail winced.

Swiss bare mice/enter 1.106(figure 9) or 1.107(figure 10) PFU of each virus. Lesions of the tail calculate once a week. In mice, which impose 1.106PFU VVTK-F2L-/FCU1, there is less than 1 pock/mouse, compared with mice that enter VVTK-/FCU1 with an average number equal to 8 pockmarks on the mouse at day 13 after infection (p<0,001) as shown in figure 9 (A). The results are similar at day 34 after infection with an average number equal to 4 pockmarks with VVTK-/FCU1 on sravnenie is from about 1 to VVTK-F2L-/FCU1 (p< 0,0001) as shown in figure 9 (B). In mice, which impose 1.107PFU VVTK-F2L-/FCU1, marked, respectively, the average number equal to 3.5 pock marks/2 mouse and pockmarks/mouse, compared with mice that enter 1.107PFU VVTK-/FCU1, with an average of 10 pock marks/mouse at day 15 after infection (figure 10 (a)). At day 31 after infection in mice, which impose VVTK-F2L-/FCU1, marked respectively on average 3.5 pock marks/mouse, compared with mice that enter VVTK-/FCU1, with an average of 7 pock marks/mouse (figure 10). The difference in the number of pockmarks between VVTK-/FCU1 and VVTK-F2L-/FCU1 is statistically significant (p<0,05). The formation of pock marks correlates with viral replication in the tail, as well as virulence and toxicity. Injection/VVTK-F2L-/FCU1 is less toxic than single remote virus TK.

The statistical analysis.

Conduct statistical studies using nonparametric U test Mann-Whitney and the software STATISTICA 7.1 (StatSoft, Inc). P<0.05 are considered statistically significant.

Links

- US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15.11.1994

- WO 2004/014314 (DAVID KIRN (US)) 19.02.2004

- WO 2004/014314 (DAVID KIRN (US)) 19.02.2004

- US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15.11.1994

- WO 93/01281 (US HEALTH)

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- EP 0998568 A

- EP 0998568 A

- EP 0998568 A

- WO 96/16183

- EP 0998568 A

- EP 0998568 A

- WO 2006/048768

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1. Poxvirus ospowiki with oncolytic activity and including defective F2L gene and the target nucleic acid, comprising a suicide gene and a defective F2L gene leads to the inability of the virus to produce any protein having the activity of a protein produced by the unmodified gene.

2. Poxvirus according to claim 1, where the specified poxvirus further includes a defective gene J2R.

3. Poxvirus according to claim 1, where said poxvirus is a strain WR Vaccinia virus.

4. Poxvirus according to claim 1, where said poxvirus is a strain Copenhagen Vaccinia virus.

5. Poxvirus according to claim 1, wherein said suicide gene encodes a protein that has at least the activity sitoindosides.

6. Poxvirus according to claim 5, wherein said suicide gene is a FCY1, FCA1, or CodA, or the equivalent.

7. Poxvirus according to claim 5, wherein said white is, has at least the activity sitoindosides is a polypeptide FCU1-8, presented in the sequence identifier SEQ ID no:2 and its analogs.

8. Poxvirus according to claim 1, wherein said suicide gene encodes a protein that has at least one activity sitoindosides and one uralspetstransmash.

9. Poxvirus of claim 8, wherein said suicide gene encodes a polypeptide comprising the amino acid sequence is almost the same as presented in the sequence identifier SEQ ID no:3 (coda::upp), SEQ ID no:1 (FCU1), or amino acid sequence FCY1 ::FUR1.

10. Poxvirus according to claim 1, wherein said poxvirus further includes nukleinovokisly sequence comprising the gene encoding permease.

11. Poxvirus of claim 10, in which permeate is a purine or chitosanpharmacia S.Cerevisiae.

12. Poxvirus according to claim 11, in which permeate chosen from the group comprising FCY2 and Fur4 and their equivalents.

13. Poxvirus according to claim 1, wherein said poxvirus further includes the elements necessary for the expression of a target nucleic acid.

14. Poxvirus of claim 10, wherein said poxvirus further includes the elements necessary for the expression of nucleic acid sequence comprising the gene encoding permease.

15. The use of dormancy the virus according to any claims 1 to 14 for the treatment of proliferative diseases or diseases with increased activity of osteoclasts.

16. The application of clause 15, wherein the poxvirus further includes a target nucleic acid.

17. The application of clause 16, wherein the target nucleic acid contains at least one target sequence, encoding a therapeutic molecule.

18. How to play poxvirus according to claim 1, where in the way:
i. poxvirus according to claim 1 is introduced into the cell;
ii. the specified cell is cultivated under conditions which are appropriate to afford to produce the specified poxvirus, and;
iii. specified poxvirus isolated from the cell culture.

19. The composition having oncolytic activity, including poxvirus according to any one of claims 1 to 14 in combination with a pharmaceutically acceptable auxiliary substance.

20. The use of poxvirus according to any one of claims 1 to 14, to obtain drugs in the treatment of proliferative diseases or diseases with increased activity of osteoclasts.

21. The application of claim 20, to obtain drugs for cancer treatment.

22. A method of treating proliferative diseases or diseases with increased activity of osteoclasts, wherein the poxvirus according to any one of claims 1 to 14 or a composition according to claim 19 administered in an effective amount in the body is the master or the cell that needs such treatment.

23. The method according to item 22, where sableman the eat is cancer or restenosis.

24. The method according to item 22, where the disease is rheumatoid arthritis or osteoporosis.

25. The method according to item 22, wherein said poxvirus or composition is administered system path.

26. The method according to item 22, comprising the additional step, in which the pharmaceutically acceptable amount of a prodrug is administered to a specified master-organism or cell.

27. The method according to p, in which the introduction of this prodrug is conducted preferably after at least 3 days, more preferably at least 4 days and even more preferably at least 5 days after the introduction of the specified poxvirus or composition.

28. The method according to item 27, in which the introduction of this prodrug carried out after 7 days after injection of the specified poxvirus or composition.

29. The method according to item 22, wherein the poxvirus or composition is administered in combination with one or more substances that patentiert cytotoxic effect of 5-fertilizin.

30. The method according to clause 29, in which these substances potentiate the cytotoxic effect of 5-fertilizin, are drugs that inhibit the enzymes of the pathway for de novo biosynthesis of pyrimidines, preferably selected from the group consisting of PALA, Leflunomide and A.

31. The method according to item 30, in which a specified substance, potentiate the cytotoxic effect of 5-tortitas is on, is methotrexate.



 

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FIELD: biotechnologies.

SUBSTANCE: created is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype containing expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain haemagglutinin gene with pre-optimised for expression in human being cells nucleotide sequence was used providing overexpression of haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain. Haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain with optimised nucleotide sequence was cloned in expressing cassette under promoter control and contains polyadenylation signal. Promoter is cytomegalovirus promoter, and polyadenylation signal is SV40. Expressing cassette is located in zone of E1 deletion of human being adenovirus genome of the 5-th serotype. Also method of use of recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype for induction of specific immunity to influenza virus B.

EFFECT: possibility of use in pharmaceutical industry for production of vaccine preparations.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use. Provided particle contains expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus, haemagglutinin gene of A/Perth/16/2009(H3N2) strain with pre-optimised for expression in human being cells nucleotide sequence presented in SEQ ID NO:2. The specified haemagglutinin gene of influenza virus of A/Perth/16/2009(H3N2) strain is cloned in expressing cassette containing polyadenylation signal SV40 under control of cytomegalovirus promoter. Presented invention may be used for induction of specific immunity to influenza virus A of H3N2 subtype during injection in efficient quantity.

EFFECT: providing intense expression of recombinant haemagglutinin of the specified influenza virus.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use as a component for production of vaccine for influenza virus A of H1N1 subtype. Presented recombinant particle contains expressing cassette including SV40 polyadenylation signal and cytomegalovirus promoter with influenza virus haemagglutinin gene being included. As influenza virus haemagglutinin gene, haemagglutinin gene of strain A/California/07/2009(H1N1) is used with pre-optimised for expression in human being cells nucleotide sequence provided in SEQ NO:2. These inventions allow raising specific immunity to influenza virus A of H1N1 subtype by provision of overexpression of haemagglutinin gene of influenza virus A/California/07/2009(H1N1).

EFFECT: improvement of the method.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: poxvirus includes defective gene I4L and/or F4L and target nucleic acid containing suicide gene. Besides, a composition containing such a poxvirus, its use for obtaining medical preparation and a treatment method of proliferative disease or disease with increased activity of osteoclasts with its application are described. The proposed group of inventions can be used in medicine.

EFFECT: poxvirus of variolovaccine has oncolytic activity.

34 cl, 18 dwg, 5 tbl

FIELD: medicine.

SUBSTANCE: there are presented two recombinant plasmid DNA pFastBac 1 -G2R-dSECRET and pQE-60-TNFR-CrmB-Ind-67 coding TNF-binding CrmB protein domain. Said recombinant plasmid DNA pQE-60-TNFR-CrmB-Ind-67 is designed for transformation in cells of the strain E.coli SG13009[pRep4] - a producer of TNF-binding CrmB BHO protein domain.

EFFECT: presented group of inventions is applicable for preparing drugs used in therapy of severe human diseases caused by hyperproduction of tumour necrosis factor and may be used in medicine.

3 cl, 3 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: chimeric flavivirus contains at least one mutation in a coat protein of chimeric flavivirus, and one or more mutations in (i) 3'-untranslated region (3'-UTR) of a chimeric flavivirus genome and/or (ii) a capside protein of chimeric flavivirus.

EFFECT: chimeric flavivirus shows reduced viscerotropism as compared to mutation-free chimeric flavovirus; virus is completely attenuated.

32 cl, 8 dwg, 7 tbl

FIELD: chemistry.

SUBSTANCE: displayed polypeptides include at least one unnatural aryl-azide amino acid such as, for example, para-azido-L-phenylalanine, which is incorporated into the phage-displayed hybrid polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA.

EFFECT: invention enables to modify protein in physiological conditions in which phage activity and survival is maintained.

19 cl, 16 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, virology and veterinary. An attenuated recombinant classical swine fever virus (CSFV) is described. Modification is carried out by progressively mutating a portion of the E2 gene of the highly pathogenic strain Brescia. As a result, two to six amino acids on the section 829-837 are replaced with two to six amino acids which are characteristic for the heterologous E2 glycoprotein of the Bovine Viral Diarrhea Virus (BVDV). A classical swine fever vaccine is also obtained based on the obtained attenuated CSFV. The invention can be used in veterinary.

EFFECT: classical swine fever vaccine is obtained.

7 cl, 7 dwg, 4 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and genetic engineering. The method of producing a target virus or one or more target proteins different from adenovirus involves culturing an expression spinal cell and extracting the end product. The cell is a spinal cell containing a gene which is stably integrated into its genome, where said gene codes PIX adenovirus or its functional version, as heterologous regulatory protein. The cell stably expresses said regulatory protein or its functional version via a target virus which is not adenovirus or via a vector carrying nucleic acid sequences which code said virus or via a vector carrying nucleic acid sequences which code said one or more target proteins.

EFFECT: disclosed method enables to increase output of the end product.

24 cl, 18 dwg, 15 ex

FIELD: medicine.

SUBSTANCE: chimeric adenovirus genome comprises the nucleotide sequence SEQ ID N0:1 provided a transcription site E2B therein comprises the nucleotide sequence SEQ ID NO:3.

EFFECT: adenovirus presented under the present invention shows a high therapeutic index.

9 dwg, 1 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: an in vitro generation method of antigen-specific cytotoxic cells with activity against ovarian carcinoma cells is proposed. Simultaneously, a non-adhesive fraction of mononuclear cells (MNC) and mature dendritic cells (DC) are cultivated in presence of recombinant human interleukine-12 and recombinant human interleukine-18. MNC are extracted from peripheral blood of patients having ovarian carcinoma. Mature DC are obtained from monocytes of adhesive MNC fraction after two-day cultivation, and first, activation by lysate of autologous ovarian carcinoma cells, and then maturation of DC loaded with lysate during one day in presence of recombinant human TMF-α (tumor necrosis factor).

EFFECT: use of the invention provides reduction of a stage for obtaining mature DC, in vitro increases cytotoxicity of antigen-specific cytotoxic cells with antitumor activity and provides an immune response via T-helper to type 1 in respect to ovarian carcinoma, which can be used in ovarian carcinoma therapy.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is disclosed is a vaccine representing four RNAs coding a prostate-specific antigen (PSA), a prostate-specific membrane antigen (PSMA), a prostate stem cell antigen (PSCA) and a six-transmembrane epithelial antigen of the prostate (STEAP). The vaccine is applicable for treating prostate carcinoma, preferentially neo-adjuvant and/or hormone resistant prostate carcinoma, as well as related diseases or disorders. Using the vaccine and a kit are also disclosed. The invention can be used in medicine.

EFFECT: preparing the vaccine for treating prostate carcinoma.

16 cl, 23 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a kit for lung cancer cell sensitisation to cisplatin. The declared kit comprises a first composition containing a therapeutically effective amount of sodium metaarsenite, and a second composition containing a therapeutically effective amount of cisplatin. Also, the invention refers to using the therapeutically effective amount of sodium metaarsenite for lung cancer cell sensitisation in a patient treated with cisplatin.

EFFECT: invention provides increasing the therapeutic effectiveness with a reduced risk of side effects.

10 cl, 4 tbl, 3 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented: a combination for treating a proliferative disease containing (a) the phosphoinositide3-kinase inhibitor 5-(2,6-dimorpholin-4-yl-pyrimidin-4-yl)-4-trifluoromethylpyridin-2-ylamine (compound B) or a pharmaceutically acceptable salt thereof and (b) a compound that modulates the Ras/Raf/Mek pathaway specified in a group consisting of (i) a compound that modulates Raf kinase activity that is Raf265, SB590885, XL281 or PLX4032; (ii) a compound that modulates Mek kinase activity that is PD325901, PD-181461, ARRY142886/AZD6244, ARRY-509, XL518, JTP-74057, AS-701255, AS-701173, AZD8330, ARRY162, ARRY300, RDEA436, E6201, RO4987655/R-7167, GSK1120212 or AS703026, wherein the active ingredients in each case present in a free form or as a pharmaceutically acceptable salt or a hydrate thereof, and used simultaneously, separately or sequentially, a respective pharmaceutical composition or a combination drug, and a method of treating a proliferative disease in a homoithermic animal, principally a human.

EFFECT: what is shown is a synergism of the antineoplastic action of the declared combinations.

11 cl, 6 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a compound CL168 of general structural formula I where R is oxygen. The invention also relates to a method of producing a compound of formula I and use of the compound of formula I to produce a medicinal agent for preventing or treating tumorous and immunological diseases.

EFFECT: compound of formula I for producing a medicinal agent for preventing or treating tumorous and immunological diseases.

4 cl, 11 tbl, 19 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a novel compound - N-(2-(dimethylamino)ethyl)-1-(3-((4-((2-methyl-1H-indol-5-yl)oxy)pyrimidin-2-yl)amino)phenyl)methanesulphonamide of formula A and/or a pharmaceutically acceptable salt thereof, having KDR and/or FGFR1 inhibitor properties. The compounds can be used to treat disorders associated with KDR mediated angiogenesis, such as cancer and age-related macular degeneration, or various cancerous diseases which respond to FGFR1 inhibition. Compound (A) corresponds to the structural formula (A) given below.

EFFECT: invention relates to crystalline forms I and II of said compound A and methods for production thereof, pharmaceutical compositions and a method of treatment.

38 cl, 8 dwg, 14 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, oncology, therapy of patients suffering lung cancer and having contraindications to the surgical management. There are prescribed autohemochemotherapy (AHCT) that is administering chemopreparations incubated with autoblood, and radiation therapy (RT). Pre-therapeutic blood prolactin and progesterone are measured, and before the beginning of the AHCT, the patient starts taking bromocriptine 2.5 mg once a day with food; besides, oxyprogesterone capronate 1 ml is administered intramuscularly twice a week every 3 days. That is followed by the AHCT course consisting of 1-3 administrations of autoblood CP, and if observing a complete tumour resorption, the surgical management to the extent of pneumoectomy is supposed to follow, while a partial resorption observed two weeks after the last auroblood CP administered, implies the RT: at first 2 Gy twice a day every 4-5 hours starting from 5 days a week to achieve a basic dose of 28 Gy. That is followed by a 2-week pause, then 4 Gy daily, 3 radiation fractions a week, 6 fractions in total, up to a total radiation dose of 52 Gy for the whole RT course. Throughout the treatment, the patient keeps taking bromocriptine and oxyprogesterone capronate with controlling the blood prolactin and progesterone values: as compared to the pre-therapeutic values, prolactin is expected to fall to the end of the treatments, while progesterone - to rise.

EFFECT: method provides improving the conservative therapeutic effect in the patients of the given group: downsizing the tumour and lymph nodes until the primary tumour regresses completely by 30%, and the patients change to the resectable state; improving the patient's quality of life.

2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to experimental studies in oncology and may be used for assessing the anti-tumour action of metal nanoparticles (NP). An iron nanoparticle suspension 1.25 mg/kg is introduced into grafted Pliss lymphosarcoma intratumourally. That is followed by the paratumoural introduction of methotrexate 0.2 mg/kg. The tumour is locally heated to temperature 42-43°C using an electromagnetic UHF emission at frequency 12.7 MHz for 10 minutes. The therapy requires 5 such sessions in total every 48 hours. That is followed by calculating an effectiveness index, a complete regression percentage and Pliss lymphosarcoma growth inhibition percentage.

EFFECT: method intensifies the antitumor effect of the thermal chemotherapy without increasing body toxicity.

2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology and radiology, and can find application in treating the patients suffering cerebral malignancies. The method for determining the indications for a radiation therapy in the patients by prediction of the effectiveness including blood sampling, gamma-irradiation of a portion of this sample in vitro, incubation of the irradiated and non-irradiated portions of the blood samples, staining of the DNA components of both blood portions with a DNA-specific fluorescent dye, leukocyte count in the irradiated portion of the blood sample, leukocyte count in the non-irradiated blood sample, staining of all DNA containing blood components, determination of DIi that is the DNA amount in all the DNA containing blood components at one leukocyte of the irradiated portion of the sample and DIn that is the DNA amount in all the DNA containing blood components at one leukocyte of the irradiated portion of the blood sample, calculation of DIn/DIi. Blood is sampled additionally, added with an aqueous solution containing ferrous iron ions in the concentration of 50-75 mg/l in the amount of 8-14% of the blood sample amount. The additional blood sample is then incubated for 15-30 minutes that is followed by gamma irradiation of a portion of the additional blood sample. Then the irradiated and non-irradiated portions of the additional blood sample are incubated for 2.5-3.5 hours with leukocyte counting in the irradiated and non-irradiated portions of the additional blood sample, staining of the all DNA containing components of the portions of the additional blood sample and determination of DIi add that is the DNA amount in all the DNA containing components of the additional blood sample at one leukocyte of the irradiated portion of the blood sample and DIn add that is the DNA amount in all the DNA containing components of the additional blood sample at one leukocyte of the non-irradiated portion of the blood sample. Then, the relation DIn add/DIi add is calculated, and if observing DIn add/DIi add> DIn/DIi being 20-35% and DIn/DIi>1, the radiation therapy is considered to be indicated.

EFFECT: invention provides higher effectiveness of the method used for determining the indications for the radiation therapy in the patients suffering glioblastomas.

2 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: peptide of DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD structure is used for suppression of allergic inflammation of respiratory passages, for prophylaxis and treatment of arthritis, as well as for pain relief. A peptide is effective as an adjuvant and for stimulation of IL-12 products in a cell.

EFFECT: peptide allows increasing IL-12 products by 10 times relative to normal levels of IL-12 cellular production.

19 cl, 25 dwg, 10 ex

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