Method of obtaining poxviruses and compositions of poxviruses

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.

EFFECT: obtaining pharmaceutical compositions, which include poxviruses.

11 cl, 3 dwg

 

The present invention relates to such compositions, and also to pharmaceutical compositions that include the poxviruses, and more specifically, to those that include extracellular enveloped viruses. The present invention also relates to a method, which is suitable for producing poxviruses and poxviruses obtained using this method. In addition, the present invention also relates to the use of these poxviruses and the specified composition to obtain a drug.

The emergence of these new threats (avian flu, West Nile virus, anthrax, etc.) as well as the development of gene therapy has increased over time, the need for production and purification of poxviruses in prophylactic or therapeutic purposes. This is especially the case with a virus which is a virus Ankara mammals (MVA). This poxvirus, which originally was used to vaccinate patients with immunodeficiency States against smallpox, now also used as a vector for the purposes of gene therapy. For example, MVA is used as a vector for gene MUC 1 in order to vaccinate patients against the expression of tumor Muc 1 (Scholl et al., 2003, J Biomed Biotechnol., 2003, 3, 194-201). MVA gene transfer, which� encodes the HPV antigens, also used as a vector for therapeutic treatment of ovarian carcinoma.

The poxviruses are a group of enveloped complex viruses, which differ mainly unusual morphology, their large genome DNA and their replication site, which is located in the cytoplasm. The genome of several members of the Poxviridae, including the Copenhagen strain of vaccinia virus (W) (Goebel et al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993, Virol. 196. 381-401) and modified vaccinia virus strain Ankara (MVA) (Antoine et al., 1998, Virol. 244, 365 - 396), was mapped and sequenced. At the Copenhagen strain of vaccinia virus has double-stranded genome DNA, consisting of approximately 192 KB designed for the coding of approximately 200 proteins, of which approximately 100 are involved in the Assembly of the virus. MVA is a highly attenuated strain of vaccinia virus, which is produced by more than 500 serial passages of the strain of vaccinia virus Ankara in the culture of chicken embryo fibroblasts (Mayr et al., 1975, Infection 3, 6-16). The MVA virus was deposited at the National Collection of Cultures of Microorganisms (CNCM) under Deposit number N602I - 721. Determining the complete sequence of the MVA genome and comparison with the genome of the Copenhagen strain of vaccinia virus allows to precisely identify the changes that occurred in viral �enome and identify seven deletions (I to VII), as well as the many mutations that lead to fragmenting the ORFs (open reading frame) (Antoine et al., 1998, Virology 244, 365-396).

Natural metabolic pathway of intracellular capture of enveloped viruses involves a number of stages, which consist of fixing of the viral polypeptide-based virus to cell surface receptor, and the point of fusion between viral and cellular membranes, which leads to the fact that the viral genome is released into the cytoplasm of the infected cells.

However, in the particular case for poxviruses, the exact metabolic pathway analysis is complicated by the fact that there are two morphologically different forms of infectious virus, which have names of the intracellular Mature virus (IMV) and extracellular enveloped virus (EEV). For the form IMV, among other features, characteristically surrounded manolidou shell of the viral core and is predominantly localized in the cytoplasm of infected cells, although it may be for extracellular released after lysis of infected cells. Many of the natural polypeptides are presented on the surface of lipid membranes IMV, were identified, such as, for example, R 14 kDa and p 21 kDa proteins respectively encoded A27L gene (Rodriguez and others, 1985, J. Virol. 56, 482-488; Rodriguez and Estaban, 1987, J. Virol. 61, 3550-3554), and A17L gene, so that the encoding�et proteins A14L, D8L, A9L (Yeh et al., 2000, J. Virol. 74. 9701-9711), E10R (Senkevich et 2000, Virol. 5, 244-252) and H3L genes. Compared with the IMV form have additional EEV outer surface of a lipid membrane (a double layer of lipid), which is derived from the Golgi complex. This corresponds to a viral form that is dated outside of infected cells. Membrane surface EEV contains approximately 10 proteins, which are absent on the surface of IMV, such as, for example, encoded In 5R, A34R and the (HA) gene products. The coexistence of the specified IMV and EEV forms have been described for most strains of smallpox (e.g., Copenhagen and MVA strain), as well as for other poxviruses, such as poxviruses poultry (Boulanger et al., 2000, J. Gen Virol. 81, 675-687).

Because they are the most stable in the environment, IMVs play the dominant role in the host cell, in order to be ready for transmission (Hooper et Virology 2003, 306, 181-185). With this in mind, IMV particles were traditional vectors for the purposes of gene therapy. Therefore, the available process conserved poxvirus purification processes only those viruses that are presented on the cells (i.e. IMV), whereas from a pool of EEV particles in the culture medium it is impossible to get rid of. Because of the presence on its surface of greater diversity of polypeptides than on the surface of IMV, the use of recombinant EEVs was wearelegion and known in the assignment for infection (US 20050208074; Galmiche, etc. J. Gen. Virol., 1997, 78, 3019-3027). However, even for this particular use, IMV particles are particularly preferred (US 20050208074, page 4, Chapter 29).

Unexpectedly, the author found that EEVs designed without infection specifically has a greater therapeutic and/or prophylactic efficacy compared with IMV.

In this aspect, the present invention relates to poxviruses and preferably the recombinant poxviruses, with poxviruses EEV is, without identified specifics of infectivity. The present invention also relates to compositions, preferably pharmaceutical compositions, which include recombinant EEV without identified specifics of infectivity.

In the meaning as used elsewhere in this description, the term "one" is used in the sense that it implies "at least one", "first", "one or more" or "many" components that are referenced, or stages, unless the context clearly does not proceed otherwise. For example, the term "cell" includes a variety of cells, including mixtures of cells.

The term "and/or" everywhere, where used herein, includes the meaning "and", "or" and "all or any other combination of items associated with the specified term.

The term "about" or "approximately" as used in this description, �means within 20%, preferably within 10% and more preferably within 5% of this interval or range.

As used in this description, the term "comprising" is intended to clarify that the products, compositions and methods include components that are referenced, or steps, but not excluding others.

"Comprising essentially of" is used when it is necessary to determine the products, compositions and methods, and it should mean that no other components or stages, with any other essential. Thus, a composition consisting essentially of the specified components, does not exclude trace amounts of contaminants and pharmaceutically acceptable carriers. "Including" shall mean that no more than trace elements of other components or steps.

The family of poxviruses includes viruses Chordo - poxvirus and subfamily Entomo - poxvirus. Among them poxvirus is presented according to the invention and preferably selected from the group which includes Ortho - poxviruses, Para - poxviruses, Avi poxviruses, Capri - poxviruses, Lepori - poxviruses, Sui - poxviruses, Mollusci - poxviruses, Yata - poxviruses. According to a preferential embodiment, poxviruses is Ortho - poxvirus.

Ortho - poxvirus - preferably a vaccinia virus, and more preferably they yavl�is a modified vaccinia virus Ankara (MVA), more precisely MVA 575 (ESAS VO0 120707) and MVA-BN (ESASS V 00083008).

As previously indicated, the IMV particle consists of a viral core, including the viral genome surrounded by a surface monolayer of lipids. The term "EEV" refers to the IMV particle surrounded by additional surface consisting of a double lipid layer on its cell surface, the viral polypeptides.

The term "without identified specific infectivity" in the sense in which it is used here, refers to a controlled infectious specificity, in which conserved poxvirus particle is oriented in such a way as to identify new or enhanced affinity to the target cells, compared with related and not modified conserved poxvirus particles.

As a result conserved poxvirus particle is identified with a specific infectivity shows that the tendency is to infect specific target cells in the same and related modified conserved poxvirus particles, which means that conserved poxvirus particle is identified with a specific infectivity infects more efficiently and/or more quickly to its target cells is presented on the surface of inteligent identified by half of the ligand displayed on the surface conserved poxvirus particles according to the invention) than untargeted cells, and cells intended for (to�did not reveal on their surface such inteligent), while the associated conserved poxvirus particle without identified specific infectivity infects, as revealed, target cells, with lower or similar efficiency, in comparison, to be not intended for cells.

The term "recombinant virus" refers to such a virus that comprises an exogenous sequence inserted in its genome. The value used here, the exogenous sequence refers to nucleic acids, which in nature is not present in the parent virus.

In one of the embodiments of this invention the exogenous sequence encodes a molecule that is directly or indirectly mediated cytotoxic activity. By "directly or indirectly" cytotoxic, we mean that the molecule encoded by the exogenous sequence may itself be toxic (for example ricin, tumor necrosis factor, interleukin 2, interferon gamma, ribonuclease, deoxyribonuclease, Pseudomonas exotoxin A), or specified can be metabolized, in order subsequently to form a toxic product, or specified may have an impact on something else to form a toxic product. The sequence of ricin cDNA disclosed Lamb, etc. (Eur. J. Biochem., 1985, 148, 265-270) and incorporated herein by reference.

In the advantage of�the essential embodiment of the invention, the exogenous sequence is the gene is able to self-destruction. A gene that is able to self-destruction, encodes a protein that is able to convert relatively non-toxic drug into a toxic drug. For example, the enzyme cytosine deaminase converts 5-fertilizin (5 FC) to 5-florouracil (5 FU) (Mullen and others (1922) PNAS 89, 33); the enzyme thymidine kinase of herpes simplex makes sensitive cells to treatment antiviral agent ganciclovir (G-CV) or acyclovir (Mooiten (1986) Cancer Res. 46, 5276; Ezzedine et (1991) New Biol 3, 608). Cytosine deaminase any organism, e.g., E. coli or Saccharomyces cerevisiae, can be used here also.

Thus, in a more preferential embodiment of the invention, a gene that encodes a protein that has a cytosine deaminase activity and, more preferably, a protein, such as described in patent applications WO 2005007857 and WO 9954481.

Other examples of Pro-drug enzyme combinations disclosed include Bagshawe and others (WO 88/07378), namely various agents of alkylation and Pseudomonas spp. The CPG enzyme 2, and disclosed Epenetos & Rowlinson-Busza (WO 91/11201), namely CYANOGEN prodrug (such as amygdalin), and those derived from plants, in particular beta-glycosidase.

Enzymes, which are applicable in this embodiment of the invention, include, but are not limited to, alkaline phosphatase, Kotor� is applicable for converting phosphate prodrug into the free drug; the arylsulfatase, which is applicable for the conversion contains the sulfate salt of the prodrug into the free drug; proteases, such as sulfuric protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins b and L), which are useful for converting containing peptide prodrug into free drugs; D-alanismorissette, which is useful for converting a prodrug, which contain the substituents D-amino acids; hydrating carbohydrate enzymes such as beta-galactosidase and neuraminidase, which is applicable for converting glucoselowering prodrug into free drugs; beta-lactamase, which is useful for the conversion of derivatives of drugs with beta-lactams into free drugs; and amidase penicillin, such as penicillin V amidase or amidase penicillin G, which is useful for the conversion of derivatives of drugs in their nitrogenous amines with phenoxyacetamide or phenylacetylamino groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the prior art as abzyme can be used to convert the prodrugs of the invention into free active drugs (R. Massey, etc., Nature, 1987, 328, 457-458).

Similarly, prolec�of rsta include, but are not limited to the above-mentioned prodrug is listed prodrug, for example, containing a phosphate prodrug, thiophosphate containing prodrugs containing sulfate prodrugs containing peptide prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide containing prodrug or arbitrarily replaced phenylacetamide containing prodrugs, 5-fertilizin and other 5-fluorinated prodrug that can be converted. Examples of cytotoxic drugs that can be converted into a form of preprepared for use in this invention include, but are not limited to, etoposide, teniposide, adriamycin, daunomycin, carminomycin, to produce remissions in childhood, dactinomycin, mitomycin, cisplatin and analogues of cisplatin, bleomycin, spiramycin (see, e.g., US 4,675,187), with 5-florouracil, melphalan and other related nitrogen mustard.

In a further embodiment the exogenous gene encodes a ribozyme, which is able to decompose and is intended for RNA or DNA. Designed RNA or DNA will be cleaved may be RNA or DNA, which are essential for cell functioning, and splitting them leads to necrosis glue�OK or RNA, or DNA will be cleaved may be RNA or DNA that encodes an undesirable protein, such as oncogenic product, and cleavage of this RNA or DNA may prevent the cells become malignant.

In all of the following further embodiments of the exogenous gene encodes antisense RNA.

By "antisense RNA" means a RNA molecule that hybridizes and interferonbeta with the expression of an mRNA molecule which encodes a protein, or other RNA molecules within the cell, such as pre-mRNA or tRNA or rRNA, or hybridizes and interferonbeta with gene expression.

In another embodiment of the invention the exogenous sequence replaces the function of the defective gene in the target cell. There are several thousands of inherited genetic diseases in mammals, including humans, which are caused by such defective genes. Examples of such genetic diseases include cystic fibrosis, in which, as we know, there is a mutation in the gene CFTR; musculary dystrophy Duchenne in which, as we know, there is a mutation in dystrophic gene; the disease of sickle cell, in which, as we know, there is a mutation in the gene for the HbA. Many types of cancer is caused by defective genes, especially protooncogene, and genes of tumor suppressor gene that has undergone mutation.

Examples of proton�of Oganov - ras, src, bcl, and so forth; examples of genes of the tumor suppressor gene p 53 and Rb.

In a further embodiment of the invention the exogenous sequence encodes a tumor associated antigen (TAA). TAA refers to a molecule that is detected at a higher frequency or density in tumor cells than in cells of neophile the same type of fabric. Examples of TAA include, but are not limited CEA, MART - 1, MAGE - 1, MAGE - 3, GP - 100, MUC - 1, MUC - 2, the indicated mutated ras oncogene, normal or point mutated p 53, expressed over R 53, CA - 125, PSA, C - erb / B2, BRCA I, BRCA II, PSMA, tyrosinase, TRP - 1, TRP - 2, NY - ESO - 1, TAG 72, KSA, HER-2/neu, SG - aI. time - fkhr, ews - fli - 1, preserved and LRP. According to a preferential embodiment is presented in this invention TAA MUC 1.

Recombinant gene poxvirus may include more than one exogenous sequence, and each of the exogenous sequence can encode more than one molecule. For example, it may be suitable to associate in the same recombinant gene poxvirus exogenous sequenced encoding TAA with exogenous sequence that encodes a cytokine.

In another embodiment of the invention, the exogenous gene encodes an antigen.

In the meaning used herein, "antigen" refers to a ligand that can be bound by antibody; antigen to�wives not directly to be immunogenic.

Preferably, the antigen derived from a virus such as, for example, HIV - 1 (such as gp 120 or gp 160), any feline immune deficiency virus, human or herpes virus of animals, such as gD or derivatives, or directly early protein such as ICP 27 from HSV 1 or HSV 2, cytomegalovirus (such as gB or derivatives thereof). Varicella Zoster virus (such as gp 1, II or III), or from a hepatitis virus such as hepatitis b virus, such as the surface antigen of hepatitis b or a derivative of this, hepatitis a virus, hepatitis C virus (preferably non-structural protein from genotype 1b strain ja) and hepatitis E virus, or from other viral pathogens, such as respiratory scientically virus, the human papilloma virus (preferably E-6 and protein E 7 from HPV strain 16) or influenza virus, or derived from bacterial pathogens, such as Salmonella, Neisseria, Borrelia (for example, Osp A or Osp b or derivatives of these), or Chlamydia, or Bordetella, such as 69, PT and FHA, or derived from parasites such as Plasmodium or Toxoplasma.

In a particularly advantageous embodiment of the invention, the recombinant gene of poxvirus encodes the same protein as that of TG 4010 (Rochlitz et J Gene Med 2003 Aug; 5 (8): 690-900) and TG 4001 (Liu et Proc Nan Acad Sci USA. 2004, October 5; 101 Suppl 2: 14567-14571). In another particularly advantageous waples�Institute of invention of poxvirus according to the invention has the sequence which is more than 90% homologous relative to the sequence TG 4010 or TG 4001.

Mainly that the recombinant gene of poxvirus further comprises the elements necessary for expression of the exogenous sequence. Elements required for expression include the set of such elements that allow you to implement the transcription of the nucleotide sequence to RNA and the transformation of the mRNA into a polypeptide, in particular the promoter sequences and/or regulatory sequences which are effective in the cell to be infected by the recombinant genome of poxvirus according to the invention, and arbitrary sequences required for selection or expression on the surface of the cells for said polypeptide. These elements can be inducible or constitutive. Of course, the promoter can be adapted to the recombinant gene of poxvirus and matched to the host cell. There may be mentioned, by way of example, the promoters of the vaccinia virus p To 7.5 p H5R, p K1L, R 28, R 11, or a combination of these promoters. Literature allows to obtain a large amount of information relating to such promoter sequences.

The necessary elements can also include additional elements which improve the expression of ekzohennozelezhnoi or her existence in the host cell. There may be mentioned, in particular, the sequence of intron (WO 94/29471), sequence secretion signal, a nuclear localization sequence, internal plots for preinitialize broadcast IRES type, poly A sequences, intended to complete the transcription.

Available methods of production of poxviruses include replication of the virus in cell lines (e.g. Hela S 3), in embryos of eggs or in chicken embryos fibroblasts. After replication of the virus to the culture medium is unsuitable, the cells are lysed, and poxvirus, which is freed from the cells, purified by centrifugation in a density gradient of sucrose (Kotwal and Abraham; Poxvirus growth, Purification and tittering in Vaccinia Virus and Poxvirology, 2004, 101-108, Humana Press Inc., Totowa; NJ; USA). According to these methods, no EEVs are not present in the purified composition.

However, available methods viral production are not satisfactory. First, they include the use of compositions derived from animals such as whey and enzymes. When using compositions derived from animals, in the production methods have several drawbacks. For example, the chemical composition of these compositions may vary between batches, even if the manufacturer is the one and the same person. Compositions could�t also be contaminated with infectious agents (e.g., Mycoplasma and viruses) which can seriously affect the condition of the cultured cells when these contaminated impurities used in the compositions of media for cell cultures.

In addition, the use of serum to culture media can complicate and increase the cost of purification of the target substance from the culture media due to non-specific purification of serum or extractable proteins. Most importantly, the use of indeterminate compositions may hinder the endorsement of the medical agencies of the pharmaceutical compositions obtained by this method.

The present invention further describes a method of producing conserved poxvirus particles according to the present invention, which comprises the following steps:

(a) preparation of culture Packed cells

(b) infection with specified cell culture,

(c) the cultivation of these infected cells for an appropriate period of time,

(d) restoration of conserved poxvirus particles produced in the culture supernatant and/or cells with the packaging defect of nucleotide sequences, and

(e) arbitrary, cleaning restored conserved poxvirus particles.

Method according to the present invention preferably is free from animal products.

Method according�of the present invention can also be used for production of wild-type, and/or attenuated poxvirus.

In the meaning, as used here, the term "attenuated poxvirus" refers to any poxvirus that was changed so that the relative pathogenicity of the infected object was significantly reduced. Preferred is that poxvirus is attenuated in essence, is a non-pathogenic from a clinical point of view, in other words, this means that identified poxvirus does not show a statistically significant increased level of pathological changes with respect to the reference objects.

According to the preferred embodiment of this invention, the attenuated virus is an attenuated vaccinia virus such as MVA.

The term "infection" refers to the transfer of viral nucleic acid to the cell, and the viral nucleic acid is replicated, viral proteins are synthesized or going new viral particles.

In the meaning, as used here, the term "Packed cell" refers to a cell that can be infected by poxviruses to be produced. The cage with the packaging defect of nucleotide sequences may be a primary cell, a recombinant cell and/or cell line. For example, can also be used recombinant cell that contains the elements necessary�required for the production of recombinant virus, which are not present in a recombinant viral vector.

In one embodiment of the invention the cell is defective packing of nucleotide sequences is a continuous line of cells of birds.

In one embodiment of the invention the Packed cell - cell DF1 (US 5,879,924), which is a spontaneous transplantable chicken cell line derived from 10-day-old eggs of the Eastern Line of Lansing (ELL - 0).

A continuous cell line of birds can be obtained from embryonic stem cells by progressive dilution of growth factor in the growing layers of cells, thereby supporting the growth characteristics and the feature of continuous lifetime undifferentiated line. For example, a chicken cell line Ebx (WO 2005007840) was obtained by this method.

According to another preferential embodiment, duck embryo can also be used as a permanent cell line. For example, the cell line is defined as DEC 99 (Ivanov et Experimental Pathology And Parasitology, 4 / 2000 Bulgarian Academy of Sciences), has been cultivated for more than 140 consecutive passages, and it was not oncogene for birds. Cell lines DEC 99 - standard system cell culture that was used for the study and can be used for biotechnological purposes. According to a preferential embodiment, the cell is defective pack�VKI nucleotide sequences - the cell line which is obtained by the method disclosed in the patent application EP 06360001.9.

According to another preferential embodiment, the cell is defective packing of nucleotide sequences is chicken embryos fibroblasts (CEF). Preparation and use of the CEF for the production of viruses is known to the specialist skilled in the art.

CEF are preferably extracted from free from specific pathogens eggs (SPF). The SPF eggs are commercially available, for example from Charles River laboratories (Wilmington, mA, USA). These eggs preferably more than 9 days, more preferably between 10 and 14 days and even more preferably 12 days.

Before removing the embryo egg preferably disinfected. Many methods and products for disinfection of eggs available in the prior art. Incubation in a solution of formol (for example, 2% formol, 1 min) followed by rinsing in 70% ethanol is especially preferred.

The cells of embryos after these procedures are separated and purified. According to the preferred embodiment of this invention, the cells are subjected to a step of enzymatic digestion, which allows to destroy the extracellular matrix. To this end, the use of enzyme able to degrade the extracellular matrix �especially useful. Such an enzyme can be selected from the group which includes but is not limited to trypsin, collagenase, pronasol, disposal, hyaluronidase, neuraminidase. This enzyme can be used alone or in combination. In a particularly advantageous embodiment of the invention dispose and trypsin (eg. Tryp LE selected from Gibco™), are used in combination. Any person skilled in this art, is able to determine the concentration of enzyme, temperature and incubation time that allows for the effective separation of cells.

According to a preferential embodiment of the method according to the present invention is free from animal products (except cells with the packaging defect of nucleotide sequences). With this in mind, the enzyme(s) used for the preparation of CEF, has(s) preferably of recombinant origin. The value used here, the "animal products" means any composition or collection of songs, which was produced or cage of animals, or in vivo.

Preparation of CEF may further include a step of filtering and/or phase centrifugation, to remove the pollutants.

This primary CEF cell can be used either directly or after one further passage the cells as a secondary cell CEF.

Any person kvalificerad�TES in this prior art, able to choose the most appropriate cell for the production of a specific virus. According to another preferential embodiment, the method according to the present invention includes the use of CEF cells or according to EP 06360001.9 for the production of MVA.

The cage with the packaging defect of nucleotide sequences used in the method according to the invention grown in appropriate culture media. It is possible to use more than one culture medium in the method according to the invention. For example, the first culture medium can be used during the preparation of the cells with the packaging defect of nucleotide sequences (i.e., during stage a) and the second cell culture medium for infection (that is, during stage C and/or step b).

According to the preferred embodiment the culture medium according to the present invention is free from animal products.

Many culture media free of animal products, have already been described, and some of them are commercially available. For example, 293 SFM II; 293 - F Cells, SFM Adapted; 293 - H Cells, SFM Adapted; 293 fectin™ Transfection Reagent; CD 293 AGT™; CD 293 Medium; FreeStyle™ 293 Expression System; FreeStyle™ 293 Medium; FreeStyle™ 293 - F Cells, SFM Adapted; Adenovirus Expression Medium (AEM) Growth Medium for PER.C6® Cells; CD 293 AGT™; CD 293 Medium; COS - 71 - Cells, SFM Adapted; EPISERF® Medium; OptiPro™ SM; VP - SFM; VP - SFM AGT™ (all are available and are commercially available) can be used as culture media in the method according to the invention.

Cases, when a cell with a packaging defect of nucleotide sequences is CEF, VP - SFM (invitrogen) for stage (a) and minimal medium for eggs (invitrogen) is a culture medium for step (b) and (C) are particularly preferred.

A specific embodiment are represented according to the invention those cases when cells are defective packing of nucleotide sequences are CEF, and medium for cell culture is selected from between 0.5 to 1.5 and is preferably selected between 1.1 and 1.3 and the more preferred case is approximately 1.2 embryos per liter of cell culture medium.

In this embodiment of the CEF are preferably grown between 1 and 5 days, more preferably are grown between 1 and 2 days and even more preferably are grown for 2 days prior to infection.

A specific embodiment according to the present invention, are those cases when poxvirus, which is suitable in order to produce, is presented MVA, wherein said virus is introduced into the cell culture medium in MOI, which is preferably located between 0.01 and 0.1, more preferably is located between 0.03 and 0.07 and more preferably is located approximately 0.05.

A specific embodiment according to the present invention are those cases when, during step C) the cells with the packaging defect of nucleotide sequences grown at a temperature that is lower than 37°C, preferably at temperatures between 30°C and 36.5°C or at a temperature between approximately 32°C and 36°C, more preferably at a temperature between 33°C and 35°C, most preferably at a temperature which is equal to 34°C.

A specific embodiment according to the present invention are those cases, when the step (C) lasts from one to six days, more preferably lasts between two and four days and most preferably lasts approximately 72 hours.

After the stage of the infection is perfect, the cells that are in culture medium, and those that will be used to grow the cell with a packaging defect of nucleotide sequences, collect. The cells from the culture medium include EEV particles, eliminated from the infected cells with the packaging defect of nucleotide sequences.

According to the preferred embodiment of this invention, after the stage of the infection, conduct e�up collecting cells from the culture medium and cells defective packing of nucleotide sequences. The environment in which cells were grown, and the cells with the packaging defect of nucleotide sequences can be joined or separated separately.

In order to get the poxviruses, which are presented on the cell with the packaging defect of nucleotide sequences, the method according to the present invention may include a stage that allows them to destroy. This phase leads to the release of poxviruses from cells with the packaging defect of nucleotide sequences. The destruction of the cell membrane with the packaging defect of nucleotide sequences may be produced by various methods known to those persons who are skilled in the art. These methods include, but are not limited to, the destruction by sonication, freeze/thaw conditions, hypotonic lysis, and microfluidizer.

According to the preferred embodiment of this invention the cell membrane with the packaging defect of nucleotide sequences destroy when using high speed homogenizer. The high speed homogenizers are commercially available from the company Silverson Machines Inc. East Longmeadow, USA) or firm Ika - Labotechnik (Staufen, Germany). According to a particularly advantageous embodiment of the invention presents a high speed homogenizer can yavl�tsya homogenizer SILVERSON L4R.

In the case where the cell is defective packing of the nucleotide sequences and the cell culture medium are combined, the destruction of the cell membrane with the packaging defect of nucleotide sequences is not carried out by the method of freezing/thawing, for the reason that this technique leads to the destruction of EEV particles (Ichihashi. and others 1996, virology, 217 (2), 478-85).

According to a preferential embodiment of the presented invention, the step (d) further includes the step of clarificatio, which allows to obtain the separation of cell debris. According to a preferential embodiment of this invention, the above step of clarificatio can be stage depth filtration.

Process depth filtration includes but is not limited to, using one or more of the following commercially available products: deep filters of a number of AR CUNO Incorporated (examples of which include AR 01), CUNO, deep filters CUNO Incorporated stimulants (examples include SR 10, SR 30, SR 50, SR 60, SR 70, SR 90), depth filters CUNO Incorporated of a number, for example, which include 10 HP, 30 HP, 50 HP, 60 HP, 70 HP, 90 HP), depth filters CUNO Incorporated Caliph series (examples include CA 10, CA 30, CA 50, CA-60, CA-70, CA-90), depth filters, CUNO Incorporated SP series (examples of which include the SP 10, SP 30, SP-50, SP 60, SP-70, SP 90), depth filters, CUNO Delipid and Delipid Plus filters, the Corporation Mllipore, the number of COE (examples of which include CE-15, CE 20, CE 25, CE 30, SE 35, SE 40, SE 45, SE 50, SE 70, 75 CE), depth filters, Millipore Corporation number of DE (examples of which include DE 25, DE 30, DE 35, DE 40, DE 45, DE 50, DE 55, DE 60, DE 65, DE 70, DE 75), filters NA Millipore Corporation (examples of which include A1 NS B1 NS, WITH NS), filters CUNO polynet (examples of which include Polynet - PB, Millipore Clarigard and Polygard filters, CUNO Life Assure, deep filters Association Man Cel (examples of which include, but are not limited, PR 12 UP, 12 PR, PR 5 UP), and PALL or SeitzSchenk Incorporated filters. To improve the ability to clarificatio units available depth filters, it may be useful to join two or more units of such size of pores are reduced. In this embodiment of the invention, the mixture, which will klasifitsirovan, passes through the first unit depth of filter, where they remain the largest contaminants, and subsequently passes through the second unit depth of filter. According to a preferential embodiment of the invention presents, sartopure® (sartorius) with a pore size of 8 microns, coupled with sartopure® with a pore size of 5 μm, can also be used for stage depth filtration.

According to another preferential embodiment of the invention presented herein, the method includes the stage of concentration. More preferably, the specified stage of concentration allow further�splash zones to eliminate those proteins located in the mixture obtained in the previously described stages.

According to a preferential embodiment of the presented invention, the specified stage of concentration is the stage microfiltration. Microfiltration is a means of applying pressure, relative to the membrane means, whereby can be implemented concentration, and this method allows you to clean large molecules. More advantageously, the solution is supplied through paleodepositional the membrane, the pore size of which is chosen in a way to keep large particles (viruses) in the layers of filter material, and allow small molecules (e.g., proteins) to pass through the membrane in permit. Microfiltration reduces the volume of the solution, which is affected by concentration.

According to another preferential embodiment of the invention presents, stage microfiltration stage accompanied by diafiltration. These two stages can be carried out with the same membrane filter. The diafiltration is an improvement on the microfiltration process and allows you to delayout concentrate solution and cause effect of reducing the concentration of impurities in the concentrate. Dissolution of the concentrate allows you to wash more impurities from the concentrate. This refers to the fact that diafil�tration can be carried out in batch mode in the semi-continuous method or continuous method. Stage of diafiltration can be usefully used to change the buffer containing the virus. For example, it may be useful to change the buffer used in the purification method, on a buffer that is acceptable for pharmaceutical products.

Preferably, filtration membranes used in microfiltration and/or in the stage of diafiltration are membranes that include a pore size between 0.01 and 0.15 μm, and more preferably that include a pore size of approximately 0.1 microns.

The nucleic acid may be contained in the obtained particles cells, such as viruses. With this in mind, the method according to the present invention, can further include a step consisting in removing nucleic acids are, as contaminants in the solution. This purpose can be used nucleases. Examples of nucleases include benzonase or any other DNase or ABC RN, which are commonly used for the purposes of this branch of engineering, among them benzonase is especially preferred.

Benzonase destroys nucleic acid and has no proteolytic activity. Thus benzonase quickly destroys the nucleic acid, hydrolizer internal phosphodiester bonds between specific n�cleotide. After total destruction, is the entire free pool of nucleic acids in solution oligonucleotides reduced to a size of 2-4 bases in length. Benzonase can be used at the final concentration comprising between 50 and 400 U/ml, preferably comprised between 100 and 300 Units/ml and more preferably comprised between 150 and 250 U/ml. Treatment benzonasos can last for 1 h and 4 h and even more preferably within 1.5 h and 2.5 h.

However, when depth filtration, microfiltration and diafiltration, previously described, are used, the use of nucleases, and more specifically, the use of benzonase is not necessary. With this in mind, the present invention also relates to a method of such production of poxvirus in which nucleases, and more specifically, no benzonase, not used.

At this stage of poxvirus obtained according to the method previously described, is sufficiently purified. It is possible to change the buffer containing the virus. For example, it may be appropriate to replace the buffer used in the purification method, pharmaceutically acceptable buffer. A variety of techniques suitable to the application in order to replace the buffer, known to the person skilled in the art. Among these methods, particularly preferred�th is method of tangential filtration.

Method cross-flow filtration is a filtration technique in which the suspension to be filtered at this stage, at high speed are oriented along the direction that is parallel to the direction of the surface of the filter, to create turbulence, which prevents the formation of a lump of filtration, and also quite frequent blocking of the filter.

While the flow of slurry is directed at high velocity, which is parallel relative to penetrating the surface, the solution passes through and penetrates the surface of the hole due to the pressure difference and continuously when it is removed. Surface filtration must be such in order to avoid mechanical resistance to give possible to occur the pressure gradient in different parts of the filter surface (i.e., the difference of concentration of suspension) and concentrate on the surface (clear filtered solution free from suspended particles).

The various stages of the method according to the present invention, can be implemented on separated workplaces and/or in different time periods. For example, infection of cells with the packaging defect of nucleotide sequences and purification of poxviruses can be implemented �and various workplaces. Also, cells with defective packaging of nucleotide sequences and/or until be realized until you will need them to clear.

The present invention also relates to such compositions which can be obtained according to the method previously described, and to the compositions that include poxvirus according to the invention. Preferably, the composition according to the invention comprises more than 1%, preferably more than 5%, even more preferably more than 10% and most preferably at least 20% of poxvirus, which is specified composition, wherein the virus are EEV.

According to another preferential embodiment of the invention presents, for the compositions according to the invention are suitable titers of at least 105preferably at least 106, more preferably at least 107even more preferably at least 108viral particles per ml.

According to another preferential embodiment of the presented invention, the compositions are suitable titers of at least 103preferably at least 104more predominantly at least 3∗103viral particles per microgram of protein.

The present invention also has relation�to the amount of such pharmaceutical compositions which include poxvirus obtained by the method according to the present invention and/or compositions according to the invention. In that value, which is used here, the term "pharmaceutical composition" is a composition that includes a pharmaceutically acceptable additive.

Such additives are preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as, for example, a sucrose solution.

Moreover, such supplements may contain any solvent, or an aqueous or partly aqueous liquid, such as sterile non-pyrogenic water. the pH factor of the pharmaceutical compositions, moreover, can be Adjaran and buffered in order to meet the requirements of use in natural conditions.

Pharmaceutical composition may also include pharmaceutically acceptable solvent, adjuvant or excipient, as well as solubilizers, stabilizing and preserving agents.

For such routes of administration as injection, the preferred composition of the aqueous, non-aqueous or isotonic solutions. This can be achieved in a single dose or in multidose for the form of liquid or dry (powder, lyophilisate, etc.) that can be reconstituted at the time of direct�CSOs use with a suitable solvent.

The present invention also relates to poxviruses and compositions obtained by the method according to the present invention.

The present invention also relates to the use of the method according to the invention for the production of virus, compositions and/or pharmaceutical compositions.

The present invention also relates to the use of poxvirus, compositions and/or pharmaceutical compositions according to the invention for preparing medicines.

According to the preferred embodiment of this invention the drug, according to the invention, is medicine for therapeutic or prophylactic treatment of cancer.

Among those disorders which may be provided, may be mentioned cancers of the breast, uterus (especially caused by papilloma viruses), prostate, lung, bladder, liver, colon, pancreas, stomach, esophagus, larynx, Central nervous system (advantage glioma) and blood (lymphomas, leukemia, etc.).

The composition according to the invention can be produced traditionally for such routes of administration as local, parenteral or enteral route.

May be provided for the following routes of administration: intragastric, subcutaneous,�atriarchy, intramuscular, intravenous, intraperitoneal, intratumoral, intranasal, intralesional or intratracheal routes of administration.

For the last three cases, the introduction of a spray or by instillation is more favorable.

Route of administration may be presented as a single dose, or as a single dose or as a dose several times after a certain time interval. The appropriate route of administration and dosage vary as a function of various parameters, such as humans, the disease to be treated, or the gene (s) of the disease to be transmitted.

According to the first possibility, the medication may be administered directly in vivo (for example, intravenous injection available in the tumor or in its periphery, primarily for therapeutic or prophylactic vaccination). It is also possible to take it ex vivo approach, which consists in the fact that receive cells from the patient (stem cells from bone marrow, peripheral blood lymphocytes, muscle cells, etc.), transported them or infecting them in vitro according to the prior methods known in the art, and administered them to the patient.

It's also possible to envisage ways, where respectively, and not deviating from the subject of an existing invention, to perform simultaneous or PEFC�coherent way of introduction, in different ways, the various components contained in the pharmaceutical composition or compositions according to the invention.

According to a preferential embodiment of the presented invention, a therapeutic use or method of treatment may be combined with other treatment of the patient by surgery (partial or total removal of the tumor), radiotherapy or chemotherapy. In this case, the treatment according to the invention may be applied before, simultaneously or following the specified other treatment.

Preferably, this treatment will be applied as follow-up after the specified second treatment.

According to another preferential embodiment of the present invention is a drug for therapeutic or prophylactic treatment of infectious diseases, especially diseases of viral origin, caused hepatitis virus, HIV, herpes, retroviruses, etc.

The examples that follow are intended to illustrate various existing objects of the invention and, therefore, not limit it, in fact.

Fig.1: depicts the survival rate of the mice 57BL / 6, which is implanted with TA 1 cells expressing HPV 16 E6 and E7, the oncogene C-Ha-ras. Different groups of mice clicks�sativali with the compositions, which include IMV and EEV encoding HPV antigens, IMV encoding HPV antigens or IMV and does not encode any antigen.

Fig.2: depicts the percentage of mice 57BL / 6 free of tumor after making with TC1 cells expressing HPV 16 E6 and E7 and the oncogene C-Ha-ras. Different groups of mice were treated with the compositions that include IMV and EEV that encode the antigens of HPV, IMV and which encode antigens of HPV or IMV, not expressing any antigen.

Fig.3: depicts the survival rate of the mice 57BL / 6, which were implanted with TA 1 cells, which Express MUC-1. Different groups of mice were treated with compositions which include IMV and EEV, which encodes MUC 1 and IL 2, IMV, which encodes MUC 1 and 2 or IL IMV, which does not expresses any antigens.

Examples

1. The preparation of compositions which include IMV and EEV.

A. Preparation of CEF.

Sixty-six of the SPF eggs were incubated for 60 with a 2% solution of formol. After rinsing in 70% ethanol, the eggs were opened and embryos were extracted and subjected to analysis. Obtained tissues were processed at 36.5°C for 120 minutes disposal (IU/ml) and triple tselection (IU/ml). The mixture was filtered to remove the raw fabric, and CEF collected by centrifugation (2300 rpm, for 15 min).

V. To�livelounge CEF and infection.

CEF were incubated in 55 l of VP-SFM (Invitrogen) for 2 days at 36.5°C. the culture Medium for the cells was selected, and was further added poxvirus (0.05 MOI) in 55 l of minimal medium for eggs (invitrogene). The infected cells with the packaging defect of nucleotide sequences were further incubated for three days.

C. Cleaning poxvirus.

Cells defective packing of the nucleotide sequences and the environment of cell culture were collected.

The mixture was further homogenized for 15 min in a high speed homogenizer Silverson® L 4 R. the Mixture, which was obtained, was further klasifitsirovan the method of depth filtration on cells 8 μm (sartorius) connected with cells 5 µm at a flow rate of 1 liter/min.

The mixture was further concentrated 18 times through 0.1 µm module Prostak microfiltration (relatively: PSWAG 021, Millipore).

Conserved poxvirus composition was further affected diafiltration on the same module relative to the desired pharmaceutically acceptable alternate agent.

2. Therapeutic efficacy relative to compositions that include EEV and IMV, and compositions that include only IMV.

A. Therapeutic treatment of mice bearing the tumor that Express HPV antigens.

The name and a brief description of each vector design

<> TG 4001: MVA vector carrying the coding sequences for HPV proteins, E6 and E7, (under the control of promoter p 7.5) and IL 2 (under the control of the pH promoter 5R). Were tested two batches, one of which included the IMV and EEV (prepared as previously disclosed) and one of which included only IMV.

# 33: an empty vector MVA, which expresses neither HPV proteins E6 and E7 or IL 2, while it was used as a negative control.

Model animal

Animals - female mouse model S BI / 6 at the age of 6-8 weeks were used throughout this entire study. These mice were obtained from Charles River (Rouen, France).

Specification: the animals were 6 weeks on the day of arrival. In the beginning of the experiment the animal was less than 8 weeks.

Environment: the animals were placed in a single, exclusive room, which was air-conditioned, to ensure a minimum of 11 air volumes per hour. The ranges of temperature and relative humidity were located within 18°C and 22°C and 40-70%, respectively. Lighting was controlled automatically to obtain a cycle of 12 hours light and 12 hours of darkness.

Specific pathogen free status has been verified by regular monitoring of the environment.

Meals: during the entire time of the study the animals had free access to sterile type d�ety D 04 (UAR, Epinay sur Orge, France). Water was fed freely through the bottle.

Acclimatization and procedures health:

All animals was carried out a clinical examination of health at the time of arrival. They were acclimatized in free from specific pathogens to animals (SPF) between one and two weeks before the experiment, in order to guarantee their suitability for the study.

Features of tumor cells and conditions of use:

Line TC-1 was derived from primary lung epithelial cells of mice S BI / 6, which were cotransformation with HPV - 16 E6 and E7 and c-Ha-ras oncogenes These cells grow in DMEM with glutamine (2 mm), fetal calf serum (10%), essential amino acids (0.1 mm), pyruvate (1 mm), paramecoptera (3 mkmm, Hygromycine (0.2 mg/ml) and G-418 (0.5 mg/ml). After thawing, the cells were amplified twice, the last passage was performed two days prior to cell injection.

Cellular injection

Cell injection was performed on the first day of the experiment, cells TA 1 were introduced under the skin of mice at a dose of 2.0 E + 05 cells per mouse.

Viral infection

After 7 days after injection of cells 5.0 E + 05 pfu / 50 μl/mouse tested parties (TG 4001 IMV/EEV or IMV only) MVATGN 33 (empty vector) were introduced into mice. Twenty mice were used as the tested party�I.

Viral injection was performed under the skin, but in a remote section from the site of injection of cells was performed 3 times with 7-day intervals.

Monitoring parameters:

Tumor growth was monitored in a period of 90 days after injection of cells with the help of calipers. Mice were killed for ethical reasons when the tumor size exceeded 25 mm in diameter, or when they felt pain, even if the tumor was smaller.

Registered surviving mouse.

Results

All groups that were treated with MVA vector that encodes an HPV antigen, showed a higher rate of survival than the group that was treated with an empty vector MVA. Mice that were treated using compositions that include EEV and IMV showed a higher rate of survival than those mice that were treated with compositions that included only IMV. In addition, 35% of mice that were treated with a composition comprising EEV and IMV were free of tumor after 77 days after being injected compared to only 10% of mice that were treated using compositions comprising IMV only.

V. Therapeutic treatment of mice carrying a tumor expressing MUC1.

The name and a brief description of each vector design

TG 4010: MVA Vector carrying the coding posledovatelno�for proteins and MUC 1 (under the control of promoter p 7.5) and IL 2 (under the control of the pH promoter 5R). Were tested two parties, one the inclusion of IMV and EEV and the inclusion of only one IMV.

An empty vector MVATGN 33, which expresses neither MUC 1 or IL 2, was used as a negative control.

Model mouse and varieties system of animal experiments

Strains and supplier: female mice WITH 57BI / 6 at the age of 6-8 weeks were used throughout this study. These mice were obtained from Charles River (Rouen, France).

Specification: the animals were housed at the age of 6 weeks on the day of arrival. At the beginning of the experiment was less than 8 weeks.

Environment: the animals were placed in a single, exclusive room, which was air-conditioned to provide a minimum of 11 air volumes per hour. The ranges of temperature and relative humidity were located within 18°C and 22°C and 40-70%, respectively. Lighting was controlled automatically to give a cycle of 12 hours light and 12 hours darkness. The animals were housed in groups of 10 per cage 43×27×15 cm, with a closed area was 1161 cm2.

Meals: during the entire time of the study the animals had free access to sterile diet type D 04 (UAR, Epinay sur Orge, France). Water was fed freely through the bottle.

Acclimatization and procedures health: All animals was carried out a clinical examination �La health at the time of arrival. They were acclimatized in free from specific pathogens to animals (SPF) between one and two weeks before the experiment, in order to ensure their suitability for the study.

Features of tumor cells and conditions of use:

Line RMA tumor was obtained from a lymphoma With 57 In 1 / 6th. Cells RMA-MUC 1 were obtained after transfection with the expression plasmid containing the gene MUC 1A. These cells were grown in DMEM with glutamine (2 mm), fetal calf serum (10%), nonessential amino acids (0.1 mm), pyruvate (1 mm), paramecoptera (36 μm) and gidrolizina (550 µg/ml). After thawing, the cells were amplified twice, the last passage was fulfilled on the day before the test.

Immunization

Mice were immunized with 104 or 1.0 3.0 104 viral particles/mouse of virus TG 4010 and 3.0 104 viral particles/mouse for MVATGN 33.

20 mice were used as the tested party.

Viral immunization was performed under the skin and is performed 3 times at 14-day intervals.

The study of tumors

Two weeks after the last immunization, mice were injected under the skin, in the same area of skin, but in a remote section from the site of viral injection, 1.0 E + 06 RMA - MUC 1 viable cells / 50 μl/mouse.

Monitoring parameters:

Tumor growth was measured over 6 h�del after injection of the tumor, using the calipers. Mice were killed for ethical reasons, in the case when the tumor size exceeded 25 mm in diameter or when they were hurting, even if the tumor was smaller.

Registered surviving mouse.

20 mice were used as the dose.

Results

All groups that were treated with MVA vector that encodes an antigen MUC-1, showed a lower tumor growth and a higher rate of survival than the group that was treated with an empty vector MVA. Mice that were treated using compositions that include EEV and IMV showed a lower tumor growth than those mice that were treated using the compositions, which included only IMV.

1. A method of producing wild type or attenuated poxvirus, with no identified infectious specificity, which comprises the following steps:
(a) preparing a culture of cells with the packaging defect of nucleotide sequences,
(b) the specified infection of culture cells to obtain infected cells,
(c) the cultivation of these infected cells during the interval of time between one and six days to obtain the conserved poxvirus particles,
(d) allocation EEV and IMV produced conserved poxvirus particles from the culture supernatant and cells with defecto� packing nucleotide sequences, to obtain a mixture of conserved poxvirus particles,
e) the stage of purification of this mixture conserved poxvirus particles, which allows you to release her from the cell residue, and the specified treatment stage is the stage depth filtration, to obtain a purified mixture of conserved poxvirus particles,
(f) the stage of concentration of the indicated purified mixture conserved poxvirus particles, and the specified stage of concentration is the stage microfiltration to obtain a concentrated mixture conserved poxvirus particles, and
(g) the stage of diafiltration,
wherein said method is free from animal products.

2. A method according to claim 1, wherein the cells of the defective packaging of the nucleotide sequences represented by CEF.

3. A method according to claim 1 or 2, wherein step (C) lasts from two to four days.

4. A method according to claim 1 or 2, in which step d) includes the destruction of the specified cell with a packaging defect of nucleotide sequences when using high speed homogenizer.

5. A method according to claim 1 or 2, which does not use any nuclease and, more specifically, which does not use any benzonase.

6. A method according to claim 1 or 2, wherein the mixture poxvirus particles obtained by this method includes EEV and IMV with the content EEV more than 1%.

7. A method according to claim 1 or 2, further comprising the step cm�HN buffer, used in the purification method, in particular in step (g), pharmaceutically acceptable buffer.

8. A method according to claim 7, in which the specified stage is carried out using a tangential filtration.

9. A method according to claim 1 or 2, where the indicated wild-type or attenuated poxvirus is a recombinant poxvirus.

10. A method according to claim 1 or 2, where the indicated wild-type or attenuated poxvirus is a vaccinia virus.

11. A method according to claim 10, wherein the indicated vaccinia virus is modified vaccinia virus Ankara (MVA) virus.

12. A method according to claim 1 or 2, where the indicated wild-type or attenuated poxvirus is a recombinant vaccinia virus.

13. A method according to claim 12, where the indicated recombinant vaccinia virus is a recombinant modified vaccinia virus Ankara (MVA) virus.

14. A method according to claim 12, where the specified recombinant MVA virus comprises at least one exogenous sequence selected from:
gene-destruction,
exogenous sequence encoding a tumor associated antigen (TTA),
exogenous sequence encoding a tumor associated antigen (TTA) and the exogenous sequence encoding the cytokine, and
the exogenous sequence encoding the antigen.

15. The use of conserved poxvirus particles obtained by�OSU the method according to claim 1 or 2, for preparing medicines.

16. The use according to claim 15 for therapeutic or prophylactic treatment of cancer.

17. The use according to claim 15 for therapeutic or prophylactic treatment of infectious diseases.



 

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18 cl, 44 dwg, 1 ex

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4 tbl

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1 tbl, 3 ex

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4 tbl

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3 tbl, 2 ex

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SUBSTANCE: invention relates to field of biotechnology. Disclosed are methods and technologies of hepatitis C virus neutralisation, as well as antibodies against hepatitis C virus. It is suggested to apply completely human homogeneous antibodies RYB1, RYB2 and RYB3, as well as compositions on their base for prevention and treatment of hepatitis C. Said antibodies are obtained by cultivation by hybrid BIONA-RYB1, BIONA-RYB2 and BIONA-RYB3. Efficiency of antibodies is conditioned by the fact that they bind epitopes, respectively, E1, E2, E3 of protein E2 of hepatitis C virus envelope. Neutralising activity of antibodies on modal system of human cells infection in culture is demonstrated. It is shown that application of claimed group of inventions makes it possible to increase reliability of binding hepatitis C virus by antibodies.

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9 cl, 9 dwg, 8 tbl, 13 ex

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SUBSTANCE: presented invention refers to immunology. There are described versions of antibodies or antigen-binding fragments binding to human 4-1BB. One of the versions is characterised by the presence of respective 3 CDR light chain sites and 3 CDR of heavy chain sites. The other version is characterised by the presence of the heavy and light chain with respective amino acid sequences. There are described versions of a pharmaceutical composition for reducing tumour growth or for treating cancer in an individual, as well as methods for reducing the tumour growth or treating cancer in the individual using the versions of antibodies or antigen-binding fragments in a therapeutically effective amount. What is described is a method of treating cancer with using a combination of the antibody and an immunotherapeutic agent. There are disclosed: versions of coding nucleic acids, an expression vector and a host cell containing the antibody expression vector. What is disclosed is a method for producing the antibody with using the cell.

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19 cl, 8 dwg, 11 tbl, 9 ex

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SUBSTANCE: invention refers to betulinic acid derivatives, producing them and using for cancer. For producing derivatives of formulas

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1.R1=OAc; R2=H; R3=Me; X=Br 2. R1=H; R2= OAc; R3=Me; X=Br 3. R1=OH; R2= OAc; R3=Me; X=Br 4. R1=H; R2= OH; R3=Me; X=I 5. R1=OAc; R2=H; R3=H; X=Br 6. R1=OAc; R2=H; R3=Bn; X=I betulin is transformed into betulonic acid, then into dihydrobetulonic acid and respective dihydrobetulonates to be acetylated; the produced compounds are hydroborated and oxidised, then transformed into respective halogen derivatives; that is followed by hydrogenolysis and triphenylphosphine reaction to produce the derivatives of the above formulas. Cytotoxic activity of all the derivatives exceeds anticancer activity of betulinic acid substantially.

EFFECT: there are presented new effective anticancer agents.

6 cl, 1 dwg, 3 tbl, 11 ex

FIELD: chemistry.

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EFFECT: obtained new crystalline forms of ABT-263 with useful biological properties.

35 cl, 2 dwg, 22 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to the field of obtaining 5,9-eicosadienoic acid of formula (1), demonstrating an inhibiting effect on human topoisomerase (5Z,9Z)-5,9- The essence of the method consists in the following: tetrahydropyran ether of 5,6-heptadien-1-ol (4) and 1,2-tridecadiene (5) interact with the Grignard reagent RMgX (R=Me, Et, Pr, Bu, Oct; X=Cl, Br, I) in diethyl ether in the presence of metal Mg (powder) and the catalyst titanocenedichloride Cp2TiCl2, with the molar ratio of (4):5): RMgX:Mg:Cp2TiCl2=10:12:(30-50):32:(0.4-0.6), in an argon atmosphere at a temperature of 0-35°C and atmospheric pressure for 6-10 h, after which the reaction mass is processed with a 5% water HCl solution with obtaining 2-[(5Z,9Z)-5,9-eicosadien-1-yloxy]tetrahydro-2H-pyrane (6), which is oxidised with the Jones reagent.

EFFECT: eicosadienic acid is promising as the medication, possessing anti-tumour, antiviral and antibacterial action.

2 cl, 1 tbl, 1 dwg, 15 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.

EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.

20 cl, 42 ex, 8 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to neurosurgery, neurooncology, and can be used for the treatment of glial brain tumours of a supratentorial localisation. For this purpose photodithazine in a dose of 1 mg/kg of body weight is introduced to a patient 2 hours before the tumour ablation. After that, surgical access to the tumour is performed. The operation wound is illuminated by blue colour with a wavelength of 400 nm, and the tumour boundaries are determined by means of fluorescence of photodithazine, selectively accumulated in the tumour tissue. The tumour is ablated under control of the tumour luminescence in blue colour with the application of an operation microscope. After that, a flexible light guide from a radiation source with a wavelength of 662 nm and power of 2.0 W with a light dispersing nozzle is placed into the tumour bed and the perifocal zone of the tumour is irradiated. The dose of irradiation is determined by the disappearance of fluorescence.

EFFECT: method provides an increase of the treatment efficiency due to the reliable clear determination of the tumour tissue boundaries with the normal brain substance independent on the malignancy degree and character of the tumour growth, with an increase of its ablation radicality, as well as due to the destruction of cells, located in the perifocal zone.

2 cl, 1 ex

Abt-263 capsule // 2550956

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutics, in particular, described is a capsule, containing a capsule envelope, which includes an encapsulated liquid solution of N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)3-(morpholin-4-yl)-1-(phenylsulphanyl)methyl)propyl)-amino)-3-((trifluoromethyl)sulphonyl)benzenesulphonamide (ABT-263) or its bis-hydrochloride salts in a non-ethanol carrier. As filling agents used are: a phospholipid, a solubilising agent for the phospholipid, selected from glycols, glycolides, glycerides and their mixtures, a surface-active substance of a non-phospholipid type and a sulphur-containing antioxidant in an amount, effective for the reduction of oxidising ABT-263 degradation in storage. The sulphur-containing antioxidant is selected from sulphites, bisulphites, metabisulphites and thiosulphites and their mixtures. A method of the capsule obtaining is also described. The capsule is used for treating a disease, characterised by the overexpression of one or several anti-apoptotic proteins of the Bcl-2 family, for instance, cancer.

EFFECT: invention provides a long storage term for the said capsule.

33 cl, 3 dwg, 20 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to oligopeptides, containing the sequence NLSSAEVVV (SEQ ID NO:6), in which one or two amino acids can be substituted, possessing the inducibility of cytotoxic T-cells, their pharmaceutical compositions and application for the production of anti-cancer vaccines.

EFFECT: obtaining pharmaceutical compositions for the production of anti-cancer vaccines.

20 cl, 6 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new chemical compounds of general formula I wherein LA, LB, LC, cycle A, cycle B, RA, RB, RC, RD, RE and RF have the values specified in the patent claim. The compounds of formula (I) are protein kinase inhibitors.

EFFECT: invention refers to pharmaceutical compositions containing the above compounds, as well as to using the above compounds for treating and/or preventing the diseases related to aberrant protein kinase activity, particularly oncological diseases.

10 cl, 14 tbl, 25 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel derivative of purinylpyridinylamino-2,4-difluorophenylsulphonamide of formula 1 and its pharmaceutically acceptable salt. Compounds have properties of inhibiting Raf-kinase super activity and can be applied for prevention and treatment of diseases, mediated by activity of Raf-kinase, such as cancer, in particular melanoma. In formula 1 R stands for methyl; ethyl; propyl; isopropyl, butyl, isobutyl; cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; C5-C6aryl, unsubstituted or substituted with one or more substituents, selected from the group, which consists of chlorine, fluorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, methoxy, ethoxy, propoxy, butoxy, trifluoromethoxy, fluoromethoxy, difluorimethoxy and trifluoroethoxy; C5-C12heteroaryl, which consists of one or two rings, non-substituted or substituted with one or more substituents, selected from the group, which consists of chlorine, fluorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, tret- butoxycarbonyl and dioxolanyl; C5-C6heterocycloalkyl, non-substituted or substituted with one or more substituents, selected from the group, which consists of chlorine, fluorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl; or C5-C6aryl-linear or branched C1-C6alkyl, non-substituted or substituted with one or more substituents, selected from the group, which consists of chlorine, fluorine, bromine, nitro, methyl, ethyl, propyl, isopropyl, butyl and isobutyl, and heteroaryl and heterocycloalkyl contain in ring one or more heteroatoms, selected from the group, which consists of N, O and S. Invention also relates to methods of obtaining formula 1 compounds.

EFFECT: improvement of characteristics.

15 cl, 3 tbl, 51 ex 1 dwg

FIELD: biotechnology.

SUBSTANCE: invention relates to novel strains of Bacillus thuringiensis B-1272 and Bacillus thuringiensis B-1273 deposited in the collection of bacteria, bacteriophages and fungi of the Federal State-Funded Institution of Science "State Research Centre of Virology and Biotechnology "Vector". The index of neutralisation of the infectious activity of virus A/H3N2 while using the preparations based on the culture fluid of any of the proposed strains is 0.5-3.2 lg.

EFFECT: strains have the ability to neutralise the infectious activity of the human influenza virus.

2 cl, 1 tbl, 8 ex

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