Dna composition for inducing immune response on tumour-associated macrophagues

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns a pharmaceutical composition for inducing an immune response on tumour-associated macrophagues containing a mini-gene DNA construct which codes a polypeptide in a pharmaceutically acceptable carrier wherein the polypeptide contains three immunogenic legumain fragments bonded together with linker peptides, and wherein each linker peptide consists of the AAA or AAY sequence.

EFFECT: invention provides effective control of breast carcinoma, non-small-cell lung cancer and colorectal carcinoma.

8 cl, 2 ex, 26 dwg

 

CROSS-REFERENCE TO RELATED APPLICATIONS

In the present application claims the priority of provisional patent application U.S. No. 60/849927, filed October 6, 2006, the contents of which are incorporated into this description by reference.

GOVERNMENT RIGHTS

This invention was made with support from the government of the United States, grants # DAMD17-02-0137 and DAMD17-02-0562 from the Ministry of Defence and grants # W81XWH-05-1-0091 and W81XWH-05-1-0318-controlled Congress program of medical research (Congressionally Directed Medical Research Program). The government has certain rights to this invention.

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention relates to compositions of deoxyribonucleic acid (DNA)coding for suitable molecules effective to produce an immune response against tumor-associated macrophages. More specifically, the present invention relates to compositions of DNA encoding at least one epitope of endopeptidase, such as legumain (legumain), which is expressed in the tumor-associated cells, such as tumor-associated macrophage. The present invention also relates to methods of using the DNA composition for inhibiting tumor growth and tumor metastasis.

The prior art INVENTIONS

Tumor-associated macrophages(TAM) are associated with tumor development and metastasis. A new anticancer strategy is to immunize against molecules sverkhekspressiya THERE, and, through this, the changes in the microenvironment of the tumor, which attracts these macrophages and mediates their function (see Oosterling et al. 2005 J. Pathol. 207:147-155; Emens et al. 2005 Endocr. Relat Cancer 12:1-17). THERE are preferably of the population of polarized macrophages M2 (CD206+, F4/80+) with low cytotoxicity towards tumor cells is limited due to their production of nitric oxide and proinflammatory cytokines (see Mills et al. 2000 J. Immunol. 164:6166-6173).

THERE also have low antigen-presenting ability and effectively suppress the activation of T cells. In fact, these macrophages to the M2 phenotype is indeed contribute to the proliferation of tumor cells and metastasis by secretion of a wide range of growth factors and Pro-angiogenic factors, and metalloproteinases, with the involvement of signal circulation that regulates the function of fibroblasts tumor stroma (Mantovani et al. 2004, Novartis. Found. Symp. 256:137-145).

Currently, anti-THERE effects caused by low molecular weight inhibitors, reportedly contribute to tumor suppression (see Lewis et al. 2005 Am. J. Pathol. 167:627-635; Mantovani et al. 2004, Eur. J. Cancer 40:1660-1667). For example, "yondelis", antineoplastics agent, has a selective cytotoxic effect on THERE, thus Zn is significantly reducing the production of their IL6 and CCL2, what contributes to the inhibition of growth of human cancers associated with inflammation (see Allavena et al. 2005, Cancer Res. 65:2964-2971). Another similar example is illustrated biphosphonates connection, zoledronate acid, which inhibits the secretion of MMP9 THERE, thereby inhibiting tumor metalloproteinase activity and reducing the binding of VEGF-c and its tyrosinekinase receptors on proliferating endothelial cells (see Giraudo et al. 2004, J. Clin. Invest. 114:623-633).

As has been shown in various experimental models, chemokine CCL5 is very important to replenish THERE. The antagonist of chemokine reduced tumor infiltration and slowed tumor growth (see Robinson et al. 2003, Cancer Res. 63:8360-8365). Therefore, although therapeutic targeting, THERE is still a fairly new approach, initial clinical results are encouraging, as they suggest that targeting THERE can complement more traditional anticancer therapeutic schema.

Legumain - this brand new evolutionary branch of the family C13 cysteine proteases (see Ishii 1994, Methods Enzymol. 244:604-615),and it is well preserved in plants and in mammals, including humans. First legumain was identified in plants as enzyme processing spare proteins during seed germination and subsequently discovered the parasites, and then mammals. Legumain is kislotnost uchiwa the cysteine endopeptidase with unusually narrow specificity, necessarily require the presence of asparagine at site P1 substrate sequence (see Chen et al. 1997, J. Biol. Chem. 272:8090-8098).

In this application the choice legumain as a target for antitumor therapy based on the fact that the gene encoding this endopeptidase, as it turned out, highly active in many murine and human tumor tissues, but absent or represented at very low levels in all normal tissues from which these tumors developed. Importantly, overexpression of legumain occurs in such stressful conditions as tumor hypoxia, which leads to increased tumor growth, angiogenesis and metastasis.

Legumain represents a particularly preferred target endopeptidase for compositions and methods of the present invention due to the observation that legumain intensively sverkhekspressiya THERE in the tumor tissues of the mammary glands in mice, as evidenced by the analysis of gene expression and immunohistochemical studies. THERE are extremely high expression legumain in the stroma of the tumor. In contrast, classical macrophage phenotype M1, which perform important functions in immune surveillance and prezentowania antigen, does not Express legumain. Therefore, targeting THERE that sverkhekspressiya legumain, does not interfere with the biological the Kim functions of M1 macrophages, including cytotoxicity and prezentowanie antigen. The present invention is a DNA composition to induce an immune response against THERE sverkhekspressiya legumain or other endopeptidase also sverkhekspressiya in THERE, and which is suitable for treatment of tumors and tumor metastases.

The INVENTION

The DNA composition of the present invention contains the structure of DNA which encodes a polypeptide comprising at least immunogenic fragment of cysteine endopeptidase, sverkhekspressiya in tumor-associated cells (e.g., tumor-associated macrophages). Design DNA includes structural elements that contribute to the expression of the polypeptide in immune cells of the subject, which introduced the design DNA. Preferably, the endopeptidase includes legumain or at least one immunogenic fragment of (e.g., epitope). Design DNA is enclosed in a pharmaceutically acceptable carrier, so that it can be administered to the patient. The composition may encode only immunogenic fragment endopeptidase (for example, the epitope legumain), the polypeptide comprising two or more immunogenic fragments of endopeptidase (i.e. immunogenic polypeptide), a protein endopeptidase or any portion of it, which will induce an immune response in the human bhakta. Preferably, the design of DNA encodes the human legumain having the sequence of amino acid residues consisting of SEQ ID NO: 2, a protein having at least 80% sequence identity with SEQ ID NO: 2 (e.g., human legumain, pork legumain or mouse legumain), or immunogenic fragment expressed in immunogenic cells of the subject, which introduced the design DNA. The design DNA of the present invention are suitable for inhibiting tumor growth and tumor metastasis.

Design DNA can be "bare", preferably in the form of plasmids. Such "bare" design DNA can be enclosed in a liposomal carrier, the polymeric carrier or be introduced by electroporation, gene gun, and the like, if desired. In some preferred embodiments, the design of DNA incorporated in an attenuated viral vector or attenuated bacterial vector.

In a preferred embodiment, the design of DNA incorporated in an attenuated bacterial vector, such as attenuated Salmonella typhimurium, for example, double-attenuated (AroA-dam-) strain of Salmonella typhimurium.

Optionally, the DNA composition may also include the construction of DNA encoding immune effector protein such as a cytokine. Preferred is itokin include CCL21, IL-2 and CD40LT.

In a particularly preferred embodiment, the DNA composition according to the invention includes minigene design DNA, which encodes the immunogenic polypeptide that includes many immunogenic fragments (e.g., epitopes) of cysteine endopeptidase (for example, legumain), which is actively expressed in tumor-associated cells. Immunogenic polypeptide capable of inducing an immune response against tumor-associated cells, is expressed in immune cells and enclosed in a pharmaceutically acceptable carrier. Immunogenic fragments joined together sequentially peptide-linker with each subsequent fragment of the polypeptide. The peptide linkers, basically, a length of at least three amino acid residue and preferably include amino acid sequence AAA or AAY. Used in the present description, the term "peptide-linker" refers to a sequence of at least two amino acid residues, preferably at least three amino acid residues that form the sequence of amino acid residues associated with immunogenic fragments endopeptidase and different from the natural endopeptidase. Typically, a combination of peptide linkers and immunogenic fragments of endopeptidase will include the polypeptide of length m is niche than about 100 amino acid residues, more preferably, a length of approximately from 19 to 62 amino acids (for example, from two to approximately five immunogenic fragments of 8-10 amino acids each, joined together one to four peptide-linkers three amino acids each). Preferably, minigonna design DNA encodes immunogenic fragments legumain human (SEQ ID NO: 2).

The composition of the DNA of the present invention can act as vaccines, focused on tumor-associated macrophages, which Express a cysteine endopeptidase, such as legumain, providing a highly selective target for T-cell-mediated cancer immunotherapy. The approach of targeting the endopeptidase, such as legumain expressed by tumor-associated macrophages, has several advantages over methods of treatment directed against antigens that are expressed exclusively by tumor cells. For example, legumain sverkhekspressiya in THERE and, therefore, is not damaged suppressed the expression of MHC-antigen, as often happens in tumor cells. In addition, tumor cells often become excessively resistant mediated T-cell destruction due to defects in apoptotic signaling pathways, increased regulation of anti-apoptotic proteins or immunosuppress the different effects on cytotoxic T-lymphocytes (CTL). Targeting THERE, expressing legumain allows therapeutic composition to treat a variety of malignant tumors, in contrast to methods of treatment involving antigens expressed exclusively specific types of tumors.

In one preferred embodiment, the composition of the DNA of the present invention violate peripheral T-cell resistance to autoantigen legumain, passing their cDNA DNA encoding one or more immunogenic fragments, as oral DNA composition with tenuiorum bacterial vector delivery (e.g., attenuated strain of Salmonella typhimurium). In such scenarios, the implementation of the DNA composition in contact with antigen presenting cells (APC) in the secondary lymphoid organ, i.e. of Meyerovich plaques of the small intestine. Prevention of T-cell-mediated antitumor immune response induced by vaccination with DNA composition according to the invention, inhibited tumor growth in numerous murine tumor models. These compositions DNA also significantly inhibit the dissemination of established pulmonary metastases in a therapeutic model carcinoma CT26 colon.

The preferred composition of the DNA consists of attenuated Salmonella carrier, such as double-attenuated ..... the m S. typhimurium, for example strain, called RE 88, which includes dam-and AroA-mutations and comes Remedyne Corporation (Goleta, CA). In the present invention attenuated Salmonella carrier transfirieran so that it includes the construction of DNA encoding the endopeptidase (for example, legumain) or a polypeptide containing immunogenic fragment. Endopeptidase or polypeptide is expressed in the immune cells of the mammal in which they are entered. Itself bacterium expresses not legumain or polypeptide, and only delivers DNA in these immune cells, like macrophages or dendritic cells (DC), which in turn Express endopeptidase or polypeptide, including its immunogenic fragment. Such compositions can provide prolonged antitumor effects in mouse models. Moreover, experiments in vivo depletion of T cells showed the involvement of CD8+and not CD4+T cells in the immune response associated with the compositions, encoding legumain and polypeptides comprising immunogenic fragments legumain. The observed cytotoxic effect mediated CD8+T-cells in vitro, was specifically directed against THERE are targets that sverkhekspressiya antigen legumain.

The composition of the DNA of the present invention can also include design DNA that encode immune eff is chornye molecules as adjuvants for the composition. Such immune effector molecules include, for example, IL-2, the inductor proliferation of T cells, CCL21, chemokine, which is chemically attracted to Mature dendritic cells, and naive T cells, as well as CD40LT, a known inducer of maturation of dendritic cells. Nucleic acids encoding the immune effector proteins, preferably built into the plasmid. Design DNA legumain and immune effector protein can be incorporated into the same plasmid or in two separate plasmids. CTL response induced against THERE, can slow the growth of some tumors and is not specific in relation to specific types of tumors.

The present invention also provides a method of inhibiting tumor growth and tumor metastasis in a mammal, including introduction to the mammal the composition of the DNA according to the invention in a quantity sufficient to produce an immune response against THERE expressing legumain.

Another aspect of the present invention is an effective combination regimen that combines chemotherapy and treatment with DNA composition according to the invention. In this mode of implementing the present invention, various chemotherapeutic agents such as doxorubicin, paclitaxel and/or cyclophosphamide, which do not cause bone marrow suppression with the introduction of the maximum permissible dose (DMD/MTD), was introduced is the patient in combination with the DNA composition according to the invention, containing design DNA encoding THERE is expressed the endopeptidase, such as legumain or polypeptide encoding its immunogenic part, preferably containing minigene design, which includes at least two immunogenic fragment endopeptidase, serially connected together by peptide-linkers between each of the successive immunogenic fragments of the polypeptide.

Another preferred method of implementation is a method of inhibiting tumor growth or tumor metastasis in a mammal (e.g. human), including the introduction phase mammal the composition of the DNA according to the invention in an amount sufficient to elicit an immune response against THERE sverkhekspressiya the endopeptidase, such as legumain, with the subsequent introduction of a mammal an effective antitumor amount of antitumor chemotherapeutic agents.

Preferably, mammals subjected to treatment by the methods of the present invention, were people.

In the implementation method of the present invention, the composition of the DNA can be introduced enterline by oral administration or by parenteral injection or intravenous infusion. It is preferable to oral administration of the compositions. Songs may be the ü Packed in airtight containers and provided information for clinicians on the effective introduction of the composition.

The composition of the DNA of the present invention are suitable for the treatment and prevention of a number of illnesses. For example, a patient suffering from colorectal cancer, breast cancer, lung cancer, and the like, may benefit from immunization compositions of the present invention. The compositions of the present invention is also useful to study the role legumain in various forms of cancer.

Brief description of drawings

FIGURE 1. Legumain is expressed to a high degree on tumor-associated macrophages in tumor stroma. (A) Expression of legumain in THERE was clearly expressed, as shown in panel A. the Macrophages infiltrating the tumor, were visualized by using a G/e staining, as shown by the arrows. Expression legumain identified by double staining antilegomena antibodies in combination with anti-CD68 antibodies. (Magnification ×35) (B) Increased expression of legumain THERE was confirmed by flow cytometrical analyses of double-positive populations CD206+/F4/80+of M2 macrophages that were isolated from fresh tumor tissue. (C) Multicolor flow cytometry demonstrated increasing regulation of the marker CD206 of M2 macrophage RAW cells after culturing with IL-4, IL-10 and IL-13 (10 ng/ml). It was shown that legumain actively expressed n the F4/80 +/CD206+positive RAW cells cultivated with IL-4, IL-10 and IL-13, as shown in the top photo, while compared with the wild type RAW cells shown in the bottom photo. (D) Confirmation of expression of legumain on RAW cells using Western blotting, following stimulation with IL-4, IL-13 and IL-10, separately or in combination.

FIGURE 2. Selection leguminosarum cells leads to suppression of tumor growth. (A) Scheme of the composition of the DNA according to the invention, constructed with pCMV/myc/cyto vector backbone, where the gene legumain was attached to the C-end of the mutant polyubiquitin. Was included a fragment, and expression of the protein was demonstrated by Western blotting. (C) Preventive model: vaccination schedule was calculated for three immunization with one-week intervals, subjugated for intravenous injections of approximately 2×105cell lung cancer D121, approximately 50×104of colon cancer cells CT26, and injection of approximately 7×103of breast cancer cells T in the fatty tissue of the breast. The mass of the lungs were measured after 24 days (D121 or CT-26) or 30 days (C) after injection of tumor cells and analyzed in each group. The difference between the two control groups (PBS and/or empty vector) and treatment group was statistically significant **P<0,005. Normalnyo easy = 0,2, (C) Therapeutic model: groups of BALB/c mice (n=8) in the fatty tissue of the breast initially introduced approximately 7×103of breast cancer cells 4T1 and later were vaccinated three times at 3, 7 and 11 days, PBS, empty vector or pLegumain composition of the DNA, respectively, and the primary tumor was removed on day 12. Graphs of the survival show the results for treatment and control groups of 8 mice each. The difference between the control empty vector group and treatment group was statistically significant **P<0,005.

FIGURE 3. The population THERE in the tumor stroma decreased by specific CD8+CTL induced by the composition of DNA, based on legumain. (A) RAW mikrofalowe cells actively Express legumain after culturing with IL-4, IL-10, IL-13 10 ng/ml and serve as target cells in a 4-hour analysis release51Cr. Splenocytes isolated from mice immunized with pLegumain vaccine, effectively destroyed the RAW cells treated in vitro by these cytokines at various ratios of effector cells target, but were not able to induce cytotoxic destruction of unstimulated RAW cells lacking expression legumain. **P<0,005 compared with control groups. (C) Flow cytometry detects the percentage of the population THERE with specific mikrofalowe markers (CD206 and F4/80) in the tumor tissue of the donkey vaccination. Showed a decrease in the percentage of the population THERE among cells in the tumor tissue isolated from mice treated with DNA composition according to the invention. No reduction of the populations THERE, isolated from mice treated with either empty vector or pLegumain after depletion of CD8+T cells (**P<0,005). (C) the Results of flow cytometry were confirmed by immunohistochemical staining assessed by confocal microscopy. The population THERE in the tumor stroma was significantly reduced after vaccination. Magnification ×5 (G/e) ×35 (Control, Empty Vector and pLegumain).

FIGURE 4. Response-specific CD8+T cells, restrictively on MHC antigens first class against cells expressing legumain. (A) Graphs of FACS show that DNA therapy increases the expression of co-stimulatory molecules due to DC. Lymphocytes from Meyerovich plaques obtained after 3 days after vaccination, were painted with FITC labeled antibody to CD11c in combination with PE-conjugated antibodies to CD80, anti-MHC first class or anti-CD40 antibodies. (*P<0,05 compared to control groups). (C) Vnutripoliticheskoi the release of INF-gamma CD8+T-cells was assessed by FACS analysis. **P<0,005 compared with control groups. (C) Producing specific INF-gamma was controlled at the level of individual cells using ELISPOT. Display the woman a number of immune spots per well for lymphocytes from immunized mice restimulating or legumain+cells 4T1 tumor tissue, or legumain-the 4T1 cells. **P<0,005 compared with the treatment group without stimulation. ##P<0,005 compared with control groups. (D) Splenocytes isolated from treated mice, effectively destroyed THERE, as demonstrated using the test with the release of51Cr (*P<0,01 compared to control groups). Inhibiting experiments with antibodies against MHC antigens first class H2Kd/H-2Dd showed that T-cell, mediating the lysis of tumor cells was restriction on MHC antigen of the first class. Depletion in vivo T-cell CD4+or CD8+showed that lymphocytes isolated from vaccinated mice were depleted of T-cells CD8+, failed to induce cytotoxic lysis of target cells, depletion of T-cells and CD4+not suppressed cytotoxic lysis of these target cells. *P<0,01 compared with PBS group, or empty vector.

FIGURE 5. Neutralization of THERE leads to a decrease in the release of growth factors, migration of tumor cells and metastasis. (A) the composition of the DNA according to the invention decreases the release of growth factors THERE. The tumor tissue of the breast T and mouse serum were collected 12 days after vaccination and the introduction of tumor cells. After 24 hours or 48 hours of culturing supernatant cell tumor who's tissues were collected, and the concentration of TGF-beta, TNF-alpha and VEGF serum or supernatants were measured using ELISA. The difference between treatment and control groups were significant. *P<0,01, **P<0,005. (C) Was performed immunohistochemical staining to detect the expression of these growth factors in the microenvironment of the tumor. Vaccinated treatment groups showed that after the reduction THERE is decreased release of VEGF, TGF-beta and MMP-9 compared with empty vector groups. (C) was conducted transwell analysis of migration (transwell migration assay), in order to determine the migration of tumor cells after vaccination. The number of migrating cells clearly decreased after vaccination. ***P<0,001 compared with the empty vector group. (D) experiments Were carried out in vivo, to determine the possibility of the formation of tumor metastasis in mice. Mice were treated with the vaccine for therapeutic scheme, as described above. Indicators of tumor metastasis and weight of the lungs was measured 25 days after excision of the primary tumor. The rate of metastasis was expressed as % of the lung surface covered with fused metastatic lesions: 0 = absent; 1 = <5%, 2 = 5% to 50% 3 = >50%. The difference in the mass of light between the group of mice treated with the vaccine, and all control groups were statistically significant (**P<0,005).

6. Elimination THERE etc which leads to reduction of tumor angiogenesis. Suppresse VEGF-induced angiogenesis: mice BALB/C were vaccinated S.typhimurium, transtitional either empty vector or pLegumain or pLegumain after elimination or CD8+or CD4+T cells in vivo, respectively. A week after the last immunization subcutaneously in the middle line of the abdomen of the mice was introduced Matrigel (Matrigel). Vascularization was induced by VEGF or bFGF. (A) Images were taken using a digital camera later, 6 days after implantation of Matrigel tube. Additionally, a slice of Matrigel plugs, painted trichromat by Masson, shows an increase of blood vessels in traffic of Matrigel, as highlighted by the arrows (magnification ×5). (C) Quantitative analysis of vascular growth was performed after staining of the endothelium in vivo FITC-labeled isolectin B4 and evaluation using fluorometry. The decrease in VEGF-induced neovascularization occurred only after the vaccine vector encoding legumain, but not after vaccination with empty vector or pLegumain after depletion of CD8+T-cells. **P<0,005, *P<0,01 compared to Leguminosae medical group. (C) Immunohistochemical staining was performed and analyzed using confocal microscopy. Cross sections of Matrigel plugs were stained to determine the type of cells, sprouted or migrated to these plugs. The images show what the endothelial cell marker CD31 or macrophages with marker CD68 has invaded or migrated into the Matrigel tube, as shown by the arrows (magnification ×35). Control was G/e staining (magnification ×5).

7. Cell line T was steadily transliterowany a retrovirus containing Leguminosae plasmid, and then used as target cells for splenocytes from immunized mice (Panel A, left photo has a 5× magnification; right photo has 35× magnification), images were taken after 2 days after transfection, and positive cells are marked by arrows. Data analysis release51Cr is shown in Part C. Splenocytes isolated from mice immunized with pLegumain composition of the DNA, effectively destroyed T cells, transfetsirovannyh legumain (*P<0,01 compared with empty vector groups). Tumor-associated mediated T-cell destruction was specific to legumain as normal T cells without Leguminosae expression were not lysed.

FIG shows the nucleotide sequence of nucleic acid that encodes legumain human (SEQ ID NO: 1).

FIG.9 shows the sequence of amino acid residues (SEQ ID NO: 2) legumain person.

FIGURE 10 shows the nucleotide sequence (SEQ ID NO: 3) nucleic acid that encodes legumain mouse.

11 shows a sequence of amino acid residues (SEQ ID NO: 4) legumain mouse.

FIG showing the characteristic nucleotide sequence (SEQ ID NO: 3) nucleic acid, encoding IL-2.

FIG shows the sequence of amino acid residues of IL-2 (SEQ ID NO: 6).

FIG shows the nucleotide sequence (SEQ ID NO: 7) a nucleic acid that encodes a human CCL21.

FIG shows the sequence of amino acid residues of human CCL21 (SEQ ID NO: 8).

FIG shows the nucleotide sequence (SEQ ID NO: 9) a nucleic acid that encodes CD40L person.

FIG shows the sequence of amino acid residues of human CD40L (SEQ ID NO: 10).

FIG shows the nucleotide sequence ubiquitinylation legumain mouse (SEQ ID NO: 11).

FIG shows the sequence of amino acid residues ubiquitinylation legumain mouse (SEQ ID NO: 12).

FIG shows the sequence of amino acid residues of the epitope sequences of the mouse legumain.

FIG shows a schematic depiction of plasmid encoding legumain of minigenes containing immunogenic fragments legumain.

FIG shows that pCMV-Kb/Kd minigonna composition protects mice from infection cells D2F2 breast carcinoma. Groups of BALB/c mice (n=8) were immunized three times at weekly intervals twice attenuated Salmonella typhimurium RE-88 carrying these vectors. One week after the last immunization mice were infected with SIP is utilized in the injection cell carcinoma of the breast D2F2 in the amount of 2×10 5. A. the Protocol Scheme of the experiment. C. the value of the tumor volume of mice after 5-25 days after infection of tumor cells. C. the Weight of the tumor mice 25 days after infection, *P<0.05 compared with the control group empty vector.

FIG shows that pKd minigonna vaccine prevents metastasis of breast carcinoma D2F2 near isogenic BALB/c mice. Groups of mice (n=8) were immunized 3 times at 1-week intervals with forced feeding attenuated Salmonella typhimurium RE-88 carrying these vectors. Later, 2 weeks after the last immunization mice infected with a/in the injection cell carcinoma of the breast D2F2 in quantities of 1×105. A. the Protocol Scheme of the experiment. Century, the Average mouse lung metastasis of each experimental group after 25 days after infection of tumor cells. Indicators of pulmonary tumor metastasis was established by defining % covered metastases surface area: 0, metastases were absent, 1, <20%, 2, from 20 to 50% and 3 >50% were represented by individual symbols for each treatment group. *P<0.05 compared with the control group empty vector.

FIG illustrates the release of IFN-gamma in legumain-specific T-cells, called pCMV-Kb/Kd minigenes composition. A. Expression legumain fresh is obtained from tumor tissues T cells, used as a cell stimulant. To show the level of expression of legumain on these cells was performed by flow cytometry. Century, the Production of IFN-gamma was controlled using ELISPOT at the level of individual cells, such as lymphocytes from immunized mice, restimulating or legumain+T tumor cells from their tumor tissue, or legumain-T culture cells. The release of INF-gamma shows the number of immune spots per well. *P<0.05 compared to groups of mice whose cells were not stimulated legumain+tumor cells. #P<0.05 compared with control groups.

FIG illustrates specific CTL destruction legumain positive mikrofalowe cells caused by the use of pCMV-Kb/Kd minigenes composition. A. Shows the expression legumain mikrofalowe RAW cells lines after culturing with IL-4, IL-10 and IL-13. To show the expression of legumain on these cells was carried out Western-blot analysis. Century Later, 2 weeks after the last immunization were euthanized groups of BALB/c mice (n=4), and extracted from them splenocytes were stimulated irradiated for 5 days T legumain+cells. This was followed by cytotoxic analysis either with wild type RAW legumain-cells (lower panel)or with RAW legumain+cells is as target cells (bottom panel). *<0.05 compared with the empty vector group, where as a target, we used RAW legumain+cells.

FIG illustrates the suppression of angiogenesis in isogenic BALB/c mice caused pCMV-Kb/Kd minigenes composition. A. the Result Madrigalejo analysis. Matrigel was implanted mice vaccinated with either empty vector or pCMV-Db/Dd, or pCMV-Kb/Kd vaccines. The measurement of the concentration of hemoglobin (Hb) in matijevich traffic was conducted to measure the growth of blood vessels. The average concentration of Hb matijevich tubes in each group of mice is shown in the histogram (n=4; mean + standard deviation). *P<0.05 compared with the control group empty vector. Century Painting by trichromat the Freemason plot of Matrigel was performed after 7 days of implantation Madrigalejo tube. Arrows indicate blood vessels in Madrigalejo tube.

A DETAILED DESCRIPTION of the PREFERRED embodiments

The present invention provides the composition of the DNA, which is directed to tumor-associated cells, such as tumor-associated macrophages (TAM). The DNA composition includes outputting a pharmaceutically acceptable carrier design DNA encoding a cysteine endopeptidase, which sverkhekspressiya in tumor-associated cells, or at least one immunogen is the first fragment, able to induce an immune response against tumor-associated cells. Preferably, the composition of DNA was included minigene design DNA encoding expressed in immune cells immunogenic polypeptide that includes many immunogenic fragments of cysteine endopeptidase (for example, legumain), which is intensively expressed in tumor-associated cells, where the polypeptide is able to induce an immune response against tumor-associated cells. Misogeny an implementation option - many immunogenic fragments endopeptidase linked together sequentially by peptide-linkers between each of the successive fragments of the polypeptide.

Preferably, cysteine endopeptidase - legumain (for example, legumain person; SEQ ID NO: 2). The design of the DNA can encode a single immunogenic fragment of cysteine endopeptidase (e.g., epitope), but preferably encodes a polypeptide including two or more immunogenic fragments of endopeptidase.

The term "design DNA"as used in this description and in the appended claims, refers to the structure of DNA which encodes a protein of interest or polypeptide, such as legumain or immunogenic fragment legumain (epitope), collectively called "DNA legumain", as well as be the key, such as IL-2, CCL21, CD40L and the like. Preferably, each immunogenic fragment included approximately 8-10 amino acid residues in length. Design DNA include any DNA that can be transferred to labeled cells, including linear DNA and plasmid DNA as well as DNA, which was included in the genetic material of a cell or virus. Preferably, the design of DNA - DNA, which was included in a viral or bacterial vector delivery, such as non-pathogenic attenuated viral or bacterial vector. In the process of treatment of the subject the composition according to the invention, DNA legumain delivered in immune cells (e.g. macrophages and dendrites cells), which then Express the protein or peptide, including its immunogenic fragment. Viral or bacterial carriers of DNA legumain do not Express legumain.

In some preferred embodiments, the implementation of the design DNA is minigonna design, which encodes the immunogenic polypeptide that includes many (e.g., 2-5) immunogenic fragments of endopeptidase that are actively expressed in THERE (for example, legumain). Used in the present description, the term "minigun" refers to the structures of the DNA coding for the many parts (fragments) of interest of the protein, which are connected together, etc is doctitle small peptides of at least three amino acids. Such minigene encode polypeptides comprising immunogenic fragments of the protein of interest, but do not encode a protein of interest. Preferably, minigonna design encodes the polypeptide, comprising about 2-5 immunogenic fragments (for example, the peptide sequence of the sections of the epitope of the protein), preferably connected by peptide bonds. Minigen may also include sequences that encode a leader sequence and/or other sequences used to facilitate expression or transport of the polypeptide.

As noted above, immunogenic fragments of the polypeptides are interconnected preferably by peptides, linkers or spacers between each piece. The peptide linker preferably comprises at least three amino acid residues (e.g., AAA or AAY). Preferably, the design of the DNA encoding immunogenic fragment legumain, also encodes a leader sequence such as a leader sequence endoplasmic reticulum (ER), attached by a peptide linker to the N-Termini of the polypeptide encoded by design DNA. When the design of the DNA encodes a polypeptide comprising two or more immunogenic fragments legumain, leader sequence, prefer the Ino associated with the first immunogenic fragment from its N-Terminus. Preferably, the immunogenic fragments of endopeptidase consist of 8 to 10 contiguous amino acid residues of one or more epitopes.

Immunogenic fragments of endopeptidase, such as legumain, including legumain person can be identified well-known in the field of ways, such as program HLA Binding Predictions presented on the NIH website www website of the Department of Bioinformatics and Molecular Analyses (BIMAS) of the National Institute of Health (NIH), the description of which is given in the present description by reference.

The composition of the DNA of the present invention stimulate the production of CTL that are effective against tumor-associated macrophages expressing the endopeptidase. Such tumor-associated macrophages actively targeted CTL, which are produced in response to immunization compositions DNA according to the invention. Elimination or neutralization of THERE changes the microenvironment of the tumor, which leads to the suppression of tumor growth and tumor metastasis.

Used in the present description, the term "immunity" refers to the long-term terms of immune protection against viral forms of infectious agents or tumor antigen. The term "immunization" refers to the impact of the antigen of the pathogenic agent, obtained from Neverwinter source, which leads IMM is nice subject, subjected to treatment to pathogenic factor.

The design of the DNA used in the composition of the DNA of the present invention, preferably includes nucleic acid encoding the polypeptide containing legumain (for example, legumain person) or immunogenic fragments legumain, functionally linked to regulatory elements necessary for gene expression in immune cells. In a preferred embodiment, the design of DNA encodes the full-size protein legumain human (SEQ ID NO: 2) or the polypeptide, while maintaining a high degree of homology of at least 80% (e.g., legumain pigs, legumain mouse and the like), or immunogenic fragment that can induce an immune response against cells sverkhekspressiya legumain.

In a particularly preferred embodiment, the design of DNA includes minigene encoding 2-5 immunogenic fragments legumain (for example, legumain person)related to three amino acid peptide-linkers (e.g., AAA or AAY), with one peptide-linker between each immunogenic fragment.

Suitable design DNA, including minigene design, preferably comprise the regulatory elements necessary for expression of the nucleotide. Such elements include, for example, a promoter, start codon, stop codon and C is the cash polyadenylation. In addition, the enhancers are often required for the expression of sequences encoding immunogenic protein target. As is well known in this field, these elements are preferably functionally linked to a sequence that encodes a desired protein. Regulatory elements selected preferably such which will be functional for species in which they will be introduced. It is desirable that the design of the DNA was in the form of plasmids or incorporated into a viral or bacterial vector. Construction of DNA encoding legumain initially can be integrated into the bacterial expression vector transfection, using methods well known in the field. Then the transformed bacteria can be cultured to provide a finished bacterial stock to enable DNA legumain into the genetic material of bacteria. Cultures of these transformed bacteria provide a ready source compositions DNA of the present invention.

Start and stop codons, it is desirable to include as part of a nucleotide sequence that encodes an endopeptidase or immunogenic polypeptide in the composition of the DNA of the present invention. In the matrix with the coding sequence should be the start and termination codons.

Promoters and polyadenylation signals included in the composition according to present the invention, selected preferably maintaining functionality in the cells of immunized subjects.

Examples of promoters that are applicable in the compositions of the present invention, especially in the production of the composition of the DNA genetic vaccine for humans, include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as HIV Long Terminal Repeat (LTR) promoter, Moloney virus, Cytomegalovirus (CMV)such as the CMV pretani promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), as well as promoters from human genes, human actin, human myosin, human hemoglobin, human muscle creatine and human metallothionein.

Examples of polyadenylation signals applicable in the compositions of the DNA of the present invention, especially in the production of compositions DNA for humans, include but are not limited to, the SV40 polyadenylation signals and LTR polyadenylation signals.

In addition to the regulatory elements necessary for expression of DNA, other elements may also be included in the DNA molecule. Such additional elements include enhancers. Enhancers can be, for example, human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers as CMV, RSV and EBV.

The regulatory sequence and the codons is generally vidosevic, therefore, in order to maximize the production of regulatory protein sequence and the codons preferably are selected on the effectiveness of the immunized species. Any average person will be able to produce design DNA, functional for the species affected.

Design DNA used in these compositions can be "naked" DNA, by definition, Restifo et al. Gene Therapy 2000; 7:89-92, a message which is given in the present description by reference. Preferably, the design of the DNA is a plasmid form or DNA incorporated into the genetic material of an attenuated virus or attenuated bacteria. Used carriers or carriers include biodegradable microcapsules, immunostimulating complexes (ISCOM), and liposomes for the bare structures of DNA and various physiologically available buffers for genetically engineered live virus or bacteria.

Examples of suitable attenuated live bacterial vectors which can be transformed to enable FAP design DNA, include Salmonella typhimurium, Salmonella typhi, Shigella species, Bacillus species, species of Lactobacillus, Bacille Calmette-Guerin (BCG), Escherichia coli, Vibrio cholerae, Campylobacter species, Listeria species, or other suitable bacterial vectors known in this field. A preferred vector is atten the new vector live Salmonella typhimurium, especially when the composition is intended for oral administration. Preferred attenuated live Salmonella typhimurium includes AroA-strains, such as SL7207, or double-attenuated AroA-dam-strains, such as RE88.

Methods of transformation of live bacterial vectors with exogenous construct DNA are described well in this area. See, for example, Joseph Sambrook and David W. Russell, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001) (Sambrook and Russell). After the transformation, the exogenous genetic material is incorporated into the genetic material of bacteria so that the bacteria reproducerea, exogenous DNA is replicated along with the native DNA of the organism. Thus, once transformed bacteria of normal reproductive processes provide a ready supply of exogenous DNA.

Presents viral vectors will include Bacteriophages, Herpes virus, Adenovirus, Adeno associated virus, Sindbis virus, Polio virus, Vaccinia virus and Avipox. Methods of transformation of viral vectors of exogenous design DNA is also well documented in this area. Cm. Sambrook and Rassell above.

Used liposomal carriers are single-layer or multilayer vesicles with the membrane portion formed of lipophilic substances, and inland water part. The water part is used in the present invention with the spruce content polynucleotide substances, intended for delivery to the selected cell. In General, it is preferable that the substance forming the liposomes had a cationic group as a Quaternary ammonium group, and one or more lipophilic groups, as saturated or unsaturated alkyl group having about 6-30 carbon atoms. One group of suitable materials are described in European Patent Publication No. 0187702 and further discussed in U.S. Patent No. 6228844 Wolff et. al., appropriate messages are given in the present description by reference. The literature describes many other suitable liposurgery cationic lipid compounds. See, for example, L. Stamatatos, et al., Biochemistry 1988; 27:3917-3925; and H. Eibl, et al., Biophysical Chemistry 1979; 10:261-271. Alternatively, it may be used microsphere, such as polylactic-coglycolide biodegrability microsphere. As is well known in this field, the polynucleotide construct is enclosed in a capsule or in other words, forms a complex with a liposome or microsphere delivery polynucleotide in the fabric.

Other suitable carriers include polymeric microspheres containing biodegradable poly(artaphernes) materials, such as described by Wang et al., Nat. Mater., 2004; 3(3):190-6. Epub 2004 Feb. 15, the corresponding messages are given in the present description by reference.

Composition, realizes the e present invention, preferably include design DNA, such as immunogenic fragments legumain person or its functional homologue. Functional homologues legumain preferably cover at least 80% identity to the sequence of amino acid residue with a human legumain, more preferably at least about 90%, most preferably at least 95% identity with human legumain.

GenBank (GenBank) is a database of genetic sequences from the National Institute of Health (NIH), which is annotated collection of all available publications of DNA sequences. GenBank is part of the International Cooperation on a Database of Nucleotide Sequences, combining the efforts of the DNA Database of Japan (DDBJ), European Molecular biology Laboratory (EMBL) and GenBank National Center for Biotechnology Information.

The nucleotide sequence of a nucleic acid that encodes a human legumain, SEQ ID NO: 1 (FIG) was published in the Catalogue of GenBank No. BC026250, the message described in the present description by reference. The corresponding sequence of amino acid residues of human legumain - SEQ ID NO: 2 (FIG.9).

The sequence of the nucleic acid DNA, teruya mouse legumain, SEQ ID NO: 3 is shown in FIGURE 10. The corresponding amino acid sequence of murine legumain - SEQ ID NO: 4 (11).

Chen et al., J. Biological Chem. 1997, 272(12): 8090-8098 (in the present description by reference) has published data on the structure and characteristics of pork legumain, which is about 83% sequence identity with human legumain.

Owing to the natural degeneracy of the genetic code, other DNA sequences encoding the amino acid sequence of human legumain can be used in the practical implementation of the invention. Such DNA sequences include those that are capable of gibridizatsiya with human legumain.

The DNA sequence encoding the human legumain and which can be used according to the invention include nucleic acids that have deletions, additions or substitutions of different nucleotide residues, for such in SEQ ID NO: 1, which lead to a sequence that encodes the same gene product legumain. DNA molecules encoding functionally equivalent homologues of human legumain, can also be used in the compositions of the DNA of the present invention.

Gene product encoded by the nucleic acid may also contain deletions, additions or substitutions of amino acid OS is Atkov inside a sequence of amino acid residues legumain, which result in a silent change, thus leading to functionally equivalent legumain. Such amino acid sequences can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipatic nature of the residues involved. For example, negatively charged amino acids include aspartic and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar terminal groups having similar hydrophilicity value, include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, tyrosine. As mentioned in the present description, functionally equivalent legumain refers to a protein that includes one or more epitopes, which, when recognized by T-cells, thereby allow T-cells to recognize epitopes legumain, indicating that cells expressing legumain. In preferred embodiments, the implementation is functionally equivalent legumain has a sequence of amino acid residues having at least 80% sequence identity with the sequence of amino acid residues of human legumain (SEQ ID NO: 2), for example, at least about 90% identity placentas the activity or at least about 95% identity to the sequence.

Construction of DNA encoding legumain, can be designed with the aim of changing the sequence coding legumain (relative to native DNA legumain, SEQ ID NO: 1) for a variety of endings, including, but not limited to, alterations which modify processing and expression of the gene product legumain. For example, mutations can be introduced into DNA by using technologies that are widely known in this field, such as site-directed mutagenesis to introduce new restriction sites, alter the nature of glycosylation, phosphorylation, etc.

In a preferred embodiment, the composition of the DNA of the present invention includes the construction of DNA encoding immunogenic polypeptide that includes many immunogenic fragments of human legumain, and design DNA, functionally encoding at least one immune effector protein, both of which are expressed in immune cells. Used in the description and in the accompanying claims, the phrase "immune effector protein" means a protein involved in the regulation of the way the immune system. Preferably, the immune effector protein is a cytokine.

Cytokines are proteins and polypeptides produced by cells, which may affect the behavior of other cells, such as cell about iterate, cell differentiation, regulation of immune responses, hematopoiesis and inflammatory responses. Cytokines can be classified into many families, including chemokines, geopoetika, immunoglobulins, factors, tumor necrosis, and many do not belong to any family of molecules. Cm. the main Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, Printing house, Oxford University, 2000; and ..Janeway, P.Travers, M.Walport and M.Schlomchik, Immunobiology, Fifth Edition, Garland Publishing, 2001 (hereinafter, "Janeway and Travers"). A brief classification of cytokines presented in "Janeway and Travers", in Appendix III, pages 677-679, the description of which is included in the present description by reference.

Hemopoietin include, for example, erythropoietin, interleukin-2, (IL-2, a protein of 133 amino acids, produced by T-cells and is involved in T-cell proliferation), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-15 (protein of 114 amino acids, such as IL-2, which stimulates the growth of the epithelium of the small intestine, T-cells and NK-cells), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophagecolony colony-stimulating factor (GM-CSF), oncostatin M (OSM) and the factor, leukemia inhibitory (LIF).

Interferons include, for example, IFN-alpha, IFN-beta and IFN-gamma (homodimeric protein of 143 amino acids produced by T-cells and NK-cells involved in activate the macrophages, increases expressio molecules MHC and antigen-processorbased components, switching of IG class and suppression of TH2).

Immunoglobulins include, for example, B7.1 (CD80) and B7.2 (CD86), each of which together stimulate T-cell responses.

Family factors tumor necrosis (TNF) includes, for example, TNF-alpha, TNF-beta (lymphotoxin), lymphotoxin-beta (LT-beta), CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, a ligand, 4-1BB, Trail and OPG ligand.

The biological role of CD40 ligand (CD40L), in particular its interaction with CD40 expressed on antigen presenting cells during co-stimulation of T-cell activation is well known in this field. CD40 is a glycoprotein 48 kDa expressed on the surface of all Mature b cells, the majority of malignant tumors of Mature b-cells and some early b-cell acute leukocytic leukemia, but is not expressed on plasma cells, Clark, Tissue Antigens 1990, 35:33-36. CD40L, membrane protein type II approximately 35 kDa, is expressed on the surface of T cells results in antigen recognition. Members of the TNF family, the most biologically active, when they are expressed as homotrimer. It is not excluded that CD40L can be expressed as homotrimer (CD40LT) by modification of the motif latinboy "lightning" 33 amino acids fused to the N-end extracellular just to the s of this ligand. DNA CD40LT reportedly Gurunathan et al. J.Immunol. 1998, 161:4563 stimulates cellular immune responses, such as induction of IFN-gamma and cytolytic activity of T-cells, resulting in vaccination of mice with DNA that encodes a highly immunogenic model antigen beta galactosidase.

CD40LT is an important factor in the activation of T cells, necessary for the development of effective protective immunity against tumor proteins. As soon as MHC-1 antigens class:peptide complexes are absorbed dendritic cells (DC) and the naive T-cells, the first antigenic signal is delivered through the T-cell receptor (TCR), followed by activation CD40LT. On the surface of T cells CD40LT then call co-stimulating activity of DC through the interaction of CD40-CD40LT. Bromirovannye thus, these antigenpresenting cells can Express the co-stimulating molecules W (CD80) and V (CD86), which send the second co-stimulating signal to T cells via interaction with CD28, an event required for full activation of T cells for the simultaneous production of proinflammatory cytokines INF-gamma and IL12 and implementation of effector functions.

A variety of cytokines, not a separate group include, for example, growth factor tumor-beta (TGF-beta), IL-1alpha, IL-beta, IL-1RA, IL-10, IL-12 (stimulating factor natures who's killer cell; heterodimer having a chain of 197 amino acids and chain of 306 amino acids which are involved in activation of NK-cells and the induction of differentiation of T cells in TH1-like cells), the factor inhibiting macrophages (MIF), IL-16, IL-17 (factor, inducing the production of cytokines, which induce the production of cytokines in epithelium, endothelium, and fibroblasts) and IL-18.

Chemokines are a family of cytokines, which are relatively small chemoattractant proteins and polypeptides that stimulate the migration and activation of various cells, such as migration of leukocytes (e.g., phagocytes and lymphocytes). Chemokines play a role in inflammation and other immune responses. Chemokines are divided into many families, including C chemokines, CC chemokines, CXC chemokines and chemokines CX3C. In the title indicate the number of cysteine residues (C) and the distance between them in molecules; C chemokines contain one cysteine, CC chemokines contain two consecutive cysteine, CXC contain two cysteines separated from each other by one amino acid residue, and chemokines CX3C contain two cysteines separated from each other by three amino acid residues. Chemokines interact with a large number of chemokine receptors present on the cell surface. Cm. Janeway and Travers, Annex IV, page 680, to the categories included in the present description by reference.

In addition, chemokines may have immunomodulatory activity and to be involved in immune responses in cancer. For example, it was reported that mouse 6Ckine/SLC, murine analogue of chemokine secondary lymphoid tissues (SLC), currently referred to CCL21, induces antitumor response in tumor cell lines carcinoma of the colon C-26. Cm. Vicari, et al. J. Immunol. 2000; 165 (4): 1992-2000. CCL21 person and its murine counterpart, 6Ckine/SLC, refer to the CC chemokines that interact with the chemokine receptor CCR7. Vicari et al. it was also reported that mouse 6Ckine/SLC (muCCL21) is a ligand of the chemokine receptor CXCR3. CCL21 person, muCCL21 mouse and many other chemokines are involved in the regulation of various immune system cells, such as dendritic cells, T cells and natural killer cells (NK).

Mig and IP-10 are CXC chemokines that interact with the receptor CXCR3 associated with activated T-cells. Lymphotactin is a chemokine C, which interacts with the receptor XCR1 associated with T-cells and NK-cells. Fractalkine is a chemokine CX3C, which interacts with the receptor CX3CR1 associated with T-cells, monocytes and neutrophils.

Especially preferred immune effector proteins encoded by the compositions of the DNA of the present invention, including the indicate the cytokines IL-2 (hemopoietin), CCL21 (chemokine), and CD40 ligand, such as legenday the trimer (CD40LT), a cytokine of the TNF family.

DNA and protein sequences for IL-2 were published in GenBank, Accession No. BC070338, the description of which is incorporated into this description by reference. DNA and protein sequence of IL-2 mice were published in GenBank Accession no NM 008366, the description of which is incorporated into this description by reference.

The nucleic acid sequence that encodes IL-2, presented at FIG (SEQ ID NO: 5)and corresponding amino acid sequence (SEQ ID NO: 6) shown in FIG.

DNA and protein sequences for human CCL21 were published in GenBank, Accession No. AB002409, the description of which is incorporated into this description by reference.

The sequence of the nucleic acid that encodes a human CCL21 presented on FIG (SEQ ID NO: 7), and the corresponding amino acid sequence (SEQ ID NO: 8) is shown in FIG.

The ligand of human CD40 (CD40L) is a protein of 261 amino acids, in their most active form existing as a trimmer (CD40LT). The DNA sequence encoding human CD40L (also known as CD154), was published in GenBank Accession no NM 000074, the description of which is incorporated into this description by reference (FIG, SEQ ID NO: 9). The corresponding sequence of the protein CD40L shown nafig (SEQ ID NO: 10).

Aspects of the methods of the present invention involve the administration to a mammal of a composition containing DNA structure DNA encoding legumain, which is expressed in the immune cells of the mammal. Preferably, the mammal is human. The composition may be administered orally, intramuscularly, intranasally, intraperitoneally, subcutaneously, intradermally or topically, depending on the particular dosage form, in which the released song. Preferably, the composition is produced in used oral dosage form such as solution, suspension, emulsion, capsule, tablet and the like.

The DNA composition according to the invention can be used to provide long-term inhibition of tumor growth and/or tumor metastases in the patient's treatment composition. In a preferred embodiment, the composition of DNA was administered in combination with an antitumor chemotherapeutic agent. The composition of the DNA can be introduced together with a chemotherapeutic agent in a combined dosage form or composition and the chemotherapeutic agent can be administered in separate dosage forms and separate intervals between receptions of drugs developed for pharmacology entered chemotherapeutic agents.

a Chemotherapeutic drug, used in combination with compositions of the DNA of the present invention include antineoplastic agents, such as doxorubicin, paclitaxel, cyclophosphamide, etoposide, 5-fluorouracil, methotrexate, and so on.

The composition of the DNA of the present invention is prepared preferably with pharmaceutically acceptable carriers or excipients, such as water, saline, dextrose, glycerol, and so forth, and combinations thereof for the purposes of the preparation and introduction of the composition. The composition may also contain auxiliary substances such as wetting components, emulsifying agents, buffers and other excipients that are well known in the pharmaceutical industry.

The compositions of the present invention preferably orally administered to mammals, such as man, in the form of a solution or suspension in a pharmaceutically acceptable carrier with DNA concentrations ranging from about 1 to about 10 micrograms per milliliter, depending on the mass of DNA that encodes legumain. The most preferred dosage form for the composition of the DNA of the invention is a suspension of attenuated legumain-transtitional bacteria in a suitable buffer solution, which can be prepared for oral administration. Suitable dosage comp is the exposure will depend on the subject, receiving the vaccine, the activity of the composition and in part the decision of the attending physician performing the introduction or purpose of the composition.

Input mammal, the dosage and mode of administration, in case of appointment of more than one injection will depend on the mammal and medicines. An effective amount of dosage and modes of introduction can be determined empirically through a clinical study of the effect of dose, as is well known in this field. The dosage and mode of administration chosen to provide sufficient expression legumain in immune cells with the aim to induce an immune response in a mammal against a tumor-associated macrophages expressing legumain. Preferably, the dosage of the composition introduced mammals, caused the expression of antigen legumain in the immune cells of the mammal in an amount sufficient to maintain the immune response against legumain-presenting tumor-associated macrophages, which will continue for a period of at least a month, for example, at least 6 months or at least about 1 year. In some preferred embodiments, implementation, dosage legumain-transformed cells, injected into a patient, is approximately 1×108cells transfected is approximately 0.3-0.8 µg legumain DNA.

The compositions of the present invention can be Packed into suitable sterile containers, such as ampoules, vials or vials, in the form of either multiple or single dosage form. After filling composition of the DNA, the vessel is preferably pressurized. Preferably, the compositions are packaged in containers with attached label, which marks the composition and which presents a record in the form prescribed by a government Agency such as the Department for supervision over the quality of the food and drug administration of the United States, reflecting the approval of the composition in accordance with the relevant laws, information about the dosage and the like. The label preferably contains information about the composition, which can be used for introducing the composition to the patient by medical personnel. The package also preferably contains printed information materials relating to the introduction of the composition, instructions, guidance, and any necessary warnings.

The following examples are intended to further illustrate the characteristic features and implementations of the present invention and not intended to be limiting thereof.

Materials and methods

Animals, bacterial strains and cell lines. Female mice BALB/C and C57BL/6, 6-8 weeks is on age were obtained from the Scripps Research Institute Rodent Breeding Facility. Double-attenuated strain RE88 (aroA-dam-) S. typhimurium was obtained from Remedyne Corporation, Goleta, CA. Cell line CT-26 colon cancer mouse was kindly provided by Dr. I. J. Fidler (MD Anderson Cancer Center), and the cells in non-small cell lung carcinoma mouse D121 - a gift from Dr. L. Eisenbach (Weizmann Institute of Science, Rehovot, Israel). Cell breast carcinoma mouse T were kindly provided by Dr. Suzanne Ostrand-Rosenberg (University of Maryland).

Immunohistochemical analyses. Immunohistochemical analyses were performed on tumor tissues T and slices matijevich tubes. Expression legumain macrophages was determined on sections of tumor tissue T using biotinylated rat protivorechivyh CD68 mAb (BD Bioscience Pharmingen) with GFP-conjugated-streptavidin as a secondary reporter reagent. Rabbit antilegomena anticavity was prepared by immunization with purified human legumain produced in E.coli. (Ishii, Methods Enzymol. 1994; 244:604-615). The reaction was visualized using conjugated with tejasvi red streptavidin. Additionally, sections of tumor tissue T and slicers Madrigalejo tube were fixed and stained MMP-9, VEGF, TGF-beta and antibody F4/80 (eBioscience, San Diego, CA) for sections of tumor tissue T, while CD68 antibodies and CD31 (BD Bioscince Pharmingen) was used for slices Madrigalejo tube. All fabric is new sections were visualized using Texas red or conjugated with GFP streptavidin as a secondary reporter reagent, and preparations were analyzed by laser scanning using confocal microscopy (Bio-Rad Laboratories). All images were recorded SPOT™ cooled color digital chamber system (Diagnostic Instruments. Inc).

Immunization and introduction of tumor cells. Preventive model: mouse BALB/C or C57BL/6 each were divided into three experimental groups (n=8) and immunized with PBS, empty vector or S. typhimurium, transtitional pUb-legumina. All mice intravenously (i.v.) introduced approximately 5×104cells CT-26 (BALB/C), approximately 2×105the D121 cells (C57BL/6) or fat injection breast with approximately 7×103cells T (BALB/C), 1 week after the last immunization to induce either experimental or spontaneous pulmonary metastases. The weight of the lungs in experimental and control groups were determined after 24 days after injection of tumor cells. therapeutic model: mice BALB/C were divided into three experimental groups (n=8), which is first in the fat body introduced approximately 7×103cells T in zero day and then three times were immunized with a DNA composition according to the invention 3, 7 and 11 days. After 24 days the primary tumor was cut to determine the mass of the lungs of mice and evaluation of metastasis, or survival rate of mice.

Depletion of CD4 cells+Il the CD8 +in vivo cytotoxicity and ELISPOT assay data. Depletion of CD4+or CD8+in vivo were presented, as previously described (Ceredig et al. 1985, Nature 314:98-100). Cytotoxicity was measured and calculated using a standard test with radioactive51Cr, as previously reported (Zhou et al. 2005, Blood, 106:2026-2032). The ELISPOT assays were performed using ELISPOT set (BD Bioscience Pharmingen) according to the instructions given by the manufacturer.

Analysis of angiogenesis in Matrigel in vivo. Matrigel was used to assess suppressive angiogenesis after vaccination. Briefly, mice BALB/C over 2 weeks after the last immunization subcutaneously (s.c.) in the region of the sternum was introduced rostamizadeh factor Matrigel (BD Bioscience)containing VEGF or bFGF-2 (approximately 200 ng/tube) and tumor cells T (approximately 5×103/tube), which were irradiated with gamma radiation in 1000 Grams (approximately 100000 rad). The endothelium was stained after 6 days after implantation of Matrigel with/in injection using lectin I Bandiera simplofica (Isolectin B4)conjugated to fluorescein (Vector Laboratories). This was done in parallel with staining of endothelium in the control animals. After approximately 30 minutes the mice were euthanized, matrimelee tube removed, and the fluorescence assessed using fluorimetry. Additionally, madrigali the e tubes were removed after 6 days after implantation of Matrigel, fixed in solution Buena within 24 hours and then embedded in paraffin. From all tissues were made cuts in the number of drugs and painted trichrome Masson. All pictures were SPOT™ cooled color digital chamber system, as described above.

Flow cytometry (FACS). Activated markers of T cells were evaluated using analysis dvuhslotovoj flow cytometry analyzer BD Biosciences FASCALIBUR@. Markers DC-cells were identified by staining vegeculture lymphocytes from successfully vaccinated mice and control mice with anti-CD11c Ab in combination with FITC-conjugated anti-CD40, CD-80 and Ab against MHC antigens class II. Macrophages that have high levels of CD206 and F4/80 were counted using dvuhcvetnaja flow analysis. Tumor cells were cultivated from successfully vaccinated mice BALB/C and then stained with anti-CD206 antibody, conjugated with PE (Cell Science, Inc.), antibodies anti-F4/80, conjugated with APC, and antilegomena antibodies conjugated with FITC, followed by FACS analysis. All antibodies were purchased from Pharmingen, San Diego, CA. IFN-gamma released from intracellular level was determined in lymphocytes of Meyerovich plaques obtained after 3 days after a period of immunization and stained with antibodies anti-CD8 labeled with APC. Cells were fixed, permeabi siruvani and later painted PE-labeled anti-IFN-gamma antibodies to determine the intracellular expression of IFN-gamma.

Migration analysis. Analysis of cell migration was performed using a modified camera Boyden (Transwell, Corning Inc., NY). Tumor cells were obtained from the tumor tissue either treated or control groups of mice to perform transwell-migration analysis. After culturing for 4 hours, the cells on the lower membrane surface were fixed with 1% paraformaldehyde, stained with 1% crystal violet and counted (Shi et al. 2004, Mol. Cancer Res. 2:395-402).

Statistical analysis. The statistical significance of the difference in the results between the experimental and control groups was determined student's t test. The results were regarded as significant if the bilateral values of R were <0,05. Analysis of Kaplan-Meier was used to estimate the survival rate of mice.

EXAMPLE 1

Vector construction, protein expression and transformation of S. typhimurium by plasmid DNA. Two designs were prepared based on the vector pCMV, which is commercially available from Invitrogen, Carlsbad, CA. Design pUb-legumain included polyubiquitination reprezentirovanii mouse legumain. DNA reprezentirovannoe mouse legumain has the nucleotide sequence shown in FIGURE 10, SEQ ID NO: 3 (the sequence of amino acid residues of murine legumain shown at 11, SEQ ID NO: 4). Control serve the and design of the empty vector. Mouse legumain was obtained from cells T breast cancer using total RNA as the matrix method PCR. The expression vector was obtained on the basis of pCMV-cell vector (Invitrogen)containing polyubiquitinated sequence, cloned in front of Leguminosae sequence. The sequence of the nucleic acid polyubiquitination mouse legumain shown in FIG (SEQ ID NO: 11). The sequence of amino acid residues ubiquitarian mouse legumain shown in FIG (SEQ ID NO: 12). Expression of protein legumain was demonstrated by Western blotting with polyclonal rabbit antibodies against mouse legumain and protivorechivymi antibodies to beta-actin (Santa Cruz Biotechnology, Inc.) as a loading control. The specific protein was detected by using goat protivoryechiya HPR-antibodies conjugated with IgG (Bio-Red Laboratories). Attenuated Salmonella typhimurim were transducible by plasmid DNA vaccines using electroporation as described in Luo et al. 2003, Proc. Natl. Acad. Sci. U.S.A. 100:8850-8855 and Xiang et al. 2000, Proc. Natl. Acad. Sci. U.S.A. 97:5492-5497.

EXAMPLE 2

Immunogenic fragments of murine legumain. Two plasmids, including each of minigun legumain encoding three immunogenic fragment legumain connected together by a spacer of 3 amino acids (AAY) between each fragment is, were obtained by the inclusion of minigene legumain in plasmid pCMV/myc/ER (MCS), which is commercially available from Invitrogen, Carlsbad, CA (see FIG and 21). The vector includes a segment encoding an ER signal peptide, myc-epitope and the ER-retention signal (see FIG). Insert minigene was made between site BssH II in part of the ER signal peptide and the site Xho I, as shown in FIG. First Leguminosae minigonna plasmid (pCMV-Db/Dd; also denoted pH-2Dd in the figures) includes a spacer AAY, immunogenic Leguminosae fragment legu137the spacer AAY, immunogenic Leguminosae fragment legu238the spacer AAY and immunogenic Leguminosae fragment legu223. Second Leguminosae minigonna plasmid (pCMV-Kb/Kd; also denoted pH-2Kd in the figures) includes a spacer AAY, immunogenic Leguminosae fragment legu405the spacer AAY, immunogenic Leguminosae fragment legu180the spacer AAY and immunogenic Leguminosae fragment legu229. On FIG shows the sequence of amino acid residues for immunogenic leguminose fragments, as well as indicators of binding MHC first class and identification number of the sequence (SEQ ID NO) for each fragment. The expression of the peptide was verified using Western blotting cells COS-7, transfected with monoclonal anti-myc antibody (Invitrogen). As soon as the expression of the peptide was confirmed, before the myc epitope is a sequence of vector immediately inserted a stop codon. Each of the obtained minigene plasmid vectors (pCMV-Db/Dd and pCMV-Kb/Kd) was checked by determination of the nucleotide sequences, and then transpirirovat in attenuated bacterium S. typhimurium RE88 using electroporation to deliver in the composition of the DNA according to the invention minigene encoding legumain. As described in detail below, the compositions are effective against carcinoma of the breast T and D2F2 in BALB/c mice. The observed epochality response was mediated by CD8 T-cells are selectively destroyed legumain+tumor-associated cells macrophages, which resulted in a significant suppression of tumor angiogenesis.

Oral immunization and introduction of tumor cells. Groups of BALB/c mice (n=8) 3 times with one week interval forced oral was administered 100 μl of PBS containing approximately 5×108CFU twice attenuated S.typhimurium containing either an empty vector, pCMV-Db/Dd plasmid, or pCMV-Kb/Kd plasmid. Later, 2 weeks after the last vaccination, the mice were injected intravenously or subcutaneously cell lines D2F2 carcinoma.

Cytotoxic analysis. Cytotoxicity was determined by standard analysis release51Cr, as described above. The percentage of specific lysis of target cells was calculated by the formula [(E-S)/(T-S)]×100, where E is the average ve is ichino experimental release S represents a mean value of spontaneous release and T represents the average value of the full release.

The ELISPOT analysis. Splenocytes were isolated in all the experimental groups of BALB/c mice 2 weeks after injection D2F2 tumor cells were cultured for 24 h with irradiated (gr) T cells or T cells from their T tumor breast tissue. The study was conducted in accordance with the manufacturer's instructions (BD Bioscience, San Jose, CA).

Analysis antiangiogenic activity. Suppression of angiogenesis was determined by analysis of Matrigel, as described above. The growth of blood vessels in the Matrigel was determined by measuring the concentration of hemoglobin using reagent Drabkina (aqueous solution containing 1 g/l NaHCO3,0.05 g/l KCN and 0.2 g/l K3Fe(CN)6). Tube Matrigel were extracted after 6 days after implantation of Matrigel, fixed in a solution Buena (15 weight parts of a saturated aqueous solution of picric acid, 5 parts by weight of 37% aqueous formalin solution and 1 weight part glacial acetic acid) for 24 hours and embedded in paraffin. From all tissues were made cuts in the number of drugs and painted trichrome Masson. All pictures were SPOT™ cooled color digital chamber system. Using minigene approach n the present invention, isogenic mice BALB/c minigene anti-TAM compositions DNA inhibited both tumor growth and angiogenesis. Six immunogenic peptides legumain were identified as N-2Dd-1,2,3, or H-2Kd-1,2,3 restrictively minigene based on the prediction of binding of these molecules MHC-antigen class 1 HLA peptide using predictions of binding HLA peptide provided by the Department of Bioinformatics and Molecular Analyses (BIMAS) on the NIH website. Amino acid sequences of these peptides and their binding activity, as predicted by the program DNASTAR@(DNAStar, Inc., Madison, WI), listed on FIG.

Minigonna vaccine pCMV-Kb/Kd protects the mouse from injection of tumor cells D2F2 breast cancer and prevents pulmonary metastasis. Initially, minigene composition of DNA were tested in a prophylactic model of breast carcinoma, when mice were first vaccinated minigenes composition, and then subcutaneously them were introduced cell carcinoma of the mammary gland of mice D2F2 (FIG). Significant slowing of tumor growth was observed in isogenic BALB/c mice vaccinated withpCMV-Kb/Kd, but notpCMV-Db/Dd. In contrast, all mice vaccinated with empty control vector was identified rapid subcutaneous tumor growth.

Minigonna vaccine legumainpCMV-Kb/Kd hearth is employed, pulmonary metastasis D2F2, destroying THERE, as proven by a noticeable suppression of experimental metastases were detected in BALB/c mice with intravenously introduced cells, breast carcinoma D2F2 2 weeks after the third vaccination. In this model, the metastases were prevented, unlikepCMV-Db/Dd. Mice vaccinated with empty control vector, was found the same fast metastasized growth of lung tumors (FIG).

Minigonna vaccine causes a CTL response, which is able to destroy legumain+cells. To represent a specific response of T-cells obtained after minigene vaccines, T-cell activation was demonstrated by the specific release of INF-gamma activated T-cells (FIG, panel B). These cells were stimulated cells derived from fresh T tumor tissue, abundantly expressing legumain, as was shown by analysis using flow cytometry (FIG, panel B). It is important that the cytotoxic assays clearly demonstrated significant CTL activity only in immunized mice. Target cells were cells mouse makropoulou cell line RAW cultivated with IL-4, IL-10 and IL-13 cytokines to induce the expression of legumain. This was also confirmed by Western-blot analyses (FIG, Panel A), when it was shown that h is of the wild type RAW cells is legumain negative. Splenocytes isolated from control mice, which were treated with the empty vector showed similar background RAW destruction of cells positive or negative for the expression legumain (FIG, Panel B). However, splenocytes from mice that were treated with pCMV-Kb/Kd, caused a significantly stronger action against legumain+target cells than against the same cells isolated from mice treated with pCMV-Db/Dd or empty vector (FIG, Panel B). These data demonstrate the selectivity of pCMV-Kb/Kd managementservices CTL activity and their ability to selectively destroy legumain+macrophages.

pCMV-Kb/Kd minigonna vaccine induces antiangiogenic effects. In order to analyse the extent to which etiogenesis plays a key role in tumor protection induced by the vaccine pCMV-Kb/Kd, were held matrimelee analyses, which caused the formation of blood vessels inside the Matrigel using recombinant bFGF. The difference in the formation of blood vessels between the different treatment groups were determined by measuring the concentration of hemoglobin (Hb) in matrimelee tube, obtained from immunized or control mice. Thus, mice that were treated with vaccine pCMV-Kb/Kd, showed on average a clear decrease in the concentration of Hb (FIG, panel A). More t the th, when sections of Matrigel from immunized and control mice were analyzed using three-color staining for Mason, sections from mice of the control group empty vector was found abundant numerous blood vessels. In contrast, the blood vessels were clearly reduced in sections of Matrigel from mice that were treated with PCMV-Kb/Kd (FIG, Panel B). These data demonstrate that immunization with a vaccine PCMV-Kb/Kd leads to reduced tumor vascularization. Taken together, these findings suggest that pCMV-Kb/Kd minigene composition caused significant antiangiogenic effects, which contributed to the protection of BALB/c mice from the introduction of D2F2 tumor cells of the breast in the preventive model.

In separate experiments with non-small cell lung cells D121 carcinoma isogenic C57/BL mice, as pCMV-Kb/Kd, and pCMV-Db/Dd vaccines provide effective anti-THERE immune responses.

DISCUSSION of RESULTS

Legumain serves as a target for destruction sverkhekspressiya THERE during tumor development. It is well known that they play an important role in tumor development and metastasian. Thus, the allocation of these M2 macrophages is a new anticancer strategy. Legumain was originally identified as a reliable marker molecules THERE because of its high levels of expression e is of their cells in the surrounding microenvironment of the tumor and stroma. THERE have been isolated from mouse T tumor breast tissue. Flow cytometry (FACS) spleen showed that legumain sverkhekspressiya on CD206 and F4/80 double-positive M2 macrophages, especially when compared with the normal M1 macrophages (FIGURE 1, Panel B). The result was also confirmed using immunohistochemical analyses showed that THERE may be visualized H/E staining, and overexpression of legumain was further detected by double staining antilegomena antibodies (Ab) (green) in combination with anti-CD68 Ab (red) (FIGURE 1, Panel A). These data show that infiltrated THERE are a disproportionately large cell subpopulation in T tumor tissue and that legumain is a potentially effective target for destruction THERE.

Induction of expression of legumain on THERE with Th2 cytokines. Mouse mikrofalowe cell line, RAW, jointly cultivated with these cytokines was used to assess the extent to which the expression legumain on THERE was called Th2 cytokines, such as IL-4, IL-10 and IL-13. Simultaneously with increased regulation legumain saw a significant increase in the expression of CD206+, F4/80+these RAW cells (FIGURE 1, Panel C). These results were confirmed using Western blotting (FIGURE 1, Panel D). Was not receiving the data about the expression of legumain tumor cell lines, cultivated with these cytokines. These results suggest that Th2 cytokines, such as IL-4, IL-10 and IL-13, released tumors and other tumor stromal cells and accumulate in the surrounding microenvironment of the tumor. In this environment, cytokines have the potential to cause proliferation and transformation from M1 macrophages to the population of M2 phenotype, sverkhekspressiya legumain.

Targeting THERE suppresses tumor development. Tumor growth and tumor metastases correlate strongly with the presence, and thus targeting this subpopulation of macrophages leads to the suppression of tumor growth and metastasis. With the aim to induce an immune response against these THERE was prepared expressing vector for the composition of the DNA encoding legumain. Attenuated bacterium Salmonella typhimurium (strain RE88) was transliterowany plasmid vector encoding legumain to ensure the delivery of DNA legumain in immune cells. The control composition was prepared by incorporating the empty plasmid vector in the same strain of attenuated bacteria. FIGURE 2, Panel And schematically shows the card design vector, based on pCMV/myc/cyto vector backbone comprising encoding legumain DNA. The gene encoding legumain, was attached to the C-end of the mutant polyubiquitin (pLegumain) in order to improve the processing of antigen in the proteasome. Then a fragment was inserted between > PST and NotI restriction sites of the plasmid. Expression legumain was demonstrated using Western blotting. In prophylactic as well as therapeutic models by reducing the number THERE with the Composition of DNA, based on legumain, the present invention effectively inhibits spontaneous metastases T breast tumor growth and metastasis of non-small cell lung carcinomas D121 lung and colon cancer CT26 on mouse models.

In the preventive model, mice C57BL/6J was immunotherapies three times with saline phosphate buffer (PBS; control group 1), attenuated S.typhimurium, with built-in empty vector (control group 2) or DNA composition according to the invention (pLegumain-transfusiona attenuated S.typhimurium; treatment group). One week after the last immunization, these mice intravenously (i.v.) introduced approximately 2×105cells D121 non-small cell lung tumor. After 24 days was measured and analyzed pulmonary metastases. In the two control groups, the average weight of the lung was significantly greater than that of the treatment groups (FIGURE 2, Panel B). Similar results were obtained in the model CT26 tumors of the colon and in the model T cancer of the breast isogenic BALB/c mice (FIGURE 2, Panel B).

In more complex terapeutiche the coy model BALB/c mice were first injected cells T breast cancer, and then, as described above, were immunized three times pLegumain based Composition DNA (treatment group), PBS (control) or composition with empty vector (control). Twelve days after injection T tumor cells, primary tumor is surgically removed. Graph of life expectancy for the control and treatment groups showed that 75% (6/8) of mice immunized pLegumain, survived for 3 months. On the other hand, mice from the control group all died within one month (FIGURE 2, Panel C). These data show that legumain composition of DNA, based on legumain, the present invention effectively inhibit tumor cell growth and metastasis in mouse models T breast cancer, D121 non-small cell lung cancer and CT-26 carcinoma of the colon. In combination with surgery, this treatment can, in fact, significantly increase the lifespan of mice by inhibiting tumor cell metastasis in these very serious (prospective) therapeutic murine tumor models.

Targeting legumain cause-specific CD8+ CTL response, reducing the population THERE in the tumor stroma. Protivochumnaja immunization induces specific T-cell response against THERE, the stable expressing this aspartic endopeptidase. Specific T-cell response was demonstrated by analyzing the release of51Cr, where splenocytes isolated from mice, successfully immunized by DNA composition according to the invention, effectively destroyed the RAW macrophages expressing high levels legumain after cultivation with the cytokines IL-4, IL-10 and IL-13. These splenocytes were not able to induce cytotoxic destruction of cells that do not Express tropic legumain (FIGURE 3, Panel A), indicating a high level of specificity of this T-cell response against legumain. In addition, the same result was obtained using legumain transfected cells as targets in the analysis release51Cr (FIG.7). Moreover, the results displayed in FIGURE 3, Panel B, show a sharp reduction in the population of F4/80+/CD206+macrophages after treatment with DNA composition according to the invention, based on legumain. These data were also confirmed by immunohistochemical staining, as shown in FIGURE 3, panel C.

Restrictively on MHC first class CD8+CTL specific active against THERE. Not limited to any theory, suggest that transfetsirovannyh bacteria are absorbed by Peyrovani plaques intestine, where DNA legumain then embedded in immune cells such as macrophages and dendritic cells (DC), which p is that Express legumain DNA. It was found that the DK in Meyerovich plaques successfully immunized mice are activated after 3 days after vaccination pLegumain, as shown by the activation markers, CD40, CD80 and MHC-I, activating DC (FIGURE 4, panel A). It was also found that CD8+T-cell activation is specific to legumain, as evidenced by double staining of INF-gamma and CD8 on splenocytes obtained from successfully immunized mice (FIGURE 4, Panel C), and specific release of INF-gamma activated T-cells, stimulated legumain-positive cells (FIGURE 4, panel C). Moreover, in vivo immune depletion of CD4+or CD8+T cells showed that only CD8+T cells play a significant role in specific cytotoxic destruction THERE, so as soon as their depletion completely annulled devastating effect. Specific cytotoxicity was restrictionon the MHC antigens of the first class, because the destruction was specific ingibirovalo anti-H-2Dd/H-2Kd antibody, (FIGURE 4, panel D). Considered together, these results indicate that the DNA composition according to the invention, based on legumain first activated DC in Meyerovich plaques, after which these cells presented Leguminosae peptides towards MHC antigen of the first class to T-cell receptors (TCR) on activated CD8+T-cells that drive the lo to a specific cytotoxic response of CD8 +T cells, neutralizing THERE.

Neutralization THERE in the tumor stroma reduces the release of factors of tumor growth and factors of proangiogenic, and also inhibits the migration of tumor cells and metastases. THERE can influence tumor metastases in a number of ways, as they secrete a large variety of growth factors, tumor factors proangiogenic and tumor-associated enzymes that stimulate tumor angiogenesis, and tumor growth and metastasis. To assess the extent to which elimination THERE really reduces the release of some of these factors from vaccinated mice and matched control animals were obtained serum and cells of the tumor stroma. Their tumor cells were cultivated, and their air-conditioned environment (supernatant) were collected after 24 and 48 hours, respectively. ELISA performed to determine the amount of TNF-alpha, VEGF and TGF-beta showed a significant reduction in TNF-alpha and VEGF in both air-conditioned environments tumor cells and serum. TGF-beta was lowered only in cell-conditioned media, but not in serum (FIGURE 5, Panel A).

Immunohistological staining confirmed the reduced expression of these factors in tumor tissue (FIGURE 5, Panel B). In addition, a significant reduction migrationology cells was observed when comparing treatment and control groups (FIGURE 5, Panel (C) in the analysis of migration and invasion. These results show that the characteristics of tumor cells changed after caused by the vaccine changes in the microenvironment of the tumor that caused suppression THERE. The ability to control tumor metastasis was confirmed by analysis of in vivo using the definition of the indicator of metastasis and weight of the lungs after 24 days after removal of the primary tumor in therapeutic model. The rate of metastasis and light weight in treated mice was significantly decreased compared with two control groups (FIGURE 5, Panel D).

Off THERE in the tumor stroma leads to a reduction of tumor angiogenesis. Significant antiangiogenic effect was observed after elimination THERE in the tumor stroma, which could partially occur due to the fact that these M2 macrophages produce a wide range of factors proangiogenic. Analyses of Matrigel revealed new blood vessels grown in vivo, which was quantified by staining endothelial FITC labeled by isolectin B4. These results clearly show that the growth of blood vessels was significantly reduced after vaccination with the DNA composition of the present invention (6, Panel B). It has been observed that the greatest number of blood vessels was increased in Madrigalejo tube at stake is control mice, immunized with empty vector, compared with mice immunized with the composition of the DNA that was detected digital image and colouring with paint trichromat by Masson (6, Panel A). Moreover, immunohistochemistry was performed histological analysis in order to assess the type of cells that are actually migrated to matijevich traffic jams. Photos confocal microscope showed that endothelial cells expressing CD31, or macrophages expressing CD68, grew and migrated largely in matijevich traffic jams in the control group empty vector, but this occurred to a much lesser extent in the treatment group (6, Panel C).

Transgenic tumor cells expressing legumain, marked by splenocytes of mice immunized with the DNA composition according to the invention. In the studied model cell line T was stable transliterowany a retrovirus containing Leguminosae plasmid, and then used in the selection of cells (7, panel A, left photo has a 5-fold increase, right photo has a 35-fold increase). Microphotography images were taken after 2 days after transfection. Positive cells are highlighted by arrows (7, panel A). Was presented analysis release51Cr; data shown figure 7, Panel C. Splenocytes isolated from we the her immunized pLegumain composition of DNA, were effective in killing cells T, transfected with legumain (*P<0,01 compared to control groups empty vector). Tumor-associated mediated T-cell destruction was specific to legumain, as the cells T, not expressing legumain, not literally.

The present invention represents a new paradigm (pattern) in anticancer treatment, i.e. the decrease in the density THERE in the tumor stroma reduces the release of factors that enhance tumor growth and angiogenesis, which alters the microenvironment of the tumor and significantly inhibits proliferation of tumor cells, vascularization and metastasis. The selection is THERE in the stroma of the tumor could strengthen fears that neutralization of these cells can interfere with normal immunological functions of these important components of the natural immune system. However, not in this case.

Circulating monocytes is a versatile precursors that can differentiate into various forms of specialized macrophages. In fact, citochina environment has strong influence on the differentiation and function of tissue macrophages. Macrophages activated by bacterial products and cytokines Th1, considered as a manifestation of the phenotype of M1, i.e. the classification is a mini-activated macrophages with increased bacterial activity and cytotoxic activity against tumor cells. On the other hand, macrophages activated by Th2 cytokines, such as IL-4 and IL-13 or immunosuppressive drugs, such as vitamin D3 and IL-10, are classified as having the M2 phenotype, characterized by low cytotoxic function, but high timeismoney activity.

Cells M1 have immunostimulatory quality and protect the body from pathogenic infections, while M2 cells weaken acute inflammatory reaction, actively destroy the decay products of the cells and produce many factors that stimulate growth and angiogenesis, especially for repair of damaged tissues. In addition, macrophages derived from healthy or inflamed tissue, able to lyse tumor cells to Express immunostimulatory cytokines and presentation of tumor-associated antigens for stimulation of proliferation and antitumor functions of T - and NK-cells. M2 macrophages, such as THERE, show reduced levels of these activities. This may be a result of their exposure to anti-inflammatory molecules, originating from the tumor, such as IL-4, IL-10, TGF-beta and prostaglandin E2. In fact, Mantovani with colleagues expressed the view that exposure to IL-4 and IL-10 can induce monocytes in the tumor to develop in the polarized type II or M2 macrophages (Mantovani et al. 2002, Trends Immunol. 23:549-555).

To the extent that they were is still studied, differentiated Mature THERE are phenotype and function similar to the type II macrophages (Mantovani et al. 2004, Eur. J. Cancer 40:1660-1667). Thus, the cytokines present in the surrounding microenvironment of the tumor, have the ability to cause and to determine the differentiation of the resulting mononuclear phagocytes. In fact, a growing body of data shows that THERE are inclined in the direction of M2 macrophages in the surrounding microenvironment of the tumor and produce many factors that promote tumor growth and angiogenesis, as well as immunosuppressive molecules. So, THERE in the localization of the tumor and the continuation of the expression and release of their products may be more likely to support than oppose the tumor development and metastasis.

THERE expresses numerous lines CD206, mannosyl receptor, activated M2 macrophages with subsequent effects on IL-4 and IL-13 (Porcheray et al. 2005, Clin. Exp. Immunol. 142:481-489). As here shown, this population of macrophages expresses a high level legumain. It is important that the Th2 cytokines IL-4, IL-10 and IL-13 activate the expression of CD206 and legumain on makropoulou cell line RAW. This discovery can be easier understood if we assume that the macrophages from the peripheral blood differentiate into M2 macrophages as soon as they arrive in the localization of the tumor, where IL-4, IL-13 and IL-10 released tumor is mi cells and the cells of the tumor stroma (Stein et al. 1992, J. Exp. Med. 176:287-292). Thus, the allocation of M2 macrophages expressing legumain, not only begins to suppress tumor growth and metastasis, but also supports the normal functioning of macrophages M1 phenotype.

On the relationship between infiltration THERE and forecasting in relation to tumor patients also indicated in several studies, which showed that increased infiltration of macrophages worsened forecasts (see, for example, Wyckoff et al. 2004, Cancer Res. 64:7022-7029). Several groups of signs show that in the tumor stroma, there is a symbiotic relationship between cancer cells and THERE, in which cancer cells are attracted THERE and increase their survival rate, while THERE are responsible opuholepodobnoe molecules, providing an important growth factors and extracellular matrix enzymes. This, in turn, stimulates tumor proliferation, angiogenesis and invasion of surrounding tissues (see, for example, Wyckoff et al. 2004, Cancer Res. 64:7022-7029). Thus, attenboroughii THERE in the tumor environment provides an effective change strategy tumor stroma and alters the microenvironment of the tumor.

The composition of the DNA of the present invention cause a sustained CTL response against legumain, which represents an aspartic endopeptidase, which functions as a stress protein, and abundantly sverge prescirbed THERE. It was shown that protivoshumovye immune response is a response-specific CD8+T cells, restrictively on MHC antigens of the first class. Importantly, the present invention shows that after immunization with DNA composition according to the invention, based on legumain, significantly reduced the density of double-positive CD206+and F4/80+macrophages, for example THERE. Additionally, a variety of factors, such as VEGF, MMP-9 and TGF-beta, which are released THERE, as has been shown, the low levels in the supernatant of cultured tumor cells and in mouse serum. It is well known that VEGF and metalloproteinase MMP-9 play an important role in the formation of vascular tumors and stimulation of tumor angiogenesis. In this respect THERE are important because they produce both VEGF and MMP-9. Increasingly enhancing angiogenesis in combination with an active expression of VEGF and extracellular proteases, such as MMP-9, whereas TGF-beta is known as an important growth factor involved in the migration of tumor cells through the blood vessels. In fact, TGF-beta can give proliferative and anti-apoptotic signals in tumor cells as well as activating plasminogen activator urokinase type (uPa), which may contribute to the destruction of the extracellular matrix, which is necessary for in which znackovania sprouting vessels.

Importantly, the present invention demonstrates that as soon as THERE nejtralizovyvat in the tumor specific cytotoxic T-lymphocytes CD8+(CTL), tumor cells change their character, becoming less malignant and less invasive. The formation of new blood vessels in tumor tissues also significantly reduced in the treatment composition of the DNA of the present invention. Additionally, according to available data, THERE are involved in immunosuppression and tolerance (endurance) in the tumor microenvironment and can inhibit T-cell responses inducyruya apoptosis of activated T-cells via activation of the release of NO, PGs, TNF-alpha and arginase activity (see, for example, Saio et al. 2001, J. Immunol. 167:5583-5593). After neutralization activity THERE specific T-cells CD8+significantly increased even more by showing that the approach against THERE according to the present invention provides an effective strategy in a sudden change of immunological tolerance against tumors.

In a murine model of spontaneous metastasis of breast carcinoma T was achieved remarkably long duration of life, in which 75% (6 of 8) mice lived 3 months after the inoculation of tumor cells T in the mammary gland, when it was made surgical resection of the primary tumor with subsequent treatment of the composition of the DNA of the present invention, based on legumain. More unexpectedly, in 62% (5 of 8) mice did not reveal pulmonary metastases. Similar results were obtained in preventive models in the other two tumor models, i.e. non-small cell lung carcinoma D121 lung and carcinoma of the colon CT-26. These additional confirming data show that the selection is THERE for changes in the microenvironment of the tumor is potentially universal anticancer strategy, the overwhelming invasion of tumor cells and metastasis by decreasing the concentration factors released THERE that enhance tumor growth and metastasis.

In example 2, to create minigenes composition of the DNA of the present invention was used expressing vector mammals with ER signal (pCMV/Myc/ER). ER signal peptide directs the protein to the Department of secretion. The vector also included a C-terminal peptide, which retains the protein in the endoplasmic reticulum (ER). Peptides encoded by the composition of the DNA was transformed into ER the immune cells of the intestine, and then joined the antigenic binding site of MHC class first class for the final prezentowania to T-cell receptors, which induce leguminaceae T-cell response against murine THERE, expressing legumain. These designs create effective minigen the s vaccine, which may cause the effective CD8+T-cell response against tumors of various organs in a murine tumor models.

Additional experiments with D121 cells non-small cell lung carcinoma in isogenic C57/BL mice showed that both minigene constructs pCMV-Db/Dd and pCMV-Kb/Kd did not provide an effective immune response against THERE. On the other hand, the results with D2F2 cells of BALB/c mice showed that the construct pCMV-Kb/Kd was significantly more effective than either pCMV-Db/Dd, or pCMV-Kb/Kd. A possible explanation of this finding is that there is a much larger number leguminose peptides designed to join H-2Dd/Kd with higher affinity than those joining H-2Dd/Kb molecules. According to the BioInformatics &Molecular Analysis Section (BIMAS) of the NIH, the predicted values of peptides legumain, communicating with H-2Dd and H-2Kd, significantly more than the reported values for H-2Db and H-2Kb, and connecting the indicator H-2Kd the most high (FIG).

Assuming that the spectrum of receptor T-cells are almost unlimited, it may be a much larger number of complexes of peptide-H-2Dd/Kd, which can be recognized by CTL mice BALB/C. moreover, the peptide H-2Dd had a higher index of antigenicity than two or three peptide N-2K (FIG). Based on this information, the antigenicity less predictable than linking indicator MTL with respect to the immune response against THERE. Based on legumain minigene compositions of the present invention showed effective protection against tumors by destroying THERE in models of mammary tumors. Antitumor protection was invoked based on Kd minigene vaccines, leading to effects on THERE in the surrounding microenvironment of breast carcinoma D2F2 in syngeneic mice BALB/C. This protective immune response was detected, mediated by T-cells CD8+that sighting destroy legumain+THERE, and also leads to a pronounced inhibition of tumor angiogenesis. It is important that minigene design DNA according to the invention provide efficiency, a similar vaccine that encodes a gene legumain. Just like Dd and Kd-based designs provide answers against THERE in models of cancer D121 mice C57/BL. Taken together, these data represent the first protivolavinnuju minigene composition effective against THERE, this strategy may be particularly useful for individuals with different genetic backgrounds and thereby provides a simple, more reliable and flexible alternative strategy telegenic vaccines in the treatment of breast cancer.

Numerous changes and modifications of the above described implementations of the invention can be made without departing from the nature and volume of new traits invented who I am. In no way intended to be and should not be deemed limiting of the invention is given here for illustrative options for its implementation.

1. Pharmaceutical composition for invoking an immune response against tumor-associated macrophages containing minigene design DNA that encodes the polypeptide in a pharmaceutically acceptable carrier, where the polypeptide contains three immunogenic fragment legumain connected together by linker peptides, and where each of the linker peptide comprises the sequence AAA or AAY, and each immunogenic fragment legumain selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

2. The composition according to claim 1, where each of the linker peptide has the sequence AAY.

3. The composition according to claim 1, where minigonna design included cytomegalovirus (CMV) plasmid expression vector that encodes the sequence of the ER signal peptide sequence ICC-epitope and the sequence of the ER-retention signal, where the ER signal peptide consists of SEQ ID NO: 19, ICC-epitope comprises SEQ ID NO: 20, ER-retention signal consists of SEQ ID NO: 21.

4. The composition according to claim 3, where minigonna design included in the vector between the site BssH II in the sequence ER signal peptide and the site Xho I.

5. The composition according to claim 1, which further is entrusted contains design DNA encoding immune effector protein expressed in immune cells.

6. The composition of the DNA according to claim 5, where the immune effector protein is a cytokine.

7. The DNA composition of claim 6, where the cytokine is CCL21, IL-2 or CD40LT.

8. The composition according to claim 1, where minigonna design incorporated in an attenuated bacterial vector AroA-, dam - Salmonella typhimurium.



 

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FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.

EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.

5 cl, 2 ex, 2 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: adherent fraction of mononuclear cells (MNC) is recovered from peripheral blood of patients suffering colorectal cancer and cultivated in the medium of certain composition for producing immature dendritic cells (DC). In the process of cultivation, the latter are added with a tumour antigen (autologous tumour cell lysate), and then rhTNF-α for maturation of the immature DC. The produced mature antigen-activated DC are cultivated with the non-adherent fraction of the MNC in the presence of the proinflammatory cytokines rhIL-12 and rhIL-18.

EFFECT: use of the method of generating the antigen-specific cytotoxic cells with anticancer activity provides high cytotoxicity of anticancer cells, and as a consequence the efficiency of a T-helper type 1 immune response on colorectal cancer antigens.

1 tbl

New compounds // 2458920

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a compound of formula or to its pharmaceutically acceptable salts wherein -A-(R1)a means a group; -B-(R2)b means a group specified in the patent claim 1, R3 means hydrogen; X means CH2 or O; and Y means CH2. Also, the invention refers to a pharmaceutical composition exhibiting FGFR inhibitor activity on the basis of the declared compound.

EFFECT: there are produced new compounds and based pharmaceutical composition which can find application in medicine for preparing a cancer drug.

8 cl, 1 tbl, 180 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula (VI): or its pharmaceutically acceptable salts; wherein n is equal to 0, 1, 2 or 3; R1 means -OH, H; R2a means OH, -CH3, provided at least one of R1 and R2a means -OH;R3 means Cl, Br, cyclopropyl, branched C3-5alkyl R4a means H; R8 means H; wherein the fragment: may be one of the groups B8, B35, B36, B37, B38, B39, B40, B41, B42, B43, B45, B46, B48, B54, B56, B58, B59, B61, B62, B71, B72, B74, B75, B76, B77, B78, B79, B80, B81, B82, B84, B86, B87, B88, B89, B90, B91, B93, B94, B95, B96, B97, B98, B99, B100 and B101 wherein the values are disclosed in the patent claim 1.

EFFECT: compounds show Hsp90 inhibitory activity that enables using them for treating the diseases caused by abnormal cell growth in mammals.

26 cl, 8 dwg, 2 tbl, 82 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to homo- and hetero-polyaminoacid fullerene derivatives of formula C60(H)x{NH(CH2)nCOO-}x{NH3+(L)COOH}x, wherein n - 2-5, x - 3, L = -(CH2)m, wherein m = 1-5, or -CO(CH2)kCH(NH2)-, wherein k = 1-2, which show activity against herpes virus, hepatitis C virus, influenza viruses of various nature, HIV, as well as anticancer and antipsoriatic activity.

EFFECT: developing the method for preparing said homo- and hetero-polyaminoacid fullerene derivatives and based pharmaceutical compositions.

7 cl, 19 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, oncology, and can be used in surgical treatment of tongue cancer. At the first stage under local anesthesia with novocaine solution into femoral vein from the side of injury guide is introduced and brought under radioscopy control into corresponding carotid artery, guide is installed at the orifice into corresponding lingual artery. Chemical embolisation of lingual artery is performed and guide is removed. At the second stage total biopsy of tumour ulceration is performed. At the third stage high-frequency hyperthermia of tongue is performed by thermal impact at temperatures 60-80 degrees Celsius during two minutes, with application of multiple introduction of apparatus antenna, which contains active electrode, into tongue tissue by punctures in area of lateral - free- edge of that half of tongue where tumour was located, from tongue tip to its root, to the depth not less than 1-2 cm.

EFFECT: such total and uniform impact ensures death of tumour cells, their "blocking" on tumour boundary, prevention of hematogenic and lymphatic cancer spread, intraoperation complications, in particular, bleeding; makes it possible to carry out complete histological analysis on tongue tissues which were not modified by hyperthermia.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of: a SSMA monoclonal antibody and its antigen-binding fragment which are bound with a mark or a cytotoxic agent. There are described versions of a pharmaceutical composition and a diagnostic kit based on such antibodies. There are disclosed methods for identification in vitro of tumour cells, as well as for diagnostic identification of tumour cells based on such antibodies. What is described is an isolated polynucleotide for producing monoclonal antibodies.

EFFECT: using the invention provides the antibodies which can bind SSMA in its native form on the surface of tumour cells, are bound with LNCAP, but are not bound with cells with lost SSMA expression that can find further application in prostate cancer therapy.

15 cl, 21 dwg, 3 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to to new compounds of formula (1) or their pharmaceutically acceptable salts, optionally in the form of (1S)-isomers showing the properties of polo-like kinase (serine-threonine kinase) PLK1 inhibitor. In the compounds of formula (1) , R1 represents a halogen atom; a lower alkyl group having 1-2 carbon atoms, which can be substituted by 3 fluorine atoms; or a cyclopropyl group; R2 represents a hydrogen atom; one of R3 and R4 represents a hydrogen atom while the other one of R3 and R4 represents: a) a lower alkyl group substituted by NRaRb wherein each Ra and Rb, which can be identical or different, represent a lower alkyl group, or each Ra and Rb, which can be different, represent a hydrogen atom, a lower alkyl group or a cycloalkyl group having 3-6 carbon atoms wherein a cycloalkyl group can be substituted by one ore more substitutes which can be identical or different and specified in a group 1): a lower alkyl; b) a 4-6-member aliphatic heterocyclic group specified in an azetidinyl group, a pyrrolidinyl group and a piperidinyl group; c) a lower alkyl group substituted by a 4-6-member aliphatic heterocyclic group specified in an azetidinyl group, a pyrrolidinyl group and a piperidinyl group; d) a 6-member aromatic heterocyclic group specified in a pyridyl group wherein each of an aliphatic heterocyclic group and an aromatic heterocyclic group can be substituted by substitutes specified in a group 1) described above; R5 represents a hydrogen atom, a cyano group, a halogen atom or a lower alkyl group.

EFFECT: compounds can find application in treating oncological diseases.

10 cl, 4 dwg, 8 tbl, 42 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new hydrated N-fullerene-amino acids of general formula C60(H)3{NH(CH2)nCOOH}3·xH2O, wherein C60 represents fullerene, n = 5-7, x = 8-10, which possess herpes virus, influenza virus, HIV activity, as well as anticancer and antipsoriatic activity. The invention refers to a method for preparing said fullerene amino acids and based pharmaceutical compositions.

EFFECT: preparing new hydrated N-fullerene-amino acids.

5 cl, 28 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely oncology and radiology, and may be used for treating stage IIA Hodgkin's lymphoma. That is ensured by 3-4 therapeutic courses of chemotherapy by the scheme ABVD - adriablastine, bleomycine, vinblastine, dacarbazine. It is followed by chemotherapy by the scheme AVB - adriablastine, vinblastine, dacarbazine. From the 2nd day of the AVD course, the radiation therapy covering initially involved zones is applied at single basic dose 1.2 Gy, daily 2 times a day every 4 hours. If the initial tumour lesion is less than 5 cm, the radiation therapy is applied to total basic dose 30 Gy, while the lesions more than 5 cm requiring dose up to 36 Gy.

EFFECT: method provides reducing total treatment length at least by 2 months, reducing a risk of recurrences, reducing a probability of secondary tumours and late complications of the radiation therapy ensured by the intensified combined chemoradiation treatment.

2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to versions of applying an antisecretory protein which corresponds the amino acid sequence SEQ ID NO:6, its homologue, and/or a fragment containing the amino acid sequence SEQ ID NO:4 showing equivalent activity, and/or its pharmaceutically active salt for preparing a pharmaceutical composition and/or nutritional care for treatment and/or prevention a dysfunction, e.g. a pathological function, lipid raft, receptor and/or small pit hypo- or hyperfunction. The lipid raft, receptor and/or small pit dysfunction can be induced by or cause a number of the other conditions, such as vascular and pulmonary dysfunctions and/or endocrine disorders, e.g. diabetes and related disorders.

EFFECT: group of inventions enables controlling intracellular transport and cell product release, as well as normalising tissue component distribution in various diseases.

13 cl, 12 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely gene therapy. What is proposed for consideration is a method for stimulating spinal regeneration by the introduction into an involved area of a vector on the basis of two-cartridge plasmid pBud-VEGF-FGF2 simultaneously expressing the combination of two cloned genes of neurotrophic and angiogenic human factors VEGF and FGF2.

EFFECT: use of the invention provides more effective spinal functional recovery that can find further application in treating the patients with traumatic spinal injuries.

3 cl, 3 ex

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