Composition for treating lung cancer, first non-small cells lung cancer (nsclc)

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine. What is described is an active immunostimulating vaccine containing at least one RNA, preferentially iRNA coding at least two antigens evoking the immune response in a mammal and used for treating lung cancer, first non-small cells lung cancer (NSCLC), preferentially specified among three primary subtypes, squamous cell carcinoma, adenocarcinoma and large-cell lung carcinoma, or NSCLS-related disorders.

EFFECT: there are produced kits, first containing the active immunostimulating vaccine.

21 cl, 34 dwg, 1 tbl, 8 ex

 

The present invention relates to an active (immunostimulatory) composition containing at least one RNA, preferably mRNA, encoding at least two (preferably different) antigens capable of inducing (acquired) immune response in a mammal. The present invention also relates to a vaccine containing specified active (immunostimulirutuyu) composition and to the use of the specified active (immunostimulatory) composition (for vaccines) and/or vaccine for the induction of (acquired) immune response for the treatment of lung cancer, especially non-small cell lung cancers lung (nmcrl), preferably selected from the three main subtypes: squamous cell lung carcinoma, adenocarcinoma and both carcinoma of the lung or related disorders. Ultimately, the present invention relates to sets, especially sets or sets of components containing active (immunostimulirutuyu) composition and/or vaccine.

25% of all malignant tumors registered bronchial carcinoma (lung carcinoma). Worldwide it is the most common cause of death from cancer in men and second most common in women. In Germany this type of cancer is the third most common, then carcinoma of t is positive cancer and colorectal carcinoma. Lung carcinoma is the cause 1.3 million deaths worldwide annually. In Central Europe the incidence is approximately 60 per 100,000 population, and the number of patients with a primary diagnosis of lung cancer has been steadily increasing (making currently in Germany-approximately 50,000 a year). After diagnosis of lung cancer, the average survival rate for a period of 5 years is less than 5%. However, the average life expectancy of each individual patient depends entirely on the stage of the disease (according to the classification TMN) and installed subtype carcinoma (lung cancer) (see below).

The main subtypes of lung cancer, classified by size and localization of malignant cells that can be recognized under the microscope, are small cell lung cancer (20%) and non-small cell lung cancer (nmcrl) (80%). Although this classification is based on the simple histological criteria, it is extremely important in clinical practice and the prediction of the development of the disease, and small cell lung cancer are usually treated with chemotherapy, while for the treatment of non-small cell lung cancer in most cases as a first line treatment using surgery.

Non-small cell lung cancer lung (nmcrl) unite on the basis that impregnation and treatment is about the same. There are three main subtypes: squamous cell lung carcinoma, adenocarcinoma and both carcinoma of the lung. The main treatment is surgery, but only a quarter of patients successfully transferred resection, the recurrence rate is 50%. Methods of treatment of the disease at a later stage include, after surgical intervention, auxiliary chemotherapy and/or auxiliary radiation therapy, and it was found that when using as monotachi (first-line therapy) was observed unsatisfactory results. When comparing the four commonly used courses of combination chemotherapy none has been quite effective. The efficacy of the treatment was changed from 15% to 22%, while the survival rates were for the first year from 31% to 36% (see, for example, article O'mahony D., S. Kummar and other, "Non-small-cell lung cancer vaccine therapy: a concise review", J. Clin. Oncol. 23(35); 9022-9028 (2005)). Thus, it appeared that even preoperative chemotherapy does not lead to the expected increase in life expectancy, and auxiliary chemotherapy used in combination with radiation therapy, also does not lead to a significant increase in life expectancy.

One of the chemotherapeutic approaches currently, the PR is dstanley a combination of substances on the basis of platinum, for example, gemcitabine, used even as a first-line therapy, when this is used, for example, pemetrexed as therapy of the second row.

Another option, used to treat nmcrl, is the so-called "targeted therapy", i.e. an attempt to strengthen the effectiveness of classical cytotoxic chemotherapy when exposed to the specific structure of the target in the tumor at the molecular level. These substances include bevacizumab (angiogenesis inhibitor) or erlotinib that affects a receptor tyrosine kinase of the epidermal growth factor (REFR).

Even though, undoubtedly, there is a positive trend in the use of modern therapeutic approaches to the treatment of lung cancer, primarily nmcrl, there are still serious difficulties associated with high mortality rates, and the urgent need for further, alternative or improved methods of treatment.

Thus, in the present invention it is proposed to use the immune system in the treatment of nmcrl. The immune system plays an important role in the treatment and prevention of numerous diseases. In accordance with the current state of science mammals provided by various defense mechanisms of the body that can recognize and kill them, for example, tumor cells. These tumor cells need to identify and distinguish from normal cells and tissues of the body.

The immune system of vertebrates, such as humans consists of many types of proteins, cells, organs and tissues, which interact in a complex and dynamic system. On one of the stages of this extremely complex immune response, the immune system over time adapts to a more efficient detection of certain pathogens or tumor cells. In the adaptation process creates immunological memory that provides more effective protection during subsequent encounters with pathogens. This process is adaptive or acquired immunity forms the basis of methods of vaccination.

The acquired immune system is antigen specific and requires the recognition of specific "native" or "foreign" antigens during a process called antigen presentation. Antigenic specificity contributes to the formation of responses aimed at specific pathogens or infected with the pathogen cells or tumor cells. The ability to generate these targeted response is supported in the body of the so-called "memory cells". If the pathogen infects the body several times, then the specification is specific memory cells are used for the rapid elimination of the pathogen. Thus, the acquired immune system provides a stronger immune response and immunological memory, with each pathogen or tumor cell memorized in the form of one or more specific antigens.

The main components of the acquired immune system in vertebrates mainly include lymphocytes at the cellular level and antibody at a molecular level. Lymphocytes as components of the acquired immune system include b cells and T cells, which are derived from hematopoietic stem cells in the bone marrow. B-cells involved in the humoral immune response, whereas T-cells involved in the cellular immune response. As b-cells and T-cells include receptor molecules that recognize specific targets. T-cells recognize the "alien" target, such as pathogenic structure is a target only once formed versions of the antigens (i.e. low molecular weight fragments of the pathogen) and the in combination with the "native" receptor, called the major histocompatibility complex (MHC). In contrast, B-cell antigen receptor is a molecule antibodies on the surface of b cells and recognizes pathogens such as when antibodies on their surface contact with the specifications the Czech foreign antigen. Such a complex antigen/antibody is absorbed In the cell and undergoes processing to proteolysis with the formation of peptides. Then In the cell presenting these antigenic peptides on their surface molecules MHC class II. This combination of MHC and antigen attract the appropriate helper T-cell that releases lymphokines and activates the B-cell. As activated b-cell then starts to share her descendants secrete millions of copies of antibodies that recognize the antigen. These antibodies circulate in blood plasma and lymph, bind pathogens or tumor cells expressing the antigen, and mark them for later destruction through the activation of complement or uptake and destruction by phagocytes.

As the cellular component of the acquired immune system, cytotoxic T cells, CFC (CD8+) can form TCA response. Cytotoxic T cells (CD8+) can recognize peptides from endogenous pathogens and native antigens associated with MHC molecules of type I. CD8+T-cell function killers in the release of cytotoxic proteins in the cell.

The mechanisms of the immune system form a target intended for the treatment. Appropriate methods, usually based on the introduction adjuvants for the induction of innate immune response or the introduction antigen is or immunogens for the induction of acquired immune response. Since antigens are usually specific components of pathogens (e.g., surface proteins or their fragments, the introduction of patient nucleic acid, with subsequent expression of the desired polypeptides, proteins, or antigens, can be considered as a promising treatment method.

Up to the present time, standard methods for the induction of an immune response, immunization or vaccination, based on the use of DNA, with the aim of embedding the required genetic information in the cell. Developed various methods for introducing DNA into cells, such as transfection by calcium phosphate, transfection of polyprenol, the fusion of protoplasts, electroporation, microinjection and lipofection, and lipofection, as it was found, is the most suitable way. Similarly, you can use the DNA-containing viruses as carriers of DNA. Thanks to their infectious properties, such viruses are characterized by an extremely high rate of transfection. Used the viruses are genetically modified to prevent the formation of functional infectious particles in transfectional cell. However, despite these precautions, it is impossible to eliminate the risk of uncontrolled propagation of the introduced gene and viral genes, for example, because of potential cases Ryoko is Binali. Risk is the possibility of introducing DNA into a healthy gene into the genome of the host cell, for example, as a result of recombination with subsequent mutation of this gene, and thus, there is a complete or partial deactivation of the gene, or it may contain incorrect information. In other words, there is a possibility of complete suppression of the synthesis of the genetic product, which is vital to the cells, or, conversely, the ability of expression of the modified or incorrect genetic product. Special risk arises when the DNA is introduced into a gene, which is involved in the regulation of cell growth. In this case, a host cell may begin to degenerate, which leads to the formation of cancer or tumor. In addition, if the DNA introduced into the cell, to be expressed, there is a need for appropriate vector DNA contains a strong promoter such as the promoter of the CMV virus. The incorporation of such promoters in the genome of affected cells may lead to undesirable deviations in the regulation of gene expression in the cell. Another risk when using DNA as an agent to enhance the immune response (for example, in the form of a vaccine) is the induction of pathogenic antibodies against DNA from the patient who was introduced foreign DNA that can induce the SQL immune response (which can lead to death).

In General, therefore, there is a need to develop an effective system that can be used for effective stimulation of the immune system, making the treatment of lung cancer, especially non-small cell lung cancer (nmcrl), in addition to eliminating the problems associated with uncontrolled reproduction of the introduced gene in the use of compositions based on DNA.

Thus, the aim of the present invention is a composition which (a) provides for the treatment of lung cancer as a result of stimulation of the immune system and at the same time b) is devoid of the above disadvantages.

This goal is achieved by designing the object of the present invention, first of all the active (immunostimulatory) composition containing at least one RNA encoding at least two (preferably different) antigens selected from the group consisting of antigens: hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 and/or MAGE-C2.

Unexpectedly, it was found that a specific combination of at least two antigens, antigenic proteins or antigenic peptides of the above-listed groups, the composition of the active (immunostimulatory) composition according to the present invention, can effectively stimulate the (acquired) immune system, providing treatment of lung cancer, primarily nmcrl. In this the context, the terms "antigen, antigenic proteins or antigenic peptides" are used as synonyms. In the context of the present invention, the term "active" (immunostimulirutuyu) the composition according to the present invention refers to a composition that induces an immune response, preferably acquired immune response, as defined in this context, with one component (or components) contained in the composition or encoded components of the active (immunostimulatory) composition, preferably at least one RNA, preferably mRNA, encoding at least two (preferably different) antigens.

At least one RNA in the composition of the active (immunostimulatory) composition may encode hTERT. In the context of the present invention, the term "hTERT" means telomerase reverse transcriptase person, and the preferred sequence RNA, preferably mRNA, encoding "hTERT", if it is used in the active (immunostimulatory) composition according to the present invention, shown in Fig.7 (SEQ ID NO:7), and even more preferably, at Fig (SEQ ID NO:8). In article Minev, B., J. Hipp and others, "Cytotoxic T cell immunity against telomerase reverse transcriptase in humans", Proc. Natl. Acad. Sci. USA, 97(9): 4796-4801 (2000) indicated that telomerase is a ribonucleoprotein enzyme that is associated with malignant transformation in human cells. T is somaratna activity increases in the vast majority of human cancers, that leads to the fact that its genetic product is the main molecule, characteristic of all human cancers. Therefore, the accumulation of endogenous protestirovannyx telomerase peptides associated with MHC molecules of class I can send cytotoxic T lymphocytes (CTLs) to tumors of different origin. Thus, according to these data, this approach may provide a promising method for cancer vaccine therapy, which is provided by the possibility of increasing the number of precursor CTLs that recognize telomerase peptides in healthy adults and patients with cancer, as a result of immunization. In the above article also described that the majority of healthy subjects and patients with prostate cancer, in vitro immunized against two peptides to HLA-A2.1, the resulting enzyme telomerase reverse transcriptase (hTRT), produced hTRT-specific LTC. In article Carpenter, E. L. and R. H. Vonderheide, "Telomerase-based immunotherapy of cancer", Expert. Opin. Biol. Ther. 6(10): 1031-1039 (2006) described a progressive research, starting with cloning telomerase reverse transcriptase person (hTERT) in 1997 to the first clinical trials. Protein hTERT sverkhekspressiya in the vast majority of human cancers, while it has limited expression in healthy human tissues. It plays important is th role in carcinogenesis and may be expressed in cancer stem cells. However, despite the fact that he is a native antigen, hTERT shows immunogenic properties both in vitro and in vivo. The results of several clinical and immunological tests in the first phase during immunotherapy with the use of hTERT in patients with cancer of the breast, prostate, lung and other cancers, are quite encouraging. Immunotherapy stimulated patients the formation of functional antitumor T cells in the absence of clinical toxicity. There is also the possibility of vaccination of subjects as a strategy for immunization during treatment on the basis of hTERT. In the article by S. K. Nair, A. Heiser and others, "Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells", Nat. Med. 6(9): 1011-1017 (2000) described the development antimisting TERT-immunity in mice vaccinated with dendritic cells, transfectional murine TERT RNA. According to a preferred variant implementation of the present invention at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the hTERT antigen selected from the sequences as shown in Fig.7 (SEQ ID NO:7), and more preferably, pig (SEQ ID NO:8). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) compositeimage alternative or additionally encode the antigen, hTERT, selected from a fragment, variant or epitope sequence hTERT, as shown in Fig.7 (SEQ ID NO:7), and more preferably, as shown in Fig (SEQ ID NO:8).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode WTI. In the context of the present invention, the term "WT1" means Wilms tumor stage 1 and the preferred sequence RNA, preferably mRNA, encoding "WT1", provided its use in the composition of the active (immunostimulatory) composition according to the present invention, as shown in figure 9 (SEQ ID NO:9), more preferably, as shown in figure 10 (SEQ ID NO:10), and most preferably, as shown in figure 11 (SEQ ID NO:11). Article Y. Oka, A. Tsuboi, etc., "Induction of WTI (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WTI peptide vaccine and the resultant cancer regression", Proc. Natl. Acad. Sci. USA 101 (38): 13885-13890 (2004) found that protein Wilms tumor sverkhekspressiya with lung cancer. This article describes the vaccination of 10 patients with lung cancer peptide of WT1. It was found that the clinical response correlates with antitumor CD8+-T-cell activity. Gene Wilms tumor WT1 sverkhekspressiya with leukemia and various types of solid tumors, and it was found that WT1 protein is a promising target antigen for immunotherapy against these malignancies. In the article the Thiey Oka and others (2004, see above) describes the results of phase I clinical trials during immunotherapy, based on the use of WT1 peptide in patients with breast cancer and lung disease, myelodysplastic syndrome or acute myeloid leukemia. Twelve of the 20 patients in whom it was possible to assess the effectiveness of vaccination with WT1 was observed clinical response, such as reducing the number of leukemic blast cells or the size of the tumor and/or tumor markers. There has been a clear correlation between increased frequency of WT1-specific cytotoxic T-lymphocytes after vaccination with WT1 and clinical response. Thus, it was found that WT1 vaccination can induce the production of WT1-specific cytotoxic T-lymphocytes and leads to remission of the cancer in the absence of damage to normal tissues. According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the WT1 antigen selected from the sequences as shown in figure 9 (SEQ ID NO:9), and more preferably, as shown in figure 10 (SEQ ID NO:), and most preferably, as shown in figure 11 (SEQ ID NO:11). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additional is but encode the antigen WT1, selected from a fragment, variant or epitope sequence WT1, as shown in figure 10 (SEQ ID NO:10), and most preferably, as shown in figure 11 (SEQ ID NO:11).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode MAGE-A2. In the context of the present invention, the term "MAGE-A2" denotes melanoma antigen family A, 2B, and the preferred sequence RNA, preferably mRNA, encoding a "MAGE-A2, provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, shown in Fig (SEQ ID NO:14), and most preferably, at Fig (SEQ ID NO:15). In the article, Gillespie, A. M. And R. E. Coleman, "The potential of melanoma antigen expression in cancer therapy", Cancer Treat. Rev. 25(4): 219-227 (1999) described the expression for bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, cancer of the jaw, melanoma, cancer of the esophagus, and cancer osteosarcoma ovarian cancer. According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the antigen MAGE-A2, selected from the sequences as shown in Fig (SEQ ID NO:14), and more preferably, as shown in Fig (SEQ ID NO:15). According to another preferred variant, at least one RNA of the active composition (immunity Wirayuda) compositions may alternatively or additionally encode the antigen MAGE-A2, selected from a fragment, variant or epitope sequences of MAGE-A2, as shown in Fig (SEQ ID NO:14), and more preferably, as shown in Fig (SEQ ID NO:15).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode T. In the context of the present invention, the term "T" means trophoblast glycoprotein, and the preferred sequence RNA, preferably mRNA, encoding "T", provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, is shown in figure 3 (SEQ ID NO:3), and more preferably, figure 4 (SEQ ID NO:4). In article R. Harrop, N. Connolly and others, "Vaccination of colorectal cancer patients with modified Vaccinia Ankara delivering the tumor antigen T (TroVax) dosage immune responses which correlate with disease control: a phase 1/11 trial", Clin. Cancer Res. 12 (11 Pt 1): 3416-3424 (2006) found that uncomprehending antigen T is a membrane glycoprotein with a high content of leucine and weight of 72 kDa, which is expressed at high levels in the placenta, as well as in a number of human carcinomas, including colorectal cancer, gastric cancer, kidney and ovarian cancer, but is rarely found in healthy tissues. Overexpression of T associated with poor prognosis in patients with colorectal cancer, gastric cancer and ovarian cancer. Despite this combination of factors, T-specific cellular and/or g is a moral immune response was induced in the majority of patients (16 of 17, 94%), after immunization TroVax, which is considered to be promising compared with the test results of many other methods of cancer immunotherapy. Thus, in the above article describes the safety and immunogenicity of delivery TroVax intramuscular and intradermal introduction methods. Article Y. Zhao and Y. Wang, "5T4 oncotrophoblast glycoprotein: janus molecule in life and a novel potential target against tumors". Cell Mol. Immunol. 4(2): 99-104 (2007) indicated that ecotrophology 5T4 glycoprotein is a transmembrane protein expressed in embryonic tissue and on the surface of cells of various malignant tumors. He plays a vital role in numerous biological and pathological processes, including a very extensive migration of cells during embryogenesis, cell invasion, caused by implantation and tumor metastases in the process of carcinogenesis. In article Kopreski, M. S., F. A. Benko and others, "Circulating RNA as a tumor marker: detection of 5T4 mRNA in breast and lung cancer patient serum", Ann. NY Acad. Sci. 945: 172-178 (2001) indicated that 5T4 is trophoblast glycoprotein, which sverkhekspressiya with high frequency in epithelial tumors, which is a potential target in the treatment of cancer. Selected sera from 19 patients with advanced breast cancer (5 patients) or non-small cell lung cancer (14 patients), and 25 healthy volunteers used in the image quality is as control patients with amplificare RNA. RNA extracted from the serum, amplified RT-PCR using palugaswewa two-stage reaction, the products were detected by electrophoresis. 5T4 mRNA was reproducibly detected in 8 of 19 (42%) sera of patients with cancer, including 2 of 5 sera of patients with breast cancer and 6 of the 14 patients ' sera of lung cancer patients, but only 3 of 25 (12%) healthy control sera (p=0,035). According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the antigen T selected from the sequences as shown in figure 3 (SEQ ID NO:3), and more preferably, as shown in figure 4 (SEQ ID NO:4). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode the antigen T selected from a fragment, variant or epitope sequence T, as shown in figure 3 (SEQ ID NO:3), and more preferably, as shown in figure 4 (SEQ ID NO:4).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode MAGE-A3. In the context of the present invention, the term "MAGE-A3" indicates melanoma antigen family A, 3, a preferred sequence RNA, pre is respectfully, mRNA encoding a "MAGE-A3", provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, shown in Fig (SEQ ID NO:16), and most preferably, at Fig (SEQ ID NO:17). In the article, Gillespie, A. M. and R. E. Coleman, "The potential of melanoma antigen expression in cancer therapy". Cancer Treat. Rev. 25(4): 219-227 (1999) described the expression for bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioma, head and neck cancer, lung cancer, cancer of the jaw, melanoma, neuroblastoma, cancer of the esophagus and ovarian cancer. The article W. Sienel, S. Varwerk and others, "Melanoma associated antigen (MAGE)-A3 expression in Stages I and II non-small cell lung cancer: results of a multi-center study", Eur. I. Cardiothorac. Surg. 25(1): 131-134 (2004) described the results of a study conducted to determine the degree of expression of MAGE-A3 at an early stage nmcrl. Were samples of the primary tumor from 204 patients with nmcrl in resectable stages I and II, and determined the stage of development of pathology. The expression of MAGE-A3 were analyzed tissue samples for the detection of transcripts of the MAGE-A3 RT-PCR. The expression of MAGE-A3 was observed in 80 of 204 (39,2%) samples of primary tumors at stages I and II. In article Atanackovic D., N. K. Altorki and others, "Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients", J. Immunol. 1 72(5): 3289-3296 (2004) described that the MAGE-A3 is a tumor-associated antigen, originally identified in melanoma, as well as in tumors of p and non-small cell lung cancer. During the clinical trial of 9 patients with nmcrl were vaccinated with protein, it was observed 3 detectable humoral immune response. Seven of the 8 patients who were injected MAGE-A3 in combination with adjuvant ASO2B, was developed antibodies against MAGE-A3. Several of these patients was also observed T-cell immune responses against the protein. According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the antigen MAGE-A3 selected from the sequences as shown in Fig (SEQ ID NO:16), and more preferably, as shown in Fig (SEQ ID NO:17). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode the antigen MAGE-A3 selected from a fragment, variant or epitope sequence of the MAGE-A3, as shown in Fig (SEQ ID NO:16), and more preferably, as shown in Fig (SEQ ID NO:17).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode MUC1. In the context of the present invention, the term "MUC1" refers to the mucin 1, and the preferred sequence RNA, preferably mRNA, encoding "MUC1", provided that it is used in the composition of the active (immunostimulatory) composition is in accordance with the present invention, shown in figure 1 (SEQ ID NO:1), and most preferably, as shown in figure 2 (SEQ ID NO:2). Tumor-associated mucines are a potential target in immunotherapy. These molecules are believed to contribute to metastasis by accelerating adhesion of malignant cells to the surface of endothelial cells. In the article, Denda-Nagai K, and So Lrimura, "MUC1 carcinoma in-host interactions", Glycoconj J., 17 (7-9): 649-658)described that MUC-I sverkhekspressiya in 90% of all cases of adenocarcinoma, including lesions of the breast, lung, pancreas, prostate, stomach, colon and ovarian cancer. In the article, Kontani, K., O. Taguchi, and others, "Modulation of MUC1 mucin as an escape mechanism of breast cancer cells from autologous cytotoxic T-lymphocytes", Br. J. Cancer. 84(9): 1258-1264 (2001) described that MUC-1 is expressed in 60% of cases of lung cancer, while in the article, Kontani, K., O. Taguchi, and others, "Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin", Int. J. Mol. Med., 12(4): 493-502 (2003) described the results of studies using loaded MUC1 antigen pulsed dendritic cells (DC) for the induction of cellular immunity in cases of MUC1-positive cancer, this has been clinically established that seven of the nine MUC1-positive patients have a response to appropriate treatment, which is expressed either in reducing levels of tumor markers, or in the disappearance of malignant pleural effusion. Moreover, three of these p the patients with a positive response was diagnosed nmcrl. Article M. Palmer, J. Parker, and others, "Phase I study of the BLP25 (MUC1 peptide) liposomal vaccine for active specific immunotherapy in stage IIIB/IV non-small-cell lung cancer", Clin. Lung Cancer, 3(1): 49-57 discussion 58 (2001) indicated that in phase I clinical trials using peptide MUC1 at stages III/IV nmcrl was observed safety and immunity of the specified agent. In 5 of 12 patients (42%) was observed immune response in 4 out of 12 patients (33%) was observed stabilization of the disease. In addition, article Wierecky J., M. Mueller and others, "Dendritic cell-based cancer immunotherapy targeting MUC-1", Cancer Immunol. Lmmunother., 55(1): 63-67 (2006) described the identification of two HLA-A2-binding new nonapeptide of the tumor-associated antigen MUC1, which sverkhekspressiya at various hematological and epithelial tumors. Cytotoxic T cells generated under the action of pulsating loaded with these peptides DK, caused lysis of tumor cells expressing MUC1 by antigen specific and HLA-restricted mechanism. According to two clinical trials found that vaccination of patients with advanced cancer using DC loaded with peptides of MUC1 was observed quite a high tolerance without serious side effects, and formed the immune response. Of the 20 patients with metastatic renal cell carcinoma in 6 patients showed regression of metastases three objective what occurred (1 CR and 2 PR). According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the MUC1 antigen, selected from the sequences as shown in figure 1 (SEQ ID NO:1), and more preferably, as shown in figure 2 (SEQ ID NO:2). According to another preferred variant of the present invention at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode the MUC1 antigen selected from a fragment, variant or epitope of MUC1 sequence, as shown in figure 1 (SEQ ID NO:1), and more preferably, as shown in figure 2 (SEQ ID NO:2).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode Her-2/neu. In the context of the present invention, the term "Her-2/neu" refers homolog 2 viral oncogene erythroblastic leukemia, v-erb-b2, and the preferred RNA sequence encodes "the Her-2/neu", preferably mRNA, encoding a "Her-2/neu", provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, is shown in figure 5 (SEQ ID NO:5), and most preferably, as shown in Fig.6 (SEQ ID NO:6). In article Baxevanis CN, P.A. Sotiropoulou and others, "Immunobiology of HER-2/neu oncoprotein and its potential application in cancer immunotherapy". Cancer Immunol. Lmmunother., 53(3): 166-175 (2004) indicated that HR-2/neu (also known as HER2, or c-erb-B2) is a protein receptor mass of 185 kDa, characterized tyrosinekinase activity and a high degree of homology in comparison with the receptor for epidermal growth factor (EGF). HER-2/neu is expressed in many epithelial tumors and is known to sverkhekspressiya approximately 20-25% of all cases of ovarian cancer and breast cancer, 35-45% of all cases of adenocarcinoma of the pancreas and up to 90% of cases of colorectal carcinoma. Overexpression of HER-2/neu is a marker of poor prognosis. Overexpression of HER-2/neu is observed in malignant tumors of the breast, ovary, pancreas, colon, lung and other tissues. Normal Her-2 is expressed at low levels in various human tissues (skin, epithelium of the digestive tract, mammary gland, ovaries, hepatocytes). In the article, Bernhard H., L. Salazar, and others, "Vaccination against the HER-2/neu oncogenic protein", Endocr. Relat. Cancer, 9(1): 33-44 (2002) indicated that the preliminary results of clinical trials suggest that cancer patients actively immunized against HER-2/neu, can induce immunity and that the immune response persists for some period of time. Modern clinical trials of a vaccine directed only to the use of vaccines based on the epitopes or peptides, mainly in connection with the data that such a strategy on the basis of pepti the Noah vaccination can prevent new non-specific tolerance in rodent models. The next generation of vaccines, as described in article Bernhard and others (2002) (see above), will likely include vaccines based on proteins, drugs DK, loaded with antigen HER-2/neu, and compositions for nucleic acids. Preclinical trials of these approaches in rodent models proved to be quite encouraging. The growth of HER-2/neu-specific T cells ex vivo after active immunization or their cultivation in vitro with the use of DC expressing HER-2/neu, is thus a promising method for the treatment of HER-2/neu-sverkhekspressiya tumors at later stages. In article Baxevanis CN, N.N. Sotiriadou and others, "Immunogenic HER-2/neu peptides as tumor vaccines", Cancer Immunol. Lmmunother. 55(1): 85-95 (2006) found that in humans, although there were cases of immune response to the peptides used for vaccination, no described cases of response in clinical trials. In article M.L. Disis, Gooley TA, etc., "Generation of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based vaccines", J. Clin. Oncol. 20(11); 2624-2632 (2002) described that Her-2/neu is a member of the family of EGF receptors. In most cases, it sverkhekspressiya in breast cancer, ovarian cancer, prostate cancer, colorectal cancer and lung cancer. In phase I clinical trial of 38 patients (2 with diagnosis nmcrl) were vaccinated with the peptide Her-2/neu. In 92% of patients were observed development is the development of T-cell immunity to the Her-2/neu. According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the antigen Her-2/neu, selected from the sequences as shown in figure 5 (SEQ ID NO:5), and more preferably, as shown in Fig.6 (SEQ ID NO:6). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode the antigen Her-2/neu, selected from a fragment, variant or epitope sequence of the Her-2/neu, as shown in figure 5 (SEQ ID NO:5), and more preferably, as shown in Fig.6 (SEQ ID NO:6).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode NY-ESO-1. In the context of the present invention, the term "NY-ESO-1" refers to the antigen 1 In testicular cancer, and the preferred sequence RNA, preferably mRNA, encoding NY-ESO-1", provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, shown in Fig (SEQ ID NO:20), and most preferably, at Fig (SEQ ID NO:21). In article Y.T. Chen, M.J. Scanlan and others, "A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening", Proc. Natl. Acad. Sci. USA 94(5): 1914-1918 (1997) described mediated mRNA expression of NY-ESO-1 in various human cancers detected by the method Of the RT-PCR melanoma 23/67, ovarian cancer 2/8, breast cancer 10/33, thyroid cancer 2/5, prostate cancer 4/16, bladder cancer 4/5, colorectal cancer 0/16, Burkitt's lymphoma 1/2, glioma 0/15, basal cell carcinoma 0/2, stomach cancer 0/12, leiomyosarcoma 0/2, lung cancer 2/12, other sarcomas 0/2, kidney cancer 0/10, pancreatic cancer 0/2, lymphoma 0/10, seminoma 0/1, hepatoma 2/7, swelling of the spinal cord 0/1. In article Jager E., J. Karbach and others, "Recombinant vaccinia/fowl pox NY-ESO-1 vaccines induce both humoral and cellular NY-ESO-1-specific immune responses in cancer patients", Proc. Natl. Acad. Sci. USA 103(39): 14453-14458 (2006) indicated that NY-ESO-1 is an antigen testicular cancer, expressed in a number of malignant tumors in humans, and developed numerous methods of vaccination, in which targets are used NY-ESO-1. In these trials evaluated the safety and immunogenicity of recombinant vaccines based on the NY-ESO-1 and recombinant vaccines based on the chicken plague-NY-ESO-1, with the participation of a group of 36 patients with various types of tumors. Each construct was initially tested individually, using two different doses, and then the secondary injection of recombinant vaccines NY-ESO-1, and then the recombinant vaccine based on the chicken plague-NY-ESO-1. The vaccine was characterized by a fairly high tolerance, as after the introduction of separately and in mixture. Response, mediated NY-ESO-1-specificname antibodies, and/or specific CD8 and CD4 T-cell responses against a broad set of NY-ESO-1 epitopes, induced at least after a course of four vaccinations with a month interval in most patients. It was found that clones of CD8 T cells obtained from five vaccinated patients are lysed target cells from NY-ESO-1-expressing melanoma. In some cases, the results obtained with the participation of a number of patients with melanoma testified to the fact that vaccination has had a beneficial effect on the natural course of the disease. In the article, Davis I.D., W. Chen et al, "Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant dosage broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans." Proc. Natl. Acad. Sci. USA 101 (29): 10697-10702 (2004) indicated that HLA-A2 restriction peptides NY-ESO-1 with intradermal characterized by safety and immunogenicity. Despite the fact that these tests were carried out only for the purpose of evaluation of safety and immunogenicity, some patients were observed tumor regression or stabilization of the disease. In addition, article Jager E., S. Gnjatic, etc., "Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1 + cancers", Proc. Natl. Acad. Sci. USA 97(22): 12198-12203 (2000) indicated that the expressed NY-ESO-1-specific immune response, including the induced antibody response, as well as CD4 and CD8 T-cell response was observed after immunization with recombinant b is lcom NY-ESO-1 in combination with adjuvant ISCOMATRIX (CSL Ltd., Parkville, Victoria, Australia) in patients with remote NY-ESO-1-expressing melanoma. It was found that the immune response to vaccination was accompanied by a lengthy period of remission. In addition, article Odunsi K., F. Qian and others, "Vaccination with an NY-ESO-1 peptide of HLA class 1/11 specificities dosage integrated humoral and T cell responses in ovarian cancer", Proc. Natl. Acad. Sci. USA 104(31): 12837-12842 (2007) indicated that vaccination with NY-ESO-1 peptide causes combined humoral and T-cell immune response in ovarian cancer. According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the antigen NY-ESO-1, selected from the sequences as shown in Fig (SEQ ID NO:20), and more preferably, pig (SEQ ID NO:21). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode antigen NY-ESO-1 is selected from a fragment, variant or epitope sequence of NY-ESO-1, as shown in Fig (SEQ ID NO:20), and more preferably, as shown in Fig (SEQ ID NO:21).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode CEA. In the context of the present invention, the term "CEA" means embryonal tumor antigen (SESAM, i.e. the cell adhesion molecule, wired is I with embryonal tumor antigen 5), while the preferred sequence RNA, preferably mRNA, encoding "CEA", provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, shown in Fig (SEQ ID NO:12), and most preferably, at Fig (SEQ ID NO:13). In the article World S., "The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues", Semin. Cancer Biol. 9(2): 67-81 (1999) indicated that CEA is oncofetal glycoprotein mass of 180 kDa, which shows the properties of the cell adhesion molecules and sverkhekspressiya in 70% of cases nmcrl. In article Berinstein N.L., "Carcinoembryonic antigen as a target for therapeutic anticancer vaccines: a review", J. Clin. Oncol. 20(8): 2197-2207 (2002) indicated that CEA has a number of promising properties for use as targets in the development of methods of active immunization against cancer. It is characterized by suitable mechanism of expression, and is expressed in more than 50% of human cancers. It can play a role in oncogenesis by itself, and, thus, its expression can be evaluated and stop in the course of cancer progression. There is strong evidence that CEA is subjected to processing and prezentuetsya on different molecules MHC class 1. In addition, immunological tolerance to CEA is not absolute. There is much evidence that T-cells can recognize, activate the I and lyse cancer cells, which Express CEA. Evaluated several different methods of therapeutic vaccination using CEA as a target antigen, and the degree of safety of these methods. In addition, we registered the presence of a humoral and/or cellular immune response to CEA. Despite the fact that most of the patients selected for these tests, she was diagnosed with progressive refractory metastatic colon cancer, signs of clinical activity, i.e. the suspension of the development of the disease and even objective responses in some patients. Dendritic cells loaded with peptide binding to CEA agonist MHC class I (CAPI-6D), and the vectors on the basis of poxvirus-enabled CEA, in the presence or in the absence of Pro-stimulation molecules, showed the greatest activity when activated CD8 T-cell response. Unfortunately, the application of methods of using dendritic cells is limited by difficulties in obtaining samples of dendritic cells that are specific to the tissue of the patient. Describes the results of four trials phase I system using vector Canary poxvirus to impact the CEA. The results testified to the fact that these methods are safe and are characterized by mild toxicity 1 and 2, depending on the Asda introduction. In addition, the test results revealed that most patients can activate specific T-cell response to the presence of CEA. Such responses can be enhanced by the inclusion in the vector together stimulatory molecules V, or adding recombinant GMCSF (granulocyte-macrophage colony stimulating factor) in the area of introduction. Although not observed objective clinical responses, a significant number of patients in this phase I trials registered cases stabilization of the disease. Developed methods of vaccination in order to further increase the frequency of detection of CEA T-cells to improve the clinical efficacy of these vaccines. Published evidence that at least some of the vaccines are more effective in the initial stages of the disease. In article Ueda Y., T. Itoh and others, "Dendritic cell-based immunotherapy of cancer with carcinoembryonic antigen - derived, HLA-A24-restricted CTL an epitope: Clinical outcomes of 18 patients with metastatic gastrointestinal or lung adenocarcinomas", Int. J. Oncol. 24(4): 909-917 (2004) described tests in which 18 patients with metastatic cancer of the gastrointestinal tract or lung cancer have introduced their own dendritic cells loaded with peptide derived from the CEA. Immune response, as determined by skin test and T-cell in vitro assays, was observed in most patients. Despite the absence of the influence of data on objective clinical response, some patients showed stabilization of disease during a specified immunotherapy. According to a preferred variant of the present invention at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the antigen CEA, selected from the sequences as shown in Fig (SEQ ID NO:12), and more preferably, pig (SEQ ID NO:13). According to another preferred variant of the present invention at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode the antigen CEA selected from a fragment, variant or epitope sequence of CEA, as shown in Fig (SEQ ID NO:12), and more preferably, pig (SEQ ID NO:13).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode survivin. In the context of the present invention, the term "survivin" refers to protein-5 inhibitor of apoptosis (IAP), including replays of baculovirus inhibitor (survivin), and the preferred sequence RNA, preferably mRNA, encoding survivin", provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, shown in Fig (SEQ ID NO:18), and most preferably, at Fig (SEQ ID NO:19). In article Grube M., S. Moritz and others,"CD8+ T cells reactive to survivin antigen in patients with multiple myeloma", Clin. Cancer Res. 13(3): 1053-1060 (2007) described survivin. Survivin is a member of the family of inhibitors of apoptosis and overexpression in the development of various types of malignant neoplasms. Cytotoxic T cells that recognize epitopes of survivin, can be obtained in vitro or by vaccination of patients with a diagnosis of leukemia, breast cancer and melanoma. We examined the level of survivin-specific CD8+T-cells in patients with multiple myeloma were identified T cells that recognize peptide HLA-A2.1 binding to survivin, in 9 of 23 patients and 1 in 21 healthy volunteers. Survivin-reactive T cells were identified as terminal differentiated effector T cells (CD8+, CD45RA+ CCR7-). Positive expression of survivin in myeloma cells in the bone marrow was observed in 7 of 11 patients. Survivin is expressed at high levels in most human cancer cells of epithelial or hematopoietic origin, and its overexpression is associated with development of cancer, poor prognosis, resistance to treatment and low survival of patients. In article M.J. Duffy, N. O Donovan and others, "Survivin: a promising tumor biomarker". Cancer Lett. 249(1): 49-60 (2007) described survivin, which is a protein mass of 16.5 kDa, sverkhekspressiya most of all malignant tumors, but rarely detected Zdorovya differentiated adult tissues. It was found that functionally survivin is an inhibitor of apoptosis, stimulates cell proliferation and enhances angiogenesis. Considering its importance in these processes, survivin attributed a key role in the development of cancer. Due to the significant difference in its expression in healthy and malignant tissues and its important role in cancer development, survivin currently intensively studied as a potential tumor marker. Taking into account the accumulated data suggest that determination of the level of survivin can be used for early diagnosis of bladder cancer, to predict the development of various types of cancer and to predict the immune response when selecting anticancer therapy. In article Zeis M., S. Siegel and others, "Generation of cytotoxic responses in mice and human individuals against hematological malignancies using survivin-RNA-transfected dendritic cells", J. Immunol. 1 70(11): 5391-5397(2003) found that survivin-specific cytotoxic T-lymphocytes (CTLs) human generated from mononuclear cells of peripheral blood (MCPC) as a result stimulate your own dendritic cells, transfectional survivin-RNA, exhibit cytotoxicity against malignant hematopoietic cell lines and primary tumor cells isolated from tissues of patients with acute myeloid leukemia. It was also found that vaccines what I mice, survivin-RNA transfected dendritic cells leads to prolonged resistance to survivin-expressing lymphoma, that suggests the possibility of using survivin as antigen rejection of the tumor. It has also been proved that survivin can be used as structure-target for the development of immunotherapy strategies for the treatment of hematological neoplasms. According to a preferred variant implementation of the present invention at least one RNA in the composition of the active (immunostimulatory) composition may thus encode survivin-antigen selected from the sequences as shown in Fig (SEQ ID NO:18), and more preferably, pig (SEQ ID NO:19). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode survivin-antigen selected from a fragment, variant or epitope sequence of survivin, as shown in Fig (SEQ ID NO:18), and more preferably, pig (SEQ ID NO:19).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode MAGE-C1. In the context of the present invention, the term "MAGE-C1" denotes melanoma antigen family C,1 and the preferred sequence RNA, preferably mRNA, encoding "MAGE-C1", provided that it is used in the composition of the active (immunostimulatory) composition according infusion is he to the invention, shown in Fig (SEQ ID NO:22), more preferably, pig (SEQ ID NO:23), and most preferably, as shown in Fig (SEQ ID NO:24). Recently in the article, Lucas, S., S. De Smet and others, "Identification of a new MAGE gene with tumor-specific expression by representational difference analysis", Cancer Res. 58(4): 743-752 (1998) described the identification of MAGE-C1, according to the representational differential analysis (RDA). MAGE-C1 is not expressed when testing on a panel of healthy tissues, except testis. Among tumor tissues, MAGE-C1 often expressed in seminoma, melanomas and carcinomas of the bladder. It is also expressed in many cases of carcinoma of the head and neck, with carcinomas of the breast, non-small cell lung carcinomas, adenocarcinomas of the prostate and sarcoma. In the article, Jungbluth, A. A., Y. T. Chen et al, "ST7 (MAGE-C1) antigen expression in normal and neoplastic tissues", Int. J. Cancer 99(6): 839-845 (2002) described expression in breast cancer, ovarian cancer, liver cancer, testicular cancer, bladder cancer, melanoma and non-small cell lung cancer (39%). In article Gure S.A., R. Chua and others, "Cancer-testis genes are coordinately expressed and are markers of poor outcome in non-small cell lung cancer", Clin. Cancer Res. 11 (22): 8055-8062 (2005). Gure, Chua and others (2005) described the analysis of tumors in 523 patients with non-small cell lung cancer, the level of expression of antigens of tumors of the testis (CT-antigens). MAGE-C1 detected in 18.8% of cases. In article Scanlan M.J., N.K. Altorki and others, "Expression of cancer-testis antigens in lung cancer: definition of bromoomain testis-specific gene (BRDT) as a new CT gene, CT9", Cancer Lett. 150(2): 155-164 (2000) also described the expression of CT antigens in 33 cases of non-small cell lung cancer, including MAGE-C1 in 30% of cases. According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode MAGE-C1 antigen selected from the sequences as shown in Fig (SEQ ID NO:22), and more preferably, pig (SEQ ID NO:23), and most preferably, at Fig (SEQ ID NO:24). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode the antigen MAGE-C1, selected from a fragment, variant or epitope sequences of MAGE-C1, as shown in Fig (SEQ ID NO:22), and more preferably, pig (SEQ ID NO:23), and most preferably, at Fig (SEQ ID NO:24).

At least one RNA in the composition of the active (immunostimulatory) composition may also encode MAGE-C2. In the context of the present invention, the term "MAGE-C2" denotes melanoma antigen family C2, and the preferred sequence RNA, preferably mRNA, encoding "MAGE-C2, provided that it is used in the composition of the active (immunostimulatory) composition according to the present invention, shown in Fig (SEQ ID NO:25), and most preferably, at Fig (SEQ ID NO:26). Recently in a hundred is rd Lucas S., E. De Plaen, and others, "the MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: four new members of the MAGE family with tumor-specific expression", Int. J. Cancer 87(1): 55-60 (2000) described the identification of MAGE-C2 according to the representational differential analysis (RDA) on cell lines of melanoma (see). MAGE-C2 is not expressed when testing on a panel set healthy tissues, except testis. Among tumor tissues, MAGE-C2, often expressed in seminoma, melanomas and carcinomas of the bladder. It is also expressed in most cases of carcinoma of the head and neck, with carcinomas of the breast, non-small cell lung carcinomas and sarcomas. In article Scanlan M.J., N.K. Altorki and others, "Expression of cancer-testis antigens in lung cancer: definition of bromodomain testis-specific gene (BRDT) as a new CT gene, CT9", Cancer Lett. 150(2): 155-164 (2000) described the expression of CT antigens in 33 cases of non-small cell lung cancer, including MAGE-C2, in 30% of cases. According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may thus encode the antigen MAGE-C2, selected from the sequences as shown in Fig (SEQ ID NO:25), and more preferably, pig (SEQ ID NO:26). According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition may alternatively or additionally encode the antigen MAGE-C2, selected from a fragment, variant or epitope consistently the ti MAGE-C2, as shown in Fig (SEQ ID NO:25), and more preferably, pig (SEQ ID NO:26).

Antigens, antigenic proteins or antigenic peptides as defined above, which can be encoded at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention may contain fragments or variants of these sequences. Such fragments or variants, as a rule, can include a sequence that is characterized by sequence homology compared with one of the above-mentioned antigens, antigenic proteins or antigenic peptides or compared with nucleotide sequences encoding them, nucleic acids, and the homology is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, also more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or even 97%, compared to the full nucleotide and amino acid sequences of the wild type.

Fragments of antigens, antigenic proteins or antigenic peptides in the context of the present invention can include the sequence of the antigen, antigenic protein or antigenic peptide as defined above, which is a shortened p is compared with the amino acid sequence (or coding it the nucleotide sequence of the nucleic acid), N-terminal, C-terminal and/or Central region of the sequence, compared with the amino acid sequence of the original (natural) protein (or nucleotide sequence coding him nucleic acid). These shortened fragments, therefore, applies to both amino acid and, accordingly, the nucleotide sequences. Therefore, homology of sequences in relation to the fragment, as defined above, may preferably be treated to a full-sized antigen, antigenic protein or antigenic peptide as defined above, or a full-sized nucleic acid sequence that encodes such an antigen, antigenic protein or antigenic peptide.

Fragments of antigens, antigenic proteins or antigenic peptides in the context of the present invention may also include the sequence of the antigen, antigenic protein or antigenic peptide as defined above, with a length of about 6 to 20 or even more amino acid residues, for example, the length of the fragments, which are formed in the processing and presentation on MHC molecules of class I, preferably, ranges from 8 to 10 amino acid residues, for example 8, 9 or 10 (or 6, 7, 11, or 12 amino acid residues), or the length of the fragments, which are formed in the process is nga and presentation on MHC molecules of class II, preferably, is approximately 13 or more amino acid residues, for example 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acid residues, and the data fragments can be selected from any part of amino acid sequence. These fragments usually are recognized by T-cells in the form of a complex consisting of the peptide fragment and MHC molecules, i.e. the fragments in native form, as a rule, are not recognized.

Fragments of antigens, antigenic proteins or antigenic peptides in the context of the present invention may also include epitopes of these antigens, antigenic proteins or antigenic peptides. Epitopes (also known as "antigenic determinants"), in the context of the present invention are, as a rule, the fragments located on the outer surface (native) antigens, antigenic proteins or antigenic peptides, as defined in this context, preferably comprising from 5 to 15 amino acid residues, more preferably comprising from 5 to 12 amino acid residues, most preferably, from 6 to 9 amino acid residues, which can be recognized by antibodies or receptors of b-cells in their native form. Such epitopes of antigens, antigenic proteins or antigenic peptides can also be selected from any mentioned this to the option of such antigens, antigenic proteins or antigenic peptides. In this context antigenic determinants can indicate conformational or discontinuous epitopes, which consist of segments of antigens, antigenic proteins or antigenic peptides, as defined in this context, which are discontinuous amino acid sequence of antigens, antigenic proteins and antigenic peptides, but together form a three-dimensional structure or a continuous or linear epitopes that consist of a single polypeptide chain.

"Variants" of antigens, antigenic proteins or antigenic peptides as defined above, can be encoded at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention, when the nucleic acid is at least one (m)RNA, encoding the antigen, antigenic protein or antigenic peptide as defined above, can be changed. Thus, there can be formed an antigen, antigenic protein or antigenic peptide, which differ from the original sequence by the content of one or more mutations, such as one or more substitutions, insertions and/or deletions of amino acids (amino acids). Preferably, these fragments and/or variants must have the same biological function or specific activity compared to the full the size of the native antigen or antigenic protein, for example, specific antigenic properties.

At least one RNA in the composition of the active (immunostimulatory) composition according to the present invention may also encode an antigen or antigenic protein, as defined above, with the encoded amino acid sequence comprises a substitution (replacement) conservative amino acid residues, compared with the physiological sequence. These encoded amino acid sequence as encoding their nucleotide sequence, first of all, included in the term "variants", as defined above. Conservative substitutions are substitutions of amino acid residue on the amino acid residue of the same type. First of all, these amino acids include aliphatic side chains, positively or negatively charged side chains, aromatic groups in side chains or residues of the amino acid side chains which can form hydrogen bonds, such as side groups containing a hydroxyl functional group. Such conservative substitution refers to, for example, that the amino acid containing a polar side chain is replaced by another amino acid with a polar side group, or, for example, an amino acid containing a hydrophobic side group, replaced by another amine is a acid, also containing a hydrophobic side group (for example, serine (threonine) to threonine (serine) or leucine (isoleucine) to isoleucine (leucine)). Insertion and replacement can be done first of all in such positions in the sequence that do not result in changing the three-dimensional structure or no effect on the binding site. Changes in the three-dimensional structure due to insertion(s) or deletion(s) can be identified by a simple method, for example, the spectra of circular dichroism (CD) (spectra circular dichroism) (Urry, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Ed. Neuberger, Elsevier, Amsterdam (1985)).

Moreover, variants of antigens, antigenic proteins or antigenic peptides as defined above, which can be encoded at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention, can also contain the specified sequence, where the nucleic acid is at least one (m)RNA, replaced, due to the degeneracy of the genetic code, and as a result of such replacement does not change the corresponding amino acid sequence of the antigen, antigenic protein or antigenic peptide, i.e. amino acid sequence or at least part thereof may not differ from the original sequence by one or more mutations, you mentioned the e value.

To determine the degree of homology in % two sequences (nucleic acid sequences, for example sequences of RNA or mRNA, as described in this context, or amino acid sequences, preferably coded for amino acid sequences, such as amino acid sequences of the antigens, antigenic proteins or antigenic peptides as defined above), these sequences can overlay each other and to compare one sequence with another. Thus, for example, you can include spaces in the first sequence and then compare the component in the corresponding position of the component in the second sequence. If in a certain position in the first sequence is the same amino acid residue, which is in the same position in the second sequence, then there is a homology in the two sequences in this position. The degree of homology (identity)% two sequences is a function of the ratio of the number of identical positions by the total number of provisions. The degree of homology in % of two sequences can be calculated using a mathematical algorithm. Preferred, but not limited to them, an example of a mathematical algorithm that you can use for this is, described in articles Karlin and others, PNAS USA, 90:5873-5877 (1993) or Altschul and others, Nucleic Acids Res., 25:3389-3402 (1997). This algorithm is compatible with the program BLAST. Sequences identical to the sequences of the present invention, to a certain extent can be identified with the help of this program.

Active (immunostimulirutuyu) the composition according to the present invention as defined above comprises at least one RNA encoding at least two (preferably different) antigens selected from among any of the antigens of the above groups, because, according to the present invention, a specific combination of at least two (preferably different) antigens in the composition of the above groups are able to effectively stimulate (acquired) immune system, providing treatment of lung cancer, especially non-small cell lung cancer (nmcrl). However, in the present invention are also offered such an active (immunostimulatory) composition containing at least one RNA encoding three, four, five, six, seven, eight, nine, ten, eleven or even twelve (preferably different) antigens selected from the above group, and provided by any combination of these antigens.

According to a preferred variant implementation of izopet is of at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention encodes at least two antigen (preferably different), selected from any of the antigens of the following subgroups:

hTERT,

WT1,

T,

NY-ESO-1,

survivin and/or

MAGE-C2.

More preferably, in the present invention it is also proposed active (immunostimulirutuyu) a composition comprising at least one RNA encoding at least three, four, five or six antigens (preferably different)selected from the group or subgroup described above, it is possible that any combination of these antigens.

In another preferred embodiment, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention encodes at least two (preferably different) antigens selected from any of the antigens of the above group (s) or subgroup (subgroup)that includes at least any one of the following combinations of antigens:

hTERT and WT1 or

hTERT and C, or

hTERT and NY-ESO-1, or

hTERT, survivin, or

hTERT and MAGE-C2, or

WT1 and T,or

WT1 and NY-ESO-1, or

WT1, survivin, or

WT1 and MAGE-C2, or

T and NY-ESO-1, or

T, survivin, or

T and MAGE-C2, or

NY-ESO-1 and survivin, or

NY-ESO-1 and MAGE-C2, or

survivin and MAGE-C2,

or

hTERT, WT1 and T, or

hTERT, WT1 and NY-ESO-1, or

hTERT, WT1, survivin, or

hTERT, WT1 and MAGE-C2, or

hTERT, T and NY-ESO-1, or

hTERT, C, survivin, or

hTERT, C and MAGE-C2, or

hTERT, NY-ESO-1 and survivin or

hTERT, NY-ESO-1 and MAGE-C2, or

hTERT, survivin and MAGE-C2, or

WT1, T and NY-ESO-1, or

WT1, T, survivin, or

WT1, T and MAGE-C2, or

WT1, NY-ESO-1 and survivin, or

WT1, NY-ESO-1 and MAGE-C2, or

WT1, survivin and MAGE-C2, or

T, NY-ESO-1 and survivin, or

T, NY-ESO-1 and MAGE-C2, or

T, survivin and MAGE-C2, or

NY-ESO-1, survivin and MAGE-C2,

or

hTERT, WT1, T and NY-ESO-1, or

hTERT, WT1, T, survivin, or

hTERT, WT1, T and MAGE-C2, or

hTERT, C, NY-ESO-1 and survivin, or

hTERT, C, NY-ESO-1 and MAGE-C2, or

hTERT, NY-ESO-1, survivin and MAGE-C2, or

WT1, T, NY-ESO-1 and survivin, or

WT1, T, NY-ESO-1 and MAGE-C2, or

WT1, T, survivin and MAGE-C2, or

T, NY-ESO-1, survivin and MAGE-C2,

or

hTERT, WT1, T, NY-ESO-1 and survivin, or

hTERT, WT1, T, NY-ESO-1 and MAGE-C2, or

WT1, T, NY-ESO-1, survivin and MAGE-C2,

or

hTERT, WT1, T, NY-ESO-1, survivin and MAGE-C2.

More preferably, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention encodes at least two (preferably different) antigens, selected exclusively from any of the antigens of the above group (s) or subgroup (subgroup)that includes at least any one of the following combinations of antigens:

hTERT and WT1 or

hTERT and C, or

hTERT and NY-ESO-1, or

hTERT, survivin, or

hTERT and MAGE-C2, or

WT1 and T, or

WT1 and NY-ESO-1, or

WT1, survivin, or

WT1 and MAGE-C2, or

T and NY-ESO-1, or

T, survivin, is whether

T and MAGE-C2, or

NY-ESO-1 and survivin, or

NY-ESO-1 and MAGE-C2, or

survivin and MAGE-C2,

or

hTERT, WT1 and T, or

hTERT, WT1 and NY-ESO-1, or

hTERT, WT1, survivin, or

hTERT, WT1 and MAGE-C2, or

hTERT, T and NY-ESO-1, or

hTERT, C, survivin, or

hTERT, C and MAGE-C2, or

hTERT, NY-ESO-1 and survivin, or

hTERT, NY-ESO-1 and MAGE-C2, or

hTERT, survivin and MAGE-C2, or

WT1, T and NY-ESO-1, or

WT1, T, survivin, or

WT1, T and MAGE-C2, or

WT1, NY-ESO-1 and survivin, or

WT1, NY-ESO-1 and MAGE-C2, or

WT1, survivin and MAGE-C2, or

T, NY-ESO-1 and survivin, or

T, NY-ESO-1 and MAGE-C2, or

T, survivin and MAGE-C2, or

NY-ESO-1, survivin and MAGE-C2,

or

hTERT, WT1, T and NY-ESO-1, or

hTERT, WT1, T, survivin, or

hTERT, WT1, T and MAGE-C2, or

hTERT, C, NY-ESO-1 and survivin, or

hTERT, C, NY-ESO-1 and MAGE-C2, or

hTERT, NY-ESO-1, survivin and MAGE-C2, or

WT1, T, NY-ESO-1 and survivin, or

WT1, T, NY-ESO-1 and MAGE-C2, or

WT1, T, survivin and MAGE-C2, or

T, NY-ESO-1, survivin and MAGE-C2,

or

hTERT, WT1, T, NY-ESO-1 and survivin, or

hTERT, WT1, T, NY-ESO-1 and MAGE-C2, or

WT1, T, NY-ESO-1, survivin and MAGE-C2,

or

hTERT, WT1, T, NY-ESO-1, survivin and MAGE-C2.

In another preferred embodiment, the present invention proposes an active (immunostimulirutuyu) a composition comprising at least one RNA encoding at least two (preferably different) antigens:

a) where from a group comprising at least one p is edocfile at least two, three, four, five or even six of these antigens at least two antigens selected from:

T,

NY-ESO-1,

MAGE-A2,

MAGE-A3,

MAGE-C1 and/or

MAGE-C2, and

b) while the other antigen (antigens) selected from at least one antigen that is described in the context of, preferably, from any described in this context, combination of groups or subgroups of antigens, for example, different antigen (antigens), selected from, for example,

hTERT,

WT1,

MAGE-A2,

T,

MAGE-A3,

MUC1,

Her-2/neu,

NY-ESO-1,

CEA,

survivin,

MAGE-C1 and/or

MAGE-C2.

According to another preferred variant, at least one antigen (antigens) in paragraph (a) is selected (selected):

NY-ESO-1,

MAGE-C1 and/or

MAGE-C2.

According to another preferred variant, at least one antigen (antigens) in paragraph (a) is selected (selected):

MAGE-C1 and/or

MAGE-C2.

According to a preferred variant of the invention, the at least one antigen (antigens) in paragraph (b) selected from the antigen (antigens)that are specified in one of the following combinations:

hTERT and WT1 or

hTERT and MAGE-A2, or

hTERT and C, or

hTERT and MAGE-A3, or

hTERT and MUC1, or

hTERT and Her-2/neu, or

hTERT and NY-ESO-1, or

hTERT and CEA, or

hTERT, survivin, or

hTERT, MOE-C1, or

hTERT and MAGE-C2, or

WT1 and MAGE-A2, or

WT1 and T, or

WT1 and MAGE-A3, or

WT1 and MUC1, and

WT1 and Her-2/neu, or

WT1 HNY-ESO-1, or

WT1 and CEA, or

WT1, survivin, or

WT1 and MAGE-C1, or

WT1 and MAGE-C2, or

MAGE-A2 and T, or

MAGE-A2 and MAGE-A3, or

MAGE-A2 and MUC1, or

MAGE-A2 and Her-2/neu, or

MAGE-A2 and NY-ESO-1, or

MAGE-A2 and CEA, or

MAGE-A2, survivin, or

MAGE-A2 and MAGE-C1, or

MAGE-A2 and MAGE-C2, or

T and MAGE-A3, or

T and MUC1, or

T and Her-2/neu, or

T and NY-ESO-1, or

T and CEA, or

T, survivin, or

T and MAGE-C1, or

T and MAGE-C2, or

MAGE-A3, MUC1, or

MAGE-A3 and Her-2/neu, or

MAGE-A3 and NY-ESO-1, or

MAGE-A3 and CEA, or

MAGE-A3, survivin, or

MAGE-A3 and MAGE-C1

MAGE-A3 and MAGE-C2

MUC1 and Her-2/neu, or

MUC1 and NY-ESO-1, or

MUC1 and CEA, or

MUC1, survivin, or

MUC1 and MAGE-C1, or

MUC1 and MAGE-C2, or

HER-2/NEU and NY-ESO-1, or

HER-2/NEU and CEA, or

HER-2/NEU, survivin, or

HER-2/NEU and MAGE-C1, or

HER-2/NEU and MAGE-C2, or

NY-ESO-1, CEA, or

NY-ESO-1 and survivin, or

NY-ESO-1 and MAGE-C1, or

NY-ESO-1 and MAGE-C2, or

CEA, survivin, or

CEA and MAGE-C1, or

CEA and MAGE-C2, or

survivin and MAGE-C1, or

survivin and MAGE-C2, or

MAGE-C1 and MAGE-C2, or

hTERT, WT1 and MAGE-A2, or

hTERT, WT1 and T, or

hTERT, WT1 and MAGE-A3, or

hTERT, WT1 and MUC1, or

hTERT, WT1 and Her-2/neu, or

hTERT, WT1 and NY-ESO-1, or

hTERT, WT1 and CEA, or

hTERT, WT1, survivin, or

hTERT, WT1 and MAGE-C1, or

hTERT, WT1 and MAGE-C2, or

WT1, MAGE-A2 and T, or

WT1, MAGE-A2 and MAGE-A3, or

WT1, MAGE-A2 and MUC1, or

WT1, MAGE-A2, and Her-2/neu, or

WT1, MAGE-A2 and NY-ESO-1, or

WT1, MAGE-A2 the CEA, or

WT1, MAGE-A2, survivin, or

WT1, MAGE-A2 and MAGE-C1, or

WT1, MAGE-A2 and MAGE-C2, or

MAGE-A2, T and MAGE-A3, or

MAGE-A2, T and MUC1, or

MAGE-A2, T and Her-2/neu, or

MAGE-A2, T and NY-ESO-1, or

MAGE-A2, T and CEA, or

MAGE-A2, T, survivin, or

MAGE-A2, T and MAGE-C1, or

MAGE-A2, T and MAGE-C2, or

T, MAGE-A3, MUC1, or

T, MAGE-A3, and Her-2/neu, or

T, MAGE-A3 and NY-ESO-1, or

T, MAGE-A3 and CEA, or

T, MAGE-A3, survivin, or

T, MAGE-A3 and MAGE-C1, or

T, MAGE-A3 and MAGE-C2, or

MAGE-A3, MUC1 and Her-2/neu, or

MAGE-A3, MUC1 and NY-ESO-1, or

MAGE-A3, MUC1 and CEA, or

MAGE-A3, MUC1, survivin, or

MAGE-A3, MUC1 and MAGE-C1, or

MAGE-A3, MUC1 and MAGE-C2, or

MUC1, Her-2/NEU and NY-ESO-1, or

MUC1, Her-2/NEU and CEA, or

MUC1, Her-2/NEU, survivin, or

MUC1, Her-2/NEU and MAGE-C1, or

MUC1, Her-2/NEU and MAGE-C2, or

HER-2/NEU, NY-ESO-1, CEA, or

HER-2/NEU, NY-ESO-1 and survivin, or

HER-2/NEU, NY-ESO-1 and MAGE-C1, or

HER-2/NEU, NY-ESO-1 and MAGE-C2, or

NY-ESO-1, CEA, survivin, or

NY-ESO-1, CEA and MAGE-C1, or

NY-ESO-1, CEA and MAGE-C2, or

CEA, survivin and MAGE-C1, or

CEA, survivin and MAGE-C2, or

survivin, MAGE-C1 and MAGE-C2, or

hTERT, WT1, MAGE-A2 and T, or

hTERT, WT1, MAGE-A2 and MAGE-A3, or

hTERT, WT1, MAGE-A2 and MUC1, or

hTERT, WT1, MAGE-A2, and Her-2/neu, or

hTERT, WT1, MAGE-A2 and NY-ESO-1, or

hTERT, WT1, MAGE-A2 and CEA, or

hTERT, WT1, MAGE-A2, survivin, or

hTERT, WT1, MAGE-A2 and MAGE-C1, or

hTERT, WT1, MAGE-A2 and MAGE-C2, or

WT1, MAGE-A2, T and MAGE-A3, or

WT1, MAGE-A2, T and MUC1, or

WT1, MAGE-A2, T and Her-2/neu, or

WT1, MAGE-A2, 5T4 and NY-ESO-1, or

WT1, MAGE-A2, 5T4 and CEA, or

WT1, MAGE-A2, 5T4, survivin, or

WT1, MAGE-A2, 5T4 and MAGE-C1, or

WT1, MAGE-A2, 5T4 and MAGE-C2, or

MAGE-A2, 5T4, MAGE-A3, MUC1, or

MAGE-A2, 5T4, MAGE-A3, and Her-2/neu, or

MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1, or

MAGE-A2, 5T4, MAGE-A3 and CEA, or

MAGE-A2, 5T4, MAGE-A3, survivin, or

MAGE-A2, 5T4, MAGE-A3 and MAGE-C1, or

MAGE-A2, 5T4, MAGE-A3 and MAGE-C2, or

5T4, MAGE-A3, MUC1 and Her-2/neu, or

5T4, MAGE-A3, MUC1 and NY-ESO-1, or

5T4, MAGE-A3, MUC1 and CEA, or

5T4, MAGE-A3, MUC1, survivin, or

5T4, MAGE-A3, MUC1 and MAGE-C1, or

5T4, MAGE-A3, MUC1 and MAGE-C2, or

MAGE-A3, MUC1, Her-2/NEU and NY-ESO-1, or

MAGE-A3, MUC1, Her-2/NEU and CEA, or

MAGE-A3, MUC1, Her-2/NEU, survivin, or

MAGE-A3, MUC1, Her-2/NEU and MAGE-C1, or

MAGE-A3, MUC1, Her-2/NEU and MAGE-C2, or

MUC1, Her-2/neu, NY-ESO-1, CEA, or

MUC1, Her-2/neu, NY-ESO-1 and survivin, or

MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1, or

MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2, or

HER-2/NEU, NY-ESO-1, CEA, survivin, or

HER-2/NEU, NY-ESO-1, CEA and MAGE-C1, or

HER-2/NEU, NY-ESO-1, CEA and MAGE-C2, or

NY-ESO-1, CEA, survivin and MAGE-C1, or

NY-ESO-1, CEA, survivin and MAGE-C2, or

CEA, survivin, MAGE-C1 and MAGE-C2,

or

hTERT, WT1, MAGE-A2, 5T4 and MAGE-A3, or

hTERT, WT1, MAGE-A2, 5T4 and MUC1, or

hTERT, WT1, MAGE-A2, 5T4 and Her-2/neu, or

hTERT, WT1, MAGE-A2, 5T4 and NY-ESO-1, or

hTERT, WT1, MAGE-A2, 5T4 and CEA, or

hTERT, WT1, MAGE-A2, 5T4, survivin, or

hTERT, WT1, MAGE-A2, 5T4 and MAGE-C1, or

hTERT, WT1, MAGE-A2, 5T4 and MAGE-C2, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, or

WT1, MAGE-A2, 5T4, MAGE-A3, and Her-2/neu, or

WT1, MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1, or

WT1, MAGE-A2, 5T4, MAGE-A3 and CEA, or

WT1, MAGE-A2, 5T4, MAGE-A3, survivin, or

WT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C1, or

WT1,MAGE-A2, 5T4, MAGE-A3 and MAGE-C2, or

MAGE-A2, 5T4, MAGE-A3, MUC1 and Her-2/neu, or

MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1, or

MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA, or

MAGE-A2, 5T4, MAGE-A3, MUC1, survivin, or

MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1, or

MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2, or

5T4, MAGE-A3, MUC1, Her-2/NEU and NY-ESO-1, or

5T4, MAGE-A3, MUC1, Her-2/NEU and CEA, or

5T4, MAGE-A3, MUC1, Her-2/NEU, survivin, or

5T4, MAGE-A3, MUC1, Her-2/NEU and MAGE-C1, or

5T4, MAGE-A3, MUC1, Her-2/NEU and MAGE-C2, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and survivin, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2, or

MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, or

MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1, or

MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2, or

HER-2/NEU, NY-ESO-1, CEA, survivin and MAGE-C1, or

HER-2/NEU, NY-ESO-1, CEA, survivin and MAGE-C2, or

NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2,

or

hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, or

hTERT, WT1, MAGE-A2, T, MAGE-A3, and Her-2/neu, or

hTERT, WT1, MAGE-A2, T, MAGE-A3 and NY-ESO-1, or

hTERT, WT1, MAGE-A2, T, MAGE-A3 and CEA, or

hTERT, WT1, MAGE-A2, T, MAGE-A3, survivin, or

hTERT, WT1, MAGE-A2, T, MAGE-A3 and MAGE-C1, or

hTERT, WT1, MAGE-A2, T, MAGE-A3 and MAGE-C2, or

WT1, MAGE-A2, T, MAGE-A3, MUC1 and Her-2/neu, or

WT1, MAGE-A2, T, MAGE-A3, MUC1 and NY-ESO-1, or

WT1, MAGE-A2, T, MAGE-A3, MUC1 and CEA, or

WT1, MAGE-A2, T, MAGE-A3, MUC1, survivin, or

WT1, MAGE-A2, T, MAGE-A3, MUC1 and MAGE-C1, or

WT1, MAGE-A2, T, MAGE-A3, MUC1 and MAGE-C2, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/NEU and NY-ESO-1, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/NEU and CEA, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/NEU, survivin, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/NEU and MGE-C1, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/NEU and MAGE-C2, or

T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA, or

T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and survivin, or

T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1, or

T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2, or

MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C1, or

MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C2, or

HER-2/NEU, NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2,

or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and Her-2/neu, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, survivin, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and NY-ESO-1, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and CEA, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU, survivin, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and MAGE-C1, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and MAGE-C2, or

MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, or

MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and survivin, or

MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1, or

MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2, or

5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, or

5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1, or

5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C1, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C2, or

MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2,

or

<> hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and NY-ESO-1, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and CEA, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1. Her-2/NEU, survivin, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and MAGE-C1, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/NEU and MAGE-C2, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and survivin, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2, or

MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, or

MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1, or

MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2, or

5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C1, or

5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C2, or

MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2,

or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and survivin, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1, or

hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin,or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1, or

WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C1, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C2, or

T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2,

or

hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, or

hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MGE-C1, or

hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2, or

WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C1, or

WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C2, or

MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2,

or

hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C1, or

hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin and MAGE-C2, or

WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2,

or

hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 and MAGE-C2.

According to another preferred variant, at least one antigen (antigens) under item (b) selected from the following specific combinations of antigens:

survivin and T.

At least one RNA in the composition of the active (immunostimulatory) composition according to the present invention usually is any RNA, preferably, but not limited to, a coding RNA, a circular or linear RNA, single - or double-stranded RNA (which may also include RNA, which is formed by non-covalent Association of two single-stranded RNA) or a partially double-stranded or partially single-stranded RNA, which are at least partially semicomplete (both of these partially double-stranded or partially single-stranded RNA molecules are typically formed from the longer or the shorter one is chained RNA molecules or two single-stranded RNA molecules of the same length, one single-stranded RNA molecule is partially complementary to the other single-stranded RNA molecule and both thus form a double-stranded RNA in this region, i.e. partially double-stranded or partially single-stranded RNA in the RNA sequence). More preferably, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention is a single-stranded RNA, more preferably a linear RNA. Most preferably, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention is messenger RNA (mRNA). In this context, the term messenger RNA (mRNA) usually refers to RNA, which is (at least) of several structural elements, for example, optional 5'-UTR region, which is the binding site with the ribosome, and then encoding the plot, optional 3'-UTR region, which may be poly-A-tail fragment (and/or poly-C-terminal fragment).

In one preferred embodiment, each of at least two (preferably different) antigens of the active (immunostimulatory) composition according to the present invention can be coded one (monocistronic) RNA, preferably one (monocistronic) mRNA. In other words, active (IMM is nostaliga) the composition according to the present invention may contain at least two (monocistronic) RNA, preferably mRNA, with each of these at least two (monocistronic) RNA, preferably mRNA can encode only one (preferably different) antigens selected from one of the above groups or sub-groups, preferably one of the above combinations.

According to another preferred variant of the active (immunostimulirutuyu) the composition according to the present invention includes at least one bi - or even a polycistronic RNA, preferably mRNA, i.e. at least one RNA, which contains two or more sequences encoding at least two (preferably different) antigens selected from one of the above groups or sub-groups, preferably from one of the above combinations. These sequence encoding at least two (preferably different) antigens (at least) one bi - or even multisectional RNA interrupted by at least one IRES sequence (part of the internal landing ribosomes), as described below. Thus, the term "encodes at least two (preferably different) antigens" means, not limited to that (at least) one bi - or even a polycistronic) RNA, preferably mRNA, encodes, for example, at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve (preferably different) antigens of the above group (s) antigens or their fragments or variants according to the definitions described above. More preferably, but not limited to, at least one bi - or even a polycistronic) RNA, preferably mRNA, encodes, for example, at least two, three, four, five or six (preferably different) antigens of the above group (s) antigens or their fragments or variants according to the definitions described above. In this context, the sequence of the IRES (internal area planting ribosomes), described above, may be a function of a single binding site of the ribosome, but based on it you can also get bi - or even a polycistronic RNA, as described above, which encode distinct proteins, which are translated by ribosomes independently of each other. Examples of IRES sequences that can be used according to the present invention include sequences from picornaviruses (such as FMDV), pestiviruses (CFFV), polioviruses (PV), viruses encephalomyocarditis (ECMV), FMD virus (FMDV), hepatitis C (HCV), a virus of classical swine fever (CSFV), leukosis virus of mice (MLV), human immunodeficiency virus monkeys (SIV) or viruses paralysis cricket (CrPV).

According to another preferred variant of the active (immunostimulirutuyu) the composition according to the present invention includes a mixture of at least one monocistronic RNA, preferably mRNA, as described above, and at least one bi - or even a polycistronic RNA, preferably mRNA, as described above. At least one monocistronic RNA and/or at least one bi - or even a polycistronic RNA preferably encodes various antigens or their fragments or variants according to the definitions described above, the antigens are preferably selected from one of the above groups or subgroups of antigens, more preferably from one of the above combinations. However, at least one monocistronic RNA and at least one bi - or even a polycistronic RNA preferably encodes also (partly) the same antigens selected from one of the above groups or subgroups of antigens, preferably from one of the above combinations, provided that the active (immunostimulirutuyu) the composition according to the present invention generally provides encoding at least two (preferably different) antigens described above. This option is suitable, for example, to cycle through, for example, dependent on the time of introduction of the active (immunostimulatory) composition according to the present invention to a patient in need of such treatment. Components specified active (immunostimulatory) composition according to the present invention, before the e just different RNA encoding at least two (preferably different) antigens, can for example be part of a (different components) of a set containing a combination of components, or they can, for example, be entered separately in the form of various active components (immunostimulirutuyu) compositions of the present invention.

Preferably at least one RNA in the composition of the active (immunostimulatory) composition, encoding at least two (preferably different) antigens selected from the above group or sub-group antigens, more preferably from one of the above combinations, usually contain from about 50 to about 20000 or from 100 to about 20000 nucleotide residues, preferably about 250 to about 20000 nucleotide residues, more preferably from about 500 to about 10000, more preferably from about 500 to about 5000.

According to one variant of the invention, the at least one RNA in the composition of the active (immunostimulatory) composition, encoding at least two (preferably different) antigens selected from the above group (s) or subgroup (subgroup) antigens, more preferably of the above combinations can be modified, and m is Nisha least one RNA in the composition of the active (immunostimulatory) composition can be modified in any way. Modifications as described in this context, preferably, allow to stabilize at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention.

According to the first variant of at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention may be present in the form of a "stable RNA", preferably in the form of stable mRNA, i.e. in the form of (m)RNA, which is highly resistant to degradation in vivo (e.g., degradation under the action of Exo - or endonucleases). This stabilization is achieved, for example, through modification of the phosphate residues in the main chain of NC in at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention. The term "modification of the main chain"used in this context, refers to the chemical modification of the phosphate residues in the main chain RNA. As the preferred modified nucleotides are used, for example, nucleotides, modified phosphorothioate phosphate residues, preferably the nucleotides in which at least one of the oxygen atoms in the phosphate residue is replaced by a sulfur atom. Stable (m)RNA may also include, for example, nonionic analogues FOS is atnah residues, such as, for example, alkyl - and arylphosphonate, while the charged oxygen atom in the composition of the phosphate residue is replaced by an alkyl or aryl group, or may include fosfomifira and alkylphosphocholine, while the charged oxygen atom in the composition of the phosphate residue is present in alkilirovanny form. These modifications of the basic circuit typically include, but are not limited to, modification through the introduction of methylphosphonates, phosphoramidon and phosphorothioates (for example, citizen-5'-O-(1-thiophosphate)).

At least one RNA in the composition of the active (immunostimulatory) composition according to the present invention may additionally or alternatively also contain modified sugar. The term "modified sugar residues"used in this context, refers to the chemical modification of the sugar residues in the composition of the nucleotides in at least one RNA and typically includes, but is not limited to, the modified sugar residues selected from the group comprising 2'-deoxy-2'-ferricopiapite (2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-diaminodiphenylamine (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate), 2'-O-alkyl-oligoribonucleotide, 2'-deoxy-2'-C-alkyl-oligoribonucleotide (2'-O-me is icitizen-5'-triphosphate, 2'-methyluridine-5'-triphosphate), 2'-C-alkyl-oligoribonucleotide and their isomers (2'-aracytidine-5'-triphosphate, 2'-aruliden-5'-triphosphate) or azeotropic (2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate).

At least one RNA in the composition of the active (immunostimulatory) composition according to the present invention may additionally or alternatively also contain at least one modified base, and this RNA is suitable preferably for increasing the expression of a protein encoded by at least one RNA sequence compared to the original, i.e. the natural RNA sequence. The term "significant"is used in this context, refers to the increase in the level of expression of the protein compared to expression of natural RNA sequences by at least 20%, preferably at least 30%, 40%, 50% or 60%, more preferably at least 70%, 80%, 90% or even 100%, and more preferably at least 150%, 200% or even 300% or more. According to the present invention indicated a modified nucleotide, preferably selected from the group of modified bases, including 2-amino-6-chlorphenesin-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thio who ridin-5'-triphosphate, 5-aminoalkylation-5'-triphosphate, 5-aminomethylpyridine-5'-triphosphate, 5-brazilein-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodization-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chlorphenesin-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-usagenotes-5'-triphosphate, 8-azidoaniline-5'-triphosphate, benzimidazolinone-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. The most preferred modified nucleotides selected from the group consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-brazilein-5'-triphosphate, pseudouridine-5'-triphosphate.

According to another variant, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention can be modified in a similar way (and preferably stabilized) by introducing additional nucleotides, modified by the remnants of the ribose or the grounds. Usually at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention includes any natural nucleotide, such as guano is in, uracil, adenosine and/or cytosine or their analogues. The term "nucleotide analogues used in this context refers to non-natural variants of natural nucleotides. Thus, analogues are chemically modified nucleotides containing unnatural functional groups are preferably introduced or removed from natural nucleotides or which replace the natural functional groups of the nucleotide. Accordingly, each component in the composition of the natural nucleotides can be modified, namely the base, the remainder of the sugar (ribose) and/or a phosphoric acid residue, forming the main chain (see above) RNA. Analogues of guanosine, uracil, adenosine and cytosine include, but are not limited to, any natural or non-natural variant of guanosine, uracil, adenosine, thymidine or cytosine, which are chemically modified through acetylation, methylation, hydroxylation, etc., including 1-methyladenosine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2,6-diaminopurine, T-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyguanosine, 2'-amino-2'deoxyuridine, 2-amino-6-chlorphenesin, 2-aminopurin-ribose, 2'-aralensis, 2'-aracytidine, 2'-aruliden, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chlorid is nothin, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-fertilizin, 2-methyladenosine, 2-methylguanosine, 2-methylthio-N6-isopentenyladenosine, 2'-O-methyl-2-aminoadenosine, 2'-O-methyl-2'-deoxyadenosine, 2'-O-methyl-2'-deoxycytidine, 2'-O-methyl-2'-deoxyguanosine, 2'-O-methyl-2'-deoxyuridine, 2'-O-methyl-5-methyluridine, 2'-O-methylinosine, 2'-O-methylpseudouridine, 2-thiocytidine, 2-tocitizen, 3-methylcytosine, 4-acetylcytosine, 4-thiouridine, 5-(carboxyhydroxymethyl)uracil, 5,6-dihydrouracil, 5-aminoalkylation, 5-aminoethylethanolamine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl, 5-chloro-aracytine, 5-ferritin,5-iodouridine,5-methoxycarbonylmethylene,5-methoxyuridine,5-methyl-2-thiouridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deazaguanosine, 6-chlorphenesin, 6-mercaptopurine, 6-methylmercaptopurine, 7 deaza-2'-deoxyguanosine, 7-deazaadenosine, 7-methylguanosine, 8-usagenotes, 8-bromoadenosine, 8-bromoguanosine, 8-mercaptoquinoline, 8-oxoguanosine, benzimidazolinone, β-D-mannosidosis, dihydrouracil, inosine, N1-methyladenosine, B6-([6-aminohexyl]carbamoylmethyl)adenosine, N6-isopentenyladenosine, N6-methyladenosine, N7-methylxanthine, methyl ester N-uracil-5-exucuse acid, puromycin, koozin, uracil-5-oxiana acid, methyl ester uracil-5-exucuse KIS is the notes, wybutosine, xanthosine and xylogenesis. Methods of obtaining such analogs are known to the specialist in the art and described, for example, in patents US 4373071, US 4401796, US 4415732, US 4458066, US 4500707, US 4668777, US 4973679, US 5047524, US 5132418, US 5153319, US 5262530 and US 5700642. In the case of analogues described above, according to the present invention the most preferred analogs are variants that increase the immunogenicity of RNA in the composition of the active (immunostimulatory) composition and/or do not affect further modification of RNA, intended for injection.

According to a preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention can be modified with the inclusion of lipid. The lipid-modified RNA is typically an RNA, as described in this context, which encodes at least two antigens selected from the group or subgroup antigens described above, preferably of the above combinations. The lipid-modified RNA is typically also includes at least one linker covalently attached to a specific RNA, and at least one lipid covalently attached to an appropriate linker. In another embodiment, the lipid-modified RNA comprises at least one RNA, as described in this context, and by at least one (bifunctional) lipid, covalently attached (without a linker) to a specified RNA. According to the third variant lipid-modified RNA includes RNA, as described in this context, at least one linker covalently attached to a specific RNA, and at least one lipid covalently attached to an appropriate linker, and at least one (bifunctional) lipid covalently linked (without a linker) with the indicated RNAS.

As lipid of at least one RNA in the composition of the active (immunostimulatory) composition according to the invention (in the form of a complex or covalently attached to the RNA) is usually used lipid or lipophilic residue, which is preferably itself exhibits biological activity. These lipids preferably include natural substances or compounds such as, for example, vitamins such as α-tocopherol (vitamin E), including RRR-α-tocopherol (formerly D-α-tocopherol), L-α-tocopherol, the racemate D,L-α-tocopherol, succinate vitamin E or vitamin a and its derivatives such as retinoic acid, retinol, vitamin D and its derivatives, such as precursors of vitamin D and ergosterol, vitamin E and its derivatives, vitamin K and its derivatives, such as vitamin K and derivatives of quinone or phytyl side-chain, or steroids, such as bile acids, such as cholic acid, desoxycholic Ki the lot, dehydrocholic acid, cortisone, digoxigenin, testosterone, cholesterol or taholesterol. Other lipids or lipophilic residues according to the present invention include, but are not limited to, alkylene glycols (see Oberhauser and others, Nucl. Acids Res., 20, 533 (1992)), aliphatic group, such as, for example, With1-C20alkanes,1-C20alkenes or1-C20the alkanols, etc. such as, for example, derivatives dodecanediol, hexadecanol or undecylenate residues (see article Saison-Behmoaras, etc., EMBO J, 10, 111 (1991), Kabanov and others, FEBS Lett., 259, 327 (1990), Svinarchuk, etc., Biochimie, 75, 49 (1993)), phospholipids, such as, for example, phosphatidylglycerol, dieselpartikelfilter, phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, dihexadecyl-rat-glycerine, sphingolipids, cerebrosides, gangliosides or 1,2-di-O-hexadecyl-rat-glycero-3-H-phosphonate of triethylamine (see article Manoharan and others, Tetrahedron Lett., 36, 3651 (1995), Shea and others, Nucl. Acids Res., 18, 3777 (1990)), polyamine or polyalkylene glycols, such as, for example, polyethylene glycol (PEG) (see Manoharan and others, Nucleosides &Nucleotides, 14, 969 (1995)), hexamethyleneimine (GEG), palmitin or palmitic residues (see Mishra and others, Biochim. Biophys. Acta, 1264, 229 (1995)), octadecylamine or paxilalertsbuypaxilonlinewi residues (see Crooke, etc., J. Pharmacol. Exp. Ther., 27, 923 (1996)), as well as waxes, terpenes, alicyclic hydrocarbons, residues of saturated and mono - or polyunsaturated fatty acids, etc.

At least one RNA in the composition of the active (immunostimulatory) composition according to the present invention can be stabilized in a similar manner to prevent degradation of RNA in vivo using various methods. It is known that the instability and (fast) degradation of mRNA or RNA in vivo usually pose a serious problem for the application of compositions based on RNA. The instability of RNA is usually caused by the action of enzymes that destroy RNA, RNase (ribonuclease), and the presence in the samples of these impurities ribonuclease can sometimes lead to complete degradation of the RNA present in the solution. Thus, degradation of mRNA in vivo in the cytoplasm of cells very precisely adjusted, and before applying the above compositions impurities RNCs usually can largely be removed by using a special processing methods, primarily using diethylpyrocarbonate (DEPC). There are many mechanisms of degradation of these compounds in vivo, which can also be used. For example, the structure of the end fragment of the mRNA is usually extremely important to its operation the Oia in vivo. For example, the 5'-terminal fragment of the natural mRNA typically includes a so-called "cap structure" (modified guanosine residues), and the 3'end fragment typically includes a sequence containing up to 200 adenosine residues (so-called poly-A-tail fragment).

At least one RNA in the composition of the active (immunostimulatory) composition according to the present invention, first of all, in case of application in the form of mRNA can be stabilized to prevent degradation under the action of RNase by introducing in its structure an additional so-called "5'-cap-patterns". According to the present invention, the most preferred "5'-cap structures are m7G(5')ppp (5'(A,G(5')ppp(5')A or G(5')ppp(5')G. However, this modification carried out only if the 5'-terminal fragment (m)RNA is incorporated into immunostimulating composition according to the invention, not previously modified, for example, a lipid, or a specified modification does not affect immunostimulatory properties (unmodified or chemically modified) (m)RNA.

According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention may contain, primarily, in the case of application in the form of mRNA, 3'end fragment of poly-And the fragment containing approx the tion from 10 to 200 adenosine residues, preferably from about 10 to 100 adenosine residues, more preferably from about 20 to 100 adenosine residues, or more preferably from about 40 to 80 residues of adenosine.

According to another preferred variant, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention may contain, primarily, in the case of application in the form of an mRNA in the 3'-terminal fragment of poly-fragment, containing from about 10 to 200 cytosine residues, preferably from about 10 to 100 cytosine residues, more preferably from about 20 to 70 cytosine residues, or more preferably from about 20 to 60 or even from 10 to 40 cytosine residues.

According to another variant, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention can be modified and thus to stabilize, especially in the case of application in the form of mRNA, by changing the content of G/C in the structure of RNA, preferably in the composition of the coding region of at least one RNA.

In the most preferred embodiment of the present invention, the content of G/C in the composition of the coding region of at least one (m)RNA in the composition of the active (immunostimulatory) composition according to N. the present invention is changed, first of all increased compared with the content of G/C in the coding region (m)RNA of wild-type, i.e. unmodified (m)RNA. Amino acid sequence encoded by at least one (m)RNA, preferably is not modified, i.e. similar amino acid sequences encoded by (m)RNA of the wild type.

The specified at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention modify considering the fact that the sequence of any area broadcast (m)RNA plays an important role for efficient broadcast specified (m)RNA. Thus, the composition and sequence of the different nucleotides play an important role. First of all sequences with a high content of G (guanosine)/C (cytosine) are more stable compared to sequences with a high content of A (adenosine)/U (uracil). Therefore, according to the present invention codons (m)RNA codons differ from (m)RNA of the wild type, while retaining translated amino acid sequence, and they are characterized by a high content of nucleotides G/C. taking into account the fact that different codons encode the same amino acid (so-called degeneracy of the genetic code), you can determine the most preferred codons in order to ensure the foster stability (the so-called alternative use of the codon).

Depending on the amino acid which encodes at least one (m)RNA, there are various methods for modifying at least one sequence (m)RNA, compared with the corresponding sequence of the wild type. If amino acids are encoded by codons that contain only G or C nucleotides, then no modification of the codon is not required. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) is not required to modify, because no nucleotide a or U.

On the contrary, the codons that contain the nucleotides a and/or U, can be modified when changing to other codons that encode the same amino acids, but do not contain nucleotides and/or U. for Example,

codons CCU or CCA (Pro) can be replaced by CCC or CCG

codons CGU or CGA or AGA or AGG (Arg) can be replaced by CGC or CGG,

codons GCU or GCA (Ala) can be replaced by a GCC or GCG,

codons GGU or GGA (Gly) can be replaced by GGC or GGG.

In other cases, although the nucleotides a or U cannot be excluded from the composition of codons can reduce their content, using codons with a lower content of nucleotides and/or U. for Example,

the codon UUU (Phe) can be replaced by UUC,

codons UUA, UUG, CUU or CUA (Leu) can be replaced by CUC or CUG,

codons UCU or UCA or AGU (Ser) can be replaced by UCC, UCG or AGC,

the codon UAU (Tyr) can be replaced by UAC,

codon UGU (Cys) can be replaced by UGC,

codon CAU (His) can be replaced by SAS,

the codon CAA (Gln) can be replaced by CAG,

codons AUU or AUA (Ile) can be replaced by AUC,

codons ACU or ACA (Thr) can be replaced by ACC or ACG,

codon AAU (Asn) can be replaced by AAS,

the codon AAA (Lys) can be replaced by AAG,

codons GUU or GUA (Val) can be replaced by GUC or GUG,

codon GAU (Asp) can be replaced by GAC,

the codon GAA (Glu) can be replaced by a GAG,

the stop codon UAA can be replaced with UAG or UGA.

On the other hand, in the case of codons for Met (AUG) and Trp (UGG) possible modification does not exist.

Replace above can be used either individually or in all possible combinations with the aim of increasing the content of G/C in at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention compared with concrete (m)RNA of wild-type (i.e. the original sequence). Thus, for example, all the codons for Thr present in the sequence of the wild type, can be changed to ACC (or ACG). However, it is preferable to use combinations of the above methods substitutions, for example

substitution of all codons coding for Thr in the original sequence ((m)RNA of wild-type) to ACC (or ACG) and

replacement of all the original codons coding for Ser, UCC (or UCG or AGC),

replace all the code is new, coding is Not in the original sequence at AUC, and

replacement of all the original codons coding for Lys, AAG, and

replacement of all the original codons coding for Tyr, UAC,

substitution of all codons coding for Val in the original sequence to GUC (or GUG) and

replacement of all the original codons coding for Glu, GAG, and

replacement of all the original codons coding for Ala, GCC (or GCG), and

replacement of all the original codons coding for Arg, CGC (or CGG),

substitution of all codons coding for Val in the original sequence to GUC (or GUG) and

replacement of all the original codons coding for Glu, GAG, and

replacement of all the original codons coding for Ala, GCC (or GCG), and

replacement of all the original codons coding for Gly, GGC (or GGG), and

replacement of all the original codons coding for Asn, AAS,

substitution of all codons coding for Val in the original sequence to GUC (or GUG) and

replacement of all the original codons coding for Phe, UUC,

replacement of all the original codons coding for Cys, UGC, and

replacement of all the original codons coding for Leu, CUG (or CUC), and

replacement of all the original codons coding for Gln, CAG, and

replacement of all the original codons that encode Pro, CCC (or CCG), etc.

Preferably the content of G/C in the coding region of at least one (m)RNA in the composition of the active (immunostimulatory) composition according infusion is he to the invention is increased by at least 7%, more preferably at least 15%, most preferably at least 20%, compared with the content of G/C in the coding region (m)RNA of the wild type, which encodes the antigen, a protein antigen or a peptide antigen that is described in this context, or a fragment or variant. According to a particular variant at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90%, 95% or even 100% of the substitutable codons in the region that encodes the antigen, a protein antigen or a peptide antigen that is described in this context, or a fragment or a variant, or in the full-size sequence (m)RNA of wild-type replace with the aim of increasing the content of G/C in the sequence.

As described in this context, most preferably the maximum possible increase in the content of G/C in at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention (i.e. 100% of the substitutable codons), primarily in the region that encodes a protein, compared with the sequence of the wild type.

According to the present invention further preferred modification of at least one (m)RNA in the composition of the active (immunostimulatory) composition based on jidanni data according to which the efficiency of the broadcast is also determined by the different frequency of formation of various tRNAs in cells. Thus, if the so-called "rare codons" are present in the composition at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention in a greater degree, then modified accordingly at least one sequence (m)RNA is translated to a much lesser extent compared to the coding sequence comprising codons that are recognized by "frequent" tRNA.

According to the present invention in a modified at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention the region, which encodes the adjuvant protein, modified in comparison with the corresponding region within the (m)RNA of the wild type in such a way that at least one codon of the sequence of the wild type, which corresponds to a relatively "rare" in the cell tRNA, replaced by a codon, which corresponds to "frequent" in the cell tRNA, which carries the same amino acid as the relatively "rare" tRNA. When using this modification of the sequence of at least one (m)RNA in the composition of the active (immunostimulatory) is oppozitsii of the present invention is modified in such a way what is included in the composition of codons that correspond to the "frequent" tRNA. In other words, according to the present invention, the above modification is used to replace all of the codons within the sequence of the wild type, which correspond to relatively "rare" in the cell tRNA, codon, which correspond to relatively "frequent" in the cell tRNA, which in each case carry the same amino acid as the relatively "rare" tRNA.

Specialist in the art known tRNA, relatively "frequent" and Vice versa relatively "rare" in the cell, for example, see Akashi, Curr. Opin. Genet. Dev., 11(6): 660-666 (2001). The most preferred codons are codons encoding a particular amino acid, which correspond to the "most frequent" tRNA, for example, the codon encoding Gly, which corresponds to the tRNA, the "most common" in the cell (of a person).

According to the present invention, first and foremost, the preferred option, in which at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention modify by the addition of consecutive G/C, the content of which is increased, first of all, as much as possible, to the "frequent" codons is m, and this does not change the amino acid sequence of the protein, which is encoded by the encoding fragment (m)RNA. In the specified preferred embodiment can be obtained most efficiently transmitted stable (modified) at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention.

The design of the modified at least one (m)RNA, which is part of the active (immunostimulatory) composition according to the present invention, as described above (high content of G/C, replacement tRNA), can be performed using software, as described in the application WO 02/098443, which is incorporated into this description in full. When using the specified software nucleotide sequence of any desired (m)RNA can be modified using the genetic code, or its degenerate type in such a way as to provide maximum content of G/C, in combination with the use of codons that are recognized by the tRNA, the most frequent in the cell, while the amino acid sequence encoded by the modified at least one (m)RNA, preferably not changed in comparison with the unmodified sequence. In another embodiment, the RNA can be modified only accounts for the increase in the content of G/C or only using codon compared to the unmodified sequence. The source code in the software of Visual Basic 6.0 (Microsoft Visual Studio Enterprise 6.0, Servicepack 3) described in the application WO 02/098443.

In another preferred embodiment of the present invention, the content of a/U in the environment of the binding site of the ribosome at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention is improved compared with the contents of A/U in the environment of the binding site of the ribosome corresponding (m)RNA of the wild type. This modification (increase in the content of A/U in the environment of the binding site of the ribosome) increases the efficiency of ribosome binding with at least one (m)RNA. Effective binding of ribosomes with the binding site of the ribosome (Kozak sequence: GCCGCCACCAUGG (SEQ ID No. 27), AUG forms the start codon) in turn increases the efficiency of translation of at least one (m)RNA.

According to another variant implementation of the present invention at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention can be modified to account for items of a sequence, potentially destabilizing RNA. First of all, the coding region and/or 5' and/or 3' untranslated area of the specified at least one (m)RNA can be modified compared to the corresponding (m)RNA of the wild type so that the Onan contained destabilizing sequence elements, and when this amino acid sequence encoded by at least one of the modified (m)RNA, preferably not changed compared to the sequence encoded by the corresponding (m)RNA of the wild type. It is known, for example, that in the RNA sequences of eukaryotes meet destabilizing sequence elements (DEP), joined by signaling proteins and regulate the enzymatic degradation of RNA in vivo. For further stabilization of at least one of the modified (m)RNA, not necessarily in the region that encodes the antigen, a protein antigen or a peptide antigen, as described in this context, compared with the corresponding fields in the structure (m)RNA of wild-type, perform one or more of these modifications in the RNA does not contain or contains almost no items, destabilizing the structure. According to the present invention when implementing these modifications in at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention, the structure of the untranslated regions (3'- and/or 5'-UTR region) can be excluded by DEP.

These destabilizing sequences are, for example, sequences with high AU content that are present in the 3'-UTR fragments of many unstable RNA (see the article Caput and others, Proc. Natl. Acad. Sci. USA, 83, 1670-1674 (1986)). Therefore at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention modify preferably so that in comparison with the (m)RNA of the wild type at least one (m)RNA did not contain these destabilizing sequences. The same applies also to the sequence motifs that can be recognized by endonucleases, for example, a sequence GAACAAG, which is contained in the 3'-UTR fragment of the gene that encodes transparency receptor (see Binder and others, EMBO J., 13, 1969-1980 (1994)). These sequence motifs are also preferably removed at least from the sequence one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention.

According to the present invention preferably at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention also includes in a modified form at least one IRES fragment, as described above, and/or at least one stable 5' and/or 3' sequence in a modified form, for example, to improve the efficiency of binding of the ribosome or to provide a different expression of the encoded antigens localized in at least one (b is - or even a polycistronic) RNA in the composition of the active (immunostimulatory) composition according to the present invention.

According to the present invention at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention, also preferably contains at least one 5' and/or 3' stabilizing sequence. These stabilizing sequences in the 5' and/or 3' untranslated regions allow to increase the half-life of at least one (m)RNA in the cytosol. The homology of these stabilizing sequences compared to natural sequences of RNA viruses, bacteria and eukaryotes may be 100%, but they may be wholly or partially synthetic sequences. As an example, stabilizing sequences, which can be used according to the present invention for stabilization of RNA, can be used untranslated sequence (UTR) globin gene, such as Homo sapiens or Xenopus laevis. Another example of a stabilizing sequence is the sequence of General formula (C/U)CCANxCCC(U/A)PyxUC(C/U)CC (SEQ ID No. 28), which is contained in the 3'UTR region within the RNA, which is characterized by a very high stability, and encodes a globin, collagen I, 15-lipoxygenase or tyrosinekinase (see the article. Holcik and others, Proc. Natl. Acad. Sci. USA, 94, 2410-2414 (1997)). These stabilizing sequence is lnasty can be used separately or in combination with each other, as well as in combination with other stabilizing sequences known to a person skilled in the art. In this regard, at least one (m)RNA in the composition of the active (immunostimulatory) composition according to the present invention is present in the form of globin UTR-(untranslated region)-stabilized RNA, primarily in the form of globin UTR-stabilized RNA.

However, substitutions, insertions or deletions of bases preferably carried out in at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention, at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention receive a DNA matrix using known in the art methods site-directed mutagenesis or ligation of oligonucleotides (see, for example, the book by Maniatis and other, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, N.Y. (2001)). In said method for obtaining at least one (m)RNA was performed transcription of appropriate DNA molecules in vitro. Specified DNA-matrix preferably includes a promoter such as the promoter of T7 or SP6 suitable for in vitro transcription, which is required nucleotide sequence of at least one RNA that must be obtained, and the term is inuu signal sequence, suitable for in vitro transcription. DNA, forming a matrix of at least one of the desired RNA, can be obtained in preparative scale methods of cell proliferation in the fermenter, followed by separation of the plasmid to replicate in bacteria. According to the present invention is suitable plasmids include, for example, plasmids RTT (GenBank identification number: U26404, see Lai and others, Development, 121, 2349-2360 (1995)), the plasmid pGEM series®for example, pGEM®-1 (GenBank identification number: X65300, firms Promega) and pSP64 (GenBank identification number: X65327), see also article Mezei and Storts, Purification of PCR Products, in the book of PCR Technology: Current Innovation, edited by Griffin and Griffin, CRC Press, Boca Raton, FL (2001).

Stabilization of at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention can be made by the Association or complexing at least one RNA with a cationic compound, primarily poly-compound such as (poly)cationic peptide or protein. The most effective is the use of Protamine, nucleolin, spermine or spermidine as a poly-binding RNA-binding protein. In addition, you can also use other poly-peptides or proteins, such as poly-L-lysine or histones. These methods of stabilization of RNA described in Patan the e EP-A-1083232, which is included in the present description in full by reference. Other preferred cationic compounds that can be used for stabilization of RNA in the composition of the active (immunostimulatory) composition according to the present invention include cationic polysaccharides, for example chitosan, polybrene, polyethylenimine (PAYS) or poly-L-lysine (PLL), and other Association or complex formation of at least one RNA in the composition of the active (immunostimulatory) composition according to the invention, cationic compounds, such as cationic proteins or cationic lipids, such as oligofectamine (as lipid complexing reagent) preferably allow to improve the transfer of at least one RNA, used as pharmaceutically the active component in cells in need of such treatment, or the patient in need of such treatment. Described in this context, ways of improving the transfer of at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention due to the complex formation can also be used for stabilization of RNA.

According to another preferred variant of the invention, the at least one RNA in the composition of the active (immunostimulatory) composition may dopolnitelnoye alternative to encode secretory signal peptide. These signal peptides typically comprise from about 15 to 30 amino acid residues and preferably localized in the N-conceal plot of the encoded peptide, but not limited to this. Signal peptides described in this context, preferably designed for the transport of antigen protein antigen or a peptide antigen encoded by at least one RNA in the composition of the active (immunostimulatory) composition, in a certain part of the cell, preferably on the cell surface, in the endoplasmic reticulum (ER) or endosome, complementary mechanism. Examples of sequences of secretory signal peptides described in this context include, but are not limited to, the signal sequence of classical or non-classical MHC (for example, signal sequences of MHC class I and II, for example MHC class I HLA-A*0201), signal sequence of a cytokine or immunoglobulin, as described in this context, the signal sequence is conservative (plots) chains of immunoglobulins or antibodies, as described in this context, the signal sequence Lamp1, tapasin, Erp57, calreticulin, calnexin, as well as other proteins associated with membranes or proteins, associated with ER, or endosomes, lysosomes. According to this izobreteny the most preferred signal sequence is the MHC class I HLA-A*0201.

Any of the above modifications can be applied to at least one RNA in the composition of the active (immunostimulatory) composition according to the present invention, as well as to any other one (m)RNA described in this context and, if necessary, such modifications can be used in any combination provided that each modification does not adversely affect other modifications of at least one RNA. Specialist in the art can choose appropriate combinations of modifications.

According to other variant is active (immunostimulirutuyu) the composition according to the present invention may include adjuvant. The term "adjuvant"is used in this context, refers to any compound that is suitable for increasing the efficiency of the introduction and delivery of the active (immunostimulatory) composition according to the present invention. In addition, it is assumed that the adjuvant may initiate or exacerbate the innate immune response, i.e. non-specific immune response. In other words, the introduction of the active (immunostimulatory) composition according to the invention typically initiates an acquired immune response is at least two antigen encoded by at least one RNA in the composition of the active (immunostimulatory) composition according to the invention. In addition, the (immunostimulirutuyu) the composition according to the invention can induce the innate immune response in response to the adjuvant, added to the active (immunostimulatory) composition according to the invention. Specified adjuvant can be selected from any known adjuvant suitable for the stated purpose, i.e. which enhances the immune response in the body of a mammal. Preferably the adjuvant is chosen from the group including, but not limited to, DMT (dikarenakan trehalose,), MOS, muramyl dipeptide, pluronic, a solution of aluminum salts (alum), aluminum hydroxide, the product ADJUMERTM (polyphosphazene), gel phosphate of aluminum, glucan from algae, product algemarin, gel aluminum hydroxide (alum), gel aluminum hydroxide, characterized by a high adsorption capacity in relation to the proteins, the gel of aluminum hydroxide with low viscosity, adjuvant AF or SPT (emulsion of squalane (5%), tween 80 (0.2%)of, pluronic L121 (1,25%), phosphate-saline buffer solution, pH 7.4), product AVRIDINE™ (propandiamine), product BAY R1005™ (hydroureter (N-(2-deoxy-2-L-leucinamide-b-D-glyukopiranozil)-N-octadecyltrichlorosilane), product CALCITRIOL™ (1-α,25-dihydroxyvitamin D3), the gel of calcium phosphate, the product SRTM (nanoparticles of calcium phosphate), Vibrio holotoxin, a hybrid protein cholera toxin A1-protein-fragment D subunit In the cholera toxin, the product CRL 1005 (block copolymers R), cytokine-containing liposomes, DDA (bromide of dimethyldioctadecylammonium), DHEA (digigraphie drosterone), DMPH (dimyristoylphosphatidylcholine), DMPG (dimyristoylphosphatidylcholine), complex DOH/alum (sodium salt deoxycholic acid), full beta-blockers, incomplete beta-blockers, γ-inulin, adjuvant coat of Arms (a mixture including: 1) N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), 2) the chloride of dimethyldioctadecylammonium (DDA), 3) complex zinc-L-Proline (ZnPro-8), GM-CSF), GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine), imiqimod (1-(2-methylpropyl)-1H-imidazo[4,5-C]quinoline-4-amine), product ImmTher™ (dipalmitate N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-Glu-L-Ala-glycerol), products DRV (immunoliposome obtained by dehydration/rehydration vesicles), γ-interferon, interleukin-1β, interleukin-2, interleukin-7, interleukin-12, product ISCOMS™product ISCOPREP 7.0.3.™, liposomes product LOXORIBINE™ (7-allyl-8-oxoguanosine), oral adjuvant LT (labile enterotoxin-protoxin E.coli), microspheres and microparticles of any composition, product MF59™, (an aqueous emulsion of squalane), product MONTANIDE ISA 51™ (purified incomplete beta-blockers), product MONTANIDE ISA 720™ (metabolisable oil adjuvant), MPL™ (3-Q-diacyl-4'-monophosphorylated A), liposomes of MTP-PE and MTP-PE ((monosodium salt of N-acetyl-L-alanyl-D-isoglutamine-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyrisperidone))ethylamide), product MURAMETIDE™ (Nac-Mur-L - Ala-D-Gln-och3 ), the product MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-GIn-sn-glyzerinmanometer), product NAGO (neuraminidase-galactosidase), nanospheres or nanoparticles of any composition, products NISV (vesicula of nonionic surfactants), product PLEURAN™ (β-glucan), product PLGA, PGA and PLA (Homo - and co-polymers of lactic acid and glycolic acid microspheres/nanospheres), pluronic L121™, PMMA (polymethylmethacrylate), product PODDS™ (proteinoid microspheres)derivatives polyethylenimine, poly-rA/poly-rU (complex polyadenylate and poliuridilovoi acid), Polysorbate 80 (tween 80), protein cochleata (Avanti Polar Lipids, Inc., Alabaster, Alabama), product STIMULON™ (QS-21), product Quil-A saponin Quil-A), the product S-28463 (4-aminoacetyl-2-ethoxymethyl-1H-imidazo[4,5-C]quinoline-1-ethanol)product SAF-1™ ("composition adjuvants Syntex"), proteoliposome Sendai virus and lipid matrix, based on Sendai virus, span-85 (sarbatorile), product Specol (emulsion Marcol 52, span 85 and tween 85), squalane or product Robane®(2,6,10,15,19,23-hexamethyltetracosane and 2,6,10,15,19,23-HEXAMETHYL-2,6,10,14,18,22-tetracosactrin), sterilisation (hydrochloride octadecylamine), product Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-Glu-L-Ala-dipalmitoylphosphatidyl), product Theronyl-MDP (Termurtide™ or [thr 1]-MDP, N-acetylmuramyl-L-threonyl-D-isoglutamine), Tu-particles (Tu-HPV or virus-like particles, liposomes Walter-reed liposomes containing lipid And adsorbed on aluminum hydroxide), and lipopetides, including Pam3Cys, first of all, aluminium salts, such as adju-FOS (Adju-phos), alhydrogel, reherasal, emulsions, including CFA, SAF, IFA, MF59, PROMAX (Provax), TiterMax, montanide (Montanide), Vaxfectin (Vaxfectin), copolymers, including product OptiMax (Optivax, CRL1005), L121, poleaxes 4010), etc., liposomes, including product Stealth, cochleate, including product BIORAL, adjuvants vegetable origin including drug QS21, Quil A, Iscomatrix, ISCOM, adjuvants suitable for extra stimulation, including Tomatin, biopolymers, including PLG, RMP, inulin, adjuvants microbial origin, including reported, the product is detox (DETOX), MPL, CWS, mannose, sequence CpG nucleic acids, CpG7909, ligands TLR 1-10 person, TLR ligands 1-13 mouse, ISS-1018, IC31, imidazoquinolines, products Ampligen, Ribi529, IMOxine, IRIV, HPV, cholera toxin, thermolabile toxin, Pam3Cys, flagellin, membranosvyazannaya substances on the basis of glycosylated phosphatidylinositol, product LNFPIII/Lewis X, antimicrobial peptides, product UC-1V150, a hybrid protein of RSV, cdiGMP and adjuvants, which are preferably antagonists, including neuropeptide CGRP.

Suitable adjuvants can also choose from cationic or poly-compounds, while adjuvant preferably obtained when the formation of the complex of at least one active RNA at the image is ateneu (part of the immunostimulatory compositions with cationic or poly connection. Association or complexation RNA in the composition of the active (immunostimulatory) composition, cationic or poly-compounds, as described in this context, preferably imparts adjuvant properties and leads to increased stability of at least one RNA in the composition of the active (immunostimulatory) composition. These preferred cationic or poly-compounds chosen from cationic or poly-peptides or proteins, including Protamine, nucleolin, spermine or spermidine, or cationic peptides or proteins, such as poly-L-lysine (PLL), polyalanine, basic polypeptides, peptides, penetrating into the cell (CPD), including HIV-binding peptides, Tat, HIV-1 Tat (HIV) - derived peptides Tat, penetratin derived VP22 or peptide analogs, HSV VP22 (herpes simplex), the product MAP product or KALA domains transduction proteins (DTB, RRT, peptides enriched in Proline, peptides, enriched with arginine, peptides, enriched with lysine, MPG-peptide (peptides), PEP-1, L-oligomers, calcitonin peptide (peptides), peptides from complex locus (primarily complex locus in Drosophila), pAntp, pIs1, FGF, lactoferrin, transportan, Butorin-2, Vs-24, SynB, SynB(1), pVEC, derived peptides hCT, SAP, Protamine, spermine, spermidine or histones. Preferred cationic or poly-compounds which may also include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, such as polyethylenimine (PAYS), cationic lipids such as DOTMA [1-(2,3-soliloque)propyl)]-N,N,N-trimethylammoniumchloride), DMRIE, di-C14-amidin, DOTIM, SAINT, DC-Chol, BGTC, OLD, DOPC, DODAP, DOPE (dioleoylphosphatidylcholine), DOSPA, DODAB, DOIC, DMEPC, DOGS (diastatochromogenes), DIMRI (demorestconstruction.com), DOTAP (tileorasi-3-(trimethylammonio)propane), DC-6-14 (chloride O,O-ditetradecyl-N-(α-trimethylammonium)diethanolamine), CLIP 1 (rat-[(2,3-dictatorshipover)(2-hydroxyethyl)]dimethylammoniumchloride), CLIP6 (rat[2(2,3-dihexanoylphosphatidylcholine)ethyl]ammonium), CLIP9 (rat[2(2,3-dihexanoylphosphatidylcholine)ethyl]ammonium), oligofectamine or cationic or poly-polymers, such as modified polyaminoamide, such as polymers of β-amino acids or converted polyamides, and the like, modified polyethylene, such as HDPE (bromide poly(N-ethyl-4-vinylpyridine)) and the like, modified acrylates, such as pdmaema (poly(dimethylaminoethylmethacrylate)) and the like, modified amidoamines, such as PAA (poly(amidoamine)), etc. modified poly-β-amino esters (P-β-AE), such as modified by diamino terminal fragment copolymers of 1,4-potentialtarget and 5-amino-1-pentanol etc., dendrimers, so is e as polipropilenovye dendrimers or dendrimers based on PAA and the like, polyimid (polyimide), such as the PAYS (poly(ethylenimine), poly(propylenimine), etc., allylamine, based polymers of sugars, such as polymers based on cyclodextrin-based polymers dextran, chitosan and the like, polymers based on silanes, such as copolymers ROCHA-PDMS etc., block copolymers comprising a combination of one or more cationic units (for example, selected from cationic polymers described above) and one or more hydrophilic or hydrophobic blocks (for example, polyethylene glycol), etc.

In addition, the preferred cationic or poly-proteins or peptides that can be used as adjuvant, forming complexes with at least one RNA in the composition of the active (immunostimulatory) composition, you can choose from the following proteins or peptides of General formula (I): (Arg)l, (Lys)m, (His)n, (Orn)o, (Xaa)xwhere l+m+n+o+x=8-15, and l, m, n or o independently of one another equal to the number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn is at least 50% of all amino acid residues included in Oligopeptide, and XAA represents any amino acid selected from natural or non-natural amino acid except Arg, Lys, His or Orn, and x is any number selected from 0, 1, 2, 3, or 4, provided that the overall content of XAA is e than 50% of all amino acid residues, part of oligopeptides. According to the present invention, the most preferred are oligoaniline, for example Arg7, Arg8, Arg9, Arg7H3R9, R9H3H3R9H3, YSSR9SSY, (RKH)4, Y(RKH)2R etc.

Suitable adjuvants can also choose from nucleic acids of General formula (II): GlXmGnwhere: G denotes guanosine, uracil or analogues of guanosine or uracil, X denotes guanosine, uracil, adenosine, thymidine, cytosine or analogs of nucleotides, as described above, l is an integer from 1 to 40, provided that when l is 1, then G denotes guanosin or equivalent, if l>1 at least 50% of the nucleotides are guanosine or its analogues, m is an integer and is at least 3, and if m is equal to 3, X represents uracil or its equivalent, if m>3, at least 3 of the remainder of uracil or its analogues arranged in series one behind the other, n is an integer from 1 to 40, provided that when n is 1, then G denotes guanosin or equivalent, if n>1, at least 50% of the nucleotides are guanosine or its equivalent.

Other suitable adjuvants can also choose from nucleic acids of General formula (III): ClXmCnwhere denotes cytosine, uracil or analogs of cytosine or uracil, X denotes guanosin, ur the cyl, adenosine, thymidine, cytosine or analogs of nucleotides, as described above, l is an integer from 1 to 40, provided that when l is 1, denotes cytosine or equivalent, if l>1, at least 50% of the nucleotides are cytosine or its analogues, m is an integer and is at least 3, and if m is equal to 3, X represents uracil or its equivalent, if m>3, at least 3 of the remainder of uracil or its analogues are consistently each other, n is an integer from 1 to 40, if n is 1, denotes cytosine or equivalent, if n>1, at least 50% of the nucleotides are cytosine or its equivalent.

In one preferred embodiment, the present invention also offered the vaccine, which contains active (immunostimulirutuyu) the composition according to the invention. The vaccine according to the invention may additionally contain a pharmaceutically acceptable carrier and/or other excipients or additives and/or adjuvants. According to a more preferred variant antigens encoded by at least one RNA in the composition of the active (immunostimulatory) composition contained in the vaccine according to the invention are selected from groups or subgroups described above. According to a further preferred variant protein antigens are selected from any of the antigens of the following subgroups, Lucaya NY-ES01 (identification No. NM_001327), hTERT (identification No. NM_198253), survivin (identification No. AF077350), T (identification No. NM_006670) and WT1 (identification No. NM_000378), and/or from any of the antigens of the following subgroups, including MAGE-C1 and MAGE-C2, as described in this context, and/or from any of the antigens of the following subgroups, including MAGE-A2 and MAGE-A3 as described in this context.

The vaccine according to the invention usually contains a safe and effective amount of at least one RNA in the composition of the active (immunostimulatory) composition, as described above, which encodes at least two antigen, as described above, preferably, the coding of at least two antigens selected from any of the above groups or sub-groups, even more preferably from any of the described combinations. The term "safe and effective amount"used in this context, refers to the amount of at least one RNA in the composition of the active (immunostimulatory) composition, which is part of the vaccines described above, which is sufficient to provide a significant positive effect on state (to be treated) with lung cancer, preferably non-small cell lung cancer (NSCLC), more preferably on state when the three major subtypes of NSCLC, including, but not limited to, squamous cell carcinoma of the lung, hell is carcinoma and both the carcinoma of the lung. However, at the same time, the term "safe and effective amount" refers to a relatively small number, with the introduction of which does not have severe side effects, i.e. this number, with the introduction of which there is an acceptable ratio of benefit/risk. The specified number can be determined by a person skilled in the field of medicine. In respect of the vaccine of the present invention, the term "safe and effective amount" preferably refers to the amount of RNA (which encodes at least two antigen), which is suitable for stimulation of acquired immunity and should not develop excessive or damaging the immune response, but preferably these responses should be developed on detektiruya level. Moreover, such "safe and effective amount of at least one RNA in the composition of the active (immunostimulatory) composition, which is part of the vaccines described in this context, you can choose depending on the type of RNA, for example, depending on use monocistronic, bi - or even a polycistronic RNA, as bi - or even a polycistronic RNA causes a more effective expression of the encoded antigens (antigen) compared with an equal number monocistronic RNA. Moreover, the term "safe and effective to the number of at least one RNA in the composition of the active (immunostimulatory) composition, as described above, may vary depending on the particular condition to be treated, and the age and physical condition of the patient in need of treatment, the severity of the condition, the duration of treatment, the particular pharmaceutically acceptable carrier and similar factors that may be assessed by the attending physician. The vaccine of the present invention can be used in medicine and in veterinary medicine in the form of pharmaceutical compositions and vaccines.

The vaccine according to the invention usually contains a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier"used in this context preferably means liquid or non-liquid bases vaccine according to the invention. If the vaccine according to the invention is presented in liquid form, the carrier is usually pyrogen-free water, isotonic saline solution or buffer (aqueous) solutions, such as phosphate, citrate buffer solutions, etc. For injections and vaccines according to the invention, first of all, use water or preferably a buffer solution, preferably aqueous buffer solution containing a salt of sodium, preferably at least 50 mm sodium salt, calcium salt, preferably at least 0.01 mm salt of calcium, and optionally a potassium salt, preferably at least 3 mm potassium salt. Under the preferred alternative in the form of salts of sodium, calcium and potassium optional use halides, for example chlorides, iodides or bromides, hydroxides, carbonates, bicarbonates or sulfates of these metals and the like, Examples of the salts, but not limited to, include, for example, NaCl, NaI, NaBr, Na2CO3, NaHCO3, Na2SO4examples of optional potassium salts include, for example, KCl, KI, KBr, K2CO3, KHCO3, K2SO4and examples of calcium salts include, for example, CaCl2, CaI2, CaBr2Caso3, CaSO4Ca(OH)2. In addition, the buffer solution may contain organic salts of these metals. According to a more preferred variant of the buffer solution suitable for injection, as described above, may contain salt selected from sodium chloride (NaCl), calcium chloride (CaCl2and optionally potassium chloride (KCl), while the buffer solution may contain other anions in contrast to chloride. CaCl2you can replace with another salt, such as KCl. Usually salt in the composition of the buffer solution for injection are present in the following concentration of at least 50 mm sodium chloride (NaCl), at least 3 mm potassium chloride (KCl) and at least 0.01 mm calcium chloride (CaCl2). Buffer solution for injection may be a hypertonic, isotonic or hypotonic solution in comparison with the environment, who want to enter the specified vaccine i.e. buffer solution contains increased, a similar or lower the amount of salt in comparison with the environment in which is injected a specified vaccine, while it is preferable to use such concentrations above salts, which do not lead to damage of cells due to osmosis or other concentration effects. As the environment into which enter the specified vaccine can be considered, for example, the liquid medium used in the methods of "in vivo", such as blood, lymph, organs and other body fluids, or liquids that can be used in "in vitro", such as standard buffer solutions or liquids. These standard buffer solutions or liquids known to the person skilled in the art. The most preferred liquid base is lactate ringer's solution.

You can also use one or more compatible solid or liquid fillers or diluents or encapsulating compounds, suitable for introduction into the body of the subject. The term "compatible"is used in this context, means that the components of the vaccine according to the invention is suitable for mixing with at least one RNA in the composition of the active (immunostimulatory) composition, encoding at least two antigen described above, and in this case there is no l the Bo interactions between these components, which significantly reduce the pharmaceutical efficacy of the vaccine according to the invention under standard conditions. Pharmaceutically acceptable carriers, excipients or diluents must be sufficiently high purity and sufficiently low toxicity, i.e. to be acceptable for introduction into the body of a subject in need of treatment. Examples of compounds that can be used as pharmaceutically acceptable carriers, excipients or components of the vaccine include sugars, such as lactose, glucose and sucrose, starches, such as corn starch or potato starch, cellulose and its derivatives, such as, for example, sodium carboxymethyl cellulose, ethylcellulose, cellulose acetate, powdered traganou gum, malt, gelatin, solid fats, solid sliding substances, such as, for example, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, such as, for example, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter, polyols, such as, for example, polypropyleneglycol, glycerin, sorbitol, lures and polyethylene glycol, and alginic acid.

Generally, the choice of pharmaceutically acceptable carrier is determined by the method of administration of the vaccine according to the invention. VA is the CIN according to the invention can be entered, for example, systemic or local way. How systemic injections typically include, for example, percutaneous, oral, parenteral, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal, and intraperitoneal injection and/or intranasal routes of administration. Methods for local introduction mainly include, for example, the different methods of local administration, but also intradermal, transcutaneous, subcutaneous, or intramuscular injection or injection, injected directly into the affected tissue, intracranial, intra-lungs, intracardiac injections and sublingual. More preferably vaccines can be administered intradermally, subcutaneously or intramuscularly. Therefore, compositions/vaccines preferably processed into liquid or solid form. Suitable amount of the vaccine according to the invention can be defined in the standard experiments on animal models. These models include, but are not limited to, models, rabbits, sheep, mice, rats, dogs, and non human primates. Preferred standard dosage forms for injection include sterile aqueous solutions, saline, or a mixture thereof. These solutions are characterized by approximately pH of 7.4. Suitable carriers for injection include hydrogels, devices for kontroliruemoi slow release of the drug, polylactic acid and collagen matrix. Suitable pharmaceutically acceptable carriers for topical application include the media, suitable for use in the composition of lotions, creams, gels, etc. If the vaccine according to the invention is administered orally, then the preferred standard dosage forms are tablets, capsules, etc. In the prior art known pharmaceutically acceptable carriers to obtain a standard dosage forms. The choice of these media depends on secondary factors such as taste, cost, and stability during storage, and such storage media may choose a specialist in this field of technology, but they do not affect the essence of the present invention.

The vaccine according to the invention may additionally contain one or more auxiliary substances intended to improve immunogenet. This preferably is achieved by a synergistic effect under the action of at least one RNA in the composition of the active (immunostimulatory) composition, as described above, and excipients, which are not necessarily included in the vaccine composition according to the invention, as described above. Depending on the type of auxiliary substances can be considered different mechanisms of action. For example, compounds that accelerate Asrian the E. dendritic cells (DC), for example, lipopolysaccharides, α-TNF or CD40 ligand included in the first class of suitable auxiliary substances. Mainly as auxiliary substances you can use any agent that affects the immune system, the mechanism of "danger signal" (LPS, GP96, and so on), or you can use cytokines, such as GM-CSF, which allow you to purposefully enhance the immune response induced immune stimulating adjuvant according to the invention, or to influence it. The most preferred auxiliary substances are cytokines, such as Monokini, lymphokines, interleukins or chemokines, which in addition to the induction of acquired immunity encoded by at least two antigens, activate innate immunity, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, α], β], γ], GM-CSF, G-CSF, M-CSF, β-LT or α-TNF, growth factors, such as with HGH.

Other additives that can be included in a vaccine composition according to the invention are emulsifiers, such as, for example, twin, moisturizing agents, such as, for example, sodium lauryl sulfate, dyes, flavorings, pharmaceutical carriers, fillers to obtain tablets, stabilizers, antioxidants, preservatives.

In ccina according to the invention can additionally also contain other compounds, Immunostimulants that as ligands specifically associated with Toll-like receptors in human TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or as ligands associated with Toll-like receptors in mouse TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which optionally can be included in a vaccine composition according to the invention, as described in this context, includes CpG nucleic acid, primarily CpG-PHK or CpG-DNA. CpG-PHK or CpG-DNA can include single-stranded CpG-DNA (ISRO-DNA), double-stranded CpG-DNA (dzanc), single-stranded CpG-PHK (ISRO-RNA or double-stranded CpG-PHK (DCSA-RNA). CpG nucleic acid is preferably specified acid in the form of CpG-PHK, more preferably in the form CpG-PHK. CpG nucleic acid preferably contains at least one or more (mitogenic) cytosine/guanidinylation sequences (CpG-motif (motifs)). According to the first preferred variant, at least one CpG motif contained in these sequences, in other words, C (cytosine) and G (guanine) in the composition of the CpG motif is demetilirovanny. All other residues of cytosine or guanine, not necessarily contained in these sequences are methylated or neetilirovannyj. However, according to another preferred variant (itzin) and G (guanine) in the composition of the CpG motif may also be present in the methylated form.

According to another preferred object of the present invention is active (immunostimulirutuyu) the composition according to the invention or at least one RNA encoding at least two (preferably different antigen, as described in this context, you can use (to get the vaccine of the present invention) for lung cancer treatment, it is preferable condition associated with NSCL, more preferably of conditions associated with the three major subtypes of NSCLC, including, but not limited to, squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung.

According to another preferred object of the vaccine according to the invention or at least one RNA encoding at least two (preferably different antigen, as described in this context, can be used for lung cancer treatment, condition, preferably associated with NSCL, more preferably of conditions associated with the three major subtypes of NSCLC, including, but not limited to, squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung.

The present invention also provides methods of treating lung cancer, preferably state associated with NSCL, more preferably of conditions associated with the three major subtypes of NSCLC, VK is UCA, but not limited to, squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung, and these methods are that the patient in need of such treatment, administered pharmaceutically effective amount of the vaccine according to the invention or pharmaceutically effective amount of the active (immunostimulatory) composition. This method is usually that it is not necessary at the first stage, get active (immunostimulirutuyu) the composition according to the invention, or a vaccine according to the invention, and in the second stage to a patient in need of such treatment, injected (pharmaceutically effective amount) of the active (immunostimulatory) composition according to the invention or the vaccine according to the invention. Patient in need of such treatment, usually chosen from any mammal. According to the present invention is a mammal, preferably selected from the group including, but not limited to, for example, goat, cattle, pig, dog, cat, donkey, monkey, Primate, rodent, such as mouse, hamster, rabbit, and, above all, with the mammal usually suffers from lung cancer, a condition that is associated preferably with NSCLC, more preferably of conditions associated with the three major subtypes of NSCLC, including, but not Ogre is nicias only them, squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung or condition associated with them.

The present invention also proposes the use of active (immunostimulatory) composition according to the invention or at least one RNA encoding at least two (preferably different antigen, as described in this context (to get the vaccine according to the invention), preferably for the induction of an immune response in a mammal, preferably for the treatment of lung cancer, more preferably for the treatment of a condition associated with NSCLC, as described in this context.

The present invention also features the use of a vaccine according to the invention by itself or at least one RNA encoding at least two (preferably different antigen, as described in this context, for the induction of acquired immune response in a mammal, preferably for the treatment of lung cancer, more preferably a condition associated with NSCLC, as described in this context.

Prevention or treatment of lung cancer in a patient in need of such treatment, preferably, the condition associated with NSCLC, as described in this context, carried out with the introduction of the active (immunostimulatory) composition according to the invention and/or vaccine according to the invention in widespace or alternately, for example, in the form of sets of these components, with each component of the set includes at least one of antigens, preferably of different antigens. The introduction can be performed by any method described above. For example, it is possible to treat lung cancer, preferably, the condition associated with NSCLC, as described in this context, due to the induction or amplification of the acquired immune response in response to at least two (specially selected) antigen encoded by at least one RNA in the composition of the active (immunostimulatory) composition according to the invention. The introduction of the active (immunostimulatory) composition according to the invention and/or vaccine according to the invention can be implemented before the introduction, simultaneously and/or after administration of another active (immunostimulatory) composition according to the invention and/or vaccine according to the invention, as described in this context, which may contain a different combination of RNA encoding different antigens, each antigen is encoded by at least one RNA in the composition of the active (immunostimulatory) composition according to the invention is preferably suitable for the treatment of lung cancer, more preferably for the treatment of a condition associated with NSCLC, as described in this context. The term "treatment"used in this context, also includes modulation Zab the diseases, associated with lung cancer, preferably diseases associated with NSCLC, as described in this context.

In another embodiment, the invention features the use of active (immunostimulatory) composition (to get the vaccine according to the invention) to modulate, preferably to induce or enhance an immune response in a mammal, as described above, is more preferable to improve the treatment of lung cancer, especially NSCLC, as described in this context. As indicated in this context, supportive therapy of lung cancer, primarily NSCLC can be implemented using any combination of standard treatment of lung cancer, especially NSCLC, as described in this context, such as radiation therapy, chemotherapy, proton therapy, hormonal therapy, treatment with antibodies, auxiliary therapeutic measures, methods of treatment using other vaccines, unlike the vaccine according to the invention, methods of treatment using inhibitors of kinases or low molecular weight nucleotides and the like, or some combination of these courses of treatment and method of treatment using the active (immunostimulatory) the composition of the invention or the vaccine according to the invention, as described in this context. Supportive therapy + treatment is of lung cancer, first of all NSCLC, as described in this context, you can use any other variant of the method according to the invention, as described in this context.

The introduction of the active (immunostimulatory) composition according to the invention or at least one RNA encoding at least two (preferably different antigen, as described in this context, or vaccine according to the invention can be performed alternately, for example, by introducing active (immunostimulirutuyu) the composition according to the invention or at least one RNA encoding at least two (preferably different antigen, as described in this context, or the vaccine according to the invention before treatment (at the same time and/or after) of lung cancer, especially NSCLC, for example, when the introduction of the active (immunostimulatory) composition according to the invention or the vaccine before treatment (at the same time and/or after) or with the introduction of drugs suitable for the treatment of lung cancer, especially NSCLC, as described in this context. The specified alternate treatment can be performed using, for example, a set, preferably the set of components, as described below.

Alternate treatment may additionally or alternatively include the introduction of the active (immunostimulatory) composition or vaccine according to the invention, p is edocfile at least one RNA, encoding at least two (preferably different) antigens, as described above, in the form in which at least one RNA encoding at least two (preferably different) antigens, as described above, preferably is part of the active (immunostimulatory) composition or vaccine according to the invention, which is injected simultaneously with the introduction, before the introduction or after the introduction of another at least one RNA encoding at least two (preferably different) antigens, as described above, which is part of the same active (immunostimulatory) composition or vaccine according to the invention. Preferably the introduction of (at least one RNA) is carried out for 1 hour, more preferably within 30 minutes, more preferably within 15, 10, 5, 4, 3 or 2 minutes or even within 1 minute of the Specified alternate treatment can be performed using, for example, a set, preferably the set of components, as described below.

In the final embodiment, the invention features a set, first set of components, including active (immunostimulirutuyu) the composition according to the invention and/or the vaccine according to the invention, and optionally instructions for use, containing recommendations for the introduction and the dosage of the active (immunostimulatory) composition is according to the invention and/or vaccine according to the invention. Instructions for use may include recommendations for the introduction and the dosage of the active (immunostimulatory) composition according to the invention and/or vaccine according to the invention. These sets, preferably sets of components can be used, for example, in any of the ways described above, it is preferable to use at least one of the active (immunostimulatory) composition according to the invention (for vaccines according to the invention) for the treatment of lung cancer, especially NSCLC, as described in this context. Sets can also be used for applying at least one active (immunostimulatory composition according to the invention (for vaccines according to the invention) for the treatment of lung cancer, preferably NSCL, as described in this context, active (immunostimulirutuyu) the composition according to the invention and/or vaccine encoding at least two antigen, induces or enhances an immune response in a mammal, as described above. These sets can also be used for applying at least one of the active (immunostimulatory) composition according to the invention (for vaccines according to the invention) to modulate, preferably to the induction of, for example, to induce or enhance an immune response in a mammal, as described above, and preferably for maintenance treatment is ia lung cancer, first of all NSCLC. The feature set as a special form of sets may contain one or more identical or different active (immunostimulatory) of the compositions according to the invention and/or one or more identical or different vaccines according to the invention in the composition of the various components in the kit. The feature set can also contain (e.g., one) active (immunostimulirutuyu) the composition according to the invention (e.g., one) the vaccine according to the invention and/or at least one RNA encoding at least one antigen, as described above, in the composition of the various components of the set, for example, each component of the set includes at least one RNA encoding a different antigen. In addition, you can use a combination of both types of sets. Feature sets can be used, for example, when performing sequential treatment, for example, using different compositions and/or increasing the concentration of the active (immunostimulatory composition according to the invention, the vaccine according to the invention and/or at least one RNA encoding at least one antigen, as described above, in the course of the same treatment (in vivo). If necessary (for technical reasons) sets of components can be used with the introduction of a separate composition or the introduction of different antigens included in the Tav active (immunostimulatory) composition according to the invention (i.e. in the composition of the various components), but provided that this is achieved by the joint presence of various agents in vivo. You can use the feature set as a special form of sets in which each component contains at least one antigen, preferably a different antigen, as described above, all the components of the kit are preferably active form (immunostimulirutuyu) the composition according to the invention or the vaccine according to the invention, as described in this context. These specific sets of components can preferably be used, for example, if different antigens are presented separately in the composition of the various components of the set, but then they are administered simultaneously or sequentially to a mammal in need of such treatment. In the latter case, all the various components of the specified collection is administered for a short period of time to all the antigens present in the body of a mammal at approximately the same time after the last component set. Any of the kits described above can be used for treatment, as described above.

The advantages of the present invention

In the present invention is proposed active (immunostimulirutuyu) the composition for treating lung cancer, especially NSCLC, when this component is ice contains at least one RNA, preferably mRNA, encoding at least two (preferably different) antigens, inducing (acquired) immune response in a mammal, and antigens selected from the group including hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 or MAGE-C2. The specified active (immunostimulirutuyu) composition can be used for effective treatment of lung cancer, especially NSCLC, or as maintenance therapy in the course of standard treatment. Proposed in the present invention method, it is used for treatment RNA) eliminates the problem of the uncontrolled production of introduced DNA sequences. RNA in the composition of the active (immunostimulatory) composition according to the invention, characterized by further significant advantages compared with expressing systems based on DNA, for example, in relation to the immune response, immunization or vaccination. These benefits include, among other things, the fact that RNA, penetrating into the cell, is not integrated into its genome. This allows you to exclude specified mutation of a gene that can lead to full or partial inactivation or lead to erroneous information. In addition, it helps to reduce the risks associated with the use of DNA as an agent for inducing an immune response, (in the example, as a vaccine, such as the induction of pathogenic anti-DNA antibodies in a patient, which introduce foreign DNA that may lead to (possibly fatal) immune response. On the contrary the formation of any anti-RNA antibodies to the present time is not set.

Figures

The following figures are for illustration only and do not limit the scope and essence of the present invention.

Figure 1. The diagram shows the RNA sequence (SEQ ID No. 1) (the starting sequence of the wild type), encoding a MUC1 (HsMUC1 - 5×VNTR (Usually a sequence of wild-type contains 40 tandem repeats. The content of such repetitions is reduced to 5 in connection with the terms of cloning). The GC content is 61,27%, length 1668 BP).

Fig 2. The diagram shows (GC) stable RNA sequence (SEQ ID No. 2), encoding a MUC1 (HsMUC1 GC - 5×VNTR, 1. The maximum GC content, 2. Using codon), the GC content is 73,56%, length 1668 BP, distinct from the primary sequence (figure 1 (SEQ ID No. 1)) is 398/1668 reason = 23,86%.

Figure 3. The diagram shows the RNA sequence (SEQ ID No. 3) (the starting sequence of the wild type), encoding T (Hs5T4 (glycoprotein of trophoblasts TPBG), the GC content is 61,60%, the length of 1263 BP

Figure 4. The diagram shows (GC) stable RNA sequence (SEQ ID No. 4), encoding T (H5T4 GC, 1. The maximum GC content, 2. Using codon), the GC content is 70,47%, the length of 1263 BP, distinct from the primary sequence (figure 3 (SEQ ID No. 3)) is 247/1263 reason = 19,56%.

Figure 5. The diagram shows the RNA sequence (SEQ ID No. 5) (starting sequence of the wild type), encoding Her-2/neu (HsHer2/neu (v-erb-b2 homolog of the viral oncogene 2 erythroblastic leukemia)), GC content is 60,78%, length 3768 BP

6. The diagram shows (GC) stable RNA sequence (SEQ ID No. 6), encoding Her-2/neu (HsHer2/neu GC, 1. The maximum GC content, 2. Using codon), the GC content is 70,54%, length 3768 P.O., distinct from the primary sequence (figure 5 (SEQ ID No. 5)) is 772/3768 reason = 20,49%.

7. The diagram shows the RNA sequence (SEQ ID No. 7) (starting sequence of the wild type), encoding hTERT (HsTERT (reverse transcriptase telomerase), the GC content is 66,08%, length 3399 BP

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 8), encoding hTERT (HsTERT GC, 1. The maximum GC content, 2. Using codon), the GC content is 72,96%, length 3399 BP, the difference from the main sequence (Fig.7 (SEQ ID No. 7)) is 566/3399 reason = 16,65%.

Fig.9. The diagram shows the RNA sequence (SEQ ID No. 9) (starting on sledovatelnot wild-type), encoding a WT1 (HsWT1 (Wilms tumor 1)), the GC content is 61,78%, length 1554 BP

Figure 10.

Figa. The diagram shows the RNA sequence (SEQ ID No. 10), encoding a WT1 (HsWT1 (Wilms tumor 1)), sequence characterized by low GC content in the site 325-408 compared with the corresponding plot in the sequence of the wild type.

On figb, and D compares the relevant sections 325-408:

on figb: the sequence of the wild type, corresponding to the sequence presented in figure 9 (SEQ ID No. 9),

on figw: the sequence with the highest possible content of GC corresponding to the sequence represented by figure 11 (SEQ ID No. 11), and

on Figg: sequence with reduced GC content corresponding to the sequence shown in figure 10 (SEQ ID No. 10), all sequences are characterized by different GC composition.

11. The diagram shows (GC) stable RNA sequence (SEQ ID No. 11)encoding a WT1 (HsWT1 GC, 1. The maximum GC content, 2. Using codon), the GC content is 72,59%, length 1554 BP, distinct from the primary sequence (figure 9 (SEQ ID No. 9)) is 322/1554 reason = 20,72%.

Fig. The diagram shows the RNA sequence (SEQ ID No. 12) (starting sequence of the wild type), encoding CEA (CEA (carcinoma the national antigen) HsCEACAM5), the GC content is 52.20%, length 2109 BP

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 13), encoding CEA (SEASAM GC, 1. The maximum GC content, 2. Using codon, in known positions), the GC content is 66,24%, length 2109 BP, the difference from the main sequence (Fig (SEQ ID No. 12)) is 495/2109 reason = 23,47%.

Fig. The diagram shows the RNA sequence (SEQ ID No. 14) (starting sequence of the wild type), encoding a MAGE-A2 (HsMAGE-A2 (melanoma antigen family a, 2) HsMAGE-A2B), the GC content is 55,87%, the length of 945 BP

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 15), encoding a MAGE-A2 (HsMAGE-A2B GC, 1. The maximum GC content, 2. Using codon), the GC content is 68,57%, the length of 945 BP, the difference from the main sequence (Fig (SEQ ID No. 14)) is 187/945 reason = 19,79%.

Fig. The diagram shows the RNA sequence (SEQ ID No. 16) (starting sequence of the wild type), encoding MAGE-A3 (MAGE-A3 (melanoma antigen family A, 3) MAGE-A3), the GC content is 56,30%, the length of 945 BP

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 17), encoding MAGE-A3 (MAGE-A3 GC, 1. The maximum GC content, 2. Using codon, already known to the increased content the GC), the GC content is 69,00%, the length of 945 BP, the difference from the main sequence (Fig (SEQ ID No. 16)) is 190/945 reason = 20,11%.

Fig. The diagram shows the RNA sequence (SEQ ID No. 18) (starting sequence of the wild type), encoding survivin (survivin (baculovirus IAP containing 5 repeats BIRC5) Hs (wild-type), the GC content is 52,68%, the length of 429 BP

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 19)encoding survivin (Hs(GC), 1. The maximum GC content, 2. Using codon, already known to the high content of GC), GC content is 65,27%, the length of 429 BP, the difference from the main sequence (Fig (SEQ ID No. 18)) is 72/429 reason = 16.78 in%.

Fig. The diagram shows the RNA sequence (SEQ ID No. 20) (starting sequence of the wild type), encoding NY-ESO-1 (Homo sapiens NY-ESO-1 (NY-ESO-1 (wild type), the GC content is 67,4%.

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 21), encoding NY-ESO-1 (NY-ESO-1(GC), GC content is 79,56%, (already known to the high content of GC), in contrast to wild-type (Fig (SEQ ID No. 20)) is 112/543 reason, 20,63%.

Fig. The diagram shows the RNA sequence (SEQ ID No. 22) (starting sequence of the wild type), encoding a MAGE-C1 (HsMAGEC1 (melanoma and is then family, 1) HsMAGEC1 (wild type), the GC content is 51,86%, length 3429 BP

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 23), encoding a MAGE-C1 (HsMAGEC1 (GC), 1. The maximum GC content, 2. Using codon), the GC content is 68,73%, length 3429 BP, the difference from the main sequence (Fig (SEQ ID No. 22)) is 964/3429 reason = 28,11%.

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 24), encoding a shortened MAGE-C1 (HsMAGEC1(GC), 1. The maximum GC content, 2. Using codon), compared with the original sequence (Fig (SEQ ID No. 22)) repeating region deleted and the sequence according pig following the start-codon (ATG), starts with the amino acid residue 613 sequence of the wild type, with the highest possible content of GC (Fig (SEQ ID No. 23)).

Fig. The diagram shows the RNA sequence (SEQ ID No. 25) (starting sequence of the wild type), encoding a MAGE-C2 (HsMAGE-C2 (melanoma antigen family C, 2) HsMAGE-C2), the GC content is 50,81%, the length of 1122 BP

Fig. The diagram shows (GC) stable RNA sequence (SEQ ID No. 26), encoding a MAGE-C2 (HsMAGE-C2 GC, 1. The maximum GC content, 2. Using codon), the GC content is 66,58%, the length of 1122 BP, the difference from the main sequence (f is A.25 (SEQ ID No. 25)) is 264/1122 reason = 23,53%.

Fig. The diagram presents data on the presence of IgG1 antibodies specific against tumor antigen NY-ESO-1 in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C) in the form of a complex with Protamine (mass ratio 4:1).

Fig. The diagram presents data on the presence of IgG2a antibodies specific against tumor antigen NY-ESO-1 in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C) in the form of a complex with Protamine (mass ratio 4:1).

Fig. The diagram presents data on the presence of IgG1 antibodies specific against tumor antigen MAGE-C1 in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C) in the form of a complex with Protamine (mass ratio 4:1).

Fig. The diagram presents data on the presence of IgG2a antibodies specific against tumor antigen MAGE-C1 in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin it) in the form of a complex with Protamine (mass ratio 4:1).

Fig. The diagram presents data on the presence of IgG1 antibodies specific against tumor antigen MAGE-C2 in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C) in the form of a complex with Protamine (mass ratio 4:1).

Fig. The diagram presents data on the presence of IgG2a antibodies specific against tumor antigen MAGE-C2 in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C) in the form of a complex with Protamine (mass ratio 4:1).

Fig. The diagram presents data on the induction of antigen specific T lymphocytes directed against tumor antigen T in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C) in the form of a complex with Protamine (mass ratio 4:1).

Fig. The diagram presents data on the induction of antigen specific T lymphocytes directed against tumor antigen NY-ESO-1 in mice vaccinated with the mRNA vaccine that contains 5 components, each of which contains mRNA encoding one antigen associated with H the RL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C) in the form of a complex with Protamine (mass ratio 4:1).

Examples

The following examples are provided only to illustrate the present invention and do not limit the scope of the present invention.

Example 1

Obtaining encoding plasmids

Were experimental DNA sequence corresponding to a typical terminal sequences of mRNA coding for the following antigens:

hTERT,

WT1,

MAGE-A2,

T,

MAGE-A3,

MUC1,

Her-2/neu,

NY-ESO-1,

CEA,

survivin,

MAGE-C1 or

MAGE-C2,

which were used for transcription and transfection in vitro. When this DNA sequence corresponding to the native antigen encoded mRNA was characterized by a high content of GC and included optimized codons. Then, the coding sequence was included in the construct of RNActive (company CureVac GmbH, tübingen, Germany), which modified the labels poly-A-tag and a poly-C-tag (A70-C30).

Example 2

Transcription in vitro

RNA sequences based on the recombinant plasmid DNA obtained in example 1 was obtained by the method of in vitro transcription. When this recombinant plasmid DNA was linearizable and then carried out the in vitro transcription using T7 RNA polymerase. Then DNA template was digested with desoksiribonukleaza I, RNA was isolated during the precipitation of LiCl, the m was purified by the method GHUR (PUREMessenger ®the company CureVac GmbH, tübingen, Germany).

Example 3

The formation of complexes with Protamine

For transfection of RNA in cells and organisms RNA obtained by transcription in vitro, preferably translated in the form of complexes, more preferably in the form of a complex with Protamine, when mixing of RNA with Protamine.

Example 4

Vaccination

For vaccination RNA obtained by in vitro transcription as described above (see example 2), was transfectional in mice (mouse: S BL/6), preferably in the form of a complex with Protamine (see example 3). Transfection was performed using different groups of animals, each group consisted of 5 mice (C BL/6), which were immunized with a composition of the mRNA according to the invention, the composition was injected intradermally 8 times over 3 weeks, they injected a mixture of mRNA in the form of complexes with Protamine, when this RNA encodes at least two antigens selected from hTERT, WT1, MAGE-A2, T, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, survivin, MAGE-C1 or MAGE-C2.

Example 5

Detection of antigen specific immune response (b-cell immune response)

Detection of antigen specific immune response (b-cell immune response) was performed using antigen specific antibodies. One week after the last vaccination in vaccinated mice was selected samples of blood and received about azzy serum. Tablets MaxiSorb (Nalgene Nunc International) were coated with protein antigen that is encoded by the composition of mRNA (0.5 μg/well). After blocking the surface of the wells with buffer 1×FSB containing 0.05% tween-20 and 1% BSA, the wells were added to the diluted samples of mouse serum(1:30, 1:90, 1:270, 1:810) and the plates were incubated under appropriate conditions. Then added biotinylated secondary antibodies (antimachine IgG2a, Pharmingen company). After washing, the wells were added to the conjugate of horseradish peroxidase and streptavidin and incubated under appropriate conditions, and then assessed the level of conversion of the substrate ABTS (2,2'-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid).

Example 6

Detection of antigen specific cellular immune response (T-cell immune response) method elispot (ELISPOT)

Two weeks after the last vaccination, mice were killed, and then removed the spleen, from which was isolated splenocytes. Splenocytes re-stimulated when they incubation for 7 days in the presence of peptides of the above antigens (peptide library) or during co-culture with dendritic cells derived from bone marrow native syngeneic mice, which was transferred RNA encoding the antigen, the method of electroporation. After re-stimulation was evaluated antigen is gunning immune response by secreting γ-d'if. For detection of γ-Interfax in wells Coat Multiscreen (Millipore) was added to the buffer solution used for coating (0.1 M carbonate-bicarbonate buffer solution, pH of 9.6 (10,59 g/l Na2CO3, 8,4 g/l NaHCO3))containing antibodies against γ-if (BD Pharmingen, Heidelberg, Germany), and incubated overnight. Stimulants and effector cells were added to wells at a ratio of 1:20 and incubated together for 24 hours of the Tablet was washed 1×FSB, in the wells was added biotinylated secondary antibody and incubated under appropriate conditions. After washing, 1×FSB/O 05% tween-20, the wells were added to the substrate (5-bromo-4-chloro-3-indolylacetic/nitro-blue tetrazolium, a liquid mixture of substrates company Sigma-Aldrich, Taufkirchen, Germany), conversion of the substrate was evaluated visually.

Example 7

Inoculation of tumor

Immunization

One week after the last immunization mice were subcutaneously injected melanoma cells 1 Mio B16 or cells TRAMP-C1. After 2 weeks (B16) or 7 weeks (TRAMP-C1), respectively, were estimated volume of the resulting tumors.

Example 8

Obtaining mRNA vaccines

Specific active (immunostimulirutuyu) the composition according to the invention, which includes a combination of different antigens intended for use as a vaccine for the treatment of non-small cell lung cancer light the (NSCL), received as described above. For example, active (immunostimulirutuyu) the composition according to the invention comprises 5 components, each of which contained mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C, respectively RNA SEQ ID No. 4, 19, 21, 24 and 26 (sequences with high GC content))received in the form of a complex with Protamine (mass ratio 4:1).

Vaccination

Of C57BL/6 mice were vaccinated, while mice intradermally injected mRNA vaccine that contains 5 components, each of which contained mRNA encoding one antigen associated with NSCL (NY-ESO-1, MAGE-C1, MAGE-C2, survivin and C, respectively RNA SEQ ID No. 4, 19, 21, 24 and 26 (sequences with high GC content), in the form of a complex with Protamine (64 μg antigen/cycle, divided into 4 injections per cycle). Control vaccination was performed using the appropriate cumulative dose of RNA that encodes a LacZ (control lacZ mRNA). Vaccination consisted of three cycles of vaccination (week 1, 3 and 5). Group, the number of mice and strains of mice are shown in the following table.

GroupThe strain of miceThe number of mice
the mRNA vaccineC57BL/610
5 for analysis by the method of the Elispot and 5 for the detection of antibodies in serum by ELISA
The control lacZ mRNAC57BL/65
3 for analysis by the method of the Elispot and all 5 for detection of antibodies in serum by ELISA

Detection of antigen specific antibodies

After 6 days after the last vaccination of retroorbital sinus were taken blood samples (200 μl), and the serum was analyzed in the presence of antigen specific antibodies, subtypes IgG1 and IgG2a by ELISA. Wells of 96-well plate to the ELISA plate was coated with recombinant protein (10 μg/ml in buffer solution for coating, incubated at 37°C for 4 h) and blocked using a blocking buffer solution (200 μl/well) overnight at 4°C. Then the hole got a serial dilution of pooled serum from each group of mice) in the range from 1:3 to 1:48 and the plates were incubated for 4 h at room temperature. After incubation with specific antibodies (in blocking buffer solution, 1:300) against IgG1 or IgG2a mouse and incubation with secondary antibodies labeled with HRP (in BC is carousel buffer solution, 1:500)was added to the substrate TMB. The calorimetric reaction was detected at 450 nm on a plate reader for ELISA (Tecan Deutschland GmbH, Crailsheim, Germany).

The analysis method elispot (ELISPOT)

For detection of the response of cytotoxic T lymphocytes (CTLs) level secretion of effector cytokine γ-if in response to a specific stimulus can be registered visually by the ELISPOT method (threshold until one of the cells).

After 6 days after the last vaccination, mice vaccinated antigenaemia and control vaccine was isolated splenocytes, which was transferred into a 96-well tablets ELISPOT, on the surface of which is pre-applied anti-γ-Interfax antibodies (10 μg/ml). The cells are then stimulated for 24 h at 37°C in the presence of the peptide from the library antigenic peptides or libraries of HIV or solvent for peptides, DMSO, as a control used cells that were incubated in a clean environment. All libraries used in a concentration of 1 μg peptide/ml After incubation, the cells were washed out of the tablet and γ-Interfax, the secretory cells were detected by adding biotinylated secondary antibodies against γ-if mouse (1 μg/ml), and then streptavidin-AKP. Spots were recorded visually using the substrate BCIP/NBT, the number was determined on an automated ELISPOT reader (firm Immuospot Analyzer, CTL Analyzers LLC).

Statistical analysis

Statistical analysis was performed using the software Graph Pad Prism 5.01 (GraphPad Software, Inc.). All results were expressed as the mean (or median) ± standard deviation. For analyses by the Elispot method, due to the fact that the underlying activation is largely dependent on the body specimens, the baseline was adjusted for each individual mouse, subtracting the number of spots in the wells with culture medium from all other values. Two-factor analysis on the criterion of Mann-Whitney was used to analyze differences between the study groups (with a confidence interval of 5%).

Results and discussion

Mice were vaccinated with the mRNA vaccine that contains five components, as described above, first of all, mRNAs with a high GC content, encoding associated with NSCL antigens NY-ESO-1, MAGE-C2, MAGE-C1, survivin and C, (respectively the sequences SEQ ID No. 4, 19, 21, 24 and 26 (sequences with high GC content)), with each RNA used in the form of a complex of cationic peptide/Protamine (mass ratio 4:1). Control mice were vaccinated with any other type of RNA that encodes LacZ, in the form of a complex with Protamine (in the same ratio as described for mRNA vaccines).

In the experiments used the serum obtained from the blood of mice, the vaccine is reported to antigenaemia vaccine and control mice were assessed level of generation of specific antibodies against antigens. For three of the five studied protein, MAGE-C1, MAGE-C2, and NY-ESO-1, were detected antigen specific antibodies in the serum of mice vaccinated with the mRNA vaccine, thus it was found that mRNA are immunogenic properties in vivo. Proteins required for the detection of antibodies was obtained in E. coli cells. As the production of proteins in E. coli cells can affect post-translational modifications and are not described for the used antigens, lack of response to other proteins can explain these phenomena.

Investigated the activation of cytotoxic T cells in response to the introduction of mRNA vaccines. γ-d'if is a major mediator of Th1 responses and secreted activated CTLs. In this regard, investigated the presence of antigen specific cytotoxic T-cells as part of splenocytes obtained from the immunized mice, the analysis was carried out by the ELISPOT method. As antigens, stimulating splenocytes used libraries restriction of peptides. Because different epitopes of the antigens used man to mouse MHC (H-2Kb and H-2Db in C57BL/6 mice) are not known, had to use a hypothetical selection of peptides selected from peptides, which are characterized by a potential affinity,using the database SYFPEITHI. From peptide libraries (15-membered peptides with an overlap of 11 amino acid residues), including the full sequence of proteins, selected 15-membered peptides containing hypothetically the most effective epitopes were selected 18 peptides. However, the selected peptides could not necessarily contain the correct epitopes, and thus the detection of immune responses using such sets can lead to false-negative results. However, the stimulation of these two libraries derived from NY-ESO-1 and T, there is a high level secretion of γ-if the splenocytes obtained from mice vaccinated with the mRNA vaccine, in contrast to splenocytes obtained from control mice that were vaccinated with mRNA that encodes an unrelated β-galactosidase. No type of splenocytes did not respond to the reference library of HIV peptides. The number of γ-Interfax spots obtained when using splenocytes, inkubiruemykh in a clean environment, consistent with the basic activation of freshly isolated cells. Because the base activation largely depends on the individual, the baseline correction was performed for each case, thus subtract the number of spots in the wells with the environment of all other values.

The results of these experiments are given in Fig-34

1. Vaccine for lung cancer treatment, it is preferable condition associated with small cell lung cancer (NSCLC), more preferably of conditions associated with the three major subtypes of NSCLC, including squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung, comprising at least one RNA:
encoding the antigen MAGE-C1 and comprising a sequence selected from the group consisting of SEQ ID no:24, and a sequence at least 90% identical to the sequence shown,
encoding the antigen MAGE-C2, and comprising a sequence selected from the group consisting of SEQ ID no:26, and a sequence at least 90% identical to the sequence shown,
encoding the antigen NY-ESO-1 and comprising a sequence selected from the group consisting of SEQ ID no:21, and a sequence at least 90% identical to the sequence shown,
encoding the antigen of survivin and comprising a sequence selected from the group consisting of SEQ ID no:19, and the sequence at least 90% identical to the sequence shown,
encoding a 5T4 antigen and comprising a sequence selected from the group consisting of SEQ ID no:4, and sequences at least 90% identical to the sequence shown, and
optional advanced coding for the expansion of antigen MUC-1 and comprising the sequence selected from the group consisting of SEQ ID no:2, and sequences at least 90% identical to the sequence shown,
moreover, the sequence at least 90% identical to these sequences encode the amino acid sequence without replacement or conservative substitutions that do not affect biological activity of the antigen.

2. The vaccine according to claim 1, where the vaccine additionally contains RNA that encodes the antigen MUC-1 and contains a sequence selected from the group consisting of SEQ ID no:2, and sequences at least 90% identical to the sequence specified.

3. The vaccine according to claim 1, in which at least one RNA is mRNA.

4. The vaccine according to claim 1, in which at least one RNA is monocistronic RNA.

5. The vaccine according to claim 4, in which at least two antigen encoded monocistronic RNA.

6. The vaccine according to claim 1, in which all RNA are monocistronic.

7. The vaccine according to claim 1, in which at least one RNA is modified RNA, in particular stabilized mRNA.

8. The vaccine according to claim 7, in which the content of G/C in the coding region of at least one RNA increased compared with the content of G/C in the coding region of the RNA of wild-type, and the encoded amino acid sequence is preferably not changed compared the structure from amino acid sequence, encoded RNA wild type.

9. The vaccine of claim 8, in which the content of A/U in areas adjacent to the sites of binding to the ribosome at least one RNA, increased compared with the contents of A/U in the environment of the binding site of the ribosome in the composition of the RNA of the wild type.

10. The vaccine according to claim 9, in which the coding region and/or 5' and/or 3' untranslated region of the modified mRNA changed in comparison with the RNA wild type in such a way that they do not contain elements destabilizing sequence, while the amino acid sequence encoded by the modified mRNA, preferably not changed in comparison with the sequence encoded by the RNA of the wild type.

11. The vaccine according to claim 7, in which the modified mRNA comprises 5'-kupirovaniu structure and/or poly(A) tail fragment, which preferably includes from 10 to 200 adenosine residues, and/or poly(C) tail fragment, which preferably includes from 10 to 200 cytosine residues, and/or at least one fragment of the IRES and/or at least one 5' and/or 3' stable sequence.

12. The vaccine according to claim 1, in which at least one RNA is present in the form of a complex with one or more polycation, preferably in the form of a complex with Protamine or oligofectamine, more preferably in the form of a complex with Protamine is.

13. The vaccine according to claim 1, which additionally contains at least one adjuvant.

14. The vaccine according to item 13, in which at least one adjuvant selected from the group comprising cationic or poly-compounds including cationic or poly-peptides or proteins, including Protamine, nucleolin, spermine or spermidine, poly-L-lysine, polyalanine, basic polypeptides, peptides, penetrating into the cell, including HIV-binding peptides, Tat, HIV-1 Tat (HIV) - derived peptides Tat, penetratin derived VP22 or peptide analogs, HSV VP22 (herpes simplex), the product MAP product KALA or domains transduction proteins (PpT620), peptides enriched in Proline, peptides, enriched with arginine, peptides, enriched with lysine, MPG-peptide (peptides), Pep-1, L-oligomers, calcitonin peptide (peptides), peptides from complex locus (primarily complex locus of Drosophila antennapedia), pAntp, pIsl, FGF, lactoferrin, transportan, Butorin-2, Bac715-24, SynB, SynB(1), pVEC, derived peptides hCT, SAP, Protamine, spermine, spermidine or histones, cationic polysaccharides, including chitosan, polybren, cationic polymers, including polyethylenimine, cationic lipids such as DOTMA ([1-(2,3-soliloque)propyl)]-N,N,N-trimethylammoniumchloride), DMRIE, di-C14-amidin, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE (dioleoylphosphatidylcholine), DOSPA, DODAB, DOIC, DMEPC, DOGS (diastatochromogenes), DIMRI (generictoolbarpalette hydroxyethylammonium), DOTAP (tileorasi-3-(trimethylammonio)propane), DC-6-14 (chloride O,O-ditetradecyl-N-(α-trimethylammonium)diethanolamine), CLIP1 (rat[(2,3-dictatorshipover)(2-hydroxyethyl)]dimethylammoniumchloride), CLIP6 (rat[2(2,3-dihexanoylphosphatidylcholine)ethyl]ammonium), CLIP9 (rat[2(2,3-dihexanoylphosphatidylcholine)ethyl]ammonium), oligofectamine or cationic or poly-polymers including modified polyaminoamide, including polymers of β-amino acids or converted polyamides and modified polyethylene, including PVP (poly(N-ethyl-4-vinylpyridinium)), modified acrylates, including pdmaema (poly(dimethylaminoethylmethacrylate)), modified amidoamine, including PAA (poly(amidoamine)), modified poly (β-amino esters (P-β-AE), including modified by diamino terminal fragment copolymers of 1,4-potentialtarget and 5-amino-1-pentanol, dendrimers, including polipropilenovye dendrimers or dendrimers based on PAA, polyimid (polyimide), including the PAYS (poly(ethylenimine), poly(propylenimine), allylamine, based polymers of sugars, including polymers based on cyclodextrin-based polymers dextran, chitosan and polymers based on silanes, including copolymers PMOXA-PDMS block copolymers comprising a combination of one or more cationic blocks, select the data from cationic polymers, described above, and one or more hydrophilic or hydrophobic blocks (for example, polyethylene glycol),
or
cationic or poly-proteins or peptides selected from the following proteins or peptides of General formula (I): (Arg)l; (Lys)m, (His)n (Orn)o, (Xaa)x, where 1+m+n+o+x=8-15, and l, m, n or o independently of one another equal to the number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn is at least 50% of all amino acid residues included in Oligopeptide, and XAA represents any amino acid selected from natural or non-natural amino acid except Arg, Lys, His or Orn, and x is equal to any number selected from 0, 1, 2, 3, or 4, provided that the overall content of Xaa does not exceed 50% of all amino acid residues forming part of oligopeptides, or
nucleic acids of General formula (II): GlXmGn, where G denotes guanosine, uracil or analogues of guanosine or uracil, X denotes guanosine, uracil, adenosine, thymidine, cytosine or analogs of nucleotides, as described above, l is an integer from 1 to 40, provided that when 1 is 1, then G denotes guanosin or equivalent, if l>1, at least 50% of the nucleotides are guanosine or its analogues, m is an integer and is at least 3, and if m is 3, then X is uracil or an analogue, if m>3, at least 3 of the remainder of uracil or aghanloo arranged in series one behind the other, n is an integer from 1 to 40, provided that when n is 1, then G denotes guanosin or equivalent, if n>1, at least 50% of the nucleotides are guanosine or its analogues,
or
nucleic acids of General formula (III): ClXmCn, where C represents cytosine, uracil or analogs of cytosine or uracil, X denotes guanosine, uracil, adenosine, thymidine, cytosine or nucleotide analogues, described above, 1 is an integer from 1 to 40, provided that when 1 is 1, denotes cytosine or equivalent, if l>1, at least 50% of the nucleotides are cytosine or its analogues, m is an integer and is at least 3, and if m is 3, then X is uracil or an analogue, if m>3, at least 3 of the remainder of uracil or its analogues arranged in series one behind the other, n is an integer from 1 to 40, if n is 1, denotes cytosine or equivalent, if n>1, at least 50% of the nucleotides are cytosine or its equivalent.

15. The vaccine according to any one of claims 1 to 13, which induces the acquired immune response.

16. The vaccine according to any one of claims 1 to 13, which further comprises a pharmaceutically acceptable carrier.

17. The vaccine according to any one of claims 1 to 16 for lung cancer treatment, it is preferable condition associated with small cell lung cancer (NSCLC), more preferable conditions include the data from the three major subtypes of NSCLC, including squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung.

18. Set for lung cancer treatment, it is preferable condition associated with small cell lung cancer (NSCLC), more preferably of conditions associated with the three major subtypes of NSCLC, including squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung, comprising a vaccine according to any one of claims 1 to 16, and optionally instructions for use, containing recommendations for the introduction and the dosage of the vaccine.

19. Set for lung cancer treatment, it is preferable condition associated with small cell lung cancer (NSCLC), more preferably of conditions associated with the three major subtypes of NSCLC, including squamous cell lung carcinoma, adenocarcinoma and both the carcinoma of the lung, comprising at least one RNA:
encoding the antigen MAGE-C1 and comprising a sequence selected from the group consisting of SEQ ID no:24, and a sequence at least 90% identical to the sequence shown,
encoding the antigen MAGE-C2, and comprising a sequence selected from the group consisting of SEQ ID no:26, and a sequence at least 90% identical to the sequence shown,
encoding the antigen NY-ESO-1 and comprising a sequence selected from the group, with Toyama of SEQ ID no:21, and sequence at least 90% identical to the sequence shown,
encoding the antigen of survivin and comprising a sequence selected from the group consisting of SEQ ID no:19, and the sequence at least 90% identical to the sequence shown,
encoding a 5T4 antigen and comprising a sequence selected from the group consisting of SEQ ID no:4, and sequences at least 90% identical to the sequence shown, and
optional optional encoding the antigen MUC-1 and comprising a sequence selected from the group consisting of SEQ ID no:2, and sequences at least 90% identical to the sequence shown,
moreover, the sequence at least 90% identical to these sequences encode the amino acid sequence without replacement or conservative substitutions that do not affect biological activity of the antigen.

20. Set according to claim 19, in which at least one RNA is mRNA.

21. Set according to claim 19, in which all RNA are monocistronic.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: compounds can find application for preventing or treating cancer, lung cancer, non-small cells lung cancer, small-cell lung cancer, EML4-ALK hybrid polynucleotide-positive cancer, EML4-ALK hybrid polynucleotide-positive lung cancer or EML4-ALK hybrid polynucleotide-positive non-small cells lung cancer. In formula (I) -X-: group of formula , A represents chlorine, ethyl or isopropyl; R1 represents phenyl wherein carbon in the 4th position is substituted by the group -W-Y-Z, and carbon in the 3rd position can be substituted by a group specified in a group consisting of halogen, R00 and -O-R00; R00: lower alkyl which can be substituted by one or more halogen atoms; -W-: a bond, piperidine-1,4-diyl or piperazine-1,4-diyl; -Y- represents a bond; Z represents a monovalent 3-10-membered monocyclic non-aromatic heterocyclic ring which contains 1 to 4 heteroatoms specified in a group consisting of nitrogen, oxygen and sulphur, which can be substituted by one or more substitutes R00; R2 represents (i) an optionally bridged saturated C3-10cycloalkyl which can be substituted by one or more groups specified in -N(lower alkyl)2, lower alkyl, -COO-lower alkyl, -OH, -COOH, -CONH-RZB and morpholinyl, or (ii) a monovalent 3-10-membered monocyclic non-aromatic heterocyclic ring which contains 1 to 4 heteroatoms specified in a group consisting of nitrogen, oxygen and sulphur, which can be substituted by one or more groups specified in a group consisting of lower alkyl, -CO-lower alkyl, oxo, -CO-RZB and benzene; and RZB: phenyl which can be substituted by a group consisting of halogen and -O-lower alkyl; R3 represents -H.

EFFECT: invention refers to new compounds of formula or their pharmaceutically acceptable salts possessing the properties of a selective inhibitor of EML4-ALK hybrid protein kinase activity.

16 cl, 201 tbl, 582 ex

FIELD: medicine.

SUBSTANCE: described is biomaterial, stimulating the anti-tumour activity, which contains lyophilisate of postnatal induced lymphatic nodes (PNILN) of a patient with cancer. Described is a preparation, stimulating the anti-tumour activity, which contains as an active substance lyophilisate of PNILN of the patient with cancer, and as a solvent, water for injections, cremofor RH-40, an emulsifier T-2, peach oil, glycerol, Kollidon CL-M and benzyl alcohol with the specified component ratio.

EFFECT: obtaining a novel preparation of domestic production, which contains biomaterial, stimulating the anti-tumour activity, possessing the expressed prolonged action, and convenient in application.

3 cl, 5 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to methods for reducing B-cell count or treating a disease or disorder related to pathological activity of B-cells. That is ensured by administering a therapeutically effective amount of CD37-specific binding molecule and a therapeutically effective amount of mTOR or PI3K inhibitor into an individual. There are also presented a composition and a kit for treating non-Hodgkin lymphoma.

EFFECT: group of inventions provides a synergetic effect in administering CD37-specific binding molecules (SMIP) in a combination with mTOR or PI3K inhibitors for treating or preventing the B-cell related hyperproliferative disease.

36 cl, 9 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and concerns irinotecan liposomes or its hydrochloride containing irinotecan or its hydrochloride, neutral phospholipid and cholesterol, wherein the weight ratio of cholesterol to neutral phospholipid makes 1:3-5, and a method for preparing them.

EFFECT: liposomes have higher stability.

15 cl, 3 dwg, 10 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to peptide derivatives of general formula

,

their stereoisomers, mixtures and/or pharmaceutically acceptable salts, to methods for preparing them, to pharmaceutical compositions containing them, to using them for treating, preventing and/or diagnosing conditions, disorders and/or pathologies involving sstr1, sstr2, sstr3, sstr4 and/or sstr5 somatostatin receptor expression.

EFFECT: preparing the compositions for preventing and/or diagnosing the conditions, disorders and/or pathologies involving sstr1, sstr2, sstr3, sstr4 and/or sstr5 somatostatin receptor expression.

14 cl, 5 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compounds of formula 1.0:

,

where Q represents tetrahydropyridinyl ring substituted. R5, R1 are selected from: (1) pyridyl, substituted with substituent, selected from group, consisting of: -O-CH3, -O-C2H5, -O-CH(CH3)2, and -O-(CH2)2-O-CH3, R2 is selected from group, consisting of: -OCH3 and -SCH3; and R5 is selected from (a) substituted triazolylphenyl-, where triazolyl is substituted with one or two alkyl groups, selected from group, consisting of: -C1-C4alkyl, (b) substituted triazolylpheenyl-, wheretriazolyl is substituted on nitrogen atom with -C1-C4alkyl, (c) substituted triazolylphenyl-, where triazolyl is substituted on nitrogen atom with -C2alkylene-O-C1-C2alkyl, (d) substituted triazolylphenyl-, where triazolyl is substituted on nitrogen atom with -C2-C4alkylene-O-CH3, and (e) substituted triazolylphenyl-, where triazolyl is substituted on nitrogen atom with hydroxy-substituted -C1-C4alkyl, and where phenyl is optionally substituted with from 1 to 3 substituents, independently selected from group, consisting of halogen; and their pharmaceutically acceptable salts and solvates, which are claimed as ERK inhibitors.

EFFECT: obtaining pharmaceutically acceptable salts and solvates, claimed as ERK inhibitors.

15 cl, 2 tbl, 32 ex

Antibody to epha2 // 2525133

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.

EFFECT: invention can be used in medicine.

74 cl, 14 dwg, 14 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to compounds or their pharmaceutically acceptable salts, where compound has formula 1-a, in which R1 and R3 are absent, m represents integer number from 1 to 2, n represents integer number from 1 to 3, A represents , B represents or , where X2 represents O or S, R4a is absent, R4b is selected from the group, consisting of: , , , , and ; Rk is selected from C1-6alkyl and C1-6halogenalkyl, L and E are such as given in i.1 of the invention formula; or compound is such as given in b) of i.1 of the invention formula. Invention also relates to pharmaceutical composition, which contains said compounds.

EFFECT: compounds by i1, possessing inhibiting activity with respect to anti-apoptosis protein Bcl-XL.

27 cl, 6 dwg, 2 tbl, 126 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel cyclic compounds of general formula which possess properties of CaSR modulator. In general formula I group represents cycloalkyl, which contains 4-7 carbon atoms, optionally substituted with one or several similar or different substituents, selected from R2, R3, R4 or R5; A represents 1-naphthyl; R1 represents methyl, ethyl or n-propyl, each of which is optionally substituted with one or several, similar or different substituents, selected from halogen and hydroxy; R2 and R3 represent hydrogen; R4 represents hydrogen, halogen, hydroxy or C1-6alkyl; each R5 represents independently one or several similar or different substituents, represented by hydrogen or C1-6alkyl; G represents -C(O)NH2, C3-8cycloalkyl, C1-6heterocycloalkyl, C1-6heterocycloalkenyl, C3-8cycloalkenyl, C6-14aryl, C1-10heteroaryl, C6-10arylamino, hydroxyaminocarbonyl, C6-10arylaminocarbonyl, C1-4aminocarbonyl, C1-6heterocycloalkylcarbonyl, C1-10heteroarylaminocarbonyl, C6-10arylsulfonylaminocarbonyl, C6-14aryloxy, or C1-4alkoxycarbonyl, where said substituents are optionally additionally substituted with one or several, similar or different substituents. Other values of radicals are given in the formula of invention.

EFFECT: compounds can be applied in treatment, relief or prevention of physiological disorders or diseases, associated with impairment of activity of CaSR, such as hyperparathyreosis, and other diseases.

23 cl, 9 dwg, 3 tbl, 315 ex

FIELD: medicine.

SUBSTANCE: invention relates to the application of a solid medicinal product, which is heated under the impact of an alternating magnetic field, for further therapeutic treatment after surgical ablation of tumours and cancerous ulcers. The medicinal product represents a surgical implant, presented in the form of a physiologically acceptable fabric, sponge or film. The medicinal product contains magnetic particles, which generate heat when excited by an impact of the alternating magnetic field, and in this way, heat the medicinal product.

EFFECT: invention ensures considerable improvement of further treatment after operation on cancerous tumour in comparison with chemotherapy.

21 cl, 14 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to an agent for treatment of ischemic lesions of tissues, which is a mixture with the ratio of 1.5-3 of two cultures of mesenchymal stem cells, one of which is modified by the genetic structure based on the viral vector which provides hyperproduction of vascular endothelial growth factor, and the other is modified by the genetic structure based on the viral vector which provides hyperproduction of angiopoietin, and a method of treatment of ischemic lesions of tissues by puncture of ischemic tissue, and can be used in medicine.

EFFECT: invention enables to achieve effective vascularisation and repair of ischemic tissue.

4 cl, 4 dwg, 3 ex

FIELD: biotechnology.

SUBSTANCE: method comprises isolation of mononuclear cells (MNC) from peripheral blood of a patient, separation of cells to adherent and non-adherent fractions, addition of the adherent fraction to the MNC of growth factors, loading of the dendritic cell with antigens of tumour lysate in vitro, the stimulation of maturation of dendritic cells for the next day. At that, the obtained immature DCs are added to lysate-autologous tumour cells at a dose of 100 mcg/ml, and after 48 hours within the subsequent 24 hours the rf-tumour necrosis factor-alpha is applied at a dose of 25 ng/ml. Then, the co-culture is carried out of mature dendritic cells activated with lysate and the non-adherent fraction of MNC at a ratio of 1:10 in the presence of recombinant human interleukin-12 at a dose of 10 ng/ml and the recombinant human interleukin-18 at a dose of 100 ng/ml.

EFFECT: invention enables to improve the level of cytotoxic and interferon-producing activity of antigen-activated dendritic cells while reducing the duration of their culture.

4 tbl

FIELD: medicine.

SUBSTANCE: what is presented is a method for stimulating the post-traumatic spinal cord regeneration consisting in a single-stage transplantation of human umbilical cord blood mononuclear cells pre-transduced with recombinant adenovirus with a cloned gene of glial derived neurotrophic factor (gdnf), in the damage area.

EFFECT: using the invention enables providing a better outcome of the post-traumatic spinal cord regeneration, reduced length of staying in hospital of patients suffering from a spinal cord injury and improving the patients' quality of life.

1 ex

FIELD: medicine.

SUBSTANCE: method involves using a photosensitiser (PS) pre-detected in the tumour by its fluorescence that is followed by tumour irradiation by light emission at a wave length in the spectral range of PS peak absorption. The PS is presented by the genetically coded protein KillerRed by providing its tumour expression by gene insertion into the tumour cells. The tumour is exposed to the light emission at energy density of 180-270 J/cm2 3 times every second day or 7 times daily. The protein KillerRed is preferentially localised either in mytochondria and nuclei, or in nuclei.

EFFECT: method provides high-selectivity PS effect on the tumour, reduced toxic load on the intact organs and tissues, the absence of PS redistribution in the tumour, with no need for the constant radiation control.

2 cl, 6 dwg, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.

EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.

3 cl, 6 ex, 1 tbl, 14 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to medicine and veterinary science. What is presented is a biocomposite for performing reparative processes following an injury in a mammal, containing a carrier, at least one nucleic acid containing genes coding VEGF and/or SDF-1, and cells ensuring reparative regeneration. There are presented methods for preparing the above biocomposite and a kit for preparing the same. There are also presented a method for providing injury healing in a mammal and a method for delivering nucleic acid.

EFFECT: presented group of inventions provides effective tissue regeneration following the injury in the mammal by using the three-component biocomposite consisting of the carrier, at least one nucleic acid and the cells ensuring reparative regeneration.

16 cl, 4 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to using microRNA (miRNA) for treating pathological cardiac hypertrophy, myocardial infarction or cardiac failure. The method provides introducing a first anti-sense oligonucleotide containing a sequence that is at least partially complementary to the mature nucleotide sequence miR-208a or miR-208b, and a second anti-sense oligonucleotide containing a sequence that is at least partially complementary to the mature nucleotide sequence miR-499 into an individual suffering the cardiac activity.

EFFECT: combined introduction of the above oligonucleotides ensured by manifesting the synergetic effect enables a more effective decrease of the expression or activity of miR-208a or miR-208b and miR-499 in individual's cardiac cells.

30 cl, 7 dwg, 5 ex

FIELD: biotechnologies.

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

EFFECT: improving compound application efficiency.

31 cl, 12 dwg, 3 tbl

FIELD: biotechnologies.

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

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

19 cl, 25 dwg, 10 ex

FIELD: biotechnologies.

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

EFFECT: poxvirus of variolovaccine has oncolytic activity.

34 cl, 18 dwg, 5 tbl

FIELD: veterinary medicine.

SUBSTANCE: method for production of anti-luteolytic blood - AlB is that luteolisate is administered subcutaneously to gelding twice with an interval of 14 days at a dose of 20 ml each, containing parts of the corpus luteum of pregnancy of cows, and after 14 days after the second administration the blood is taken from the jugular vein. The method of treatment and prevention of persistent corpus luteum, subinvolution of uterus and postpartum endometritis in cows comprises at the background of general therapy the use of anti-luteolytic blood that is administered subcutaneously in the neck region twice at a dose of 10 ml each with an interval of 6 days.

EFFECT: use of anti-luteolytic blood promotes the resorption of the corpus luteum of pregnancy and shortens the duration of postpartum involution of genitals that enables to prevent and reduce the time of treatment of ill cows with persistent corpus luteum, subinvolution of uterus and postpartum endometritis.

2 cl

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