Preventive cancer vaccine

FIELD: medicine, pharmaceutics.

SUBSTANCE: there is described an immunogen for making an immunogenic cancer composition free of DNA-binding function and all domains of a zinc finger, on the basis of polynucleotide coding a nonfunctional mutant form of a related molecule ("brother") of regulator of imprint sites (BORIS) of protein, polypeptide or peptide, containing amino acid sequence presented in the description. The immunogenic cancer composition contains aforementioned immunogen and an adjuvant chosen particularly from cytokine, chemokin, a costimulating molecule. There is described an expression vector containing polynucleotide, coding above-stated protein, e.g., in bacterial systems, mammal systems, in yeast or viral systems. The cancer vaccine under the invention contains polynucleotide (immunogen), additionally the adjuvant and, if necessary, a pharmaceutically acceptable carrier. The invention describes the method for of cancer immunisation of a mammal with using said immunogen on the basis of polynucleotide.

EFFECT: invention allows improving effectiveness of cancer prevention.

28 cl, 7 dwg, 2 tbl, 1 ex



The present invention relates to compositions and methods used to obtain tumor vaccine.


Vertebrates have the ability to generate an immune response in protection from the environment and from aberrant cells, such as tumor cells developing in the body. The immune response is the result of complex interactions of different cell types and factors, but usually includes two main aspects. One of them is a cellular immune response, characterized by the fact that specialized cells directly attack the agent-offender-bearing antigen), while the second is the humoral response, in which molecules are antibodies specifically bind to the antigen and contribute to its elimination. Together, the individual elements very effectively limit the primary attack invading pathogens and distinguish them from the host body.

The primary cells involved in the development of the immune response, are lymphocytes, which in General can be divided into two main classes. Cells of the first of them, called cells or lymphocytes, usually formed in the bone marrow and, among other functions, is responsible for the production and secretion of anti-Christ. ate. Products b-cell antibodies have a tendency to react directly with foreign antigens and neutralize them or activate other components of the immune system, which then eliminates them. In particular, opsonizing antibody binds to the extracellular alien agents, thereby making them susceptible to phagocytosis and subsequent intracellular killing. On the other hand, T cells or T lymphocytes, which are usually formed or Mature in the thymus, which is responsible for mediating cellular immune response. These cells do not recognize the whole antigen, but, instead, react to his short peptide fragments that can be detected on the surface of target cells, and antigen presenting cells. More specifically, it is clear that proteins produced in the cell or captured by the cell from the extracellular space during the normal metabolism continuously broken down to peptides. The resulting short fragments associated with extracellular molecules major histocompatibility complex (MHC) and complexes of MHC-peptide transported onto the cell surface and recognized by T-cells. Thus, the cellular immune system continuously monitors the full range of proteins produced or absorbed by the cells, and forces to eliminate any cells, p is isentirely alien antigens or tumor antigens, i.e. cells, inficirovannye viruses, or cancer cells.

Structure of immunoglobulin G (IgG) is a complex protein tetramer containing two identical heavy (H) chains and two identical light (L) chain immunoglobulin. These chains are joined together by disulfide bonds with the formation of Y-shaped antibodies. However, in solution, the molecule takes the shape of globules and easily associated with foreign antigens present in the biological fluids. Analysis of the amino acid sequences of antibodies resulted in the identification of specific areas with different functional activities within the chain. Each light and each heavy chain contains a variable region (VLand VHrespectively), containing the first 110 amino terminal residues. Three-dimensional plot of the connection regions VLand VHis a plot of recognition "antigennegative customers" ("ACS") of immunoglobulin molecules. Due tetramer nature of the immunoglobulin molecule has two identical antigenspecific site. The variable domains of these circuits contain vysokovitaminnye sequence and report the diversity antigennegative sites that are highly specific in relation to a large number of antigenic structures. Heterogeneity variable domains Nera is nomemo distributed variabeln areas but is in three segments, called hypervariable regions ("CDR"), designated CDR1, CDR2 and CDR3. Read more about these structures, see the monograph Watson et al., 1987, Molecular Biology of the Gene, Forth Edition, Benjamin/Cummings Publishing Co., Inc. Menio Park, Calif.

Each of the heavy chains also includes a constant region that defines a specific isotype and lets be attributed to one of the immunoglobulin classes and subclasses of immunoglobulins. Constant region contains segments called domains (i.e. CH1CH2and so on)that are slightly different in the antibodies of the same class. The constant region is not involved in antigen binding, but may be associated with several biological activities, known as "effector functions", such as binding to Fc receptors on cell surfaces, as well as the protein binding of complement. Antigenpresenting cells, such as dendritic cells and macrophages, among other features, are usually the presence of Fc receptor. Accordingly, if the antibody is associated with a pathogen, it can then communicate with phagocyto through the Fc section. This allows phagocytes to ingest and destroy the pathogen, pathogenic antigens can processionals and identified using ARS for further stimulation of the immune response.

Unlike heavy chains light chains contain a single is the only constant region (C L). Light chain connects to a heavy chain by a disulfide bond, which binds to the constant region of the heavy chain WithNwith CL. In addition, the heavy chain also contain a hinge region separating the constant region (CH1and CH2from the rest of the molecule. This is the hinge region, which is largely responsible for the flexibility of the tetramer. Two heavy chain molecules are linked together by disulfide bonds at a point between the hinge region and CH2.

To create such a broad spectrum, receive immunoglobulin genes so that it was possible to form a large number of different proteins of the immunoglobulin from a limited number of genes, i.e. the internal polymorphism. Due to internal polymorphism mammals are able to produce antibodies, presumably, the infinite variety of antigens. Review on genetics of immunoglobulins and protein structure, see Lewin, "Genes III, John Wiley & Sons, N.Y. (1987) and Benjamin and Leskovitz, 1988, Immunology, Alan R. Liss. Inc., New York.

Over the last few years, antibodies have become extremely important in the diagnosis and therapy due to their diversity and specificity. Methods of molecular biology are increasingly used to expand the diversity and availability of antibodies for research. For example, producing a single antibody In the years can be immortality by merging with a tumor cell and multiplied with the aim of obtaining in vitro source of a single specificity, known as "monoclonal antibody" (mAb). This immortal line of cells called "hybridomas".

Until recently, the source of most of the mAb were mouse hybridoma in vitro. To have the mouse usually were injected with the selected antigen or immunogen. Then the animal was slaughtered and the selected cells of the spleen was merged with immortal myeloma cells. Mouse cells, although they are widely used for diagnostics, not very suitable for use in the treatment of most mammals, including humans. Partly due to the fact that mouse antibodies other mammals are recognized as foreign and cause an immune response, which in itself can cause disease. In order to overcome at least some of the problems associated with the immune response of invasive alien mAb, and the lack of a suitable human mAb to create (design) chimeric molecules gumanitarnogo immunoglobulin, which contain antigennegative hypervariable region of a mouse antibody, while the remainder of the molecule consists of sequences of human antibodies that are not recognized as foreign, using methods of recombinant DNA. Such antibodies are used to treat tumors, as murine variable region recognizes a tumor antigen, and GU is Anisimovna portion of the molecule can mediate the immune response and don't stand out quickly from the body. See, for example, the article by Jones et al., Nature, 321: 522-525 (1986).

Other application of such antibodies is described in detail in published international application WO 94/14847. In these cases, the epitopes of foreign antigens, such as viral or bacterial epitopes, "transplanted" in the hypervariable region of immunoglobulin with the purpose to provoke a response. That is, the generated antibodies are used as vaccines to induce an immune response and to provide long-term immunogenic memory, thereby to facilitate the victory of the subject of future infections.

These and more traditional vaccines are effective because they stimulate both components of the immune system. Despite the difficulties associated with the humoral component of the immune response, it cannot, by its nature, and without connection with other phenomena, effectively protect the animal from a myriad of pathogenic attacks to which it is exposed every day. Rather, only the presence of a strong cellular response allows higher organisms to survive and reproduce.

As indicated above, T lymphocytes, or T cells, which are formed from precursors in the bone marrow, are the main participants in the development of the immune response against damaging viruses and other microbes. Precursor stem cells migrate to the thymus (thymus), where they become special is zirovanii as a so-called thymocytes. In particular, they begin to visualize receptor molecules, which later allow Mature T cells to detect the infection. In order to be useful, T cells must be capable of receptor contact with antigen (protein markers transmit a signal about the presence of the pathogen ("invaders")). At the same time they should not react to substances produced in the body as self-reactive T cells can destroy normal tissue. Usually only those thymocytes, which form a useful receptors, ripen fully and get into the bloodstream, guard the health of the body. Other thymocytes, ineffective or those that attack the body's own tissue, healthy people are eliminated by the mechanism of apoptosis before you leave the thymus.

Mature T cells, which are eventually absorbed into the bloodstream, either in the form of cytolytic T lymphocytes, or T-helper cells, dormant, if they do not meet the antigens that can recognize their receptors. When confronted with a specific antigen, in respect of which the lymphocytes are affinity, they proliferate and perform effector functions, resulting in the elimination of foreign antigens.

T cells are divided into several subpopulations with respect to the different tasks they perform. These sub is opulatio include helper T cells (T hthat are required to stimulate or enhance T - and b-cell response; cytotoxic (or cytolytic) T lymphocytes (CTL), which directly kill their target cells using lysis of cells, and T-suppressor or regulatory T cells (Tsor Tr), which suppress the immune response. In each case, T cells recognize antigens, but only when present at the cell surface in the form of a specialized protein complex associated with the surface of antigen presenting cells. More specifically, T cells use a specific receptor, the so-called specific to the antigen T-cell receptor (TCR), which is a transmembrane protein that can recognize antigen in Association with a group of proteins, in General, called the major histocompatibility complex (MHC, MHC). Thousands of identical TCR expressed on each cell. TCR as a function, and structure, surface antibody (descretionary), In which cells used as antigen receptors. In addition, various subpopulations of T cells also Express a number of cell surface proteins, some of which are called "marker proteins", as they are specific to particular subpopulations. For example, most of the Thcells Express a protein of the cell over the spine of CD4, while most of CTL cells Express the protein on the cell surface CD8, cells and TrExpress molecules CD25 and CD4. These surface proteins play an important role in initiating and maintaining immune responses that depend on recognition of specific proteins or protein complexes on the surface of the ARS or the interaction between these proteins or protein complexes.

Recently it became known that the major histocompatibility complex, or MHC, actually contains a number of glycosylated proteins that have specific Quaternary structure. In General, the patterns fall into two types: class I MHC, which renders the peptides of proteins obtained inside the cell (such as autobake or proteins produced after viral replication), and class II MHC, which generally renders the peptides of the proteins trapped in a cage from the outside, from the side (soluble antigens, such as bacterial toxins). Recognize different antigens is provided with internal polymorphism, which continuously creates a diverse pool of MHC molecules can bind any may be formed of pathogenic peptides. Essentially all nucleated cells produce and Express proteins of class I MHC, which may be natural peptides, peptides associated with the tumor or peptides produced viral zahwah the chick". On the contrary, some other nucleated cells, and among them specialized lymphoid cells, known as antigenpresenting (antigen) cells produce and Express proteins of class II MHC. Regardless of the type of cells, both MHC class are peptides to the cell surface and present them reposing T lymphocytes. Usually Thcells recognize complexes of class II MHC-antigen, whereas CTL tend to recognize complexes of class I MHC-antigen, although it is cross - (cross-) presentation of antigens.

When that rests T cell, bearing the appropriate TCR, meets the gas station, visualizeus peptide on its surface, TCR binds to the complex peptide-MHC. More specifically, hundreds of TCR contact with numerous complexes of peptide-MHC. When contacting a sufficient amount of TCR, the cumulative effect activates T cells. The receptors on T cells that are responsible for specific recognition of the complex of MHC-antigen and the answer is, consist of a complex of several integral proteins of the plasma membrane. As previously discussed MHC complex, diverse pool of TCR is provided with internal polymorphism, leading to the somatic rearrangeable. It should be emphasized that although the pool TCR can be different(a lot)neck, each individual T cell Express the quote only the specific TCR. However, each T cell is usually exhibits thousands of copies of this receptor that is specific for only one peptide on the surface of each cell. In addition, several other types of membrane-bound proteins involved in binding and activation of T cells.

Activation of T cells leads to the appearance of a number of chemical signals (mainly cytokines)that directly cause the cells to act or to stimulate other cells of the immune system. In case of activation of the protein complex of MHC class 1 antigen CTL proliferate and destroy infected cells presenting the same antigen. Killing of infected cells deprives the virus vital support and makes it accessible for antibodies that, finally, they should be removed. In contrast, activation of Thcell protein complexes of MHC class P-antigen does not destroy the antigen presenting cell (which is part of the defense system of the host), but rather stimulates Ththe cell to proliferate and generate signals (again, mainly cytokines)that affect many cells. In addition to these results, the signal transmission causes b-cell stimulation, activation of macrophages, CTL differentiation and stimulation of inflammation. This coordinated response is relatively specific and is aimed at the alien element is s, bearing the peptide represented by the system class II MHC.

Continuous monitoring of epitopes in all of these structures in the body, under the influence of immune control, provides a very effective means of recognizing and preserving "their" and destruction of epitopes and their carriers, affecting the body or causing pathology. With the right action of the immune response causes strikingly effective elimination of microscopic pathogens and neoplastic (tumor) cells, which are believed to occur consistently in the body and, for the most part, are removed by the immune system before they can be detected. Some parts of the body such as the brain, eyes and testicles, protected from immune control, missile defense, it also says that they are immune privilege. Typically the complicated mechanisms of recognition of "his" very effective and contribute to the direction of a strong response to foreign antigens. Unfortunately, the immune system sometimes does not work correctly (denies) and directs its activity against host cells, causing an autoimmune response. Typically, autoimmune reaction occurs when antigen receptors on immune cells recognize specific antigens in healthy cells and cause cell death, supporting these specific substances. In many cases of autoimmune react and are self-limited, as they stop when the underlying antigens disappear. However, in some cases self-reactive lymphocytes are living longer than should have been, and continue to induce apoptosis or otherwise eliminate normal cells.

Recent data show that immune protection against all types of cancer requires the generation of a strong cellular immune response to a unique tumor antigen expressed in cells of malignant tumors. As a result, for a successful immune defense, first, requires a unique antigen expressed in tumor cells (tumor-specific antigen), and, secondly, the induction of potent T-cell immune response aimed at a tumor antigen.

Currently, there are several associated with tumor antigens, they are used in preclinical and clinical studies to produce vaccines. For example, PSMA, PAP and PSA are antigens expressed in tumor cells of the prostate. Her2/neu and MUC1 expressing cancer cells of breast and other cancer cells, including cancer cells of the lung, ovary, colon and pancreas. MAGE and MART-1 are antigens associated with tumor cells of melanoma, and CEA is an antigen associated with the pancreas or the color is the mental cancer. Also described other tone - and/or tumor-specific antigens. However, although these antigens are expressed in tumor cells in normal or aberrant form, they are also expressed in some normal cells and, therefore, cannot be used for preventive vaccination. In other words, these tumor antigens recognized by immune cells as "its" molecules, and therefore is not happening this activation of the immune system. This creates at least two obstacles to the application of those associated with tumors of the molecules as the basis for vaccines. The first obstacle is immunological noticement (tolerance) of the immune system to "its" molecules, which limits its ability to develop a strong cellular immune response. The second difficulty is that any developed strong cellular immune response should not be sent to normal cells that Express the antigen target. For this reason, all discussed above tumor antigens are encouraged to use only as targets for therapeutic vaccination.

Recently described a new protein, which make it possible to overcome the difficulties associated with known tumor antigens. A close relative ("brother") of the regulator of imprinted sites (BORIS) Pervy what was described as a DNA-stasisaudi protein, detected in the testis. This protein contains 11 domains of zinc finger (ZF), in common with CCCNC-binding factor (CTCF), which is a multivalent nuclear factor containing 11 zinc fingers. CTCF is conservative, ubiquitary and highly variable factor, involved in various aspects of gene regulation and forming susceptible to methylation of the insulators that regulate the inactivation of X chromosomes and the expression of imprinted genes. However, BORIS is different from CTCF on N and on the end and is mutually exclusive with CTCF in the process of development of the cells of the embryo is a male. The expression of BORIS limited to the testis, and then only the selected cell subpopulation of spermatocyte, which together with regulation (restoration) label methylation are involved in the process of development of the cells of the embryo is a male. This subpopulation of cells of the testicles is also the only type of normal cells, about which it is known that he never expresses CTCF. Because the inhibition of the expression of CTCF in cultured cells leads to apoptosis, it is reasonable to assume that BORIS is activated, keeping some vital functions BORIS in cells of the testes (Loukinov et al. (2002) Proc. Natl. Acad. Sci. 99(10):6806-6811).

Later it was shown that although overexpression of CTCF also blocks cell proliferation, expression of BORIS in normal BORIS-negatives is positive cells stimulates cell growth, which can lead to transformation (Klenova et al (2002) Cancer Biol. 12: 399-414). Human BORIS mapped in the field 20ql3, which is well-known for frequent conversion and/or amplification observed in many tumors of the same type, which is also frequently observed loss of heterozygosity (LOH) in analogichnyh genes on 16q22 where CTCF. These areas are associated with "hot spots"associated with cancer of the breast, prostate, ovary, stomach, liver, endometrial cancer, malignant glioma, colorectal cancer (colon) cancer, and esophageal cancer, and tumors Wilms ' tumor. It is important that the abnormal activation of BORIS expression is detected in a significant number of a wide range of tumors. Using Northern blotting or RT-PCR, Klenova et al (2002) analyzed the mRNA level of BORIS in more than 200 cell lines, representing most of the major forms of human tumors, and found the transcripts in more than half of the tested cell lines. Subsequent analysis of primary cancer, for a sample of breast cancer, confirmed the results obtained from these cell lines.


The present invention relates to a non-functional mutant polynucleotide, encoding a tumor antigen "brother of the regulator of imprinted sites (BORIS), and the use of such polynucleotides for preventive HAC is inali and immunotherapy of primary or metastatic cancer. Polynucleotide can be either DNA or RNA. In one preferred embodiment of the invention the tumor antigen is a non-functional mutant form of the molecule BORIS, devoid of the ability to bind to DNA. In another preferred embodiment of the invention, at least one domain zinc finger (ZF) is non-functional due to mutations or deletions, and BORIS function is eliminated. In another preferred embodiment of the invention any combination of domains "zinc finger" mutated or deleted, and the function of a protein, polypeptide or peptide BORIS eliminated. In another preferred embodiment of the invention deleted (delegated) all ZF-binding sites. In another preferred embodiment, polynucleotide encoding the mutant form of BORIS, merges with a molecular adjuvant. In another preferred embodiment, polynucleotide that encodes a non-functional mutant form of BORIS, is mixed, at least one excellent polynucleotide coding for molecular adjuvant. You can use any molecular adjuvant, which enhances the cellular immune response. Cytokines, chemokines and molecules costimulation are particularly preferred. Especially preferred chemokines, cytokines and molecules costimulation are beta defensin, IL12, IL18, MIPα3 IFNγ and CD80/86.

The present invention also relates to vectors containing polynucleotide that encodes a non-functional mutant form of BORIS. In a preferred embodiment of the invention the vector directs expression in the system of bacterial cells, mammalian cells, yeast cells or viral system.

In addition, the present invention relates to non-modified (mutant) form of the protein, polypeptide or peptide BORIS. Non-functional mutant can be obtained by any known method which introduces a sequence of deletions, substitutions or additions that result in non-functional protein. In a preferred embodiment of the invention the mutant BORIS protein, polypeptide or peptide is devoid of DNA-binding ability. In another preferred embodiment of the invention the mutant BORIS protein, polypeptide or peptide is mixed with a conventional adjuvant. In another preferred embodiment of the invention nonfunctional mutant BORIS protein, polypeptide or peptide is associated with a pharmaceutically acceptable carrier (frame). In another preferred embodiment of the invention nonfunctional mutant BORIS protein, polypeptide or peptide binds to the peptide, which modifies BORIS and retains its antigenic properties. In another preferred embodiment of the invention nonfunctional Mut is ntny BORIS protein, the polypeptide or peptide binds to the region protein transduction (PTD).

The present invention also relates to dendritic cells expressing the molecule nonfunctional mutant BORIS. In a preferred embodiment of the invention dendritic cells transfairusa using DNA that encodes a molecule mutant BORIS. In another preferred variant of the invention, dendritic cells are infected by a viral vector that encodes a molecule nonfunctional mutant BORIS. In another preferred variant of the invention, dendritic cells "load" nonfunctional mutant BORIS protein, polypeptide or peptide, or any non-modified protein form BORIS.

The present invention encompasses cellular immune responses generated against non-functional mutant form of BORIS protein, polypeptide or peptide, or any non-modified protein forms BORIS. The present invention encompasses antibodies to non-functional mutant form of BORIS protein, polypeptide or peptide, or any modified protein form BORIS.

The present invention also covers preventive or therapeutic vaccine against cancer, containing polynucleotide that encodes a non-functional mutant form of BORIS, nonfunctional mutant BORIS be the ka, polypeptide or peptide, or dendritic cells expressing nonfunctional mutant BORIS molecule.

The present invention also relates to a method for cancer treatment, which consists in the introduction to the patient (who needs preventive vaccine), effective number of polynucleotide encoding a non-functional mutant form of BORIS, non-functional mutant form of BORIS protein, polypeptide or peptide, or dendritic cells expressing or containing a nonfunctional mutant BORIS molecule. The introduction can be performed intramuscular, subcutaneous, intradermal, intravenous, nazalnam, rectal, vaginal or peritoneal way. Cancer can be primary or metastatic cancer. The patient may be several different cancer types. In a preferred embodiment of the invention the cancer is a cancer of the breast, prostate, ovary, stomach, liver, endometrial cancer, malignant glioma, cancer of the colon (colon) cancer or esophageal cancer.


In Figures 1a, b presents the results of vaccination of mice (n=10) using BORIS (immunization DNA) pIL12/IL18 (molecular adjuvant). This leads to the protection of mice from control and infected tumor cells 104T, naturally expressing murine BORIS. In Figure 1A by the C Y shows degree of survival, and on the X-axis delayed days after control of infection for pBORIS/pIL12/IL18, pIL12/IL18 and only one vector. The Figure 1b shows the relationship between tumor volume and the number of days after control of infection tumor for pBORIS/pIL12nL18 compared with pIL12/IL18 and only one vector (*P<0.001).

In the Figure 2A and b shows the relationship between survival and the number of days after infection control tumor cells (a) and between tumor volume and the number of days after infection control for mice vaccinated pBORIS (DNA-immunization), followed Ad5-BORIS (virus-like particles), and infected with 104T cells. The data demonstrate complete protection against infection control tumor cells, at least through 33 days after control of infection.

In Figure 3A, b and c shows the results of immunization of mice pBORIS plus pIFNy or pIL12/IL18 gene gun with subsequent infection control 105tumor cells T. The Figure shown For a long time of tumor growth to a volume of 2 cm3and on the Figure 3b shows a lower degree of tumor growth. Figure 3c shows a significant difference of tumor volume on day 14 between groups pBORIS/pIFNγ compared with the vector (p<0.05), pBORIS/pIL12/IL18 compared with pIL12/IL18 (P<0.05), and pBORIS/pIL12/IL18 compared with the vector (P<0.01).

In Figure 4A, b and c shown in d is ulitity vaccination using pBORIS (DNA-immunization), followed by injection of Ad5-BORIS (virus-like particles). Figure 4A shows a significantly prolonged life expectancy vaccinated mice, whereas Figure 4b shows that in these mice, there is a lower rate of tumor growth and longer tumor growth to a volume of 2 cm3after controlling for the infection of mice with tumor cells T 105. Figure 4C shows a significant difference in the tumor volume on day 15 between groups Ad5-BORIS compared to Ad5 (P<0.001).

The Figure 5 shows the alignment method of the best approximation of the human and murine BORIS polypeptide carried out using the GCG software package, the default extension gaps is assumed to be zero, and conservative area of the zinc fingers selected and listed as ZF-11.

DETAILED description of the INVENTION

Although the present invention can be embodied in many different forms, in this description reveals the specific illustrate variants of the invention, which explain with examples the principles of the invention. It should be emphasized that the present invention is not limited to a specific limiting examples.

The present invention includes the use of an antigen expressed only in immunologically privileged cells of the testis and appearing in many transformed tumor cells, to prevent the Denia effect of tolerance, which can induce other tumor antigens. The invention also includes the introduction of specific changes in the DNA encoding the antigen, for elimination of side effects and autoimmunity. In this context, the following definitions apply.

The term "tumor", "cancer", "tumor", "neoplasia" and their etymological relatives are applied alternately (on equal terms interchangeably) in the context of this application for designation as a whole abnormal proliferative diseases and related diseased cells or cell masses. Preferably, cells with abnormal proliferation relate this description to the antigen with the immune privilege.

Cytotoxic T lymphocytes (CTL) are effector T cells, usually CD8+, which can mediate lysis of target cells bearing antigenic peptides associated with MHC molecule. Other cytotoxic cells include gamma/Delta and CD4+ NK1.1+ cells.

The expression "antigen with the immune privilege" and "immunologically privileged antigen" refers to the allocation of specific sites and antigens in the body of the immune system and, therefore, associated with the antigens against which produces an abnormal immune response. Antigens with immune privilege, expressed ectopiceski (i.e. outside their usual what about the immunologically privileged sites), can cause autoimmunity or immunity to tumors. Immunological preferred antigens are expressed in some tumors, which leads to the development of immune response against tumor and non-tumor site, expressing the same immunological preferred antigens.

Antigenpresenting cells (APC) are cells, including dendritic cells, macrophages and b cells, which can processional and present (present) antigenic peptides in conjunction with MHC molecules of class I or class II and to deliver co-stimulating signal required to activate T cells.

Scope (domain) zinc finger" ("zinc fingers") refers to small independently folding ("vyvetrivayasj") domain, which is to stabilize its structure necessary coordination with one or more zinc ions. The fingers contact the subsites of three base pairs, and specific contacts mediated by the amino acids at positions -1, 2, 3 and 6 relative to the beginning of the alpha-helix.

"Nonfunctional mutant BORIS form" refers to BORIS protein, polypeptide or peptide with loss of function. "Loss of function" is supposed to denote the inability to carry out any of the important activities of the molecules of wild-type BORIS, such as DNA-binding, restoration of "paternal" ("parents is skogo") of the pattern of DNA methylation, etc.

"Non-functional mutant" refers to changes in the level of DNA or protein that disrupt the activity of the wild-type protein obtained. Such changes may include amino acid substitutions, deletions or additions in the areas of molecules that behave as catalytic sites and/or participate in DNA binding or protein. Examples of changes that can break (destroy) activity are deletions or substitutions of important amino acids involved in the catalytic or binding interaction, adding amino acids that alter the required three-dimensional structure of the website involved in catalytic and/or binding interactions, or additions or deletions of nucleotides that cause shifts the reading frame, thereby disrupting the necessary three-dimensional structure. Mutations can be performed in a normal molekulyarnymi methods, such as PCR, using oligonucleotides and so on (see, for example, Sambrook, Maniatis and Fritsch). Natural mutations also can be isolated from a population of cells (see, for example, Sambrook, Maniatis and Fritsch).

"Peptide" refers to a molecule containing at least 2 amino acids connected by peptide bond. "Polypeptide" refers to a molecule containing at least 10 amino acids connected by peptide bonds, and "protein" refers to a molecule that contains, hence, is her least 20 amino acids.

"Polynucleotide that encodes a nonfunctional mutant BORIS form" refers to any polynucleotide, the sequence of which at least 50%, 60% or 70% identical to the sequences of human (SEQ ID NO: 1) or mouse (SEQ ID NO: 3) polynucleotide BORIS, more preferably, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences of human (SEQ ID NO: 1) or mouse (SEQ ID NO: 3) BORIS polynucleotide.

"Nonfunctional mutant BORIS peptide, polypeptide or protein" refers to a molecule BORIS, who are deprived of the possibility to carry out any of the important activities of the molecules of wild-type BORIS, such as DNA-binding, restoration of "paternal" ("parent") of the pattern of DNA methylation, etc. "Nonfunctional mutant BORIS peptide, polypeptide or protein has a sequence that is at least 50%, 60% or 70% identical to the sequences of human (SEQ ID NO: 2) or mouse (SEQ ID NO: 4) peptide, polypeptide or protein BORIS, more preferably, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences of human (SEQ ID NO: 2) or mouse (SEQ ID NO: 4) peptide, polypeptide or protein BORIS.

Molecule nonfunctional mutant BORIS is recognized as "foreign" antigen expressed only in transformed tumor cells, and used as antigen to overcome the Ogre is Iceni previous prior art. The mutant form of BORIS used as an ideal non-toxic vaccine, as it should not cause any unwanted side effects caused by its activity DNA binding and/or native function. In other words, mutant BORIS used for vaccination, has no functional activity and is present only as an immunogen (antigen). Unlike other tumor-specific antigens, BORIS is not expressed in normal tissues in women. In addition, even though BORIS is expressed during puberty in normal testis in men, the introduction and/or expression of nonfunctional mutant BORIS should be harmless, because the testis is an immunologically privileged organ (not available for immune cells). In other words, anti-BORIS the immune response generated after immunization, is not dangerous for normal cells and vaccine BORIS does not cause autoimmunity. In addition, it is guaranteed the establishment of a powerful immune response, as BORIS, in contrast to other tumor-specific antigens recognized as a foreign antigen. BORIS-specific T cells are not destroyed in the thymus, recognize mutant BORIS as "foreign" antigen and produce an immune response.

In one embodiment of the invention cDNA encoding a murine BORIS (mBORIS), obtained by the method of RT-PCR on mRNA, vyd the Lenna of murine or human testis. DNA-binding domain of the molecule is delegated and replaced with a small spacer, which is known that it works well when creating antibodies, single-chain Fv domains. The correct sequence is confirmed by automated analysis of the nucleotide sequence. In the resulting molecule is missing 11 ZF domains, and it consists of the N-terminal region m BORIS (amino acids 1-258)associated with the C-terminal region (amino acids 573-636) via a spacer containing 18 amino acids.

Mutant cDNA clone in the vector pORF under the control of the promoter hEF1-HTLV, but you can use other expression vectors. Mutant cDNA functionally associated with the promoter and/or regulyatornymi molecules capable of inducing the expression in the cell host. You can use viral vectors, including vectors α viral DNA or RNA, adenoviruses and retroviruses (see Vasilevko, V., et al. (2003) Clin. Exp. Metastas. 20:489-98; Leitner, W.W. et al. (2003) Nat Med 9:33-39; Ribas, A et al (2002) Curr. Gene Ther 2: 57-58).

In addition to the above, the invention encompasses the use of virus-like particles, molecules encoding nonfunctional mutant BORIS, such as adenovirus, the virus human hepatitis b virus human hepatitis C virus, vaccinia virus, poliovirus, etc. Recombinant viral proteins of different viruses have the useful property of self-Assembly in viruspool the s particles (the VLP). These particles do not contain viral nucleic acids and therefore are dereplication, non-communicable and retain conformationally correct antigenic epitopes. Producing the VLP shown in many experimental systems, such as mammalian cells infected with the baculovirus insect cells, yeast, E. coli cell-free system and transgenic plants. It is important to note that vaccination with the VLP induces the production of not only humoral but also cellular immune responses. The VLP infect "professional" APCS, and then induce protective cellular immune response, including CD4+Th1 type CD4+T cells help cells CD8+T) and CD8+CTL responses. Thus, the VLP clearly showed exceptional ability to activate cellular immune response (T-cell response). A possible application of the VLP as a preventive vaccines currently checked in a number of different clinical trials. The results of these tests are encouraging due to the excellent tolerability and high immunogenicity reported in these studies. Getting the VLP vaccine, consisting of a portable BORIS antigen will stimulate strong cellular immune response against cancer cells expressing this tumor antigen. Cow antigen (HBcAg) virus heat is that In (HBV) and VSV are examples of suitable the VLP.

For more strong (stable) cellular immune response truncated or mutant mBORIS before cloning into the vector combine with molecular adjuvants, such as molecules costimulatory V7 beta-defensin 2/3, MIP3α, IFNγ, cytokines, chemokines, etc. Other suitable molecular adjuvants are listed in the following Table 1.

Or for DNA immunization can use conventional adjuvants, such as Tween 80, 5% ethanol or bupivacaine. Other examples of traditional adjuvants include mineral salts (such as gels, aluminum hydroxide or aluminum phosphate), oil emulsions and compositions on the basis of surface-active substances, such as MF59, QS21, AS08 [SBAS2] (oil emulsion in water+MPL+QS21), Montanide ISA-51 and ISA-720, adjuvants of particulates, such as virosome, as04 asset [SBAS4] Sol A1 with MPL), ISCOMS, a copolymer of lactide with glycolide (PLG), compounds of microbial origin (natural and synthetic), including monophosphorylated And (MPL), Detox (MPL+M.Phlei of cell wall skeleton), AGP [RC-529], DC_Chol, OM-174 (a derivative of lipid A, CpG motifs, modified LT and CT (genetically modified bacterial toxins), endogenous immunomodulators, such as GM-CSP, IL-12, immutably, as well as all other chemokines, cytokines and co-stimulating molecules listed in the Table above, and inert n the bearers, such as gold particles.

Adjuvants can be mixed with polynucleotides coding for non-functional mutant form of the protein, polypeptide or peptide family member of the regulator of imprinted sites (BORIS), nonfunctional mutant BORIS protein, polypeptide or peptide and dendritic cell expressing nonfunctional mutant BORIS peptide, polypeptide or protein.

To raise/promotion presentation nonfunctional mutant BORIS "professional" antigen presenting cells (APC) metabolic pathway of MHC class design nonfunctional mutant BORIS, you can include additional peptide molecules. One such example is the design that is created by transducible peptide domain (PTD). In General, the immune response relies on the processing and presentation of native antigen. Tumor antigens can be expressed in bacteria, yeast and mammals, however, the protein antigens expressed in these systems, apparently, does not stimulate the maximum T-cell immune response (responses tend to either CTL or Th1 responses), as soluble exogenous proteins are processed mainly using metabolic pathway of MHC class II. In fact, many cancer vaccines based on the induction of CD8+CTL, but it usually is required that the protein was synthesized in the cytosol of the APC. Unfortunately, in General the plasma membrane of eukaryotic cells impervious to most proteins. Recently it was shown that the foreign proteins, United with protein-traduzioni domain (PTD), can penetrate through the plasma membrane, which allows to accumulate the protein inside the cells. This improves the presentation of foreign peptides molecules MHC class II APC antigenspecific CD8+ T cells.


Preparations (formulations, compositions) vaccines to the present invention contain immunogenic amount of polynucleotide encoding a non-functional mutant form of the protein, polypeptide or peptide BORIS, nonfunctional mutant BORIS protein, polypeptide or peptide, or dendritic cells expressing nonfunctional mutant BORIS peptide, polypeptide or protein, in combination with a pharmaceutically acceptable carrier. You can use mimotope, which are polypeptides with an unrelated sequence, but the three-dimensional structure corresponding to the nonfunctional mutant BORIS protein, polypeptide or peptide, and which function in an identical manner. Mimetype that represent any biological molecule, an unrelated structure BORIS, but have identical(e) 3x-d(e) is pitop(s), can be recognized by anti-BORIS cells.

"Immunogenic amount" is an amount of polynucleotide encoding nonfunctional mutant BORIS protein, polypeptide or peptide; nonfunctional mutant BORIS protein, polypeptide or peptide; or dendritic cells expressing nonfunctional mutant BORIS protein, polypeptide or peptide, sufficient to induce an immune response in the subject, which is the vaccine given. The quantity entered is a number, which induces a given immune response and the desired degree of protection. Examples of pharmaceutically acceptable carriers include, but without limitation, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution.

The vaccine formulations of the present invention is suitable for patients diagnosed with at least one type of cancer, including, but without limitation, breast cancer, prostate, ovarian, stomach, liver, endometrial cancer, malignant glioma, cancer of the colon (colon) cancer or esophageal cancer. Formulations of vaccines according to this invention is also suitable for patients with a known genetic predisposition to cancer. In addition, the vaccine formulations of the present invention is suitable for the entire population, including representatives of the population, not breusegem or without a genetic predisposition to cancer, those wishing to seek protection from contact with at least one type of cancer, which is expressed BORIS protein, polypeptide or peptide.

The vaccine can be accomplished by any suitable means, including parenteral injection (such as intraperitoneally, subcutaneous or intramuscular injection, intradermal, intravenous injection, nasal, rectal, vaginal application or introduction through the respiratory tract. Topical application of the virus for insertion through the respiratory tract can be accomplished using intranasal (e.g., using a pipette, swab (torontoca) or nebulizer, which is introduced into the nose pharmaceutical drug). Topical application of the virus to introduction into the respiratory tract can also be entered by inhalation, for example, creating a pharmaceutical preparation in the form of respirable particles (including both solid particles and liquid particles)containing the replicon, and then prompting the subject to breathe respirable particles. Methods and apparatus for the introduction of respirable particles of pharmaceutical preparations are well known and can be applied to any conventional method. "Immunogenic amount" is an amount of replicon particles, sufficient to induce an immune response in the subject, which is the vaccine given.

If the qualities of the vaccines use RNA or DNA RNA and DNA can be entered directly by methods known to experts in the art, such as delivery on the gold granules (gene gun), delivery using liposomes or direct injection. One or more design or replicate RNA can be used in any combination, effectively causing immunogenic response in the subject. Generally, the amount of nucleic acids in the input vaccine may be enough to induce a given immune response and the desired degree of protection. The exact number of injected vaccines may depend on the resolution of the practitioner and can be individualized for each subject and antigen.

The vaccine can be administered according to the scheme, providing a single dose, or preferably, a scheme providing for repeated reception, when the primary course of vaccination may consist of 1-10 separate doses, and then, through successive time intervals give other doses with the aim of preserving or enhancing the immune response, for example, 1-4 months for a second dose and, if necessary, after blowing(s) dose(s) after several months. Examples of appropriate immunization schemes include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schemes that are suitable for the detection of defined immune responses, presumably, providing the x protective immunity or reduce the symptoms of the disease or reduce disease severity.

Human hBORIS can be isolated from human testis and manipulate it to him. Similarly, BORIS can be distinguished from testicular any mammal or vertebrate and use a similar manner.


1. Obtaining a plasmid encoding ZF-delegated form molecules mBORIS under the control of the promoter hEF1-HTLV

The reaction RT-PCR carried out using RNA poly-And mouse testis and the following primers:







Conditions of PCR as follows: 94°C 30 sec, 60°C 30 sec, 72°C 2 min Spend thirty (30) cycles.

The PCR products subcloning in the cloning vector PCRII-TOPO (Invitrogen). C-terminal cDNA in PCRII-TOPO subjected to restriction analysis with the enzyme BamHI, a positive clones containing the insert, assemble and then cut using enzyme HindIII. Primers (SpF and SpR) for amplification of the spacer annealed receive protruding sticky ends and are ligated into a vector cut with BamHI-HindIII. N-terminal of the encoded fragment is cut using HindIII and inserts (inserts), now separated from the vector gather and are ligated into the design, cut with HindIII containing s-end and the spacer. Then the clones with the proper orientation selected, sec is enrout (for example, see below the sequence of the molecule ZF-deletirovanie BORIS) and subcloning in plasmid pORF under the control of the promoter hEF1-HTLV (Invitrogen).

Cells SNO transferout using the design using standard methods of molecular genetics (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor).

Expression design mBORIS without zinc fingers (with tick marks zinc fingers) analyze Northern-blotting of mRNA isolated from cells SNO standard method of molecular genetics Sambrook et al. 1989).

2. Immunization of mice with DNA encoding ZF-depletirovannoi form molecules BORIS

A plasmid encoding ZF-depletirovannoi design mBORIS, allocate, using the kit EndoFree Plasmid maxi (Qiagen). The purity of the plasmid DNA to confirm the UV range (the ratio of optical densities at 260 nm/280 nm >1.7) and gel electrophoresis.

Gold granules cover DNA (1 mg/0.5 mg gold) and 5-7-week-old Balb/c mice subjected to immunization by gene gun Helios. Mice subjected to immunization (fired) in a similar way three times in two weeks. Ten days after the last "fire" in mice selected a sample of blood and infect by introducing 1.0×104or 1.0×105cells T breast cancer. Tumor size was measured every day or every two or three days with calipers.

3. Immune studies

Immunization of mice plasmas is ne vaccine:

One example of a method with the use of prophylactic cancer vaccines is the use of DNA that encodes a delegated form molecules of murine BORIS, in which no domain zinc finger, and therefore no property DNA-binding.

Purified plasmid used for coating of gold granules (2 µg plasmid / 0.5 mg of gold granules), as described previously (Ghochikyan et al. (2003) Eur. J. Immunol 33:3232-41). Immunization of mice BALB/C carried out through the shaved skin of the abdomen using a gene gun Helios (Bio-Rad, Hercules, CA), as described by Ross et al. (2000, Nat. Immunol 1: 127-131). Briefly, mice bombard 3 times the dose contains 2 micrograms of DNA per 0.5 mg of gold granules with the size of ~1 µm (DeGussa-Huls Corp., Ridefield Park, NJ) under a helium pressure of 400 psi (2758 kPa). Mice subjected to immunization and enrich (fired) by the same method every two weeks and infect control two different doses of breast cancer cells (105or 104) ten days after the last enrichment, as described (Vasilevko, V., et al. (2003). Different groups of mice subjected to immunization with plasmid that encodes a modified BORIS, mixed with DNA coding opredelennyi (e) molecular(e), adjuvant(s) (see Table 2). It is known that such molecular adjuvants enhance the cellular immune response to various antigens.

Table 2
Mice subjected to immunization every two weeks, a total of five times, using a preventive vaccine, BORIS (pORF-mBORIS)mixed with pORF-mGMCSF (encoding mouse mGMCSF), pORF-mIFNγ (encoding mouse mIFNγ) or pORF-mIL12+pIRES-mIL18 (two plasmids encoding murine IL12 and IL18, respectively). After the last firing of the mice infected with the help of 105or 104cells T breast cancer mice, which Express a modified BORIS
GroupImmunogenMolecular adjuvantControl infected cells 4T1
5- pIL12/IL18105

Getting adenoviral vector encoding ZF - depletirovannoi form molecules of murine BORIS (Ad5-BORIS), and immunization of mice

Recombinant virus Ad5-BORIS receive using adenoviral vector system AdEasy XL from Stratagene. Shuttle vector design by sublimemovies fragment of ZF-deletirovanie mBORIS in plasmid pShuttle-CMV. For this purpose, the fragment BORIS synthesized by PCR using as template a plasmid pORF-mBORIS and the following primers:



The PCR product subcloning in the cloning vector PCR4-TOPO (Invitrogen). Fragment BORIS cut using restrictase SalI and NotI. The resulting product was then purified on agarose gel and subcloning using the cloning sites SalI-NotI, vector pShuttle-CMV.

Expression in vitro ZF-deletirovanie mBORIS analyze in cells SNO by Western blot turns (see Figure 6).

Shuttle vector, n the things deleteriously BORIS, linearized with Pmel and purified on an agarose gel. Electrocompetent cells BJ-5183-Ad-l transform using the plasmid pShuttle-mBORIS, split using Pme receive recombinant Ad plasmid. Cells AD-293 transferout using DNA selected recombinant Ad-BORIS and prepare the primary source of virus solutions. Received primary viral solution (10 PFU/ml) amplified in the cells of the AD-293, and then purified in CsCl-gradient. Purified virus cialiswhat against PBS - 5% sucrose and used for immunization of mice.

Mice Balb/c mice immunized with pBORIS four times in two weeks, once injected V.M. booster injection of Ad5-BORIS (109The COMBAT). Control animals injected vectors and spend booster immunization with Ad5. Ten days after the last booster injection to control infection with two different doses of cells T breast cancer (105or 104), as described (Vasilevko, V., et al. (2003).

Lines of tumor cells

Use mammalian cancer cells, provided by Dr. F. Miller (Karmanos Cancer Institute, Detroit, MI). Cells T are resistant to Tg variant derived from 410.4 cells (cell line mammals, originally isolated from a single spontaneously grown a mammalian tumor in BALB/c mice fC3H) without affecting mutagenic cultured (37°C, 10% CO2in a basic environment, Needle, modified by way of Dulbecco (DMEM)with low glucose content and with the addition of 5% fetal bovine serum, 5% serum of newborn calves, 2 mm glutamine, 100 Units/ml penicillin, 100 mg/ml streptomycin, 0.1 mm nonessential amino acids and 1 mm sodium pyruvate (D10) (Life Technologies, Inc.).

Determination of tumor volume

The volume of tumors determine daily, making measurements in two directions and calculating according to the formula L×(W2)/2, where L represents the length, and W represents the width of the tumor. Experiments end with the death of a mouse or upon reaching a tumor size of about 1.5 cm3in the experiments, including the control of infection with 104cells, and 2 cm3in the experiments, including the control of infection with 105cells T.

Formation time (latent period) is denoted as the time before, when the tumor reached a volume of more than 0.1 cm3. To determine the growth rate of tumors analyze the dispersion curves in the periods almost linear growth of the tumor.

Statistical analysis

The results obtained in the determination of the mean time of appearance of the tumor nodules (latent period) and tumor growth (tumor volume), and the time of post-vaccination immunity, study method of analysis of variants (ANOVA) and post-test use Cree is ERI Tukey multiple comparisons. The mean and standard deviation (deviation) (SD) calculated using the software GraphPad Prism 3.0.

4. Immunology

B - and T-cell immune response against BORIS analyzed using two different immunization Protocol.

1. Getting murine BORIS protein and immunization of mice.

ZF-deleteriously fragment of murine BORIS subcloning in bacterial expressing vector pET24d(+), using NcoI - XhoI cloning sites in reading frame with the C-terminal 6His "tail". Enter both the site and remove the stop codon at the stage of cloning PCR. In addition, create plasmids encoding the molecule delegated BORIS, merged with domain protein transduction (PTD). Domain protein transduction with HIV-Tat (Tat47-57YGRKKRRQRRR) connect the N-end of delegated BORIS PCR, and then clone into the cloning sites NcoI-XhoI vector pET24d(+). The strain E. coli BL21(DE3), transformed using the obtained plasmid pET-mBORIS or pET-TATmBORIS, grown in LB with kanamycin at 28°C to achieve And6000.8. Protein synthesis induce by adding IPTG to a final concentration of 1 mm. Cells are harvested in three to five hours by centrifugation and used for protein purification by affinity chromatography on a column with Ni-NTA (nitrilotriacetic acid) (Qiagen) (see Figure 7).

Protein is analyzed by the method 10% SDS-PAGE. Some data (not shown) receive applied with the eat BORIS, United with PTD.

In addition, ZF-deleteriously fragment BORIS, merged with PTD, subcloning in expressing the yeast vector of pGAPaa in reading frame with the signal sequence into the cloning sites EcoRI-XbaI. Both sites injected, and the initiating ATG codon is removed during stage PCR cloning. Strain Pichia pastoris X33 transformed by electroporation with pGAPZ-BORIS, linearized using restrictase AvrII and positive clones selected on YPD medium containing 100 μg/ml Zeocin. For analysis of expression of selected clones grown in broth YPD/Zeocin and analyzed in the supernatant at different time points by Western blot turns.

2. Immunization of mice dendritic cells (DC).

Primary DC bone marrow derived from precursor cells in the bone marrow of mice as follows. Depleted erythrocytes of bone marrow cells from the femur and tibia bones are seeded in complete medium RPMI-10 with the addition of recombinant murine GM-CSF (100 U/ml). On day 3 is not attached to the substrate granulocytes carefully remove and add fresh medium. Not attached to the substrate DC harvested on day 7 and purified using the set for positive selection (Miltenyi) microspheres CD11c.

DC collected on day 7 of cultivation, clear positive selection and infect with Ad5-BORIS, incubare at a density of cells 107 cells/ml in RPMI-1640 PR the multiplicity of infection 1000-2000. After 1 hour of complete medium was added to dilute the DC to a final concentration of 1×106-2×106cells/ml After 24 hours, cells are harvested intensively washed to eliminate any transfer of adenoviral particles, and used for immunization. In addition, DC, which are collected at day 7 of cultivation and purified by positive selection, incubated with 10 μg/ml ZF-delegated protein mBORIS at 37°C, 5% CO2within 24 hours, washed twice by PBS. The absorption of protein by the cells DC analyze the aliquot by flow cytometry using antibodies against murine BORIS and corresponding secondary antibodies labeled with FITC. Balb/c mice subjected to immunization I.P. (IPR) three times every three weeks with 106DC and T-cell response analyzed 10 days after the last injection in culture (booster) culture of splenocytes.

5. Results

The results of immunization presented in Figures 1-4.

DNA encoding the mutant form of specific in relation to cancer cells murine BORIS antigen without DNA-binding function (deletion of the 11-zinc finger), create using expressing vectors mammals pORF (hivivogen) and adenoviral vector system AdEasy XL (Stratagene). These vaccines are used as prophylactic cancer vaccine in a murine model of breast cancer. For this model using the BALB/C mice (haplotype H-2d) and native line neoplastic mammalian cells T, which is resistant to Tg variant, formed from 410.4 cells without mutagenic treatment. It is important to note that these mouse breast cancer cells Express full-sized molecule murine BORIS, as was demonstrated by the method of RT-PCR. Therefore, there is an ideal model to study the ability of molecules to BORIS used as immune vaccines against cancer.

Conduct two different types of experiments. The first type of experiments includes the group of mice vaccinated pBORIS (a plasmid that encodes a delegated molecule murine BORIS, mixed with a DNA that encodes a different mouse cytokines (pGM-CSF; pIL12/IL18; pIFNγ) as a molecular adjuvant. Mice injected with the vector (pORF) or pIL12/IL18 as a control. Mice subjected to immunization and spend booster immunization by gene gun, and then conduct control infection by 104or 105cells T.

The second type of experiment involves the group of mice vaccinated with pBORIS, buffer immunization, which carried replication defective adenoviruses vector (Ad5), which is modified in such a way as to Express the ZF-depletirovannoi molecule murine BORIS (Ad5-BORIS). As a control using the group of mice injected with vector with subsequent booster immunization with the aid of the d Ad5. Control infected animals performed using 104or 105cells T and analyze the emergence and growth of the tumor. Note, it was previously found that the injection of such a small quantity as 104cells T in the mammary glands of BALB/c mice leads to local growth of breast tumors in 100% of animals with infection control.

Vaccination with pBORIS plus pIL12/IL18 or pBORIS followed Ad5-BORIS leads to the protection of mice from control and infected with 104unmodified T tumor cells. Although 50% of mice in the group immunized with pBORIS mixed with pIL12/IL18, formed small tumors (0.2-0.4 cm3), they are all viable on day 39. All mice died about ten days earlier.

The results obtained in mice immunized with Ad5-BORIS, are outstanding. On day 24, when the mouse in the control group died from tumors, 100% of mice immunized with Ad5 vaccine-BORIS, were not only alive, but they have not formed tumors. Actually they have not formed a tumor until day 33 after control of infection. These results show that the vaccine ZF-deletirovanie BORIS effectively protects with the introduction of 104tumor cells of the breast.

The second series of experiments conducted under more severe conditions and with control infected mice 10 5T tumor cells. Vaccination with plasmid pBORIS plus pIFNγ or pIL12/IL18 significantly prolongs the time of tumor growth to a volume of 2 cm3and increases the survival rate of BALB/c mice. Vaccination also reduces the rate of tumor growth in the control of infection with 105tumor cells T. A more profound effect find in mice vaccinated with pBORIS with subsequent booster immunization with Ad5 vaccine-BORIS before infection with 105unmodified cells T. In this case, on day 23, when all mice in the control group died as a result of tumor growth, 80% of mice immunized with Ad5 vaccine-BORIS remain alive and in surviving mice tumors was significantly smaller.

Separate groups of BALB/c mice subjected to immunization delegated mouse BORIS protein, purified from E. coli system. In this case, five mice subcutaneously injected protein (50 μg/mouse)mixed with conventional adjuvant type Quil And Th1 (Sigma). After four immunization all animals were observed significant titers of antibodies against BORIS. Another group of 5 mice simultaneously subjected to immunization administered intraperitoneally selected dendritic cells infected with Ad5 vaccine-BORIS. After three injections in mice produces T-cell response against mBORIS, which is found in vitro in the culture of splenocytes, activated protein mBORIS. SL is therefore immunization with BORIS induces b - and T-cell immune response in mice and the immune response protects the animal from the control of infection.

6. Truncated BORIS associated with PTD. as a subunit vaccine

PTD is associated with a non-functional truncated or mutant BORIS protein, and the product of the merger of forms of yeast expressing the system. The genes encoding the PTD and nonfunctional mutant BORIS, subcloning in yeast expressing vector, such as pGAPZα. Expressed and secreted protein purified by standard methods of molecular genetics. Mice subjected to immunization with antigen prepared in two different normal adjuvant, and analyze the immune response and protection against tumor antigen.

7. Virus-like particles encoding nonfunctional mutant BORIS, as a subunit vaccine

Get a subunit vaccine the VLP-BORIS-based measles antigen (HBcAg), hepatitis b virus (HBV). This antigen samsobeats" in the VLP after expression in yeast cells. Alien sequence can be embedded in some areas HBcAg, without disturbing the Assembly process. Accordingly, receive particles chimeric HBcAg-BORIS, which is used for immunization of mice.

8. The analysis in BALB/c mice and p53 KO

Analyze the immune response in BALB/c mice without infection and young mice with knockout of p53 gene, the cat is older at this age is not a tumor. Define both humoral and cellular immune response in mice immunized with different vaccines BORIS. Analyze the serum of immunized mice to detect the production of antibodies against BORIS during the 3-month experiment. Determine the proliferation of CD4+ and CD8+ T cells and activation of regulatory T cells before and after infection of BALB/c mice. At the same time analyze the activation of NK cells, which can directly induce killing of tumor cells of the breast. Demonstrated functional activity of BORIS-specific cytotoxic T-lymphocytes (CTL) before and after exposure of the tumor cells of the breast T. For detection of NK and CTL activity as target cells using tumor cells R, which naturally (in nature) Express molecules BORIS wild-type, along with T cells.

Additional references

Filippova, G.N. et al. Tumor-associated Zinc Finger Mutations in the CTCF Factor reduced Selectively Alter Its DNA - binding Specificity. Cancer Research 62: 48-52 (2002).

Kim, J.J. et al. In vivo engineering of a cellular immune response by co-administration of IL-12 expression vector with a DNA immunogen. J. Immunol 158: 816-826 (1997).

Kim, J.J. et al. CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines. Journal of Clinical Investigation 102: 1112-24(1998).

Kim, J.J. et al. Modulation of amplitude and direction of n vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens. European Journal of Immunology 28: 1089-1103. (1998).

Kim, J.J. et al. Intracellular adhesion molecule-1 modulates beta-chemokines nd directly costimulates T cells in vivo. Journal of Clinical Investigation 103: 869-77 (1999).

Kim, J.J. et al. Macrophage Colony-Stimulating Factor Can Modulate Immune Responses and Attract Dendritic Cells in Vivo. Human Gene Therapy 11: 305-321 (2000).

Lutz, M.V. et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. Immunol. Meth. 223: 77-92 (1999).

Nardelli, B. & Tam, J.P. The MAP system. A flexible and unambiguous vaccine design of branched peptides. Pharm. Biotechnol. 6: 803-819 (1995).

Resko, J.E.J. et al. Cell Growth Inhibition by the reduced Multivalent Factor CTCF. Cancer Research 61: 6002-7 (2001).

Ribas, A., Butterfield, L.H., Glaspy, J.A. & Economou, J.S. Cancer Immunotherapy Using Gene - modified Dendritic Cells. Curr. Gene Ther 2; 57-78 (2002).

Ross, R., Xu, Y., Bright, R.A. & Robinson, H.L. C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge. Nat. Immunol. 1, 127-131(2000).

Smith, M., Burchell, J.M., Graham, R., E.P., C. & J., T.-P. Expression of B7.1 in a MUC1-expressing mouse mammary epithelial tumor cell line inhibits tumorigenicity but does not induce autoimmunity in MUC1 transgenic mice. Immunol. 97, 648-655 (1999).

An ordinary person skilled in the art it is obvious that you can use various modifications of the materials and methods in the application of the above-described invention. Such modifications may be considered as included in the scope of the present invention defined by the following claims.

1. Immunogen designed for production of immunogenic anticancer composition containing polynucleotide that encodes a non-functional mutant form of the relative ("brother") of the regulator of imprinted sites (BORIS protein, polypeptide or peptide that contains the sequence of SEQ ID NO:2, lacking the function of the DNA-binding and all domains of zinc finger.

2. The immunogen according to claim 1, characterized in that the BORIS protein, polypeptide or peptide attached to a pharmaceutically acceptable carrier.

3. The immunogen according to claim 1, characterized in that the BORIS protein, polypeptide or peptide is associated with a domain, a protein transduction.

4. The immunogen according to claim 1, characterized in that the BORIS protein, polypeptide or peptide is linked to the peptide, which modifies BORIS and maintains or increases the antigenicity of the specified BORIS protein, polypeptide or peptide.

5. Immunogenic anticancer composition comprising the immunogen according to any one of claims 1 to 4 and adjuvant.

6. The composition according to claim 5, in which the immunogen is mixed or merged with adjuvant.

7. The composition according to claim 6, in which the adjuvant is chosen from the group consisting of cytokine, chemokine and to stimuliruuschey molecule.

8. Vector expresii containing polynucleotide according to any one of claims 1 to 4.

9. The vector of claim 8, which allows for expression in bacterial systems, systems, mammalian, yeast or viral systems.

10. The vector of claim 8, which is a virus.

11. A host cell transformed by polynucleotide according to any one of claims 1 to 4 or a vector according to any one of p-10 expressing non-functional mutant form of the relative ("brother") of the regulator of imprinted sites (BORIS protein, polypeptide or peptide.

12. A host cell of claim 10, which represents a bacterial cell, the cell is mammalian or yeast cell.

13. A vaccine against cancer, containing polynucleotide according to claim 1.

14. The vaccine according to item 13, additionally containing adjuvant.

15. The vaccine according to item 13, further containing a pharmaceutically acceptable carrier.

16. The method of immunization of a mammal against cancer, which consists in the introduction to the needy in this patient an effective amount of the immunogen according to any one of paragraphs 1-4.

17. The method according to clause 16, in which polynucleotide mixed or combined with a molecular adjuvant.

18. The method according to 17, in which the molecular adjuvant is a molecule that increases cellular immune response and/or antibody-based test or answers.

19. The method according to p, in which the molecular adjuvant chosen from the group comprised the soup of molecules of any cytokine, the chemokine and co-stimulating molecules.

20. The method according to clause 16, in which polynucleotide mixed with any conventional adjuvant.

21. The method according to clause 16, in which the introduction is carried out intramuscularly, subcutaneously, intradermal, intravenous, nasal, rectal, vaginal or peritoneal.

22. The method according to clause 16, in which the patient has more than one type of cancer.

23. The method according to clause 16, in which the patient has cancer.

24. The method according to clause 16, in which the patient does not have cancer.

25. The method according to clause 16, in which the patient does not have cancer, but there is a genetic predisposition to cancer.

26. The method according to clause 16, in which the patient is not sick detectable cancer, but wants to protect itself from possible malignancy.

27. The method according to clause 16, in which immunization is a consequence developed a cellular immune response involving T-cells that recognize the epitope nonfunctional mutant BORIS protein, peptide or polypeptide.

28. The use of the immunogen according to claim 1 for the manufacture of a vaccine against cancer.


Same patents:

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is offered molecule of nucleic acid inducing CEA immune response, containing a nucleotide sequence that codes a fused protein on a basis of carcinoembryonal antigen (CEA) or its functional version fused with a subunit B of thermolabile enterotoxin E coli. There are described versions thereof, as well as the related purified protein. There is disclosed an expression vector containing said molecule of nucleic acid, and a host-cell containing specified vector. There are described adenoviral vaccinal vector for inducing the immune response and a vaccinal plasmid on the basis of the specified molecule.

EFFECT: application of the invention allows to inducing the immune response in a mammal which is stronger, than that induced with natural CEA that can find application in medicine for cancer treatment.

20 cl, 62 dwg, 20 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology, specifically, to separation and identification of new genes of spiramycins biosynthesis track and to new polypeptides, which participate in this biosynthesis, and may be used to produce acyltransferase, which is responsible for modification of platenolid in position 3. Polynucleotide coding acyltransferase, which is responsible for modification of platenolid in position 3, cells of bacterium Streptomyces type are transformed, and strain-producer of end polypeptide is made.

EFFECT: increased extent of production and purity of produced spiramycin.

26 cl, 41 dwg, 44 tbl, 31 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology and immunology. Separated and cleaned DNA is presented, which codes receptor CTLA-4 (CD 152) of cat. The following is also suggested - diagnostic oligonucleotide, cloning vector, vaccine, methods of induction, strengthening and suppression of immune response in cat.

EFFECT: creation of model cat for research of retroviral infection.

24 cl, 10 dwg, 6 tbl, 8 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.

EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.

10 cl, 26 dwg, 2 tbl, 22 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.

EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.

35 cl, 13 dwg, 36 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.

16 cl, 3 tbl, 7 ex

FIELD: food industry.

SUBSTANCE: strain Streptococcus thermophilus which produces lactic acid is described. Sequence of nucleic acids made of the strain producing polysaccharides are also described as well as food or pharmaceutical composition and milk product containing such strain.

EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of genetic engineering and medicine. Described is animal, non-human, having sequence of nucleic acid encoding presenilin 1, carrying mutations, corresponding to M233T and L235P mutations in PS1 protein of mouse. Animal also contains sequence of nucleic acid, encoding whole gene or part of gene, encoding APP. APP protein represents APP751, originates from human and carries mutations Swedish and London. Animal is intended for application in fight against Alzheimer's disease. Also described are PS1 protein and encoding it nucleic acid.

EFFECT: invention can be used in medicine for discovering compounds intended for Alzheimer's disease treatment.

20 cl, 50 dwg, 1 tbl, 8 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to production of versions of the Gla domain of human factor VII or human factor VIIa, and can be used in medicine. Amino acid sequence of the FVII or FVIIa version is obtained, which differs on 1 to 15 amino acid residues with amino acid sequence of the human factor VII (hFVII) or human factor VIIa (hFVIIa), in which a negatively charged amino acid residue is introduced by substitution in position 36. Obtained variants of FVII or FVIIa are used in a composition for treating mammals with diseases or disorders, where blood clotting is desirable.

EFFECT: invention allows for producing versions of FVII or FVIIa with high clotting activity and/or high activation of factor X, compared to natural form of hFVIIa.

42 cl, 3 dwg, 5 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: cardiac hystiocytes are proliferated by inducing of cyclin and CDK expression in cardiac hystiocytes and by inhibiting function or activity of protein of Cip/Kip family or by inhibiting production of protein of Cip/Kip family. There is described expression vector that contains a cyclin gene, a cyclin-dependent kinase gene and one or several agents chosen from the group consisting of a gene coding the factor which inhibits production, function or activity of protein of Cip/Kip family and nucleic acid sequence which inhibits production of protein of Cip/Kip family. There is disclosed pharmaceutical composition containing said vector, and applied for treatment of cardiac diseases. There is offered cardiac hystiocyte produced by the declared method. There is presented method of treating a cardiac disease that implies injection of the disclosed pharmaceutical composition.

EFFECT: regeneration and recovery of cardiac hystiocytes.

31 cl, 13 dwg, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention can be used in manufacturing of vaccines for Streptococcus pyogenes - streptococci of group A (SGA) and Streptococcus agalactiae - streptococci of group B (SGB). Substance of the invention involves development of recombinant DNA pB1 derived from PCR with using chromosomal DNA of strain 090R Ia of serotype SGB, primers Pb1 and Pb2 and following cloning with using expression plasmid pQE-30 in E coli M15. Recombinant DNA pB1 codes recombinant protein PB1 expressing protective properties in relation to specified streptococci which has no enzymatic activity and causes synthesis of anti-Pb1 antibodies expressing protective properties in relation to Streptococcus pyogenes and Streptococcus agalactiae. In the invention there is developed recombinant plasmid DNA pQE-pB1 representing plasmid DNA pQE-30 that bears recombinant DNA pB1, and strain-producer E. coli M15-PB1 enabling to express recombinant protein PB1.

EFFECT: no enzymatic activity of produced recombinant protein allows application as an ingredient of the vaccine for Streptococcus pyogenes and Streptococcus agalactiae.

7 cl, 7 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention is related to the field of medicine and is related to treatment of proliferative diseases with application of antisense oligomer IAP and chemotherapeutical preparation. Substance of invention includes method for treatment of patient suffering from proliferative disease with application of antisense oligomer SEQ ID NO: 151 or its pharmaceutically acceptable salt and chemotherapeutical preparation.

EFFECT: invention advantage consists in improved efficiency of treatment.

28 cl, 15 ex, 9 tbl, 25 dwg

FIELD: medicine.

SUBSTANCE: method of cultivation of pluripotential stem cells herewith conserving undifferentiated condition and pluripotency thereof without using feeder cells. The method applies fluid medium and culture bottle on the surface of which there are immobilised molecule of fused protein containing outer domain of E-cadherin and Fc-region of immunoglobulin.

EFFECT: invention can be used in regenerative medicine.

9 cl, 11 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: method involves deactivation of definite VGC2 DNA sequence of Salmonella typhimurium positioned between ydhE and pykF genes or its part containing at least 50 nucleotides, or the DNA version of at least 85% identity, representing VGC2 DNA of any microbe out of Salmonella aberdeen, Salmonella gallinarum, Salmonella cubana and Salmonella typhi.

EFFECT: obtainment of microbe with reduced adaptability to specific environmental conditions.

6 cl, 12 dwg, 8 ex

FIELD: genetic engineering.

SUBSTANCE: invention can be used in monocotyledon plants selection for creation of novel sorts and hybrids by means of genetic engineering, in works insertional mutagenesis, separating and cloning of plant genes. In order to obtain transgenic monocotyledon plants in period of their active blooming flowers lacking own fertile pollen are selected. As object of genetic transformation, blooming female gametophyte is used, which is processed with suspension of strain Agrobacterium tumefaciens with activated vir-genes, pistil filaments being processed directly. After that said flowers are pollinated with pollen of fertile plants. For processing cells with strain Agrobacterium pistil filament sections, located near flower ovary, are used.

EFFECT: said operations allow to create transgenic monocotyledon plants preserving high frequency of their obtaining under conditions, that correspond to natural temperatures of blooming, and to simplify technology of obtaining transgenic plants.

4 cl, 1 dwg, 1 tbl

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns biotechnology. It is described bispecific antibody which binds also the factor of blood coagulation IX or the activated factor of blood coagulation IX, and the factor of blood coagulation X, and functionally replaces the factor of blood coagulation VIII or the activated factor of blood coagulation VIII which strengthens enzymatic reaction. The pharmaceutical composition containing the described antibody is revealed. The present invention can be used as an alternative agent for functional replacement of cofactor which strengthens enzymatic reaction.

EFFECT: creation of bispecific antibody which can replace functional proteins, strengthens enzymatic reaction.

14 cl, 18 dwg, 37 ex

FIELD: biotechnology, organic chemistry, biochemistry.

SUBSTANCE: invention represents a novel method of preparing optically active 4-(indole-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG) used in preparing monatine, and a method for synthesis of optically active monatine. Also, invention relates to a novel aldolase used in these methods. 4-(Indole-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid of high optical purity representing effective intermediate compound for synthesis of optically active monatine can be synthesized from indolpyruvic acid and pyruvic acid (or oxalacetic acid). Invention provides preparing 4-(indole-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid and monatine with high degree of effectiveness.

EFFECT: improved preparing method.

18 cl, 12 dwg, 12 tbl, 25 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to new aldolase which catalyzes reaction of producing substituted alpha-ketoacid from oxalacetic or racemic pyrotartaric acid and indole-3-racemic pyrotartaric acid.

EFFECT: method for production of substituted alpha-ketoacids with increased effectiveness.

FIELD: gene engineering, in particular yeast strain modified by introducing of foreign genetic material.

SUBSTANCE: claimed strain is obtained by transforming of starting culture Pichia pastoris X-33 with two albumin structural genes with signals of yeast alpha-factor secretion, transcribed under control of 5'AOX1 promoter and transcription termination region of hepatitis G virus. Strain of present invention is useful in production of albumin-containing drugs.

EFFECT: strain for production of human recombinant albumin of increased yield.

3 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a kit for an immunogenic composition, and an immunogenic composition which contains a polynucleotide adjuvant composition together with an antigen (for example as a vaccine). The disclosed adjuvant compositions (versions) have special physical properties (for example molecular mass, concentration and pH), which meet requirements for a safe adjuvant for inducing intense immune response. The invention also relates to a method of preparing an immunogenic composition and using immunogenic adjuvant compositions, particularly for inducing immune response to an antigenic compound.

EFFECT: disclosed adjuvant compositions are safe and efficiently provide the desired immunogenic effect in humans.

25 cl, 2 dwg, 15 tbl, 16 ex