Immunoprophylactic cancer vaccine
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to biotechnology and represents an immunogenic composition for preventing and treating cancer diseases, which contains the non-functional BORIS protein, a sequence of which is free from the zinc finger protein. The present invention also discloses an immunotherapeutic cancer composition containing the above non-functional BORIS protein or a bacterial, mammalian or yeast cell, or a viral particle able to express the above non-functional BORIS protein. The present invention also discloses a method for immunising a patient by administering an effective amount of the above immunotherapeutic composition, as well as using the above immunotherapeutic composition for preparing the cancer vaccine.
EFFECT: invention enables increasing the efficacy of the immunoprophylactic and therapeutic cancer vaccine.
22 cl, 7 dwg, 2 tbl, 8 ex
The SCOPE TO WHICH the INVENTION RELATES
The present invention relates to compositions and methods used to obtain tumor vaccine.
Background of the INVENTION
Vertebrates have the ability to produce an immune response as 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 cells and factors, but typically includes two main aspects. One of them is a cellular immune response, characterized by the fact that specialized cells directly attack agent-violator (bearing antigen), while the second one is the humoral response, in which the antibody molecule binds specifically to the antigen and contribute to its elimination. Working together, the individual elements very effectively limit the primary attack invading pathogens and excrete them from the body of the host.
The primary cells involved in the development of the immune response are lymphocytes, which in General are divided into two basic classes. The cells of the first of them, called cells or lymphocytes, usually formed in the bone marrow and, among other functions, responsible for the production and secretion of anti�ate. Products b-cell antibodies tend to react directly with foreign antigens and neutralize them or activate other components of the immune system, which then eliminates them. In particular, opsonizing antibodies bind to the extracellular alien agents, thus 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, are responsible for mediating cellular immune response. These cells do not recognize whole antigens 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 obvious that proteins produced in a cell or captured by the cell from the extracellular space under normal metabolism continuously are degraded to peptides. The resulting short fragments associated with extracellular molecules of major histocompatibility complex (MHC) complexes and MHC-peptide is transferred to the surface of cells and are recognized by T-cells. Thus, cellular immune system continuously monitors the full spectrum of proteins produced or absorbed by the cells and forces to eliminate any cells, p�esentire alien antigens or tumor antigens, i.e., cells infected by viruses or cancer cells.
Structure of immunoglobulin G (IgG) is a complex tetrameric protein containing two identical heavy (H) chains and two identical light (L) chains of immunoglobulin. These chains are joined together by disulfide bonds with the formation of Y-shaped antibodies. However, in solution the molecule adopts the shape of globules and easily associated with foreign antigens present in biological fluids. Analysis of amino acid sequences of immunoglobulins resulted in the identification of specific areas with different functional activities within the chains. Each light and each heavy chain contains a variable region (VLand VH, respectively), containing the first 110 amino terminal residues. Three-dimensional plot of the connection regions VLand VHis a plot of recognition "antigen-binding site" ("ACS") of immunoglobulin molecule. Due to the tetrameric nature of immunoglobulins in the molecule has two identical antigen-binding site. The variable domains of these chains contain vysokovitaminnye sequence and report the diversity of antigen-binding sites that are highly specific in relation to a large number of antigenic structures. The heterogeneity variable domains Nera�dimensional distributed variable regions, but is located 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 comprises a constant region that defines a specific isotype and classification of the immunoglobulin to one of the classes and subclasses of immunoglobulins. The constant region contains segments called domains (i.e. CH1, CH2etc.), which differ slightly in 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 the cell surface, and binding of complement proteins. Antigen-presenting cells such as dendritic cells and macrophages, among other features, are characterized usually by the presence of Fc receptor. Accordingly, if the antibody is linked to a pathogen, it can then communicate with phagocytes via Fc plot. This allows the phagocytes to ingest and destroy the pathogen, pathogenic antigens can processionals and be detected using APC for further stimulation of the immune response.
Unlike heavy chains light chains contain a common�public constant region (C L). Light chain is connected to a heavy chain by disulfide bond that binds the constant region of the heavy chain WithNwith CL. In addition, the heavy chain also contains a hinge region that separates the constant region (CH1and CH 2from the rest of the molecule. This is the hinge region, which is largely responsible for the flexibility of the tetramer. Two heavy chain molecules linked together by disulfide bonds at a point between the hinge region and CH2.
To create such an extensive range, receive immunoglobulin genes so that it was possible to form a large number of different immunoglobulin proteins from a limited number of genes, i.e. the internal polymorphism. Due to the internal polymorphism mammals are capable of producing antibodies, presumably, an infinite variety of antigens. The review of genetics of immunoglobulins and structure of proteins, 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 scientific research. For example, a single antibody producing � cell may be immortality by fusion with a tumor cell and multiplied with the aim of obtaining in vitro sources of single specificity, known as a "monoclonal antibody" (mAb). This immortal line of cells is called a "hybridoma".
Until recently, the source of most of the MABs were of murine hybridomas cultured in vitro. To eat, the mouse is usually injected with a selected antigen or immunogen. Then the animal was sacrificed and the isolated cells of the spleen were fused with immortal myeloma cells. Murine 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 murine antibodies in other mammals are recognized as foreign and provoke an immune response, which itself can cause disease. In order to overcome at least some of the problems associated with the immune response caused by alien mAb, and the lack of a suitable human mAb, to create (design) chimeric molecules, humanized immunoglobulin, which contain the antigen-binding hypervariable region of murine antibody, while the remainder of the molecule consists of sequences of human antibodies that are not recognized as alien, use the methods of recombinant DNA. Such antibodies are used for the treatment of tumors, as murine variable region recognizes a tumor antigen, and g�monitorowania plot the molecules are able to mediate an immune response and not to stand out quickly from the body. See, for example, the article Jones et al., Nature, 321: 522-525 (1986).
Other uses 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 to induce a response. We have created antibodies are used as a vaccine to induce an immune response and provide long-term immune memory, thereby to facilitate the victory of the subject on future infections.
These and more traditional vaccines are effective because they stimulate both components of the immune system. Despite the difficulties associated with humoral component of the immune response, it cannot, by its nature and without connection with other phenomena, effectively protect the animal from the myriad of pathogenic attacks to which it is exposed to 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 primary actors in creating an immune response against damaging viruses and other microbes. Precursor stem cells migrate to the thymus (thymus gland), where they become specialists�zirovanii as a so-called thymocytes. In particular, they begin to visualize receptor molecules that later allow Mature T cells to detect infection. To be useful, T cells must be able by using the receptor to bind to the antigens (protein markers convey a signal about the presence of the pathogen ("invaders")). At the same time, they should not respond to substances produced in the body, as autoreactive T cells can destroy normal tissue. Usually only those thymocytes that form helpful the buds ripen fully and enter the bloodstream, are sentinels for the health of the body. Other thymocytes, inefficient, or those that attack its own tissue, in healthy people are eliminated through apoptosis before you leave the thymus.
Mature T cells that eventually enter the bloodstream, either in the form of cytolytic T lymphocytes or T-helper cells, resting, if they do not meet the epitopes can recognize their receptors. When confronted with the specific antigen against which the lymphocytes show an affinity, they proliferate and carry out effector functions, resulting in the elimination of foreign antigens.
T cells are divided into several subpopulations with regard to the various tasks they perform. These sub�opulatio include helper T cells (T h), which are necessary for the stimulation or enhancement of T - and b-cell response; cytotoxic (or cytolytic) T lymphocytes (CTL) that directly kill their target cells through cell lysis, and T-suppressor or regulatory T cells (Tsor Tr) that 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, generally called major histocompatibility complex (MHC, GCG). Thousands of identical TCR expressed on each cell. TCR refers, both in function and in structure to surface antibody (descretionary), In which cells are used as antigen receptors. In addition, different subpopulations of T cells also Express a number of cell surface proteins, some of which are called "marker proteins", as they are characteristic for specific subpopulations. For example, most of Thcells Express the protein cell on top�spine CD4, while the majority of CTL cells Express cell surface protein CD 8, and cells of the 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 APC or interaction between these proteins or protein complexes.
Recently it became known that major histocompatibility complex, or MHC, actually contains a number of glycosylated proteins that have specific Quaternary structure. Generally patterns are of two types: class I MHC, which visualize peptides proteins found inside the cell (such as autobake or proteins produced after viral replication), and class II MHC, which generally visualize peptides of the proteins trapped in the cage from the outside, from outside (soluble antigens, such as bacterial toxins). Recognition of different antigens is provided with internal polymorphism, which continuously creates a diverse pool of MHC molecules capable of binding any which can be formed of pathogenic peptides. Essentially all nucleated cells produce and Express proteins of class I MHC, which may be natural peptides, the peptides associated with the tumor, or peptides, produced viral capture�ICOM". On the contrary, some other nucleated cells, and among them specialized lymphoid cells known as antigen-presenting (antigen presenting) cells produce and Express proteins class II MHC. Regardless of the type of cells, both classes of MHC are peptides to the cell surface and present them to resting T lymphocytes. Usually Thcells recognize complexes of MHC class II-antigen, whereas CTL tend to recognize complexes of MHC class I-antigen, although it also occurs cross - (cross-) presentation of antigens.
When resting T cell, bearing the appropriate TCR, meets the gas station, visualizeus peptide on its surface, the TCR binds to the complex peptide-MHC. More specifically, hundreds of TCR contact with numerous complexes of peptide-MHC. When sufficient contact TCR, the cumulative effect activates T cells. The receptors on T cells responsible for the specific recognition of the complex of MHC-antigen and for answering it consists of a complex of several integral proteins of the plasma membrane. As previously discussed MHC complex, diverse pool of TCR polymorphism is provided with internal, resulting in the somatic rearrangeable. It should be emphasized that although the pool of TCR may be different(a lot)neck, each individual T cell Express�ruet only specific TCR. However, each T cell typically exhibits thousands of copies of this receptor, 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.
The activation of T cells leads to the appearance of a number of chemical signals (mainly cytokines) that directly force the cells to act or to stimulate the action of other immune system cells. In case of activation of the complex proteins of MHC class 1 antigen CTL proliferate and destroy the infected cells presenting the same antigen. Killing infected cells deprives the virus of the vital support and makes it accessible to antibodies, which are finally removed. In contrast, activation of Thcells protein complexes of MHC class P-antigen does not destroy the antigen presenting cell (which is part of the protective system of the host), but, rather, stimulates Ththe cell to proliferate and to 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 concerted response is relatively specific and is aimed at alien elements�s, bearing peptide provided by class II MHC.
Continuous monitoring of epitopes in all of these structures in the organism under the influence of immune system control, provides effective ways to recognize and save "his" and destruction of epitopes and their media is affecting the body or causing pathology. With proper action, the immune response causes strikingly effective elimination of microscopic pathogens and neoplastic (tumor) cells, which are believed to occur continuously 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 testes, protected from immune control, these areas also States that they possess immune privilege. Usually complex mechanisms for the recognition of "its" very effective and help towards the strong response to foreign antigens. Unfortunately, the immune system sometimes works incorrectly (or refuses) and directs its activity against host cells, triggering an autoimmune response. Usually, autoimmune response occurs when the antigen receptors on immune cells recognize specific antigens on healthy cells and causes the death of cells that carry these specific substances. In many cases, an autoimmune response, which will�and are self-limiting, as they cease when the cause of their antigens disappear. However, in some cases autoreactive lymphocytes are living longer than should have been, and continue to induce apoptosis or otherwise eliminate normal cells.
The latest data show that the immune protection against all types of cancer requires the generation of strong cellular immune response to a unique tumor antigen expressed in cells of malignant tumors. As a result of a successful immune defense, first, require a unique antigen expressed in tumor cells (tumor-specific antigen), and, secondly, the induction of potent T-cell immune response targeting tumor antigen.
Currently there are several associated with tumor antigens, they are used in preclinical and clinical studies to obtain vaccines. For example, PSMA, PAP and PSA are antigens expressed in tumor cells of the prostate. Her2/neu and MUC1, expressed by cancer cells of breast and other cancer cells, including cells of lung cancer, ovarian, colon and pancreas. MAGE and MART-1 antigens are associated with tumor melanoma cells, and CEA is an antigen that is associated with the pancreas or the color�talinum cancer. Also describe other tissue - and/or tumor-specific antigens. However, although these antigens expressed in tumor cells in normal or aberrant form, they are also expressed in several 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 molecules as the basis for the vaccine. The first obstacle is immunological noticement (tolerance) of the immune system to "its" molecules, which limits its ability to produce a potent cellular immune response. The second difficulty is that any developed powerful cellular immune response must not be sent on 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 will help overcome the difficulties associated with known tumor antigens. A close relative ("brother") controller imprinted sites (BORIS) W� has been described as a DNA-binding protein detected in testis. This protein contains 11 domains, zinc fingers (ZF), shared with CCCNC-binding factor (CTCF), which is a multivalent nuclear factor containing 11 zinc fingers. CTCF is a conservative, ubiquitous and highly variable factor involved in various aspects of gene regulation and constitutive susceptible to methylation of the insulators that regulate the inactivation of the X chromosome and the expression of imprinted genes. However, BORIS is different from CTCF on N and on With the end and expressed mutually exclusive with CTCF in the process of development of the embryo cells of the male sex. The expression of BORIS is limited to the testicle, and then only the selected cell subpopulation of spermatocytes, which together with regulation (restoration) label methylation are involved in the process of development of the embryo cells of the male sex. This subpopulation of cells of the testicles is also the only type of normal cells, which is known that he expresses CTCF. Since inhibition of CTCF expression in cultured cells leads to apoptosis, it is reasonable to assume that BORIS is activated, keeping some vital features BORIS cells in the testes (Loukinov et al. (2002) Proc. Natl. Acad. Sci. 99 (10): 6806-6811).
Later it was shown that although CTCF overexpression also inhibits cell proliferation, expression of BORIS in normal BORIS-not�exploring cells stimulates cell growth which can lead to transformation (Klenova et al. (2002) Cancer Biol. 12: 399-414). Human BORIS mapped in the region 20q13, 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 is located. These areas are associated with "hot spots" associated with cancer of the breast, prostate, ovarian, stomach, liver, endometrial cancer, malignant glioma, cancer of the colon (colon) colon and esophageal cancer, and tumors of Wilma. It is important that abnormal activation of the expression of BORIS detected in a significant number of a wide range of tumors. Using Northern-blotting or RT-PCR, Klenova et al. (2002) analysed the levels of BORIS mRNA in more than 200 cell lines representing the most basic forms of human tumors, and found the transcripts in more than half of the tested cell lines. Subsequent analysis of primary cancer, for sample of breast cancer, confirmed the results obtained with these cell lines.
Summary of the INVENTION
The present invention relates to a non-functional mutated polynucleotides encoding a tumor antigen (brother of the regulator imprinted sites (BORIS), and the use of such polynucleotides for preventive VA�Canazei and immunotherapy of primary or metastatic cancer. The polynucleotide may 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 of the zinc finger (ZF) is non-functional due to mutations or deletions, and the function of BORIS eliminated. In another preferred embodiment of the invention any combination of domains "zinc finger" is mutated or deleted, and the function of a protein, polypeptide or peptide BORIS eliminated. In another preferred embodiment of the invention removed (delegated) all ZF-binding sites. In another preferred embodiment, the polynucleotide encoding the mutant form BORIS, merges with molecular adjuvant. In another preferred embodiment, the polynucleotide encoding a nonfunctional mutant form of BORIS, is mixed at least with one great polynucleotide, encoding a molecular adjuvant. You can use any molecular adjuvant that 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 the polynucleotide encoding a nonfunctional 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 in viral system.
In addition, the present invention relates to non-functional
modified (mutant) form of the protein, polypeptide or peptide BORIS. Non-functional mutant can be obtained by any known method which implements the sequence deletions, substitutions or additions that results in nonfunctional protein. In a preferred embodiment of the invention mutant BORIS protein, polypeptide or peptide is devoid of DNA-binding ability. In another preferred embodiment of the invention 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 associated with a peptide that modifies BORIS and retains its antigenic properties. In another preferred embodiment of the invention is nonfunctional� mutant BORIS protein, the polypeptide or peptide binds to a region of a protein transduction (PTD).
The present invention also relates to dendritic cells expressing a molecule nonfunctional mutant BORIS. In a preferred embodiment of the invention dendritic cells transfairusa when using a DNA molecule encoding a mutant BORIS. In another preferred embodiment of the invention, dendritic cells are infected with a viral vector that encodes a molecule nonfunctional mutant BORIS. In another preferred embodiment of the invention dendritic cells "load" nonfunctional mutant BORIS protein, polypeptide or peptide, or any retrofit protein non-functional form of BORIS.
The present invention encompasses cellular immune responses generated against the non-functional mutant form of the BORIS protein, polypeptide or peptide, or any non-functional modified protein forms BORIS. The present invention encompasses antibodies to non-functional mutant form of the BORIS protein, polypeptide or peptide, or any modified protein form BORIS.
The present invention also encompasses a prophylactic or therapeutic vaccine against cancer, containing the polynucleotide encoding a nonfunctional mutant form of BORIS, a nonfunctional mutant BORIS baie�ka, polypeptide or peptide, or a dendritic cell expressing a nonfunctional mutant BORIS molecule.
The present invention also relates to a method of treating cancer, comprising administration to a patient (in need of prophylactic vaccines), an effective amount of a polynucleotide encoding a nonfunctional mutant form of BORIS, non-functional forms of mutant BORIS protein, polypeptide or peptide, or dendritic cells expressing or containing a nonfunctional mutant BORIS molecule. The introduction can be carried out intramuscular, subcutaneous, intradermal, intravenous, nazalnam, rectal, vaginal or peritoneal method. Cancer can be a primary or metastatic cancer. The patient can be several different cancer types. In a preferred embodiment of the invention cancer is a cancer of the breast, prostate, ovarian, stomach, liver, endometrial cancer, malignant glioma, cancer of the colon (colon) of the colon or esophageal cancer.
BRIEF description of the DRAWINGS
In the Figures 1a, b presents the results of immunization of mice (n=10) with BORIS (DNA immunization) pIL12/IL18 (molecular adjuvant). This leads to the protection of mice from infection control tumor cells 104T, naturally expressing murine BORIS. In Figure 1A by �C Y shows degree of survival, and on the X-axis deposited days after control of infection for pBORIS/pIL12/IL18, pIL12/IL18 and only one vector. Figure 1b shows the relationship between tumor volume and the number of days after control of infection tumor for pBORIS/pIL12/IL18 compared to pIL12/IL18 and only one vector (*P<0.001).
Figure 2A and b shows the relationship between the percentage of survivors and the number of days after control of infection by tumor cells and between tumor volume and the number of days after control of infection in mice vaccinated with pBORIS (DNA immunization), followed Ad5-BORIS (virus-like particles), and infected with 104T cells. Data demonstrate complete protection against infection control tumor cells after at least 33 days after control of infection.
Figure 3A, b and C show the results of immunization of mice pBORIS plus pIFNγ or pIL12/IL18 using a gene gun with the following control infection 105tumor cells T. Figure 3A shows the a long time of tumor growth to a volume of 2 cm3and Figure 3b shows a lower degree of tumor growth. Figure 3C shows a significant difference of tumor volume at day 14 between groups pBORIS/pIFNγ compared with the vector (p<0.05), pBORIS/pIL12/IL18 compared to pIL12/IL18 (P0.05), and pBORIS/pIL12/IL18 compared with the vector (P<0.01).
Figure 4A, b and R shown�the results of vaccination with pBORIS (DNA immunization), followed by injection of Ad5-BORIS (virus-like particles). Figure 4A shows a significantly prolonged lifespan vaccinated mice, whereas in Figure 4b it is shown that in these mice is observed a lower tumor growth rate and a longer time of tumor growth to a volume of 2 cm3after control of infection of mice with tumor cells T 105. Figure 4 shows a significant difference in tumor volume on day 15 between groups Ad5-BORIS compared to Ad5 (P<0.001).
Figure 5 shows the linear best approximation of the human and murine BORIS polypeptides carried out using the GCG software package, by default, the extension gap is equal to zero, and conservative region of the zinc fingers selected and indicated as ZF-11.
DETAILED description of the INVENTION
Although the present invention may be embodied in many different forms, herein disclosed illustrating specific embodiments of the invention, which explain with examples the principles of the invention. It should be emphasized that the present invention is not limited to specific limiting examples.
The present invention involves the use of antigen expressed only in immunologically privileged cells of the testis and appears in many of the transformed tumor cells, for the openings�Denia effect of tolerance, which can induce other tumor antigens. The present invention also includes the introduction of specific changes in DNA encoding the antigen, elimination of side effects and autoimmune reactions. In this context, the following definitions apply.
The term "tumor", "cancer", "neoplasm", "neoplasia" and its etymological relatives are used alternately (on equal terms interchangeably) in the context of this application to denote, in General, abnormal proliferative diseases and related affected cells or cell masses. Preferably, cells with abnormal proliferation relate this description to the antigen with immune privilege.
Cytotoxic T lymphocytes (CTL) are the effector T cells are usually CD8+, which can mediate lysis of target cells bearing antigenic peptides associated with the MHC molecule. Other cytotoxic cells include gamma/Delta and CD4+ NK.1.1+ cells.
The expression "antigen with immune privilege" and "immune privileged antigen" refer to the allocation of specific sites and antigens in the body from the immune system and therefore are associated with antigens in respect of which produces an abnormal immune response. Antigens with the immune privilege of a murine ectopiceski (i.e. outside their Oba�but immunologically privileged sites) can cause autoimmunity or immune system to the tumor. Immunologically privileged antigens expressed in certain tumors, which leads to development of an immune response against tumor and non-tumor site expressing the same immunological privileged antigens.
Antigen-presenting cells (APC) represent cells, including dendritic cells, macrophages and b cells, which can processional and to presentation (to present) antigenic peptides in conjunction with MHC molecules of class I or class II and deliver the costimulatory signal required to activate T cells.
"The scope (domain) zinc finger" ("zinc fingers") refers to small, independently folding ("vydelyaemye") domain, which for the stabilization of its structure is necessary to coordinate one or more zinc ions. Fingers contact the subsites of three base pairs, and specific contacts mediated by amino acids at positions - 1, 2, 3 and 6 relative to the beginning of the alpha helix.
"Non-functional mutant form of BORIS" refers to BORIS protein, polypeptide or peptide with a loss of function. "Loss of function" intends to denote the inability to realize any of the important activities of wild-type molecules BORIS, such as DNA binding, restoration of the "paternal" ("bear�raising") 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 represent amino acid substitutions, deletions or additions in the areas of molecules that behave as catalytic sites and/or participating in the binding of DNA or protein. Examples of changes that can disrupt (destroy) activity are deletions or substitutions of important amino acids involved in catalytic or binding interaction, addition of amino acids that alter the necessary three-dimensional structure of the site that is involved in catalytic and/or binding interactions, or add or division of nucleotides that cause shifts in the reading frame, thus violating the necessary three-dimensional structure. Mutations will be by standard molecular methods such as PCR, using oligonucleotides, etc. (see, e.g., Sambrook, Maniatis and Fritsch). Natural mutations could also be identified from a population of cells (see, e.g., Sambrook, Maniatis and Fritsch).
"Peptide" refers to a molecule containing at least 2 amino acids, connected by peptide bonds. "Polypeptide" refers to a molecule containing at least 10 amino acids joined together with peptide bonds, and "protein" refers to a molecule containing, for men�her least 20 amino acids.
"The polynucleotide encoding a nonfunctional mutant form of BORIS" refers to any polynucleotide, the sequence of which at least 50%, 60% or 70% identical to the human sequence (SEQ ID NO:1) or mouse (SEQ ID NO:3) of the polynucleotide BORIS, preferably 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the human sequence (SEQ ID NO:1) or mouse (SEQ ID NO:3) the polynucleotide BORIS.
"Nonfunctional mutant BORIS peptide, polypeptide or protein" refers to a molecule BORIS, which is prevented from exercising any of the important activities of wild-type molecules BORIS, such as DNA binding, restoration of the "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 human sequence (SEQ ID NO:2) or mouse (SEQ ID NO:4) peptide, polypeptide or protein BORIS, preferably 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the human sequence (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 limiting�s previous level of technology. The mutant form of BORIS is used as an ideal non-toxic vaccine, as it should not cause undesirable side effects caused by its activity DNA-binding and/or native function. In other words, mutant BORIS used for vaccination, does not possess a functional activity and is present only as an immunogen (antigen). Unlike other tumor-associated antigens, BORIS is not expressed in normal tissues in women. In addition, even though BORIS is expressed in the process of puberty in normal testis in men, the introduction and/or expression of the nonfunctional mutant BORIS should be harmless, since the testis is an immunologically privileged organ (inaccessible to immune cells). In other words, anti-BORIS immune response generated after immunization, is not harmful to normal cells, and the vaccine BORIS does not cause autoimmunity. In addition, the guaranteed production of powerful immune response, as BORIS, unlike other tumor-specific antigens recognized as foreign antigen. BORIS-specific T cells are not deleted in the thymus, recognize mutant BORIS as a "foreign" antigen and generate an immune response.
In one embodiment of the invention, cDNA encoding murine BORIS (mBORIS), obtained by RT-PCR on mRNA, DLY�Lenno from murine or human egg. 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 was confirmed by automated analysis of the nucleotide sequence. In the resulting molecule is missing the 11 ZF domain and consists of the N-terminal region BORIS m (amino acids 1-258) associated with the C-terminal region (amino acids 573-636) via a spacer containing 18 amino acids.
Mutant cDNA cloned into the vector pORF under the control of the promoter hEF1-HTLV, but you can use other expression vectors. The mutant cDNA is functionally associated with the promoter and/or regulatory molecules capable of causing expression in the host cell. You can apply viral vectors, including vectors and 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 a nonfunctional mutant BORIS, such as adenovirus, 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 viruscode�nye particles (VLP). These particles do not contain viral nucleic acids, and therefore are dereplication, non-communicable and retain conformationally correct antigenic epitopes. Producing the VLP is shown in many experimental systems, such as mammalian cells infected with baculovirus insect cells, yeast, E. coli, cell-free systems and transgenic plants. It is important to note that vaccination with VLP induces the production not only humoral but also cellular immune responses. VLP infect "professional" APC, and then induce protective cellular immune responses, including CD4+Th1 (type CD4+T cells that help the cells CD8+T) and CD8+CTL responses. Thus, VLP clearly showed exceptional ability to activate cellular immune response (T cell response). The possible use of VLP as a prophylactic vaccines currently checking 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 VLP vaccine, consisting of a portable BORIS antigen that will stimulate a strong cellular immune response against cancer cells expressing this tumor antigen. From crustal antigen (HBcAg) virus hepat�that B (HBV) and VSV are examples of suitable VLP.
For more strong (stable) cellular immune response truncated or mutant mBORIS before cloning into the vector combined with molecular adjuvants, such as molecules of costimulatory V7, beta-defensin 2/3, MIP3α, IFNγ, cytokines, chemokines, etc. Other suitable molecular adjuvants are listed in Table 1 below.
|XCL1 (Impracti α, SCM-1α, ATAC)|
|XCL2 (Impracti β, SCM-1β, ATAC)|
|SS (MPIF-2, β6, eotaxin-2)|
|CX3CL1 (Fractalkine, neurotensin)|
Or for DNA immunization can be used conventional adjuvants, such as Tween 80, 5% ethanol or bupivacain. Other examples of traditional adjuvants include mineral salts (such as gels of aluminum hydroxide or aluminum phosphate), oil emulsions and formulations of the power�surface-active substances,such as MF59, QS21, AS08 [SBAS2] (oil emulsion in water+MPL+QS21), Montanide ISA-51 and ISA-720, adjuvants from particulates, such as virosomes, AS04 [SBAS4] A1 salt with MPL), ISCOMS, a copolymer of lactide with glycolide (PLG), compounds of microbial origin (natural and synthetic), including monophosphorylated A (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 immune modulators, such as GM-CSF, IL-12, immolation and all other chemokines, cytokines and costimulatory molecules listed in the Table above, and inert carriers such as particles of gold.
Adjuvants can be mixed with the polynucleotide, encoding a non-functional mutant form of the protein, polypeptide or peptide relative controller imprinted sites (BORIS), a nonfunctional mutant BORIS protein, polypeptide or peptide and dendritic cell expressing a nonfunctional mutant BORIS peptide, polypeptide or protein.
To raise/promotion presentation nonfunctional mutant BORIS "professional" antigen-presenting cells (APC) metabolic pathway of MHC class in the design nonfunctional mutant BORIS you can include additional peptide molecules. One such example is the design, with�savama using transducible peptide domain (PTD). Overall, immune response relies on processing and presentation of native antigen. Tumor antigens can be expressed in bacteria, yeast and mammals, however, protein antigens expressed in these systems apparently do not stimulate maximal T-cell immune response (responses are prone to either CTL or Th1 responses), as a soluble exogenous proteins processinputs mainly using metabolic pathway of MHC class II. In fact, many anti-tumor vaccines based on the induction of CD8+CTL, but it usually requires that the protein was synthesized in the cytosol of APC. Unfortunately, in General the plasma membrane of eukaryotic cells impervious to most proteins. Recently it was shown that the foreign proteins, coupled with protein-traduzioni domain (PTD) can penetrate through the plasma membrane, thus generating a protein inside the cells. This improves the presentation of foreign peptides by MHC molecules of class II APC antigen-specific CD8+T cells.
Preparations (formulations, compositions) vaccines to the present invention contain an immunogenic amount of a polynucleotide encoding a nonfunctional mutant form of the protein, polypeptide or peptide BORIS, nonfunctional mutant BORIS protein, polypep�IDA or peptide, or a dendritic cell expressing a 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 with a three-dimensional structure corresponding to the nonfunctional mutant BORIS protein, polypeptide or peptide, and which function in an identical fashion. Mimetype that represent any biological molecule, not related to the structure of BORIS, but all have identical(e) 3x-d(e) the epitope(s) recognized by anti-BORIS cells.
"Immunogenic amount" is an amount of polynucleotide encoding a nonfunctional mutant BORIS protein, polypeptide or peptide; nonfunctional mutant BORIS protein, polypeptide or peptide; or a dendritic cell expressing a nonfunctional mutant BORIS protein, polypeptide or peptide sufficient to induce an immune response in a subject to whom the vaccine is injected. Enter the number represents the amount that induces a defined immune response and desired degree of protection. Examples of pharmaceutically acceptable carriers include, but without limitation, sterile apyrogenic water and sterile apyrogenic physiological saline solution.The formulation of vaccines 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) of the colon or esophageal cancer. The formulation of vaccines of the present invention is also suitable for patients with known genetic predisposition to cancer. In addition, formulations of the vaccines of the present invention is suitable for the whole population, including members of a population not suffering from cancer or genetic predisposition to cancer who wish to seek protection from contact at least with one type of cancer, which is expressed BORIS protein, polypeptide or peptide.
The vaccine can be realized by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous or intramuscular injection), intradermal, intravenous injection, nasal, rectal, or vaginal administration, the introduction through the respiratory tract. Topical application of the virus for administration via the respiratory tract can be performed using intranasal (e.g., by pipette, swab (towndock) or inhaler, which is administered in the nose pharmaceutical pre�Arat). Topical application of the virus for administration to 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 causing the subject to inhale the respirable particles. Methods and apparatus for the introduction of respirable particles of pharmaceuticals are well-known, and can be used any conventional method. "Immunogenic amount" is an amount of replicon particles, sufficient to induce an immune response in a subject to whom the vaccine is injected.
If the vaccines use RNA or DNA, RNA, and DNA can be read directly by methods known to those skilled in the art, such as delivery on gold pellets (gene gun), delivery via liposomes, or direct injection. One or more design or replicate RNA can be used in any combination, effectively causing an immunogenic response in a subject. Usually the number of nucleic acids introduced in the vaccine may be such as to induce a given immune response and desired degree of protection. The precise number of vaccines administered may depend on the decision of the practitioner and can be individualized for each subject and the Antiga�.
The vaccine can be administered according to the scheme, providing a single dose, or preferably by the scheme, which involves multiple flow when the primary course of vaccination may consist of 1-10 separate doses, and then, through successive time intervals give the other doses with the aim of preserving or enhancing the immune response, e.g., 1-4 months for a second dose and, if necessary, subsequent(s) dose(s) after several months. Examples of suitable immunization schemes include: (W) 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 circuit suitable for the detection of defined immune responses, presumably providing protective immunity, or reduce disease symptoms, or reduce severity of disease.
Human hBORIS can be isolated from human testis and manipulate it to him. Similarly, BORIS can distinguish from the testicles of any mammal or vertebral and use in this way.
1. Obtaining a plasmid encoding the ZF-delegated form molecules mBORIS under the control of the promoter hEF1-HTLV
Reaction RT-PCR performed using RNA poly-And murine testis and the following primers:
MB1F 5'-CGTCACCATGGCI GCCGCTGAGGTCCCTG
The following PCR conditions: 94°C 30 sec, 60°C 30 sec, 72°C 2 min. Spend thirty (30) cycles.
PCR products subcloning in the cloning vector PCRII-TOPO (Invitrogen). The C-terminal cDNA into PCRII-TOPO was subjected to restriction analysis using the enzyme BamHI, a positive clone containing the insert, assemble and then cut when using the enzyme HindIII. Primers (SpF and SpR) for amplification of the spacer is annealed, get protruding sticky ends and be ligated into the 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 be ligated into the design, cut with HindIII containing the C-end and the spacer. Then the clones with the proper orientation is selected, sequeiros (for example, see below the sequence of the molecule ZF-deletirovanie BORIS) and subcloning in the plasmid pORF under the control of the promoter hEF1-HTLV (Invitrogen).
Cells SNO transferout with the resulting construct, 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 divisions zinc fingers) analyze the Northern-blots of mRNA isolated from cells SSS standard method of molecular genetics Sambrook et al. 1989).
2. Immunization of mice with DNA. Kodir�owns ZF-depletirovannoi form molecules BORIS
The plasmid encoding the ZF-depletirovannoi design mBORIS, allocate, using EndoFree Plasmid maxi (Qiagen). The purity of plasmid DNA confirmed by UV spectrum (the ratio of optical densities at 260 nm/280 nm >1.7) and gel electrophoresis.
Gold pellets coated with DNA (1 μg/0.5 mg gold) and 5-7-week-old mice Balb/c immunize using gene gun Helios. Mice immunizer (shelled) in a similar way three times in two weeks. Ten days after the last "attack" of mice take the blood sample and infecting, injecting 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 with plasmid vaccine:
One example of a method with the use of prophylactic cancer vaccines is the use of DNA that encodes a delegated form of the molecule murine BORIS, in which there are no domains of zinc finger and therefore the property is missing the DNA-binding.
Purified plasmid used for gold coating of the granules (2 µg plasmid/0.5 mg gold pellets), as described previously (Ghochikyan et al. (2003) Eur. J. hnmunol 33: 3232-41). Immunization of mice BALB/C is carried out through the shaved skin of the abdomen by means of gene gun Helios (Bio-Rad, Hercules, CA) as described by Ross et al. (2000, Nat. hnmunol 1: 127-131). Briefly g�Vorya, mice bombard 3 times doses containing 2 μg DNA, 0.5 mg of gold granules with a size of ~1 µm (DeGussa-Huls Corp., Ridefield Park, NJ) under a helium pressure of 400 psi (2758 kPa). Mice immunize and enrich (fired) by the same method every two weeks and control infect 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 immunize a plasmid that encodes a modified BORIS, mixed with DNA that encodes a specific(e) molecular(e), adjuvant(s) (see Table 2). It is known that these molecular adjuvants enhance the cellular immune response to various antigens.
|Mice immunize every two weeks, a total of five times, with the help of preventive vaccines BORIS (pORF-mBORIS) mixed with pORF-mGMCSF (encoding murine mGMCSF), pORF-mIFNγ (encoding murine mIFNγ) or pORF-mIL12+pIRES-mIL18 (two plasmids encoding murine IL12 and IL18, respectively). After the last attack of mice infected with 105or 104cells T breast cancer mice that Express a modified BORIS|
|Group||The immunogen||Molecular adjuvant||Control infected cells T|
|1||pBORIS||pGM - CSF||105|
Obtaining an adenoviral vector encoding the ZF - depletirovannoi the foam molecules murine BORIS (Ad5-BORIS), and immunization of mice with Recombinant virus Ad5-BORIS get using adenoviral vector system AdEasy XL from Stratagene. Shuttle vector constructed by sublimemovies fragment of ZF-deletirovanie mBORIS in the plasmid pShnttle-CMV. For this purpose, the fragment BORIS synthesized by PCR, using as template the plasmid pORF-mBORIS and the following primers:
The PCR product subcloning in the cloning vector PCR4-TOPO (Invitrogen). A fragment of BORIS cut using restrictase Sail and Notl. The resulting product was then purified on agarose gel and subcloning using the cloning sites SalI-NotI, the vector pShnttle-CMV.
Expression in vitro ZF-deletirovanie mBORIS analyzed in Cho cells by immunoblotting (Figure 6, see below).
Shuttle vector containing delegated BORIS, linearized using Pmel and purified on agarose gel. Electrocompetent cells BJ-5183-Ad-1 transformed using the plasmid pShuttle-mBORIS, split through the Mouth, get recombinant Ad plasmid. Cells AD-293 transferout when using selected DNA recombinant Ad-BORIS and prepare primary outcome�s viral solutions. The primary viral solution (10-fight/ml) amplificateur cells in AD-293, and then purified in a CsCl-gradient. Purified virus cialiswhat against PBS - 5% sucrose and used for immunization of mice.
Mice Balb/c 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 is carried out control infection of two different doses of cells T breast cancer (105or 104as described (Vasilevko, V. et al. (2003).
Tumour cell lines
Use neoplastic mammalian cells, provided by Dr. F. Miller (Karmanos Cancer Institute, Detroit, MI). Cells T.2 are resistant to Tg variant derived from 410.4 cells (the cell line is mammalian, originally isolated from a single spontaneously grown mammalian tumor in mice BALB/c fC3H) without the influence of a mutagen. The cells are cultured (37°C, 10% CO2) in the medium Needle, modified by way of Dulbecco (DMEM), low glucose and with the addition of 5% fetal bovine serum, 5% serum of newborn calves, 2 mm glutamine, 100 U./ml of penicillin, 100 mg/ml streptomycin, 0.1 mm nonessential amino acids and 1 mm sodium pyruvate (D10) (Life Technoloies, Inc.).
The definition of tumor volume
The volumes of tumors determine daily by making measurements in two directions and calculating according to the formula L×(W2)/2, where L represents the length, W denotes a width of the tumor. The experiments are over with the death of a mouse, or upon reaching a tumor size of about 1.5 cm3in the experiments, including the control infected with 104cells, and 2 cm3in the experiments, including the control infected with 105cells T.
Formation time (latent period) is indicated as the time until the tumor reaches a volume of more than 0.1 cm3. To determine the growth rate of tumors analyzed dispersion curves at periods of almost linear growth of the tumor.
The results obtained in the determination of the average time of appearance of tumor nodules (latent period) and tumor growth (tumor volume) and the time of post-vaccination immunity, study the method of analysis of variants (ANOVA) test and post-test using the Tukey criterion for multiple comparisons. The mean and standard deviation (deviation) (SD) calculated using the software GraphPad Prism 3.0.
B - and T-cell immune response against BORIS analyzed using two different immunization Protocol.
1. Obtaining murine proteins and BORIS �mmunicate mice.
ZF-deleteriously fragment of murine BORIS subcloning in bacterial expression vector pET24d(+), using NcoI-XhoI cloning sites in reading frame with a C-terminal 6His "tail". Enter both the site and remove the stop codon at the stage of cloning of PCR. In addition, create a plasmid encoding the molecule delegated BORIS, merged with a domain, a protein transduction (PTD). Domain protein transduction of HIV-Tat (Tat47-57YGRKKRRQRRR) was combined with N-end delegated BORIS by PCR, and then cloned into the cloning sites NcoI-XhoI vector pET24d(+). The strain E. coli BL21(DE3) transformed with the obtained plasmid pET-mBORIS or pET-TATmBORIS, grown in LB with kanamycin at 28°C to achieve A6000-8. Protein synthesis is induced by adding IPTG to final concentration of 1 mm. Cells are harvested after three to five hours by centrifugation and used for protein purification affinity chromatography on a column with Ni-NTA (nitrilotriacetic acid) (Qiagen). Cm. Figure 7.
In addition, ZF-deleteriously fragment BORIS, fused with PTD, subcloning in expressing vector yeast Rourkela in reading frame with the signal sequence into the cloning sites EcoRI-XbaI. Both sites are administered and the initiating ATG codon is removed during the PCR stage of cloning. The Pichia pastoris strain X33 transformed by electroporation with pGAPZ-BORIS, linearize�t using restrictase AvrII and positive clones were selected on YPD medium, containing 100 µg/ml Zeocin. For analysis of expression of the selected clones are grown in broth YPD/Zeocin and analyzed in the supernatant at different time points by immunoblotting.
2. Immunization of mice with dendritic cells (DC).
Primary DC bone marrow is obtained from precursor cells in the bone marrow of mice as follows. Depleted erythrocytes of bone marrow cells from the femur and tibia are seeded in complete medium RPMI-10 with the addition of recombinant murine GM-CSF (100 U./ml). On day 3 is not affixed to the substrate granulocytes carefully removed and add a fresh environment. Not attached to the substrate DC harvested on day 7 and purified, using a set of positive selection (Miltenyi) with CD11c microbeads.
DC harvested on day 7 of culturing purified by positive selection and infect with Ad5-BORIS by incubating with cell density of 107 cells/ml in RPMI-1640 at a multiplicity of infection of 1000-2000. After 1 hour of complete medium is added to dilute the DC to a final concentration of 1×106-2×106cells/ml After 24 hours the cells are harvested intensively washed to eliminate any transfer of adenoviral particles, and used for immunization. In addition, DC harvested on day 7 of cultivation and purified by positive selection, were incubated with 10 μg/ml ZF-deletirovanie protein mBORIS at 37°C, 5% CO2within 24 hours, twice washed with PBS. Protein uptake by cells DC analyze the aliquots by flow cytometry using antibodies against murine BORIS and appropriate secondary antibodies labeled with FITC. Mice Balb/c immunize I. p.(intraperitoneally) three times every three weeks with 106DC and T-cell response was analyzed 10 days after the last injection in culture (booster) culture of splenocytes.
The immunization results presented in the Figures 1-4.
DNA encoding the mutant form specific for cancer cells of mouse BORIS antigen without DNA-binding functions (deletion 11-zinc finger), create using expression vectors mammals pORF (hivivogen) and adenoviral vector system AdEasy XL (Stratagene). These vaccines are used as a prophylactic cancer vaccines in a mouse model of breast cancer. For this model using mice BALB/c (haplotype H-2d) and native line of tumor cells mammals T, which is resistant to Tg variant, formed from 410.4 cells without mutagenic treatment. It is important to note that these murine breast cancer cells Express the full-size molecule murine BORIS, as was demonstrated by RT-PCR method. Consequently, there is an ideal model to study the ability of the molecule BOIS be used as immune vaccines against cancer.
Conduct two different types of experiments. The first type of experiments involves the group of mice vaccinated with pBORIS (a plasmid encoding the delegated molecule murine BORIS, mixed with a DNA that encodes a different mouse cytokines (pGM-CSF; pIL12/IL18; pIFNγ) as molecular adjuvants. Mice injected with vector (pORF) or pIL12/IL18 as a control. Of mice and immunize conduct booster immunization using gene gun, and then hold control infection with 104or 105cells T.
The second type of experiment involves the group of mice vaccinated with pBORIS, buffer immunization embracing a replication defective adenoviral vector (Ad5), which is modified to Express the ZF-depletirovannoi molecule murine BORIS (Ad5-BORIS). In use as a control group of mice injected with vector with subsequent booster immunization with Ad5. Control infection of animals is carried out using a 104or 105cells T and analyze the emergence and growth of tumors. Note, it was previously found that injection of such small quantity as 104cells T.2 in mammary glands of mice BALB/c leads to local growth of mammary tumors in 100% of animals with infection control.
Vaccination with pBORIS plus pIL12/IL18 or pBORIS with the last�posing Ad5-BORIS leads to the protection of mice from infection control with 10 4unmodified T tumor cells. Although in 50% of mice from group immunized with pBORIS mixed with pIL12/IL18, formed small tumors (0.2-0.4 cm3), they are all viable at day 39. All of the experimental mice died approximately ten days earlier.
The results obtained in mice immunized with Ad5-BORIS, are outstanding. On day 24, when the mice in the control group died from tumors, 100% of mice immunized with vaccine Ad5-BORIS, were not only alive, but they are 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 carried out under more severe conditions and with control infected mice 105T tumor cells. Vaccination with plasmid pBORIS plus pIFNy or pIL12/IL18 significantly prolongs the time of tumor growth to a volume of 2 cm3and increases the survival of mice BALB/c. Vaccination also reduces the rate of tumor growth in the control infected with 105tumor cells T. Deeper effect detected in mice vaccinated with pBORIS with subsequent booster immunization vaccine Ad5-BORIS before infection with 105nomodifier�nnyh 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 vaccine Ad5-BORIS, remain alive, and the surviving mice tumors much smaller in size.
Separate groups of mice BALB/c immunize delegated murine BORIS protein, purified from E. coli system. In this case, five mice subcutaneously injected protein (50 μg/mouse) mixed with a conventional adjuvant type Qui1 A Th1 (Sigma). After four immunization all animals were observed significant titer of antibodies against BORIS. Another group of 5 mice simultaneously intraperitoneally immunize allocated dendritic cells infected with Ad5 vaccine-BORIS. After three injections in mice produces T-cell response against mBORIS which can be detected in vitro in the culture of splenocytes activated protein mBORIS. Therefore, immunization with BORIS induces b - and T-cell immune response in mice, and this immune response protects the animal from (control) infection.
6. Truncated BORIS associated with PTD, as subunit vaccine
PTD communicates with a non-functional truncated or mutant BORIS protein, and the product of the merger of forms of yeast expressing system. Genes encoding PTD and nonfunctional mutant BORIS, subcloning in yeast expressing vector, such as pGAPZα. Exp�ansirovanie and secreted protein is purified by standard methods of molecular genetics. Mice immunize with antigen prepared in two different conventional adjuvant, and analyze the immune response and protection against tumor antigen.
7. Virus-like particles encoding a nonfunctional mutant BORIS, as subunit vaccine
Receive a subunit vaccine VLP-BORIS on the basis of crustal antigen (HBcAg) of hepatitis b virus (HBV). This antigen "samsobeats" in VLPs after expression in yeast cells. Foreign sequences can be embedded in some areas of HBcAg, not breaking the build process. Accordingly, particles get chimeric HBcAg-BORIS, which are used for immunization of mice.
8. Analysis on mice BALB/c and p53 KO
Analyze the immune response in mice BALB/c without contamination and young mice with a knockout of the gene p53, which by this age, the tumor does not appear. Determine humoral and cellular immune response in mice immunized with various vaccines BORIS. Analyze the serum of immunized mice to detect antibody production against BORIS for 3x-month experiment. Determine the proliferation of CD4+ and CD8+ T cells and activation of regulatory T cells before and after infection of mice BALB/c. At the same time analyze the activation of NK cells, which can directly induce killing of tumor cells of the breast. Shown functional active�nce BORIS-specific cytotoxic T lymphocytes (CTL) before and after infection of tumor cells breast cancer T. For the detection of NK and CTL activity as target cells using tumor cells R that naturally (in nature) Express molecules BORIS wild-type, along with T cells.
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Ordinary specialist in the art it is obvious that it is possible to use various modifications of the materials and methods in the application of the invention described above. Such modifications can be considered within the scope of the present invention, which is defined below by the claims.
Description of sequence: nucleotide sequence of the molecule with BORIS ZF-deletion
Description of sequence: nucleotide sequence of mouse BORIS wild-type
Description of sequence: nucleotide sequence of the human BORIS wild-type
Description of sequence: amino acid sequence of human BORIS
Description of sequence: amino acid sequence of murine BORIS
Description of sequence: amino acid sequence of murine BORIS with ZF-deletion
1. Immunogenic composition for the prevention and treatment of cancer, containing nfunction�iny BORIS protein, and a pharmaceutically acceptable carrier, excipient or adjuvant, where nonfunctional BORIS protein consists of SEQ ID NO:2, lacking all of the zinc fingers.
2. A composition according to claim 1, in which the nonfunctional mutant BORIS is not associated with DNA.
3. A composition according to claim 1, in which nonfunctional BORIS protein attached to a pharmaceutically acceptable carrier.
4. A composition according to claim 1, in which BORIS protein is associated with the domain, a protein transduction (PTD).
5. A composition according to claim 1, in which nonfunctional BORIS protein is associated with a peptide that modifies BORIS and preserves or increases the antigenicity of the specified nonfunctional BORIS protein
6. A composition according to claim 1, wherein the nonfunctional BORIS protein is expressed in the bacterial cell, the cell of a mammal (including dendritic cell), a yeast cell or viral particle.
7. A composition according to claim 1, wherein the adjuvant is conventional or molecular and enhances its immunogenicity.
8. A composition according to claim 7, in which adjuvant or molecular adjuvant is mixed, merged or stitched with the specified nonfunctional BORIS protein.
9. A composition according to claim 7, in which the adjuvant can be molecules from the group of cytokines, chemokines or costimulatory molecules.
10. A composition according to claim 1, wherein the nonfunctional BORIS protein is expressed dendritic cell of a mammal.
11.Immunotherapeutic composition against cancer, containing nonfunctional BORIS protein, or bacterial cells, mammal cells (including dendritic cell), a yeast cell or viral particle expressing a non-functional form of the BORIS protein containing SEQ ID NO:2, lacking all of the zinc fingers, and further comprising an adjuvant, a molecular adjuvant, a pharmaceutically acceptable substance that enhance anti-tumor immune response.
12. The method of immunization of a patient, comprising the introduction of an effective amount of the immunotherapeutic composition according to claim 11, which enhances the immune response to tumors.
13. A method according to claim 12, in which the effective amount of the immunotherapeutic composition has a molecular adjuvant from the group of cytokines, chemokines or costimulatory molecules.
14. A method according to claim 12, in which the effective amount of the immunotherapeutic composition contains a pharmaceutically acceptable carrier.
15. A method according to claim 12, in which the effective amount of the immunotherapeutic composition contains nonfunctional BORIS protein associated with the peptide that modifies BORIS and retains or increases its antigenic and anti-tumor properties.
16. A method according to claim 12, in which the effective amount of the immunotherapeutic composition contains nonfunctional BORIS protein, which additionally contains� domain protein transduction (PTD).
17. A method according to claim 12, in which the introduction is carried out intramuscularly, subcutaneously, intradermally, intravenously, nasally, rectally, vaginally or peritoneal.
18. A method according to claim 12, in which the patient is sick with more than one type of cancer.
19. A method according to claim 12, in which the patient has cancer.
20. A method according to claim 12, in which the patient has a genetic predisposition to cancer.
21. A method according to claim 12, in which immunization leads to depleted cellular immune response involving T cells that recognize the epitope nonfunctional BORIS protein.
22. The use of immunotherapeutic composition according to claim 11 for the preparation of vaccines against cancer, wherein the immunotherapeutic composition comprises an effective amount nonfunctional BORIS protein containing SEQ ID NO:2, lacking all of the zinc fingers.
SUBSTANCE: invention refers to biotechnology, specifically to VEGF-A specific binding proteins, and can be used in medicine for treating pathological angiogenesis in mammals. The antiangiogenic protein contains one ankyrin recurrent domain consisting of a N-terminal capping module of ankyrin recurrence, a recurrent module presented by an ankyrin recurrent motif of the sequence 1D23G4TPLHLAA56GH7EIVEVLLK8GADVNA (SEQ ID NO:5), wherein 1 represents an amino acid residue specified in A, N, R, V, Y, E, H, I, K, L, Q, S and T; 2 is specified in S, A, N, R, D, F, L, P, T and Y; 3 is specified in T, V, S, A, L and F; 4 is specified in W, F and H; 5 is specified in P, I, A, L, S, T, V and Y; 6 is specified in W, F, I, L, T and V; 7 is specified in L or P and 8 is specified in A, H, N and Y; a recurrent module presented by an ankyrin recurrent motif of the sequence 1D23G4TPLHLAA56GHLEIVEVLLK7GADVNA (SEQ ID NO:1), wherein 1, 2, 3, 4, 5, 6 and 7 independently represents an amino acid residue specified in the group of A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y, and a C-terminal capping module.
EFFECT: invention enables producing an antiangiogenic binding VEGF-A165 with Kd less than 10-7 M protein, which inhibits binding VEGF-A165 to VEGFR-2.
12 cl, 4 dwg, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the field of biotechnology, namely to obtaining oligopeptide compounds, containing a motive, interacting with a proliferating cell nuclear antigen (PCNA) and can be used in medicine. The oligopeptide compound consists of 14-70 amino acids and contains. a PCNA-interacting motive, representing [K/R]-[F/Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R], at least one signal sequence of nuclear localisation and at least one signal sequence of penetration into a cell, with the PCNA-interacting motive being located towards an N-end relative to the signal sequence.
EFFECT: invention makes it possible to carry out the efficient treatment of hyperproliferative disorders by the application of the oligopeptide compound in cyctostatic therapy or in radiotherapy as a sensitising substance.
34 cl, 6 dwg, 4 tbl, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, more specifically to MUC1 cytoplasmic domain peptides, and can be used in the anticancer therapy. A method for inhibiting MUC1-positive cancer cell in an individual involves administering into an individual the MUC1-peptide of the length of at least 6 sequential MUC1 residues and no more than 20 sequential MUC1residues and containing the sequence CQCRRK, wherein the amino terminal cysteine from CQCRRK is closed at its NH2 terminal by at least one amino acid residue, which shall not conform with the native transmembrane sequence MUC-1. Alternatively, there can be used MUC-1 peptide of the length of at least sequential MUC1 residues and no more than 20 sequential MUC1 residues, which contains the sequence CQCRRK with all amino acid residues of the above peptide being D-amino acids.
EFFECT: invention enables inhibiting MUC1oligomerisation effectively and inducing the tumour cell apoptosis and the tumour tissue necrosis in vivo.
80 cl, 16 dwg, 1 tbl, 3 ex
SUBSTANCE: invention relates to casein succinylate of iron (III) wherein iron content varies from 4.5 wt % to 7 wt %, water solubility exceeds 92% while phosphorus-to-nitrogen ratio exceeds 5 wt %.
EFFECT: additionally, invention relates to production of iron (III) and to pharmaceutical composition containing casein succinylate of iron (III).
17 cl, 4 tbl, 9 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: presented group of inventions refers to biotechnology, and concerns a DLK1-Fc fused protein and using it for the metastases inhibition, a polynucleotide coding such a protein, an expression vector containing the polynucleotide, a host cell producing the above fused protein, a method for producing the fused protein by culturing the above host cell, a composition containing the above fused protein, and a method for the metastases inhibition. The characterised fused protein contains a DLK1 extracellular soluble domain consisting of the amino acid sequence SEQ ID NO:4 and Fc domain of a human antibody.
EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.
11 cl, 36 dwg, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to biochemistry. Application of a fused protein to obtain a composition for the body weight reduction is described. The fused protein contains a domain of transduction, a signal of mitochondrial localisation and a domain of a mitochondrial factor of transcription A, binding polynucleotide (TFAM), containing a group with high mobility. Methods of treating obesity by means of the said protein are described.
EFFECT: invention extends an arsenal of means for treating obesity.
9 cl, 5 dwg, 2 ex
SUBSTANCE: invention relates to a method of production of casein calcium chloride of technical casein by precipitation, and can be used in microbiological studies for production of components of storing media of cultures of microorganisms, and also production of calcium co-precipitates for food industry.
EFFECT: improvement of the method.
2 cl, 1 tbl, 5 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.
EFFECT: invention enables treating or preventing autoimmune disorders of skin, inflammatory diseases of skin or mucous membrane, or injured skin in an animal effectively.
16 cl, 19 dwg, 3 tbl, 12 ex
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
SUBSTANCE: invention relates to field of biotechnology, in particular to immunogens based on antigenic tau-peptide, and can be used in medicine. Obtained is immunogen, which contains antigenic tau-peptide, consisting of amino acid sequence, selected from SEQ ID NO:6, 8-19, 21-26, 105 and 108-112, covalently bound with immunogenic carrier by means of linker, represented by formula (G)nC, where n equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Linker can be located either on C-terminal (peptide -(G)nC), or on N-terminal (C(G)n-peptide) of peptide. Obtained immunogens are used as base for creation of pharmaceutical compositions for treatment of tau-associated neurological disorders.
EFFECT: invention makes it possible to induce immune response against tau autoantigen in efficient way.
12 cl, 10 dwg, 5 tbl, 16 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biochemistry. There are presented pharmaceutical compositions having a concatemer molecule and a kit, as well as using for preparing an agent for immune system modulation or for human's or animal's immune system activity modulation, and a composition according to the invention. The given invention can find further application as an immunomodulatory agent in therapy of various diseases.
EFFECT: what is presented is a concatemer molecule of non-coding nucleic acid containing at least four single-strand sites with non-methylated CG motives for human's or animal's immune system activity modulation.
20 cl, 4 dwg
SUBSTANCE: invention represents an oral anti-enteroviral and immunostimulant agent in the form of capsules containing interferon and additives, differing by the fact that a therapeutic substance is human recombinant interferon alpha-2b immobilised on polyethylene glycol, having a molecular weight of 1.5 kD by a physical method of binding by an accelerated electron flow in a dose of 1.5 Mrad. The ingredients in the agent are taken in a certain ratio.
EFFECT: extending the range of anti-enteroviral agents possessing immunostimulant properties.
6 ex, 10 tbl
SUBSTANCE: immunomodulatory agent contains 3-O-propionate allobetulenole (19beta,28-epoxy-18alpha-oleanane-3beta-yl and propionate) as an active substance. The immunomodulatory agent stimulates a humoral immune response. The T-cell immune response develops in a delayed-type hypersensitivity test of the immunomodulatory agent of 3-O-propionate allobetulenole (19beta,28-epoxy-18alpha-oleanane-3beta-yl and propionate).
EFFECT: reduced oedema.
1 dwg, 2 tbl, 4 ex
SUBSTANCE: drops possessing antiviral and immunomodulatory effects characterised by the fact that they represent a 95% ethanol infusion of wild strawberry leaves and fruit specified in: red raspberry fruit, mountain ash fruit, bilberry fruit, blood-red hawthorn fruit, cinnamon rose fruit; 15-25 mg of the substance in 1 ml of the infusion.
EFFECT: drops possess pronounced antiviral and immunomodulatory effects.
15 tbl, 5 ex
SUBSTANCE: increase in biocidal and immunobiological action due to the use of distilled water ionised with silver ions and adding into the composition of succinic, ascorbic, citric acid and aethonium.
EFFECT: increase in biocidal immunobiological properties of the antiseptic-stimulant of Dorogov ASD-2F.
6 ex, 1 tbl
SUBSTANCE: method includes carrying out complex treatment at the background of diet therapy. Intake of antihelmintic of vegetable origin and immunomodulator is carried out daily with washing down each of them with 200 ml of radon water. Intestinal lavage is performed every second day. On days of performing intestinal lavage, patient takes bath with radon water in the morning before lavage, with performing underwater hydrodynamic massage (UHM) on other days. On days, when intestinal lavage is not performed, sessions of sound therapy are carried out after UHM.
EFFECT: method provides correction of biological age of organism as prevention of premature ageing.
3 cl, 4 tbl, 2 ex
SUBSTANCE: before Mantoux test with 2 TE PDD-L, Ergoferon preparation is used in the infants infected with tuberculosis mycobacteria with the suspected early period of primary tuberculosis infection and active tuberculosis infection, in a dose of 1 tablet 20-30 minutes before or after a meal, once a day for 45 days.
EFFECT: invention provides the complex preparation of frequently and chronically ill children with an allergic pathology, for Mantoux test with 2 TE PDD-L.
3 tbl, 2 ex
SUBSTANCE: before Mantoux test with 2 TE PPD-L, general advice for phthisiologist-controlled preparation of the infants are given. That is added with prescribing the immunomodulatory preparation Anaferon for children aged 1 year and older 1 tablet 20-30 minutes before and after meals once a day for 30 days.
EFFECT: invention provides the complex preparation of frequently and chronically ill children for Mantoux test with 2 TE PDD-L.
3 tbl, 2 ex
SUBSTANCE: invention relates to biotechnology, specifically to immunostimulating compounds and may be used in medicine. An immunostimulating peptide of an amino acid sequence XLYDKGYTSKEQKDCVGI, where N-terminal X is N-acetylalanine, and may be covalently linked to fatty acids, selected from C2-C25, to form PDAG (peptidyl-2,3-diacylglycerides). The resulted compound may be included in pharmaceutical formulations for stimulating an immune response.
EFFECT: invention provides efficient stimulation of an immune response in subjects and may enhance the immunogenicity of the antigenic peptide when administered with PDAG.
29 cl, 10 dwg, 9 ex
SUBSTANCE: first stage involves a cervical uterine repair by staged dissection on the 5-7th menstruation day in a combination with laser destruction of exophytic condylomas. Laser light power is 6-9 Wt, spot diameter is 1.5 mm, and exophytic condylomas penetration is 1-1.5mm. The second stage involves the immunomodulatory therapy.
EFFECT: method enables increasing the clinical effectiveness by recovering the immunological homeostasis of the uterine cervix by potentiating two effects: recovering archtechtonics of the cervical canal and immunomodulatory therapy.
4 tbl, 2 ex
SUBSTANCE: invention relates to the field of organic chemistry, namely, to novel heterocyclic compounds of the general formula or to their pharmaceutically acceptable salts, where R1 stands for cyano, nitro, amino, -NHCOOR4 or -NHCOR4; R2 stands for a halogen, C1-alkyl, halogenC1-alkyl or C1-alkoxy; R3 stands for C1-alkyl; or both radicals R3 form a cycloalkyl, containing 3 members, together with carbon atom, which they are bound to; X stands for either an alkylene chain of 4-7 carbon atoms, linear or branched, and the said chain can contain one or several similar or different additional units, selected from -O-, -N(R5)-; either a group where n1 and p1 stand for two integer numbers, the sum of which n1+p1 is an integer number, selected from 2; R6 and R7 together form a covalent bond or R6 and R7 together with carbon atoms, which they are bound to, form a cycle or a cycloalkyl, containing 3 members; R4 stands for C1-alkyl; R5 stands for C1-alkyl. The invention also relates to particular compounds, a pharmaceutical composition based on formula (I), application of the formula (I) compound.
EFFECT: obtained are the novel heterocyclic compounds, useful in treating cancer.
23 cl, 10 dwg, 23 ex