New isoforms of vascular endothelial cell growth inhibitor

FIELD: biotechnology, molecular biology.

SUBSTANCE: invention proposes a polynucleotide VEGI-192a encoding polypeptide that inhibits growth of human vascular endothelial cells. Invention describes expressing vector comprising polynucleotide and E. coli cell-host comprising vector. Invention discloses polypeptide encoded by polynucleotide and fused protein based on indicated polypeptide. Invention describes polynucleotide encoding fused protein and expressing vector based on indicated polynucleotide. Invention discloses a pharmaceutical composition used for inhibition of angiogenesis based on polypeptide-inhibitor of growth of human vascular endothelial cells and polynucleotide encoding its. Invention describes therapeutic methods for inhibition of angiogenesis and suppression of tumor growth based on this composition. Invention describes an antibody raised to polypeptide that inhibits growth of human vascular endothelial cells. Using this invention provides novel forms of inhibitor of human growth of vascular endothelial cells and can be used in medicine.

EFFECT: valuable biological and medicinal properties of inhibitor.

27 cl, 27 dwg, 13 tbl, 34 ex

 

This application claims the priority of provisional patent application U.S. serial number 60/331190, filed November 9, 2001 This priority application is enabled thereby in the present description by reference in its entirety.

This invention was made with U.S. government support under grant of the Ministry of defense DAMD17-98-1-8093, grant from the National institutes of Health NHLBI R1 HL60660 and a grant from the National Cancer Institute SA-08. The government has certain rights to this invention.

The technical field to which the invention relates.

This invention relates to compositions that are applicable in the treatment of conditions in which it is beneficial to angiogenesis was inhibited, for example in the treatment of solid tumors, diabetic retinopathy, Kaposi's sarcoma, psoriasis and rheumatoid arthritis. In particular, this invention relates to a new isoform of the growth inhibitor of vascular endothelial cells (VEGI), their DNA and associated protein sequences, their compositions and variants and their use in treatment-driven angiogenesis diseases.

The level of technology

Under normal physiological conditions, humans and animals are exposed to angiogenesis, new blood vessels in a tissue or organ, in a very specific limited situations. E.g. the measures angiogenesis is normally observed during wound healing in embryonic development and in the formation luteum, endometrium and placenta. The term "endothelium" refers to a thin layer of flat epithelial cells that line the serous cavities, lymph vessels and blood vessels. The term "angiogenic" or "inhibiting angiogenesis activity" means the ability of a molecule to inhibit angiogenesis in General.

It is believed that both controlled and uncontrolled angiogenesis occurs in a similar manner. Endothelial cells are actively involved in inflammation, cell adhesion, coagulation, thrombosis, fibrinolysis, and angiogenesis. Endothelial cells and pericyte (perivascular cells), surrounded by a basal membrane, form a capillary blood vessels. Angiogenesis begins with the erosion of the basal membrane by enzymes released by endothelial cells and leukocytes. Then the endothelial cells that line the lumen of blood vessels that protrude through the basal membrane. Stimulators of angiogenesis induce endothelial cells to migrate through the eroded basement membrane. Migrating cells form a "sprout" from the original blood vessel endothelial cells undergo mitosis and proliferate. Endothelial sprouts merge with each friend is m with the formation of capillary loops, creates a new blood vessel.

Persistent unregulated angiogenesis occurs in many pathological conditions, metastasis of tumors and abnormal growth of endothelial cells and supports the pathological damage seen in these conditions. A variety of pathological conditions in which there is unregulated angiogenesis, were United together as angiogenesis-dependent or angiogenesis-associated diseases.

During the growth of tumor endothelial cells proliferate, invaziruyut stroma, migrate toward the source of the angiogenic stimulus, such as cancer cells, interact with perivascular cells and stromal cells and, eventually, form the capillary connecting the tumor tissue with blood flow (J.Folkman (1995) Nat.Med. 1:27-31). Although the mechanism is undoubtedly of high complexity, which regulates angiogenesis is still unclear, it is clear that the initiation or termination of this process is the result of a balance between positive and negative regulators of angiogenesis. A number of angiogenic factors, often experiencing a pronounced increase regulation in tissues of tumors has been described, including several members of the family of fibroblast growth factors such as FGF-I (G.Gimenez-Gallego et al. (1985) Science 230:1385), FGF-2 (L.Scheigerer et al. (1987) Nature 325:257), and members of the family of growth factors vascular endothelial cells (VEGF) (D.W.Leung et al. (1989) Science 246:1390), and receptors for these growth factors (L.W.Burros and ..Olwin (1989) J.Biol.Chem. 264:18647; S.Wemistrom et al. (1991) Growth Factors 4:197; Wapap et al. (1992) Biochem. Biopys. Res. Comm. 187:1579). .de Vries et al. (1992) Science 255:989). Recently it was discovered that two new protein factor, proliferin and related proliferin protein involved in regulating the initiation and termination of neovascularization in the mouse placenta (Jackson D, et al. Science 266, 1581-4, 1994). All documents cited here supra and infra, are specifically included in the description by reference in their entirety.

It was also reported about several inhibitors of angiogenesis, including thrombospondin (D.J.Good et al. (1990) Proc. Natl.Acad.Sci. USA 87:6624), angiostatin (M.S.O''reilly et al. (1994) Cell 79:315), endostatin (M.S.O''reilly et al. (1997) Cell 88:277) and factor 4 platelet count (Emaap et al. (1997) Science 247:77). Obviously, normal angiogenesis quickly activated when needed and quickly terminated when it is no longer required, whereas pathological angiogenesis, though it often continues, and it is hard to stop. This indicates that the mechanism of negative regulation, functioning in the normal process of angiogenesis, is absent or depressed during pathological angiogenesis. It has been suggested that proteolytic activity, which visuo odut inhibitors of angiogenesis from several predecessors, may be partially responsible for lower regulation of angiogenesis, as shown by proteolytic activation of angiostatin from plasminogen and proteolytic activation of endostatin from collagen XVIII (M.S.O''reilly (1997) Cell 88:277). Many known regulators of angiogenesis are pleiotropic and can act on other cell types, as well as on the cell that produces these regulators, although it is possible that endothelial cells can produce autocrine factors for suppression of angiogenesis or maintain quiescence of a Mature vascular network. Thus, the purpose of this application is the description of a new autocrine negative regulators of angiogenesis class, called inhibitors of the growth of vascular endothelial cells (VEGI), which are specifically expressed by endothelial cells.

Published PCT application WO 99/23105 describes protein VEGI (VEGI-174and splicing variant VEGI-251and their corresponding nucleotide sequences, the disclosure of which is specifically incorporated thus in this application by reference in their entirety. Was described antiangiogenic activity shortened from the N-Terminus forms VEGI-174. Protein VEGI-174found 20-30% sequence homology with TNFα, TNFβ and Fas-ligand. A protein with a molecular mass of 22 kDa was formed in the experiment is ante with a transcription and translation in vitro using cDNA clone as template, that is consistent with the predicted open reading frame of 174 amino acids. This protein is called VEGI-174. Analysis of the hydrophobicity of this protein allowed us to predict the hydrophobic region of 12 amino acids directly after the N-terminal segment of 14 negitiable amino acids. This is consistent with the structure of a transmembrane protein type II, similar to TNF (..Aggarwal and .Natarajan (1996) Eur. Cytokine News. 7:93). Was also described VEGI isoform. This protein is called here VEGI-251that, as predicted, is a membrane protein.

Recent Northern analysis of preparations of total RNA from 22 different types of cultured cells of various lines of differentiation showed that the transcripts for this protein can be detected in only two lines of endothelial cells: in HUVE cells and venous endothelial cells early passage. mRNA is not detected in venous endothelial cells later passage and were not found in blood cells of man. In sharp contradiction with this, members of the TNF family expressed mainly in immune cells (..Aggarwal and .Natarajan (1996) (supra). For example, TNFα is produced by macrophages, monocytes, neutrophils and T-cells, whereas TNFβ mainly is produced by mitogen-stimulated T-lymphocytes and leukocytes. Similarly, l is handy for Fas and other members of the TNF family, CD27, CD30, CD40, OH and 4-1 BB all expressed in the cell types of the immune system. Using Northern blots of many tissues, it was found that the transcript EGI is expressed in placenta, lung, kidney, skeletal muscle, brain, liver, thymus, testis, ovary and peripheral blood lymphocytes.

Inhibition of angiogenesis in tumors is an important approach for the treatment of cancer, such as breast cancer and other solid tumors. First of all, the growth and metastasis of tumors depend on angiogenesis. In the model system, it was shown that blocking of the capillaries of the newly formed vascular network is specifically induced coagulation causes the elimination of a tumor vascular network and leads to the suppression of tumors. In addition, an assumption was made that the endothelial cells are vysokopoligonalnymi in tumors, but mostly resting in normal tissues. This makes the newly formed vasculature of a tumor specific and attractive target. In addition, although the properties of cancer cells can greatly vary in different tumors, the population of endothelial cells in most solid tumors is, in all probability, normal, and, therefore, remains homogeneous. This applies to both man and animals. So about the time, perhaps that can be developed antiangiogenic therapeutic agent that can be applied universally for the treatment of many different cancers.

In addition to solid tumors, other important-driven angiogenesis diseases include diabetic retinopathy, sarcoma Galoshes, psoriasis, rheumatoid arthritis. Patients who suffer from these diseases can benefit from antiangiogenic therapeutic approach.

This invention identifies and describes sequences, functions, composition and therapeutic application of new isoforms of the members of the protein family VEGI. Two new isoforms, named respectively VEGI-192aand VEGI-192bcontain a new N-terminal sequence, which significantly modifies the properties of this protein is related to its expression, secretion and antiangiogenic properties.

Describes two new VEGI isoforms, called VEGI-192aand VEGI-192bconsisting both of 192 amino acid residues. Both isoforms detect specific for endothelial cells expression and have a common C-terminal segment of the 151 balance with the previously described VEGI-174and VEGI-251. These isoforms are generated from the gene size 17 TPN human alternative splicing. VEGI-251the most abundant isoform, contains the estimated signal secretion Protein VEGI detected in the conditioned media of endothelial cells, human sera and transfected VEGI-251mammalian cells. The distribution of subcellular localization VEGI-251assumes that it is a secretory protein. Overexpression of VEGI-251in endothelial cells induces dose-dependent cell death. VEGI-251-transfetsirovannyh cancer cells give rise to tumors xenografts with a reduced growth rate and reduced density of microvessels in comparison with tumors of the VEGI-174-transfectants. This invention substantiates the view that the secretory endothelial cells VEGI may function as an autocrine inhibitor of angiogenesis and existing natural modulator of vascular homeostasis.

All publications cited here included, therefore, in the description by reference in their entirety.

Disclosure of inventions

This invention relates to inhibitors of proliferation of endothelial cells and inhibitors of angiogenesis, in particular, to methods of their use. The complete nucleotide sequence of the VEGI-192a, VEGI-192band VEGI-251shown in table 1 (SEQ ID NO:1), table 2 (SEQ ID NO:2) and table 3 (SEQ ID NO:3), a predicted amino acid sequences shown in table 4 (SEQ ID NO:4), table 5 (SEQ ID NO:5) and table 6 (SEQ ID NO:6), respectively.

Thus, in one GP is owenii this invention provides an isolated polynucleotide, which contains the sequence shown in table 1 (SEQ ID NO:1)or its complement. This invention also provides an isolated polynucleotide, which contains at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100 or more contiguous nucleotides of SEQ ID NO:1, where these contiguous nucleotides within nucleotides 1-93 SEQ ID NO:1. This invention also provides an isolated polynucleotide, which contains at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100 or more contiguous nucleotides of SEQ ID NO:1, where the contiguous nucleotides include nucleotides 93 and 94 of SEQ ID NO:1.

In other embodiments, the invention provides an isolated polynucleotide, which contains the sequence shown in table 2 (SEQ ID NO:2), or its complement. This invention also provides an isolated polynucleotide, which contains at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100 or more contiguous nucleotides of SEQ ID NO:2, where these contiguous nucleotides within nucleotides 1-386 SEQ ID NO:2. This izobreteny who also provides isolated polynucleotide, which contains at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100 or more contiguous nucleotides of SEQ ID NO:2, where the contiguous nucleotides include nucleotides 386 and 387 of SEQ ID NO:2.

In some embodiments, the invention provides an isolated polynucleotide, which contains the sequence encoding the polypeptide SEQ ID NO:4. This invention also provides an isolated polynucleotide, which contains polynucleotide encoding at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:4, where these adjacent amino acids within amino acids 1-26 SEQ ID NO:4. This invention also provides an isolated polynucleotide, which contains polynucleotide encoding at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:4, where these contiguous amino acids include amino acids 26 and 27 of SEQ ID NO:4. In some embodiments, these adjacent amino acids are amino acids approximately 5-192, 10-192, 15-192, 20-192 or 25-192 the sequence shown in table 4 (SEQ ID NO:4).

In some embodiments, the invention provides an isolated polynucleotide, which which contains the sequence encoding the polypeptide SEQ ID NO:5. This invention also provides an isolated polynucleotide, which contains polynucleotide encoding at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:5, where these adjacent amino acids within amino acids 1-26 SEQ ID NO:5. This invention also provides an isolated polynucleotide, which contains polynucleotide encoding at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:5, where these contiguous amino acids include amino acids 26 and 27 of SEQ ID NO:5. In some embodiments, these adjacent amino acids are amino acids approximately 5-192, 10-192, 15-192, 20-192 or 25-192 the sequence shown in table 5 (SEQ ID NO:5).

In some embodiments polynucleotide the present invention provides a sequence encoding a functionally-safe variants of the sequences of the nucleic acids described herein, which include substitutions, additions and/or deletions of nucleic acids. Variants include naturally occurring variants of this polynucleotide sequence (e.g., degenerate variants, allelic variants, and so on).

In some embodiments of the invention which allows a stand-alone polynucleotide, having at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity with polynucleotides of the present invention described herein. One embodiment provides an isolated polynucleotide having at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity with the sequence of nucleotides 1-93 shown in table 1 (SEQ ID NO:1), or nucleotide 1-386 shown in table 2 (SEQ ID NO:2).

In some embodiments polynucleotide the present invention additionally contain detektiruya tag. In some embodiments polynucleotide of the present invention immobilized on the surface. In some embodiments of the present invention polynucleotide of the present invention is single-stranded. In some embodiments of the present invention polynucleotide of the present invention is selected from the group consisting of DNA and RNA. In some embodiments of the present invention polynucleotide of the present invention receive partly by chemical synthesis.

It is clear that (unless otherwise specified or does not require) any described embodiment of the present invention, which is redstavlena, is polynucleotides, includes both double-stranded form and each of two complementary single-stranded forms, which, as is well known, or predicted, this form of double-stranded form.

It is also clear that this invention provides embodiments, "consisting of" or "consisting essentially of" polynucleotide, polypeptides and/or antibodies described herein.

In another aspect, the invention provides vectors and expressing vectors that contain any of these polynucleotides.

In other aspects, the invention provides a cell host containing any of polynucleotides or vectors described herein. In some embodiments this a host cell is prokaryotic, such as E. coli. In some embodiments, a host cell is eukaryotic, such as cells of the Chinese hamster ovary (Cho).

The invention also includes cells containing recombinant polynucleotide that contain polynucleotide VEGI-192aor VEGI-192bor options polynucleotide VEGI-192aor VEGI-192b. In one embodiment, the invention provides genetically engineered mammal cells or bacteriology cell, such as E. coli, containing recombinante modified polynucleotide VEGI-192aor VEGI-19b so this polynucleotide sverkhekspressiya. In another embodiment, the invention provides cells containing variant of polynucleotide VEGI-192aor VEGI-192b. In another embodiment of polynucleotide VEGI-192aor VEGI-192bfunctionally linked to the inducible promoter. In other embodiments the genetically engineered cells have a gene variant VEGI-192aor VEGI-192binstead of the natural gene VEGI-192aor VEGI-192b.

The invention also provides polypeptides VEGI-192a. Thus, this invention provides an isolated polypeptide containing the sequence of SEQ ID NO:4. This invention also provides an isolated polypeptide containing a polypeptide encoded by any of polynucleotides of the present invention described herein. In other embodiments, the invention also provides an isolated polypeptide, which contains at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acid sequence depicted in table 4 (SEQ ID NO:4), where these adjacent amino acids within amino acids 1-26 of the sequence depicted in table 4 (SEQ ID NO:4). In other embodiments, the invention also provides a selected polypeptide, which contains, IU the greater extent, 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acid sequence depicted in table 4 (SEQ ID NO:4), where these contiguous amino acids include amino acids 26 and 27 of SEQ ID NO:4. In some embodiments, these adjacent amino acids are amino acids approximately 5-192, 10-192, 15-192, 20-192, 25-192 SEQ ID NO:4.

The invention also provides polypeptides VEGI-192b. Thus, this invention provides an isolated polypeptide containing the sequence of SEQ ID NO:5. This invention also provides an isolated polypeptide containing a polypeptide encoded by any of polynucleotides of the present invention described herein. In other embodiments, the invention also provides an isolated polypeptide, which contains at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acid sequence depicted in table 5 (SEQ ID NO:5), where these adjacent amino acids within amino acids 1-26 of the sequence depicted in table 5 (SEQ ID NO:5). In other embodiments, the invention also provides an isolated polypeptide, which contains at least about 5, at least 10, at least 15, at least 20, at least 25 or Bo is its adjacent amino acid sequence, shown in table 5 (SEQ ID NO:5), where these contiguous amino acids include amino acids 26 and 27 of SEQ ID NO:5. In some embodiments, these adjacent amino acids are amino acids approximately 5-192, 10-192, 15-192, 20-192, 25-192 SEQ ID NO:5.

In other embodiments, the invention provides any described herein, the polypeptide, where the polypeptide comprises an epitope. In other embodiments, the invention provides any described herein, the polypeptide, where the polypeptide immobilized on a solid substrate.

In other embodiments, the invention provides polypeptides that retain the biological activity of VEGI-192aand/or VEGI-192band/or VEGI-251. As shown in the examples, VEGI-192ainhibits the growth of vascular endothelial cells; and VEGI-251being expressed, inhibits the growth of vascular endothelial cells, formation capillaroscopy tubes in a model of angiogenesis in vitro and inhibits tumor growth of xenografts in Nude naked mice.

The invention also provides antibodies that selectively bind VEGI-192aand/or VEGI-192b. Thus, this invention provides an antibody that selectively binds to any of the polypeptides VEGI-192aand/or VEGI-192bthat is described here. In one embodiment the antibody is capable of CBE is to yatsa selectively with VEGI- 192aor VEGI-192b. In another embodiment of this antibody is capable of selectively contact with VEGI-192aand VEGI-192bbut not with other VEGI isoforms. In some embodiments, the antibody binds to a polypeptide encoded by any of the described here polynucleotides. In one embodiment, the invention provides an antibody capable of contacting the polypeptide of the present invention. In another embodiment the antibody is capable of selectively contacting the polypeptide containing (a) the sequence shown in table 4 (SEQ ID NO:4) and/or table 5 (SEQ ID NO:5); or (b) at least 10 contiguous amino acids of SEQ ID NO:4 and/or SEQ ID NO:5, where these adjacent amino acids within amino acids 1-26, shown in table 4 (SEQ ID NO:4) and/or table 5 (SEQ ID NO:5). This invention also provides an antibody that is able to contact the site of the polypeptide shown in table 4 (SEQ ID NO:4) and/or table 5 (SEQ ID NO:5), where this plot represents at least about 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:4 and/or SEQ ID NO:5, and the specified area contains amino acids 26 and 27 SEQ ID NO:4 and/or SEQ ID NO:5.

In some embodiments the antibody is a polyclonal antibody. In other embodiments the antibody is a monoclonal anti the scrap. In other embodiments, the antibody is immobilized on a solid substrate. In other embodiments, the antibody further comprises detektiruya tag.

This invention also provides compositions, including pharmaceutical compositions, containing polynucleotide, polypeptides, antibodies, recombinant vectors and cells are the owners of this invention. In some embodiments, the invention provides a pharmaceutical composition comprising the polypeptide SEQ ID NO:4 or a fragment, where this fragment contains amino acids 26 and 27, in a pharmaceutically acceptable carrier.

This invention also provides an angiogenesis inhibitor, where the inhibitor contains polynucleotide, polypeptides or derivatives VEGI-192a, VEGI-192bor VEGI-251in a pharmaceutically acceptable carrier, a pharmaceutically acceptable amount.

In another embodiment, the invention provides a composition repressor or inhibitor of cancer growth, containing essentially purified polynucleotide or polypeptides VEGI isoforms (i.e. VEGI-192a; VEGI-192bor VEGI-251) this invention.

In another embodiment, the invention provides an accelerator angiogenesis containing the antibody, antisense oligonucleotide, an antagonist, a ribozyme, a drug or agent that reduces or elimi the range function VEGI- 192a, VEGI-192bor VEGI-251when delivering in a pharmaceutically acceptable carrier, a pharmaceutically acceptable amount.

The invention also provides kits, arrays containing any of polynucleotides, polypeptides and antibodies described herein. In one embodiment this invention provides kits or arrays to estimate the number of polynucleotide in the sample containing any of these polynucleotides. In another embodiment this invention provides kits or arrays to assess the level of the polypeptide in the sample containing any of the antibodies described herein.

In another embodiment, the invention provides a method of inhibiting angiogenesis, providing for the introduction of the individual (such as human or animal) composition comprising, essentially, peeled polynucleotide, polypeptides VEGI-192a, VEGI-192bor VEGI-251this invention or a modified form of those disclosed VEGI isoforms described herein at a dose sufficient to inhibit angiogenesis. In one embodiment the composition comprises a vector for the delivery of the gene containing polynucleotide shown in table 3 (SEQ ID NO:3), or polynucleotide encoding the polypeptide SEQ ID NO:6. In some embodiments, this polynucleotide functionally linked to a regulatory sequence, to ora controls gene expression. In another embodiment the composition comprises, essentially, a purified polypeptide VEGI-192awith the sequence shown in table 4 (SEQ ID NO:4), or a functional fragment, where the fragment comprises amino acids 26 and 27 of SEQ ID NO:4 or includes at least one amino acid from amino acids 1-26 SEQ ID NO:4.

In another embodiment, the invention provides a method of treating or alleviating diseases and processes that are mediated by uncontrolled angiogenesis which the stages of the introduction of individual compositions containing polynucleotide, polypeptides VEGI-192a, VEGI-192bor VEGI-251or a modified form of those disclosed VEGI isoforms described herein at a dose sufficient to control angiogenesis. In one embodiment, the composition contains a vector for delivering genes containing polynucleotide shown in table 3 (SEQ ID NO:3), or polynucleotide encoding the polypeptide SEQ ID NO:6. In some embodiments, this polynucleotide functionally linked to a regulatory sequence that controls gene expression. In another embodiment, the composition contains essentially purified polypeptide VEGI-192awith the sequence shown in table 4 (SEQ ID NO:4), or a functional fragment, where the fragment comprises amino acids 26 and 27 of SEQ ID NO:4 or includes at least one amino is islote of amino acids 1-26 SEQ ID NO:4.

In another embodiment, the invention provides a method of treating cancer or suppression of tumor growth, providing for the introduction of individual compositions containing polynucleotide, polypeptides VEGI-192a; VEGI-192bor VEGI-251or a modified form of those disclosed VEGI isoforms described herein at a dose sufficient to suppress tumor growth. In one embodiment, the composition contains a vector for delivering genes containing polynucleotide shown in table 3 (SEQ ID NO:3), or polynucleotide encoding the polypeptide SEQ ID NO:6. In some embodiments, this polynucleotide functionally linked to a regulatory sequence that controls gene expression. In another embodiment, the composition contains essentially purified polypeptide VEGI-192awith the sequence shown in table 4 (SEQ ID NO:4), or a functional fragment, where the fragment comprises amino acids 26 and 27 of SEQ ID NO:4 or includes at least one amino acid from amino acids 1-26 SEQ ID NO:4.

In another embodiment, the invention provides a method for accelerating angiogenesis, introducing a human or animal a composition comprising the antibody, antisense oligonucleotide, an antagonist, a ribozyme, a drug or agent that reduces or eliminates the activity of VEGI-192a, VEGI-192band/or VEGI- 251.

In another embodiment, the invention provides a therapeutic method and composition for treating or alleviating diseases or processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, plagie neovascularization, coronary collaterals, ischemic angiogenesis limbs, diseases of the cornea, redness, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, uveitis, retinopathy of prematurity, macular degeneration, corneal neovascularization of the graft reaction, graft versus host, inflammatory bowel disease, mielosupression and restenosis; where angiogenesis is uncontrolled or excessive and requires inhibition, and this method involves providing an individual in need of such treatment, an effective amount of polynucleotides or polypeptides VEGI isoforms (i.e. VEGI-192a, VEGI-192bor VEGI-251) this invention so that angiogenesis is inhibited.

In another embodiment, the invention provides a therapeutic method and composition for treating or OS is Alenia diseases, such as macular degeneration, poor wound healing, peptic ulcer, fractures, keloids, education and development of blood vessels (vasculogenesis), hematopoiesis, ovulation, menstruation and the formation of the placenta in which angiogenesis is desirable, and this method comprises the administration to an individual in need of such treatment, antagonist polynucleotides or polypeptides VEGI isoforms (i.e. VEGI-192a, VEGI-192bor VEGI-251) of the present invention; antisense oligonucleotides specific to polynucleotides VEGI isoforms or anti-VEGI-antibodies, agents or medicines that reduce or helps eliminate the function of VEGI, in a pharmaceutically acceptable carrier, a pharmaceutically acceptable amount.

In another embodiment, the invention provides a method of detecting a polypeptide VEGI isoforms (VEGI-192aor VEGI-192b), involving contacting a sample from the individual with an antibody described herein, which selectively binds to the VEGI polypeptide of the present invention, and the detection of the presence or absence of a complex formed between the polypeptide in the sample and the antibody. These methods of detection are applicable also to detect any of the VEGI-192aor VEGI-192bthat is described here.

In another embodiment, the invention also provides methods for the detection of polynucleotides VEGI isoforms (VEGI- 192aor VEGI-192b), involving contacting a sample from the individual with polynucleotides (such as an oligonucleotide), which selectively binds to polynucleotides VEGI present invention; and detecting the presence or absence of a duplex formed between the oligonucleotide and polynucleotide samples. These methods are also applicable to detection of any of polynucleotides VEGI-192aor VEGI-192bthat is described here.

In another embodiment, the invention provides a method for the diagnosis of conditions involving pathological angiogenesis, and this method provides for detection of the presence or absence of polypeptides originating from VEGI-192aor VEGI-192bin the sample, providing stages:

(i) contacting a sample from the subject, who suspect the presence of pathological angiogenesis, with antibodies that are specific for polypeptides VEGI-192aand/or VEGI-192bof the present invention; and

(ii) detecting the presence or absence of a complex formed between VEGI-192aand/or VEGI-192band antibodies.

In another embodiment, the invention provides a method for the diagnosis of pathological angiogenesis, providing for detection of the presence or absence of polynucleotides VEGI-192aor VEGI- 192b(preferably RNA) in the sample, and the method comprises the stages:

(i) contacting a sample from the subject, who suspect the presence of pathological angiogenesis, polynucleotide (such as oligonucleotides)that specifically bind polynucleotide VEGI-192aor VEGI-192bthe present invention (e.g., RNA); and

(ii) detecting the presence or absence of a complex formed between polynucleotides and oligonucleotides derived from VEGI-192aor VEGI-192b.

In another embodiment, the invention provides a method for the diagnosis of pathological angiogenesis using polymerase chain reaction (PCR), providing for designing primers using the nucleotide sequence of the VEGI isoforms (i.e. VEGI-192a, VEGI-192bshown in SEQ ID NO:1 and SEQ ID NO:2, in which polymerase chain reaction specific amplificare district VEGI as the basis for detection. These primers can be used for DNA amplification or VEGI RNA VEGI, the latter amplification occurs after the conversion of RNA into complementary DNA (cDNA) by reverse transcription of this RNA. PCR assay can be made quantitative by comparing the amplified product with the standard, which can be obtained using the Institute of economy and management methods, known in this field.

In another embodiment, the invention provides a method for detecting polynucleotides VEGI isoforms (i.e. VEGI-192aor VEGI-192bin the sample, providing analysis on the presence or absence of RNA or DNA isoforms VEGI-192aor VEGI-192bin a sample using hybridization analysis.

In the following embodiment, the invention provides a diagnostic or prognostic kit containing antibodies which bind polynucleotide or polypeptides VEGI isoforms (i.e. VEGI-192aor VEGI-192b) of the present invention; oligonucleotides that hybridize to DNA or RNA VEGI; and/or PCR primers for the amplification of DNA or RNA VEGI and ancillary reagents suitable for use in detecting the presence of VEGI isoform in the sample. Because VEGI can function as a membrane protein, existing natural soluble form of the membrane-bound VEGI can function as his antagonist, and detection methods such soluble forms included in another embodiment of the present invention.

In another embodiment, the invention provides a diagnostic analysis, providing for detection of the presence or absence of mutations in polynucleotide VEGI isoforms (i.e. VEGI-192aor VEGI-192b), which leads to reduction and is and an increase in the expression or function VEGI isoforms. This analysis includes hybridization analysis, analysis of polymorphisms of restriction maps and sequencing of genes, among others.

In another embodiment, the invention provides a method for testing potential agents or drugs on angiogenesis inhibitory activity by testing, capable or incapable of remedy or agent polysoude to regulate the expression and/or activity of VEGI isoforms (i.e. VEGI-192aor VEGI-192b). Because the VEGI isoforms, like other inhibitors of angiogenesis, are activated by proteases that release the protein from the cell membrane, protease, and other agents that facilitate such activation, such as metal ions, could be useful as a means to increase expression of VEGI isoforms.

In the following embodiment of the present invention provides a method for testing potential anti-tumor agents or drugs by testing, is capable or not capable of remedy or agent to inhibit angiogenesis by increasing the regulation of expression and/or activity of VEGI isoform.

In another embodiment, the invention provides a method for testing potential drugs or agents that stimulate angiogenesis through testing, can or cannot the agent or the medicine to block and/or inhibit the function of VEGI (for example, inhibition of angiogenesis). In this case, can be used in the inhibition of the proteases that activate the VEGI isoforms, as discussed above, or agents, which are necessary or which facilitate such activation, such as metal ions, for down-regulation of VEGI with increased angiogenesis through this.

Brief description of drawings

Figure 1. The level of VEGI in the serum of healthy adults. Sera from 40 healthy volunteers (20 men, 20 women) were measured using ELISA with anti-VEGI-antibody. Each point represents a single value. To obtain the standard curve used purified recombinant VEGI. The horizontal line among the points indicate the median value for each sex group.

Figure 2. VEGI is expressed as multiple transcripts in human tissues. The expression of VEGI in the tissues of adults was determined by Northern blot analysis of multiple tissues using32P-labeled full-size cDNA VEGI-174as a probe. Found three separate related VEGI mRNA transcript of the specified size.

Figure 3. The selection of cDNA new VEGI. Scheme showing the synthesis of the 5'RACE products, followed by screening cDNA libraries for selection of full-size cDNA VEGI from various human tissues. Shaded blocks represent legirovannye 5'-adapters, prisutstvie the matter in panel RACE (method for rapid amplification of cDNA ends). PCR primers are marked by arrows with open edges. PCR products of different size were visualized by staining with ethidium bromide. These PCR products were isolated and subjected to sequencing. L=lung; U=uterus; In=brain. Marker of molecular weights of DNA (ladder) 1 TPN shown between tracks L and U. the Alignment of amino acid sequences of the three isoforms of VEGI. Estimated hydrophobic region VEGI-251and VEGI-174are underlined. The asterisk indicates the beginning of homologous sequences.

Figure 4. Differential expression of VEGI-174and VEGI-251in human tissues. Northern-blot analysis of the expression of VEGI in adult tissues was performed with cDNA fragments specific for VEGI-251and VEGI-174. Transcript 2 TPN were detected with probe VEGI-174while the transcript of 7.5 TPN were detected with probe VEGI-251. Studied human tissues were as follows: 1. The peripheral blood leukocytes, 2. Easy, 3. Placenta, 4. The small intestine, 5. Liver, 6. Kidney, 7. Spleen, 8. The thymus, 9. The colon, 10. Skeletal muscle 11. Heart, 12. Brain.

Figure 5. Analysis using the ribonuclease protection VEGI isoforms in various cultured cells. Total RNA from each culture hybridized with isoform-specific VEGI-probes and used β-actin to control for the Eseniya. Full undigested probes shown in the track probes (P), indicated by arrows with dark edges, and products protection from RNase arrows with light edges. Y=PHK yeast, NS=endothelial cells of the human coronary artery, Hm=endothelial cells of dermal microvessels man, Hu=endothelial cells of the umbilical vein person, Sm=smooth muscle cells of the human coronary artery, 3T=line embryonic mouse NIH3T3 cells, Ba=endothelial cells of the aorta of an adult cow, Bh=endothelial cells fetal calf heart, SN=cells hybridoma person EAO, bE=cells endothelioma brain mouse bEND.3.

Figure 6. The structure of the gene VEGI person and offer the generation of isoforms. The segments are numbered 1-9 represent the PCR fragments obtained during mapping of the gene pairs with specific primers, see "Materials and methods". Rectangles with Roman numerals on them represent the exons and horizontal lines represent intron sequence. The presumed site of transcription initiation is indicated by the arrow with double edge. R stands 5'-noncoding sequence that is unique for each of the corresponding transcript, engraved rectangles represent the common 3'-noncoding region. Specified priblizheniyami introns. VEGI-251, VEGI-192aor VEGI-174-specific sequence tagged as 251, 192 or 174. Exon IIIb encodes residues, as with VEGI-251and VEGI-192a. Introns in 5'-direction from exons III and IV shown by the dotted line, as the 5'-end or the sites of initiation of transcription and VEGI-192aand VEGI-174not been determined. "COM" denotes the coding region of the last exon, which is common to all three isoforms.

Figure 7. TNFα induces the expression of VEGI isoforms in endothelial cells of the microvasculature, and large vessels. Analyses of the protection of ribonuclease showing a parallel induction of the expression of VEGI. The arrows show the protected RNA. A. HMVE Cells treated with TNFα at 15, 50 and 90 ng/ml for 24 hours. C. Induction by TNFα VEGI gene expression in HUVE cells. HUVE cells were treated with 20 ng/ml TNFα for 4, 8 and 24 hours (+). Control (-) was received appropriate treatment by the media.

Figure 8. Intracellular localization of recombinant VEGI-174and VEGI-251in transfected endothelial cells. A. VEGI-174-myc and VEGI-251-myc (C) were detected in the transfected cells AWE staining with Texas red linked myc tags. C. Double staining VEGI-251-myc (red) and the factor a background of Villebranda (green) in transfected cells HUVE. Diagrammes D depicts expression design VEGI with C-terminal myc-tag. E-J. Marked by the N-end design GFP-VEGI showed a different distribution in the cells of AWE. Cells transfetsirovannyh vector plasmid (E)showed the distribution of GFP throughout the cell, whereas three designs VEGI (F, H-J) led to localized distribution of GFP. In I and J VEGI-2511-99 directed the distribution of GFP in the plasma membrane. K. GFP Tag in the expression constructs used in F-J, located on aminobenzo VEGI-fragments.

Figure 9. Detection of VEGI-251using Western analysis in the environment, air-conditioned transfitsirovannykh cells MW and nitrostilbene HUVE cells. A. air-Conditioned environment from stable transfectants MDA-MB. Track 1 = only the vector pcDNA3, tracks 2 and 3 = two independent clones expressing VEGI-251. C. Track 1 = Air HUVE cells Wednesday, track 2 = lysate of HUVE cells. In both experiments, the conditioned medium was concentrated using the column Method (limit bandwidth based on the molecular weight of 10000)was immunoprecipitated using polyclonal antibodies were then subjected to electrophoresis in LTO-page and Western detection using monoclonal antibodies 1-8F against the common C-terminal region of VEGI (residues 29-174). Both panels show VEGI-peptides with a mass of approximately 25 kDa.

Figure 10. Sverge xpressia VEGI- 251causes death of cells and inhibits the growth of tumor vessels (neovascularization). A. delivery using lentivirus secreted VEGI is lethal to HUVE cells. Dose-dependent cytotoxicity of the original solution lentivirus expressing VEGI-251and sVEGI, compared with VEGI-174. Twenty-four hours after viral infection is attached cells remaining in the culture, positively using a counter of Coulter. The levels of viral P24 was determined, the dose of virus were expressed as multiplicity of infection (MOI). Presented values are average ± standard error of three independent experiments. C. Delay of tumor growth breast cancer xenograft MDA-MB231 VEGI-251and sVEGI. Pools of stably transfected cells MDA-MB231 expressing the indicated construct were subcutaneously injected with in the fatty tissue of the mammary gland of female Nude mice and tumor size were recorded blindly. Control mice received cells MDA-MB231, transfetsirovannyh empty vector pcDNA3. Attenuation of tumor growth was observed for VEGI-251and sVEGI, but not for full-length VEGI-174. C. Transfection of VEGI-251and sVEGI leads to reduced density of microvessels in the tumor xenografts MW. Paraffin sections (5 μm) of about the of whole did for mice, described on figure 10A. Vessels identified D31-immunoablative, as described in "Materials and methods". Used univariate analysis of variance, and: P<0,0005; b: P<0,05 in comparison with the control xenografts with the vector pcDNA3.

Figure 11. A photograph of the results of Northern blot analysis of multiple tissues in the expression of VEGI in various human organs using32P-labeled cDNA VEGI-174as a probe. Visible signals of mRNA VEGI different size.

Figure 12. Diagram illustrating procedures for RACE-PCR, is used to search for possible VEGI isoforms. ADP1 and ADP2 indicate the adapter-specific primers. GSP1 and GSP2 indicate gene-specific primers.

Figure 13. A photograph of the results of agarose gel electrophoresis of the products of RACE-PCR. Four PCR product of different sizes from different human tissues were visualized using staining with ethidium bromide. PCR products were isolated and subjected to sequencing.

Figure 14. Photograph of a Western blot analysis of conditioned media of cells MDA-MB-231, transfected with empty vector (lane 1) or cDNA VEGI-251(track 2). The conditioned medium was subjected to gel-filtration chromatography, and the fraction with a range of molecular masses between 10 and 50 kDa were collected and subjected to electrophoresis in LTO-PAG. Panel A: Staining of the El, Kumasi blue. Panel b: Western blotting with a monoclonal antibody (13-2D) to VEGI.

Figure 15. Graph showing inhibition of growth of tumor xenografts formed by cells MDA-MB231, transfitsirovannykh VEGI-174, VEGI-251IL-6/VEGI or the empty vector pCDNA-3. One million stably transfected cells were injected with in the fatty tissue of the breast females Nude naked mice. Had 2 introduction places on each animal and 5 animals per group. These groups are coded, and the size of the tumor xenografts were registered blind. Statistically significant inhibition of growth of these tumors was observed for sverkhekspressiya VEGI-251or IL-6/VEGI cells. Shorecrest VEGI-174had no effect on tumor growth.

Figure 16. Immunohistochemical analysis of tumor samples obtained from experiments described in figure 7, using mAb 13-2D against VEGI person. Sverkhekspressiya VEGI cells are stained in brown color. Panels on the left are photographs of sections of tumors formed VEGI-251-transfitsirovannykh cells. Levels of education VEGI-251were clearly vysokopribylnymi, as can be seen from intense staining of some of the slices of the tumor (G9-1R), which implies high levels of production VEGI, in comparison with significantly less staining of some tumors that W is the group (G9-2R). Panels on the right are photographs of sections of tumors formed by control cells with the vector. Brown staining in sections of control of the tumor is, apparently, the result of cross-reaction of antibodies with natural molecules VEGI on the endothelium of the mouse.

Figure 17. The graph shows that the rate of growth of tumors formed VEGI-251-transfitsirovannykh cancer cells MDA-MB-231, varies in accordance with the amount of VEGI produced these cancer cells. Tumors, in which the level of VEGI is higher (G9-1R), grow much more slowly than tumors with low levels VEGI (G9-2R).

Figure 18. Northern blot analysis of transcripts of VEGI. Panel A, the expression of VEGI in human cells: Jurkat cells, T-cell leukemia person; L293, embryonic kidney cells; HL60, promyelocytic leukemia person; V.E., venous endothelial cells (10th passage); A431, epidermoid carcinoma of the person; V.E.-2, venous endothelial cells (20th passage); Raji, Burkitt's lymphoma person; A.U., arterial endothelial cells; TNR-1, monocytic leukemia person; CCD-29Lu, emphysema person; SAMA, breast cancer; AN3CA, cancer of the uterus; SK.UT.1, cancer of the uterus; MG63, osteoblastoma; HOS, osteoblastoma; MCF7, breast cancer; OVCAR-3, ovarian cancer; CAOV-3, ovarian cancer; HUVE, endothelial cell is umbilically (umbilical) Vienna person; AOSMIC, smooth muscle cells. The calculated size of the transcript 6.5 TPN Panel, expression of VEGI in adult tissues using Northern blots of multiple tissues (Clontech): conducted three separate blotting. Shown positive results in any of the three experiments.

Figure 19. A graph showing the effect of VEGI on the proliferation of endothelial cells and cancer cells of the breast. The number of cells was plotted on the graph depending on the concentration VEGI, as shown. Shows the growth inhibition of the cells AVE (dark circles), but not cells MDA-MB-231 (light circles) or MDA-MB-435 (triangles). Cancer cells and cells of AWE were sown in quantity at 2000 and 8000 cells per well, respectively, in triplicate in 24-well plates. The culture medium containing full (Gibco) and 10% FCS. In the environment of the cells AWAE added FGF-2 (1 ng/ml). The cultures were maintained at 37°C, 5% CO2within 6 days. Then the cells were trypsinization, and the number of cells was determined using the counter of Coulter. Shows one-fifth of the total number of received cells AWE to normalize the comparison with cancer cells.

Figure 20. The expression of VEGI in proliferating or resting endothelial cells. The mRNA level of VEGI in cultured cells HUVE define Northern-blotting analysis. Identical amounts of total RNA(15 µg) is applied on each track, as evidenced by the intensity of the band β-actin. Total RNA receive at specified time points (days after planting). The number of cells in each culture flask determine at the same time. This experiment is carried out in two replications. Cells were seeded at a density of 125,000 cells per flask (T-25) in full, 10% FCS, 6 ng/ml FGF-2 and cultured at 37°C, 5% CO2.

Figure 21. A graph showing the effect of VEGI on the ability of the cells AWE to form capillaroscopy tubes on collagen gels. The ability of recombinant VEGI to inhibit the formation of capillarity tubules cells AVE. The value p (t - test)are given above the columns, obtained by comparison of the degree of education capillaroscopy tubules cells AWE in the presence of various concentrations of VEGI, as shown, with the degree of their education in the absence of VEGI in the culture medium.

Figure 22. A graph showing the inhibition of VEGI angiogenesis in collagen gels placed on the chorioallantoic membrane of chicken embryo (CAM). The growth of new capillary blood vessels in the granules of the collagen gel (0.05 ml), placed on ITSELF, induce or FGF-2 (100 ng), or VEGF (250 ng), prisoners in these gels. The degree of angiogenesis in the gels is determined by evaluating the fluorescence intensity of FITC-dextran, injectable into the bloodstream ITSELF and hold the th in this gel. Shows the inhibition of growth of capillary blood vessels VEGI, which is indicated by a value less than 100. This inhibitor is also enclosed in the gels. Vertical lines represent standard deviation of three replicates experimental values.

Figure 23. A graph showing the inhibition of VEGI growth of tumor xenografts of cancer of the breast man in Nude naked mice. The mixture sverkhekspressiya VEGI or transfected with the vector cells Cho (5×106cells per injection) and breast cancer cells (1×106cells per injection) were injected with in the fatty tissue of the mammary gland of female Nude mice. The sizes of tumors were recorded after injection. Panel A: size Chart tumor xenografts of MDA-MB-231 (mm2) as a function of time after inoculation (days). Panel: Graph of the tumors xenografts of MDA-MB-435 (mm2) as a function of time after inoculation (days). Bright circles inoculated together with transfitsirovannykh vector cells SNO. Dark circles inoculated together with transfitsirovannykh secretively VEGI cells SNO.

Figure 24. A graph showing the effect of VEGI-192aon the proliferation of endothelial cells. Shows the growth inhibition of the cells AWE correctly re-laid VEGI-192abut not wrong is re-laid VEGI- 192aor buffer. Cells AWE plated at 8,000 cells per well, respectively, in triplicate in 24-well plates. The culture medium containing full (Gibco) and 10% FCS. In the environment of the cells AWAE added FGF-1 (1 ng/ml). The culture was kept at 37°C, 5% CO2within 6 days. Then the cells were trypsinization and the number of cells was determined by means of a counter of Coulter. Shows one-fifth of the total number of received cells AWE to normalize the comparison with cancer cells.

The implementation of the invention

This invention provides new isoforms of polynucleotides and VEGI polypeptides that inhibit the growth of vascular endothelial cells, and methods for treating diseases and processes that are mediated by angiogenesis or associated with angiogenesis, through the introduction of these polynucleotides, polypeptides and other agents. Polynucleotide or VEGI polypeptides of this invention can be isolated from body fluids including, but not limited to, serum, urine, and ascites, or synthesized by chemical or biological methods (e.g., using cell culture, expression of recombinant genes).

Recombinant methods include amplificatio genes from sources of DNA using polymerase chain reaction (PCR) and amplification of genes from sources RNA with what ispolzovaniem reverse transcriptase/PCR. These methods are well known in this field. VEGI inhibits the sprouting of blood vessels in tissues such as revascularisation or vascularized tumor. The invention includes a protein that has a molecular mass of approximately 22 kDa, and any modified form of this protein, including, but not limited to, a shortened form or post-translational modification, such as a glycosylated form of this protein, which is able to overcome the angiogenic activity of endogenous growth factors.

Definition

As described herein, "mutant" or "variant" polynucleotide or VEGI polypeptide is a polynucleotide or polypeptide sequence that contains a single (one) or more deletions, additions, transverse or changes in nucleic acid sequence or amino acid sequence. As described there, the mutant sequence VEGI may result in a shortened or modified polynucleotide or VEGI polypeptide, is increased or decreased expression of polynucleotide or VEGI polypeptide or any combination thereof. The mutation can be in coding, non-coding, 5'- or 3'-flanking, genomic or coding nucleotides.

"Functionally intact" option polynucleotide VEGI isoforms (i.e. VEGI-192a, VEGI-192bor VEGI-251or polypeptide isoform VEGI is a sequence of VEGI, which stores at least one aspect of the functions of the VEGI isoforms. Functionally intact variations can arise due to differences in linear sequence arising from, for example, mutations (mutations) of a single base, add (adds), deletions (deletions and/or modifications (modifications) bases. This difference may arise from changes in sugar (sugars) and/or in communications (connections) between the bases. With regard to polypeptides that are functionally intact variants can occur, for example, by conservative and/or non-conservative substitutions of amino acids, amino acid analogues and deletions. Function, which is stored depends on the relevant functions considered. For example, if polynucleotide VEGI isoform is considered as a probe, the relevant feature is the ability of the variant polynucleotide sequence to gibridizatsiya with the target. If polynucleotide considered in relation to its ability to encode a polypeptide isoforms VEGI (or its fragment), the relevant function is the ability of the variant sequences can encode the same polypeptide. If the polypeptide isoforms VEGI considered in relation to its R & d ability is to bind to a specific molecule (such as an antibody or other protein), the relevant function of the variant sequence is its ability to encode a polypeptide with equivalent binding characteristics. Polypeptide VEGI isoforms may also be considered in relation to the biological activity of the encoded gene product (e.g., biological activity ascribed to a gene product corresponding to polynucleotides VEGI isoforms as a result of the inclusion of this gene product of a family (families) proteins and/or identification of a functional domain present in the gene product). A polypeptide demonstrating "functional activity"refers to a polypeptide that is able to display one or more functional activities associated with the complete or Mature polypeptide isoforms VEGI. Such functional activities include, but are not limited to, biological activity (e.g., inhibition of angiogenesis, inhibition of proliferation of vascular endothelial cells, induction of cell adhesion, antigenicity (ability to bind or compete with one or more polypeptides isoforms VEGI for binding with the antibody against the VEGI isoforms), immunogenicity (ability to generate antibody which binds to one or more polypeptides isoforms VEGI), the ability to form polymers with other polyp is pridani VEGI and the ability to bind to a receptor or ligand for a VEGI polypeptide (for example, DR3).

In this context "expression" includes transcription and/or translation.

"Heterologous" means coming from (i.e. derived from) the body, genotypic different from the body, which make the comparison. For example, polynucleotide can be placed by means of genetic engineering in a plasmid or vector, originating from different source, so it becomes a heterologous polynucleotide. The promoter, which is associated with the coding sequence with which it is naturally associated, is a heterologous promoter.

Polynucleotide"reagent"polypeptide"reagent" or " antibody"reagent" is a substance, provide for the reaction, a substance with some famous and desirable parameters for this reaction. The reaction mixture may also contain a "target", such as polynucleotide, antibody, polypeptide or group of polypeptides which are able to react with this reagent. For example, in some diagnostic tests the presence and/or amount of target in the sample is determined by adding the reagent, allowing this reagent to interact with the target and measuring the amount of reaction product (if available). In the context of clinical use "target" can also be a cell, a mixture of cells, tissue is whether the body who are the target of an introduced substance, such as a pharmaceutical compound.

The term "stable duplex" polynucleotides or stable complex formed between any two or more components in a biochemical reaction, refers to a duplex or complex, which is fairly long-lasting for existence between education duplex or complex and the subsequent detection, including any optional stage leaching or other manipulation that can take place in this period of time.

Gene or polynucleotide is differentially expressed in the test sample when polynucleotide detects when a higher or lower level in comparison with the control sample of the same type. Usually the differentially expressed polynucleotide includes polynucleotide, which is expressed in such a way that, for example, mRNA is found at levels at least about 25%, at least about 50-75%, at least about 90%, at least about 2 times, at least about 4-fold, at least about 5 times and at least about 10 times or higher level (i.e. sverkhekspressiya), or lower (for example, under the ciently expressed). The comparison may be performed, for example, between the two tissues, if using in situ hybridization, or other method of analysis, which allows some degree of discrimination between different types of cells in this tissue. This comparison can also be made between cells, isolated from their tissues.

"Effective amount" of the drug, compound or pharmaceutical composition refers to the amount sufficient for obtaining beneficial or desired results, including clinical results, such as growth inhibition, vascular endothelial cells, inhibition of angiogenesis, stimulation of angiogenesis, reduction of tumor size, delay the growth of cancer cells, reducing one or more symptoms caused by the disease, improve the quality of life of patients suffering from the disease, decreasing the dose of other medications required to treat the disease, increases the effect of other medicines, delay disease progression and/or prolonging survival of patients, either direct or indirect. An effective amount can be administered in the form of one or more injections. As is understood in the clinical context associated with angiogenesis disease, an effective amount of a medicinal product, with the unity or pharmaceutical composition can be achieved in conjunction with another drug, connection or another pharmaceutical composition or without them. Thus, the term "effective amount" may be considered in the context of the introduction of one or more therapeutic agents, and a single agent may be considered as provided in an effective amount if, together with one or more other agents, can be obtained or achieved the desired result.

In this context, the term "treatment" is an approach for obtaining beneficial or desired results, including clinical preferably results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following results: reduced proliferation vasculary endothelial cells, inhibition of angiogenesis, stimulation of angiogenesis, the tumor size decreased, decreasing symptoms resulting from the disease, improve the quality of life of patients suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease and/or prolonging survival of patients.

"Development" or "progression" is associated with angiogenesis diseases here means initial manifestations and/or pic is abusee the progression of the violation. Development associated with angiogenesis diseases can be detektivami and estimated standard clinical methods. However, the development also applies to the progression of the disease, which can be redetection. For the purposes of this invention, the terms "development" or "progression" refers to the biological course of the pathological condition. The term "development" includes the manifestation, the recurrence and occurrence. In this context, the term "appearance" or "manifestation" is associated with angiogenesis disease involves the initial occurrence and/or recurrence.

In this context, a "developmental delay" associated with angiogenesis disease means a delay, postponement, deceleration, braking, stability and/or delay the development of this disease. This delay may vary in time depending on the history of violations and/or medical profile of the subject to treatment of the individual. As obvious to the person skilled in the art, a sufficient or significant delay can actually include a warning, namely, that the individual does not develop detective disease. A method that "delays" development of the disease, is a method that reduces the extent of the disease in these time frames compared with non-use of this innovation is on the way. Such comparisons are usually based on clinical studies, using a statistically significant number of subjects, although this knowledge may be based on episodic data. "Developmental delay" may mean that the extent and/or undesirable clinical attenuated and/or the time course of the progression is weakened or lengthened in comparison with the absence of the introduction of the agent. Thus, this term includes, but is not limited to relieving symptoms, reducing the extent of disease, stabilized (i.e. not worsening) state of disease, delay or slowing of disease progression, and remission (whether partial or total), detektiruya or redetection.

In the application here and in the accompanying claims only forms "a", "and" and "the" include plural objects, unless the context dictates explicitly to the contrary. So, for example, reference to "polynucleotide" includes many such polynucleotides, and reference to "the agent" includes reference to one or more agents and equivalents known to specialists in this field, and so on

Common methods

The practice of this invention will be used, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), Microbiology, cell biology, biochemistry immunologie, which are within its competence in this area. Such methods are fully explained in the literature, such as: "Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J.Gait, ed., 1984); "Animal Cell Culture" (R.I.Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M.Wei and C.C.Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M.Miller and M.P.Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M.Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J.E.Coligan et al., eds., 1991).

Polynucleotide this invention

This invention provides polynucleotide VEGI isoforms, including polynucleotide VEGI-192a, VEGI-192band VEGI-251. The nucleotide sequence corresponding to these new isoforms are shown in tables 1, 2 and 3 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3), and the corresponding polypeptide sequence is shown in tables 4, 5 and 6 (SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6).

Polynucleotide sequence, shown in table 1 (SEQ ID NO:1)was obtained by sequencing cDNA clone, which was deposited in the American type culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, and got the access number.

Polynucleotide sequence, shown in table 2 (SEQ ID NO:2)was obtained by sequencing cDNA clone, which was deposited in the American type culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, and received the Omer access.

With reference to the sequence alignment comparing the predicted amino acid sequence for SEQ ID NO:4, 5 and 6 (table 7), the C-terminal region of polypeptides encoded by those SEQ ID sequences is identical from Val-24 VEGI-174to-the end of this protein. However, N-ends of these four isoforms are different. The examples show that VEGI-174no inhibits angiogenesis, as it is not exported efficiently from cells after expression. In contrast, VEGI-251effectively transported into the extracellular environment after expression and is therefore effective in the inhibition of angiogenesis. Export VEGI-251leads to the elimination of the pre-sequence; it is believed that the site of proteolysis is in position 61 or 96 VEGI-251; but he may also be in another site, localized approximately between Glu-20 and Ser-57 VEGI-251. Possible sites include, but are not limited to, E, K, E77, S81, R90 and C. The purified polypeptide VEGI-192ais also effective in inhibiting the growth of vascular endothelial cells.

Thus, this invention provides isolated nucleic acid molecule containing sequences corresponding to the new VEGI isoforms shown in tables 1, 2 and 3 (SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3). Polynuclear the Chida this invention, including fragments of polynucleotides of the present invention, used as probes, primers, expression systems (including systems of expression in vivo and in vitro, described here, which can also be the basis for gene therapy) and screening systems. A particularly useful application of these polynucleotides will be discussed below.

The term "isolated" nucleic acid molecule refers to a molecule of nucleic acid, DNA or RNA, which has been removed from its natural environment. In some embodiments deleted at least 50%, preferably at least 70%, more preferably at least 80% and even more preferably at least 90% of the substances with which it is associated in nature. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of this invention. Other examples of isolated DNA molecules include recombinant DNA molecules contained in heterologous cells masters, or purified (partially or essentially purified) DNA molecules in solution. Isolated RNA molecules include RNA transcripts in vivo or in vitro DNA molecules of the present invention. In addition, the isolated nucleic acid molecule include such molecules, obtained by synthesis. Thus, "isolated" what olignucleotides or polypeptides are also called recombinant or other non-naturally occurring forms of polynucleotides or polypeptides, which, due to their origin or manipulation: (1) are not associated with all or part of polynucleotide or polypeptide with which it is associated in nature; (2) associated with polynucleotide or polypeptide other than polynucleotide or polypeptide with which it is associated in nature, or (3) does not occur in nature, or (4) in the case of polypeptides that result from expression of recombinant polynucleotides.

The invention also provides molecules of nucleic acids (including, as is well understood to a person skilled in the art and described herein, isolated and/or recombinant forms), encoding the Mature form of the polypeptide proteins described herein. Amino acid sequence of the complete polypeptide isoforms VEGI includes a leader sequence and a Mature protein. According to the hypothesis of the signal sequence is initiated as soon as the export of the growing protein chain across the rough endoplasmic network, proteins secreted by mammalian cells have a signal or secretory leader sequence that is cleaved from the complete polypeptide with education Sekretareva "Mature" form of the protein. The majority of mammalian cells and even insect cells break down secreted proteins with the same specificity. However, in some cases splitting secretarye the CSOs protein is not completely homogeneous, that leads to two or more Mature varieties of protein. Next, it's long been known that the specificity of cleavage of the secreted protein is determined, ultimately, the primary structure of the complete protein, meaning that it is inherent in the amino acid sequence of this polypeptide.

This invention also provides polynucleotide encoding the protein. As is well known in this field, fused protein or polypeptide is a polypeptide containing the parts in a different position, compared to the natural situation. These sites can in norm to exist as separate proteins and connect fused together in the polypeptide, or they may be the norm to exist in the same protein, but be placed in a new location in the slit of the polypeptide. Thus, this invention provides polynucleotide, in which the coding sequence for the Mature protein may be fused in the same reading frame to a polynucleotide sequence that facilitates expression and secretion of the polypeptide from the host cell, such as a leader sequence which functions as a secretory sequence for the regulation of the transport of a polypeptide from the cell. Polypeptide having a leader sequence is a pre-protein and can be a leader in series is here, tsepliaeva the host-cell with the formation of the Mature form of the polypeptide. Polynucleotide can also encode Pro-protein, which is the Mature protein plus additional 5'-amino acid residues. The Mature protein having proposedvalue, is a Pro-protein is an inactive form of this protein. When the removal of this proposedvalue remains active Mature protein. Thus, for example, polynucleotide of the present invention may encode a Mature protein, or a protein having proposedvalue and pre-sequence (leader sequence).

Polynucleotide the present invention provides the coding sequence fused in reading frame to a marker sequence that provides purification or detection of the polypeptide of the present invention. This marker sequence can be getservletinfo label provided by the vector pQE-9, to ensure purification of the Mature polypeptide is fused with this token, in the case of a bacterial host, or, for example, the marker sequence may be hemagglutinine () ON the label when using mammal cells as a host cell, such as COS cells. ON-label corresponds to the epitope derived from hemagglutinin protein of influenza virus (Wilson I., et al. Cell, 37:767 (1984).

Thus, the term "polynucleotide encoding a polypeptide" includes polynucleotide, which includes only coding sequence for the polypeptide and polynucleotide, which includes additional coding and/or noncoding sequence. For the purposes of this invention and in order to avoid burdensome links on the complementary chain argues that the antisense (or complementary) circuit such polynucleotide also encodes this sequence, i.e. a polynucleotide sequence that "encodes" a polypeptide that includes both conventional coding strand and a complementary sequence (or chain).

Mutant or variant of polynucleotide may be naturally occurring allelic variant of this polynucleotide or not occurring natural variant of this polynucleotide. Such nucleotide mutants or variants include deletion variants with substitutions and options with additions or insertions. The variant sequence may result in a shortened or modified polynucleotide or polypeptide, is increased or decreased expression of polynucleotide or polypeptide or any combination thereof. Option can be in coding, non-coding, 5'- or 3'-flanking, genomic or coding well is leotide.

In some embodiments polynucleotide sequence contains a sequence different from the sequence shown in tables 1, 2, or 3 (SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3), due to the degeneracy of the genetic code. The genetic code is well known in this field. The receipt of such degenerate variants is routine for a person skilled in the field. Thus, in some embodiments, the invention provides polynucleotide encoding the polypeptide SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

Further, the invention provides fragments or a shortened form of isolated nucleic acid molecules described herein. The " fragment of an isolated nucleic acid molecule having the nucleotide sequence given here of the nucleotide sequences or complementary to her chain, refers to fragments of length at least 5 NT at least 10 NT, at least 15 NT, at least 20 NT, at least 30 NT, at least, 40, 50, 100, 150, 200, 250, 300, 400 or 500 NT (contiguous nucleotides). These fragments have numerous applications, which include, but are not limited to, use as diagnostic probes and primers as discussed here. As is well understood specialists in this field, usually the probe is used to detect the project target by hybridization. In some embodiments, the probe may contain a label or means by which a label can join, either before or after the hybridization reaction. Suitable labels include, but are not limited to, radioactive isotopes, fluorochromes, chemiluminescent compounds, dyes and enzymes. Next, the experts in this field it is clear that the primer is usually extended by polymerization after hybridization with sequence-target. Of course, larger fragments with a length of 50-1500 NT also applicable in accordance with this invention. Under the fragment with a length of at least 20 NT mean fragments which include 20 or more contiguous bases from the nucleotide sequence. In some embodiments, these fragments contain nucleotides 93 and 94 of the sequence shown in table 1 (SEQ ID NO:1), or nucleotide 386 and 387 of the sequence shown in table 2 (SEQ ID NO:2). Alternatively, the fragments may have a length less 1500, 1250, 1000, 750, 500, 250, 150, 100, 50, 40 HT and include nucleotides 93 and 94 of the sequence shown in table 1 (SEQ ID NO:1), or nucleotide 386 and 387 of the sequence shown in table 2 (SEQ ID NO:2).

This invention also provides polynucleotide containing a sequence VEGI-192a(nucleotides 1-93 the sequence shown in table 1 (SEQ ID NO:1) and the and VEGI- 192b(nucleotides 1-386 the sequence shown in table 2 (SEQ ID NO:2).

In some embodiments, the invention provides polynucleotide containing at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100 or more contiguous nucleotides of SEQ ID NO:1 (which can usually also called sites), where these contiguous nucleotides within nucleotides 1-93 the sequence shown in table 1 (SEQ ID NO:1). In some embodiments, the invention provides polynucleotide containing at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100 or more contiguous nucleotides of SEQ ID NO:1, where the specified contiguous nucleotides include nucleotides 93 and 94 of SEQ ID NO:1.

In some embodiments, the invention also provides isolated polynucleotide, which contains at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100, at least 150, at least 175, at least 200, at least 250, at least 275, at least 300, at least 350, at least, 375, at least 400 or more is Minich nucleotides SEQ ID NO:2, where these contiguous nucleotides within nucleotides 1-386 the sequence shown in table 2 (SEQ ID NO:2). In some embodiments, the invention provides polynucleotide containing at least 10, at least 15, at least 18, at least 20, at least 25, at least 30, at least 50, at least 100, at least 150, at least 175, at least 200, at least 250, at least 275, at least 300, at least 350 at least, 375, at least 400 or more contiguous nucleotides of SEQ ID N0:2, where these contiguous nucleotides include nucleotides 386 and 387 of SEQ ID NO:2.

This invention provides an isolated polynucleotide containing a sequence encoding the polypeptide SEQ ID NO:4. This invention also provides an isolated polynucleotide containing a sequence encoding at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:4, where these adjacent amino acids within amino acid residues 1-26 in table 4 (SEQ ID NO:4). This invention also provides an isolated polynucleotide containing a sequence encoding at least 5, at least 10, at least 15, at least 20, at the ore, 25 or more contiguous amino acids of SEQ ID NO:4, where these contiguous amino acids include amino acids 26 and 27 of the sequence shown in table 4 (SEQ ID NO:4). This invention also provides an isolated polynucleotide containing the sequence encoding amino acid residues 5-192, 10-192, 15-192, 25-192 the sequence shown in table 4 (SEQ ID NO:4).

This invention provides an isolated polynucleotide containing a sequence encoding the polypeptide SEQ ID NO:5. This invention also provides an isolated polynucleotide containing a sequence encoding at least 5, at least 8, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:5, where these adjacent amino acids within amino acid residues 1-26, shown in table 5 (SEQ ID NO:5). This invention also provides an isolated polynucleotide containing a sequence encoding at least 5, at least 8, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:5, where these contiguous amino acids include amino acids 26 and 27 of the sequence shown in table 5 (SEQ ID NO:5). This invention also provides an isolated polynucleotide, soda is containing the sequence encoding amino acid residues 5-192, 10-192, 15-192, 20-192 or 25-192 the sequence shown in table 5 (SEQ ID NO:5).

It is clear that the area adjacent amino acids or nucleotides that are within a given pair of amino acids or nucleotides, may, but need not, include any member of the specified pair. For example, contiguous nucleotides within nucleotides 1-93 SEQ ID NO:1 can include a nucleotide 1 and/or nucleotide 93 of SEQ ID NO:1.

Embodiments of the present invention eliminate polynucleotide encoding a polypeptide consisting of amino acids 27-192 SEQ ID NO:4 or SEQ ID NO:5, or any shortened form of such polynucleotides.

This invention also provides polynucleotide containing a sequence encoding any of the polypeptides described herein VEGI.

In typical embodiments the fragments polynucleotides of the present invention encode a polypeptide which demonstrates a functional activity. Under the polypeptide demonstrating functional activity, mean polypeptide, which can exhibit one or more known functional activities associated with a full or Mature VEGI polypeptide. Such functional activities include, but are not limited to, biological activity (e.g., inhibition of angiogenesis, inhibition of proliferation of vascular & Telelink cells, the induction of cell adhesion), antigenicity (ability to bind or compete with a polypeptide VEGI-192aand/or VEGI-192bfor binding with the antibody against VEGI-192aand/or against VEGI-192b), immunogenicity (ability to generate antibody which binds to the polypeptide VEGI-192aand/or VEGI-192b), the ability of formation of polymers with other VEGI polypeptides and the ability to bind to a receptor or ligand for a VEGI polypeptide (e.g., DR3).

Similarly VEGI polypeptides encoded by any of the described here polynucleotides may have one or more functional activities VEGI described above and here.

Another embodiment of the present invention provides an isolated polynucleotide having at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity with polynucleotides of the present invention described herein. One embodiment provides an isolated polynucleotide having at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity with the sequence VEGI-192aor VEGI-192bthat show is Noah in table 1 or table 2 (SEQ ID NO:1 or SEQ ID NO:2). In other embodiments, the isolated polynucleotide additionally have less than 85%, 83%, 80%, 75%, 70% identity sequence with the above sequence VEGI-192aor VEGI-192b. The invention also includes isolated polynucleotide having at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity with fragments of at least 10 contiguous nucleotides (or more, for example, 15, 18, 20, 25, 35, 40, 45, 50, 60, 75 or 100 contiguous nucleotides) of the sequence shown in table 1 (SEQ ID NO:1) or table 2 (SEQ ID NO:2), where the contiguous nucleotides include nucleotides 93 and 94 of SEQ ID NO:1 or nucleotides 386 and 387 of SEQ ID NO:2. In some embodiments polynucleotide have at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity with fragments of at least 10 contiguous nucleotides (or more, for example, 15, 18, 20, 25, 35, 40, 45, 50, 60, 75 or 100 contiguous nucleotides) of the sequence shown in table 1 (SEQ ID NO:1), or the sequence shown in table 2 (SEQ ID NO:2), where these contiguous nucleotides within nucleotides 1-93 SEQ ID NO:1 or nucleotides 1386 SEQ ID NO:2.

When they say that polynucleotide or district polynucleotide has a certain percentage (for example, 80%, 85%, 90% or 95%) of "sequence identity" with respect to another sequence, this means that when aligning the percentage basis is the same in the comparison of these two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in this field, for example programs described in Current Protocols in Molecular Biology (F.M.Ausubel et al., eds., 1987) Supplement 20, section 7.718, Table 4.7.1. The percent identity can be determined electronically, e.g., by using the MegAlign program. TM.program (DNASTAR, Inc., Madison Wiss.). MegAlign. TM.program can create alignment between two or more sequences according to different methods, for example, using the Clustal algorithm. (See, for example, Higgins, D.G. and P.M.Sharp (1988) Gene 73:237-244.) The algorithm Clustal groups sequences into clusters by analyzing the distances between all pairs. These clusters are aligned in pairs and then in groups. The percentage of similarity between two amino acid sequences, such as sequence a and sequence B, is calculated by dividing the length of the sequence And minus the number of residues in breach of the sequence A, minus the number ostad is in breach of the sequence is At the amount of matching residues between sequence a and sequence B, and multiplying by 100. Gaps with low similarity or lack of similarity between two amino acid sequences are not included in the definition percent of similarity. The percent identity between the sequences of nucleic acids can also be counted or calculated by other methods known in this field, for example, by the method Jotun Hein. (See, for example, Hein, J. (1990) Methods Enzymol. 183:626-645.)

This invention also provides an isolated nucleic acid which hybridizes in conditions of high stringency to a nucleic acid having a sequence complementary to the nucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, or a nucleic acid having a sequence complementary to the nucleotide sequence that encodes a polypeptide SEQ ID NO:4 or SEQ ID NO:5, or its complement.

From the point of view of the hybridization conditions, the higher the required sequence identity, more stringent conditions are hybridization, if such a sequence is determined by their ability to gibridizatsiya sequence polynucleotide of the present invention. Thus, this invention also includes polynucleotide, which is capable of g is to breitowitz sequence, containing polynucleotide of the present invention, as discussed here. An example of stringent hybridization conditions is incubation overnight at 42°in a solution: 50% formamide, 1×SSC (150 mm sodium chloride, 15 mm sodium citrate), 50 mm sodium phosphate (pH of 7.6), 5X denhardt's solution, 10% textresult and 20 microgram/ml denatured, fragmented DNA, salmon sperm, followed by washing the filters in 0.1×SSC at approximately 65°C. Discussion regarding hybridization reactions, see below.

In one embodiment, the invention provides an isolated polynucleotide contain a sequence of at least 10 contiguous nucleotides (or more, for example, 15, 18, 20, 25, 35, 40, 45, 50, 60, 75 or 100 or more contiguous nucleotides), which hybridizes with polynucleotides (such as DNA or RNA)that contains the sequence depicted in table 1 (SEQ ID NO:1) or table 2 (SEQ ID NO:2), or its fragment, as described above, in the conditions in which it is not hybridized with other polynucleotide of mammalian cells, preferably human cells, or in conditions in which hybridization with polynucleotide having the sequence depicted in table 1 (SEQ ID NO:1) or table 2 (SEQ ID NO:2), is increased in relation to hybridization with other polynucleotide of mammalian cells. In some embodied eniah these fragments include nucleotides 93 and 94 of SEQ ID NO:1 or nucleotides 386-387 SEQ ID NO:2. In some embodiments, these fragments are within nucleotides 1-93 SEQ ID NO:1 or nucleotides 1-386 SEQ ID NO:2.

These embodiments are especially applicable in the diagnostic context (detection).

In another embodiment, the invention includes a polynucleotide sequence containing at least 10, preferably 15, preferably 18, preferably 20, more preferably 25, more preferably 35, more preferably 50, more preferably 75, 100, 125, 150, 200, 250 contiguous nucleotides non-coding (i.e. flanking) parts, shown in table 1 (SEQ ID NO:1) or table 2 (SEQ ID NO:2). These embodiments may be particularly useful as diagnostic probes or as primers for amplification of non-coding parts of the gene VEGI-192aor VEGI-192b.

It is clear that (unless otherwise specified or does not require) any embodiment of the present invention described herein, which is polynucleotides, includes both double-stranded form and each of two complementary single-stranded forms known or predicted that they form a double-stranded form.

The hybridization reaction can be performed in different measures. Conditions that increase stringency hybridization reaction, widely known and published in this area. See, for example,Sambrook et al. (1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperature of 25°, 37°S, 50°and 68°C; concentration of the buffer 10×SSC, 6×SSC, 1×SSC, 0,1×SSC (where SSC is a 0,15M NaCl and 15 mm citrate buffer) and their equivalents using other buffer systems; the concentration of formamide 0%, 25%, 50% and 75%; incubation periods from 5 minutes to 24 hours; 1, 2 or more stages of leaching; periods of incubation under leaching 1, 2, or 15 minutes and wash solutions of 6×SSC, 1×SSC, 0,1×SSC, or deionized water. An example of stringent hybridization conditions is hybridization at 50°With or higher and 0.1×SSC (15 mm sodium chloride/1.5 mm sodium citrate). Another example of stringent hybridization conditions is incubation overnight at 42°in a solution: 50% formamide, 1×SSC (150 mm sodium chloride, 15 mm sodium citrate), 50 mm sodium phosphate (pH 7,6), 5×denhardt's solution, 10% textresult and 20 microgram/ml denatured, fragmented DNA, salmon sperm, followed by washing the filters in 0.1×SSC at approximately 65°C. Stringent hybridization conditions are hybridization conditions, which are at least as stringent as the above representative conditions. Other stringent hybridization conditions known in this field and can also be used for ID manually is the ratification of the nucleic acids of this particular embodiment of the present invention.

This invention also provides primers and probes containing the plot of SEQ ID NO:1 or SEQ ID NO:2, where the site is located within nucleotides 1-93 SEQ ID NO:1 or nucleotides 1-386 SEQ ID NO:2. This invention also provides primers and probes containing the plot of SEQ ID NO:1 or SEQ ID NO:2, where this plot includes nucleotides 93 and 94 of SEQ ID NO:1 or nucleotides 386 and 387 of SEQ ID NO:2.

Probes from more than one polynucleotide sequence provided herein may gibridizatsiya with the same nucleic acid, if cDNA from which they were produced, which corresponds to one mRNA. Using probes, in particular labeled probes for DNA sequences, we can distinguish homologous or related genes. The source of homologous genes can be any species, for example species of Primate, dog, cat, sheep, horses, yeast, nematodes. Can be used probes from more than 10 nucleotide ("NT"), for example, probes with size in the range of about 15 NT, 18 NT, 20 NT, 25 NT, 75 NT and 100 NT, but usually the probe approximately 15 NT is sufficient sequence for unique identification.

Tm" is the temperature in degrees centigrade at which 50% of the polynucleotide duplex derived from the complementary chains linked by hydrogen bonds in antiparallel direction the result is pariani basis according to the rules of Watson-Crick, dissociated into single chains in the experiment. Tmcan be predicted in accordance with the standard formula, such as:

Tm=81,5+16,6log[X+]+0,41(%G/C)-0,61(%F)-600/L,

where [X+] denotes the concentration of cation (usually sodium ion, Na+) in mol/l; (%G/C) denotes the number of G residues and percent of the total number of residues in the duplex; (%F) is the percentage of formamide in solution (wt./about.) and L denotes the number of nucleotides in each circuit of the duplex.

As described above, the invention includes variants or modifications of polynucleotides VEGI-192aand VEGI-192bsuch as deletions, substitutions, additions or changes in the nature of any parts of the molecules of nucleic acids. Option or a modification is any difference in nucleotide sequence as compared with polynucleotides, which, as shown here, encodes a polypeptide VEGI-192aor VEGI-192band/or any difference in the nucleic acid molecules of this polynucleotide (polynucleotides). Such changes may be useful to facilitate cloning or modifying the expression of polynucleotides VEGI-192aor VEGI-192b. Such changes may also be useful to impart desirable properties to polynucleotide (polynucleotides), such as stability. The definition of polynucleotide, given the Noah here gives examples of these modifications. Thus, the invention also includes functionally intact variants of the sequences of the nucleic acids described herein, which include substitutions, additions and/or deletions of nucleic acids. Variants include naturally occurring variants of a polynucleotide sequence (e.g., degenerate variants, allelic variants, and so on). Typically, allelic variants contain 15-25% incorrect matings (mismatches) base pairs (BP) and may contain only 5-15%, or 2-5%, or 1-2% incorrect matings base pairs, as well as erroneous pairing of one base pair.

As described above, the present invention includes polynucleotide VEGI-192aor VEGI-192bincluding a full-sized (reprezentirovanii), versions of the coding, non-coding or parts thereof. Partial genomic map parcel VEGI-192ashown in figure 6, including the predicted boundaries of introns-exons. This invention may additionally include the 3'- and 5'-untranslated region, were found in the Mature mRNA, specific regulatory sequence for transcription and translation, such as promoters, enhancers, etc. including approximately 1 TPN and perhaps more flanking genomic DNA or 5'-or 3'-end of TRANS euroimage plot. This genomic DNA can be isolated in the form of a fragment 100 TPN or less and essentially free of flanking chromosomal sequence. Genomic DNA flanking the coding section with either 3'-or 5'-end, or internal regulatory sequences, such as find sometimes in introns, contains sequences required for proper tissue-specific, statespecific or specific to the state of disease expression. Embodiments are also mRNA and cDNA sequences and fragments thereof, including fragments, which include part of the coding segment VEGI-192aor VEGI-192b. Normally mRNA species have contiguous exons with intervening introns, when they are present, being removed by nuclear RNA splicing, with the formation of a continuous open reading frame that encodes a polypeptide. Varieties of mRNA may also exist with both exons and introns, where these introns can be removed by alternative splicing. In addition, different varieties of mRNA encoded by the same genomic sequences that may exist in varying amounts in the cell, and the detection of these various quantities of mRNA species may indicate differential expression of the encoded gene product is in the cell.

The invention also includes polynucleotide encoding functionally equivalent variants and derivatives of full-VEGI-192aor VEGI-192band their functionally equivalent fragments (such as deletion of amino acids from N-Terminus and/or C-end VEGI-192aor VEGI-192b), which may increase, reduce or property as not to impact significantly on the properties of the coded polypeptide. For example, changes in the DNA sequence that do not alter the encoded amino acid sequence, and which result in conservative substitutions of amino acid residues that are not harmful non-conservative substitutions, deletions or additions of one or a small number of amino acids and substitution of amino acid residue analogues of amino acids, are changes that will not significantly affect the properties of the encoded polypeptide. Nucleotide substitutions which do not alter the encoded amino acid residues, can be useful for optimization of gene expression in various systems. Suitable replacement well-known experts in this field and are, for example, to reflect the preferred use of codons in a particular expression systems. In another embodiment, polynucleotide alternative splicing can result in a functionally equivalent fragment is whether VEGI option. Alternative versions of the variants of the polynucleotide sequence defined as a polynucleotide sequence corresponding to the mRNA, which differ in their sequence relative to each other, but originate from the same genomic area, such as an mRNA, which arise due to: 1) use of alternative promoters; 2) the use of alternative polyadenylation sites, or 3) the use of alternative splicing sites.

This invention also provides a DNA insert containing the nucleic acid having the nucleotide sequence of SEQ ID NO:1 or its complement. In other embodiments, the invention provides a DNA insert containing the nucleic acid having the nucleotide sequence of SEQ ID NO:2 or its complement.

As is well understood specialists in this field, the term "polynucleotide" refers to polymeric forms of nucleotides of any length, which include deoxyribonucleotides, ribonucleotides, and/or their analogues. The terms "polynucleotide" and "nucleic acid" are used interchangeably, as is well known in this field. Polynucleotide can have any three-dimensional structure. The term "polynucleotide" includes double-stranded, single-stranded molecules and molecules in the form of a triple helix. what if another is not specified or is not required any embodiment of the present invention described herein, which is polynucleotides, includes both double-stranded form and each of two complementary single-stranded chains which are known or predicted that they form dwuhtsepochechny form. Not all communication polynucleotide must be identical.

In some embodiments polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. The use of uracil as a replacement of thymine in deoxyribonucleic acid is also considered as the use of a pyrimidine analog.

As is well known in this field, the modification of the nucleotide structure may be performed before or after Assembly of the polymer. In some embodiments, the nucleotide sequence may be interrupted dinucleotide components. As described here, polynucleotide can be further modified after polymerization, for example, by conjugation with aiming component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotide analogue, mezhnukleotidnyh modifications, such as modifications with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoamide, carbamates, etc) and with charged linkages (EmOC is emer, phosphorothioate, phosphorodithioate etc), modification containing side chains, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc), modification intercalators (e.g., acridine, psoralen, etc.), modification, containing chelators (e.g., metals, radioactive metals, boron, metals, oxidizing agents, etc), modification, containing alkylating agents, versions with modified linkages (e.g., alpha anomeric nucleic acids, etc), and well as unmodified forms of polynucleotide (polynucleotides). All these modifications are well known in this field.

Further, any of the hydroxyl groups normally present in the sugars may be replaced by phosphonate groups, phosphate groups, protected by standard protective groups or activated for more links with additional nucleotides, or may be conjugated to a solid media. 5'- and 3'-terminal Oh-group can be phosphorylated or substituted amines or organic blocking groups of 1-20 carbon atoms. Other hydroxyl can also be derivativea standard protective groups.

Polynucleotide may also contain analogues of sugars ribose or deoxyribose, which is well known in this field, including, but not t is like, 2'-O-methyl, 2'-O-allyl, 2'-fluoro - or 2'-isidoros, carbocyclic analogues of sugars, (-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugar, sedoheptulose, acyclic analogs and not containing base analogs of nucleosides, such as methylribose).

Although usually use conventional sugars and bases, replacement analogues of sugars, purines and pyrimidines may be useful in the design of the final product, as an alternative of skeletal structures such as polyamide skeleton or phosphorothioate skeleton.

This invention encompasses compositions, including pharmaceutical compositions containing the described polynucleotide. These compositions can optionally contain suitable fillers, such as pharmaceutically acceptable excipients, including buffers, which are well known in this field.

The invention also provides kits containing any of these polynucleotides. In some embodiments, these kits contain polynucleotide SEQ ID NO:1 and/or SEQ ID NO:2. In some embodiments, these kits contain polynucleotide, encoding the polypeptide SEQ ID NO:4 and/or SEQ ID NO:5. In some embodiments, these kits contain probes and primers containing at least 15 contiguous, at least 20, Myung is our least 25, at least 30 or at least 50 nucleotides of SEQ ID NO:1 or SEQ ID NO:2, and these contiguous nucleotides within nucleotides 1-93 SEQ ID NO:1 or nucleotides 1-386 SEQ ID NO:2. These kits can optionally include the reagents and instructions for detecting the presence or absence or level of expression of VEGI-192aand/or VEGI-192b. The kits of this invention are in suitable packaging and may optionally provide additional components such as buffers or instruction.

This invention also provides polynucleotide described herein attached to a solid carrier. Methods of attaching polynucleotides to a solid carrier, for example to the surface of the arrays (matrices), also known in this field. Solid carrier is any suitable material, including granules based on polystyrene and glass chips, such as the GeneChip. RTM. product (Affymetrix, Inc., Santa Clara, Calif.). Cm. International Publication numbers WO 97/10365, WO 97/29212, WO 97/27317, WO 95/11995, WO 90/15070 and U.S. Patent numbers 5744305 and 5445934.

The invention also provides arrays containing polynucleotide VEGI-192aand/or VEGI-192b. Arrays of polynucleotides provides a high performance method that can analyze a large number of polynucleotides or polypeptides in a sample. This those is technology can be used as a tool to test for differential expression. Numerous methods of making arrays, as well as variations of these methods known in this field and is suitable for use in this invention. For example, arrays can be created by applying the polynucleotide probes on a substrate (for example, glass, nitrocellulose, and so on) in a two-dimensional array or matrix carrying the associated probes. These probes can be connected with the substrate either covalent bonds or non-covalent interactions, such as hydrophobic interactions. Samples polynucleotides can be detektirano marked (for example, with the use of radioactive or fluorescent labels) and then hybridized with these probes. The double-stranded polynucleotide containing labeled polynucleotide samples associated with polynucleotide probes can be detected, as only the unbound portion of the sample will be washed. Alternatively, polynucleotide test sample can be immobilized in the array, and probes detektirano marked. The methods of constructing arrays and methods of using these arrays are described, for example, Schena et al. (1996) Proc.Natl.Acad.Sci. USA 93(20):10614-9; Schena et al. (1995) Science 270(5235):467-70; Shalon et al. (1996) Genome Res. 6(7):639-45, USPN 5807522, EP 799897; WO 97/29212; WO 97/27317; EP 785280; WP 97/02357; USPN 5593839; USPN 5578832; EP 728520; USPN 5599695; EP 721016; USPN 5556752; WO 95/22058 and USPN 5631734.

Arrays can be used to study different the approaches of gene expression and can be used to determine the function of genes. For example, arrays can be used to detect differential expression of VEGI isoform, corresponding described here polynucleotide, where the expression is compared between the experimental cell and the control cell. For example, high expression of mRNA specific VEGI isoforms in the cell from a subject who has a disease, which is not observed in the corresponding normal cell, may indicate the Association of this VEGI isoforms with this disease. Examples of applications of additional arrays are described, for example, in Pappalarado et al., Sem. Radiation Oncol. (1998) 8:217, and Ramsay Nature Biotechnol. (1998) 16:40. In addition, many variations of the methods of detection using arrays are quite qualified specialists in this field and within the scope of this invention. For example, instead of immobilizing a probe on a solid substrate on a solid substrate can be immobilized in the test sample, which is then brought into contact with the probe.

Polynucleotide VEGI isoforms, which are differentially expressed in cells from individuals with the disease may have clinical significance in relation to this disease. Polynucleotide VEGI isoforms differentially expressed in the cell, when this polynucleotide detected at higher or less high levels in the cell of the individual with the disease,compared with a cell of the same cell type, which is taken from the individual who does not have this disease. Usually screened for differentially expressed polynucleotide focuses on polynucleotide, which is expressed in such a way that, for example, mRNA is found at levels at least about 25%, at least about 50% to about 75%, at least about 90%, at least about 2 times, at least about 4-fold, at least about 5-fold, at least about 10 times or at least about 50 times or more higher (for example, sverkhekspressiya) or lower (e.g., not expressed) levels in the cell of the individual who has this disease, in comparison with a cell of the same type, which is not taken from such an individual. The comparison may be performed, for example, between the two tissues, if using in situ hybridization, or other method of analysis, which allows some degree of discrimination among types of cells in this tissue. This comparison can also be made between cells, isolated from their tissue source.

Thus, the invention provides an array containing polynucleotide VEGI isoforms described here. In some embodiments this izaberete is the Oia provides an array containing the polynucleotide sequence shown in table 1 (SEQ ID NO:1), or section polynucleotide this sequence, shown in table 1 (SEQ ID NO:1), where this plot is a plot of at least 10 contiguous nucleotides (or more, for example, 15, 18, 20, 25, 35, 40,45, 50, 60, 75 or 100 contiguous nucleotides). In other embodiments this area further includes nucleotides 93 and 94 of the sequence shown in table 1 (SEQ ID NO:1). In other embodiments, this site is located within nucleotides 1-93 the sequence shown in table 1 (SEQ ID NO:1).

In some embodiments, the invention provides an array containing the polynucleotide sequence shown in table 2 (SEQ ID NO:2), or section polynucleotide this sequence, shown in table 2 (SEQ ID NO:2), where this plot is a plot of 10 contiguous nucleotides (or more, for example, 15, 18, 20, 25, 35, 40, 45, 50, 60, 75 or 100 contiguous nucleotides). In other embodiments this area further includes nucleotides 386 and 387 of the sequence shown in table 2 (SEQ ID NO:2). In other embodiments, this site is located within nucleotides 1-386 the sequence shown in table 2 (SEQ ID NO:2).

Arrays are also applicable for detection of mutant polynucleotides VEGI isoforms. Mutant polynucleotide isoforms EGI can be detected in the genomic DNA, for example, genomic DNA isolated from the blood of an individual or from other tissue samples. Mutant polynucleotide VEGI isoforms can be detected using cDNA or mRNA of the individual with changed polynucleotide VEGI isoforms, if this mutant polynucleotide VEGI isoforms leads to mRNA, which is, for example, changed in size. Mutant gene VEGI isoforms may also lead to differential expression (increased or decreased) mRNA isoforms VEGI, which can be detected, as described here.

This invention also provides an array containing one or more selected polynucleotides that specifically hybridize to the one described here polynucleotides. In some embodiments, the invention provides an array containing one or more isolated polynucleotides, which specifically hybridize with polynucleotides shown in table 1 (SEQ ID NO:1), or section polynucleotide the sequence shown in table 1 (SEQ ID NO:1), where this plot is a plot of at least 10 contiguous nucleotides (or more, for example, at least, 15, 18, 20, 25, 35, 40, 45, 50, 60, 75 or 100 contiguous nucleotides). In other embodiments this area further includes nucleotides 93 and 94 of the sequence shown in table 1 (SEQ ID NO:1). In drugaware this site is located within nucleotides 1-93 sequence, shown in table 1 (SEQ ID NO:1).

In other embodiments, the invention provides an array containing one or more isolated polynucleotides, which specifically hybridize with polynucleotides shown in table 2 (SEQ ID NO:2), or section polynucleotide the sequence shown in table 2 (SEQ ID NO:2), where this plot is a plot of at least 10 contiguous nucleotides (or more, for example, at least, 15, 18, 20, 25, 35, 40, 45, 50, 60, 75 or 100 contiguous nucleotides). In other embodiments this area further includes nucleotides 386 and 387 of the sequence shown in table 2 (SEQ ID NO:2). In other embodiments, this site is located within nucleotides 1-386 the sequence shown in table 2 (SEQ ID NO:2).

The polypeptides of the present invention

The invention includes a polypeptide sequence VEGI-192a, VEGI-192band VEGI-251man, shown in table 4 (SEQ ID NO:4), 5 (SEQ ID NO:5) and 6 (SEQ ID NO:6). VEGI polypeptides can be extracted and purified from cultures of recombinant cells of ways, including precipitation with ammonium sulfate or ethanol, extraction with acid, anyone - or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic chromatography, affinity chromatography, hydroxylapatite chromatography and chromatography the application of lectins. Stage re-laying (refolding) protein can be used, if necessary, restore the structure of the Mature protein. Finally, for the final stages of purification can be used high-performance liquid chromatography (HPLC). Examples of ways of re-packing and protein purification are described in application for U.S. patent 20010044521 and WO 01/55174.

The polypeptides of this invention can be purified natural product or product of the methods of chemical synthesis or can be obtained by recombinant methods from prokaryotic, such as E. coli) or eukaryotic host (such as cells SNO). Depending on the host used in the method of recombinant received, the polypeptides of the present invention can be glycosylated or can be deglycosylation. The polypeptides of this invention can also include amino acid residue of the initiating methionine.

Polypeptides VEGI-192a, VEGI-192band VEGI-251the present invention (as described herein include various options, such as full-length, Mature, merged, fragments, etc.) have various applications, as described here. These polypeptides are of particular interest as genetic or biochemical markers (e.g., in blood or tissues)that indicate associated with and what geogenesis disease and/or for monitoring the effectiveness of various therapies and preventive interventions. Diagnostic methods (i.e. detection) and methods of screening described in more detail below. The polypeptides of this invention are also used in obtaining antibodies that bind to these polypeptides, to their use as an agent for screening pharmaceutical candidates (both in vitro and in vivo), their use in a rational (i.e. based on the structure) construction of medicines, as well as other applications include therapeutic applications described herein (for example, if full-VEGI-192aor VEGI-192bexerts its action by binding to another protein, polypeptide, which competitively binds to VEGI-192aor VEGI-192bcould impair the function of VEGI-192aor VEGI-192bas a competitive inhibitor and, therefore, to show therapeutic activity). Polypeptides VEGI-192aor VEGI-192bcan also be used to identify proteins, in particular proteins of the people who contact you (or interact physically with VEGI-192aor VEGI-192bwho, therefore, could themselves be targets of drugs.

This invention provides a polypeptide, truncated forms or fragments of VEGI- 192aand VEGI-192b. VEGI polypeptides of the present invention have one or more functions, as described in the previous section. In some embodiments of the VEGI polypeptide is used to bind specific antibodies. In other embodiments of the VEGI polypeptide is an immunogen. In the following embodiments of the VEGI polypeptide inhibits the growth of vascular endothelial cells and/or angiogenesis. The methods for testing the activity of VEGI polypeptide (including a shortened form VEGI) is well known in this field and are described in detail in the examples, such as the analysis to test the influence on the growth of vascular endothelial cells, formation capillaroscopy tubes, capillary growth in collagen gels placed on chorioallantoic membrane of chicken embryo tumor growth of xenografts.

The size of the polypeptide fragments can vary widely. Thus, the invention includes polypeptide fragments of the full-VEGI-192aor VEGI-192bcontaining part of amino acid sequence depicted in table 4 (SEQ ID NO:4) or table 5 (SEQ ID NO:5), in which the polypeptide VEGI-192aor VEGI-192bis at least about 5, about 10, about 15, 25, 50, 75, 100, 150 or more contiguous amino acids of the sequence shown in table 4 (SEQ ID NO:4) or table 5 (SEQ IDNO:5). It is clear that these fragments contain at least one of the amino acids within amino acids 1-26 SEQ ID NO:4 or SEQ ID NO:5, preferably of land within amino acids 1-26 SEQ ID NO:4 or SEQ ID NO:5. In some embodiments of this part of the amino acid sequence contains the amino acids 26 and 27, shown in table 4 (SEQ ID NO:4) or table 5 (SEQ ID NO:5). In some embodiments of this part of the amino acid sequence is located within amino acids 1-26 SEQ ID NO:4 or SEQ ID NO:5. As is obvious to a specialist with expertise in this area, these polypeptides, regardless of their size, can also be linked or conjugated with other substances or agents to facilitate, enhance or modulate the function and/or specificity polypeptide VEGI-192aor VEGI-192b. These fragments can be used for various purposes, including as an immunogen (either alone or together with a suitable agent or as an agent for the inhibition of angiogenesis. These fragments (and polypeptides) must have one or more biological functions described above for the VEGI polypeptide. In some embodiments, these fragments inhibit angiogenesis. Shortened form can be smaller than any number of the following: 185, 170, 160, 150, 125, 100, 80, 50, 40, 25, 20, 15 or 10 amino acids.

It is clear that the area adjacent amino acids or nucleotide is, which is restricted to a particular pair of amino acids or nucleotides, may, but need not, include any member of the specified pair. For example, adjacent amino acids within amino acids 1-26 SEQ ID NO:4 can include amino acid 1 and/or amino acid 26 of SEQ ID NO:4.

In some embodiments of the present invention, the polypeptides of the present invention contain at least 5, at least 8, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids within amino acid residues 1-26 shown in table 4 (SEQ ID NO:4) or table 5 (SEQ ID NO:5) (which can usually also be referred to as sections). The invention also provides polypeptides containing amino acid residues approximately 5-192, 10-192, 15-192, 20-192 or 25-192 the sequence shown in table 4 (SEQ ID NO:4) or table 5 (SEQ ID NO:5).

Embodiments of the present invention exclude any polypeptide consisting of amino acids 27-192 SEQ ID NO:4 or SEQ ID NO:5, or a shortened form of such polypeptides.

Further, the invention includes polypeptides that have at least 90% similarity, more preferably at least 95% similarity, and still more preferably 96%, 97%, 98% or 99% similarity to the polypeptides described above. The polypeptides of this invention also include polypeptides that t is Auda, at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described herein, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. In some embodiments, the invention provides polypeptides that have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity with the polypeptide SEQ ID NO:4 or a polypeptide SEQ ID NO:5. The polypeptides of this invention also include polypeptides that are at least 80%, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide SEQ ID NO:4 or polypeptide SEQ ID NO:5.

This invention also provides fused proteins comprising polypeptides described herein. The polypeptides described herein may be merged with such sequences as sequences that enhance the immunological reactivity, facilitate the binding of this polypeptide with the substrate or carrier or facilitate purification (e.g., sequences encoding EPI is experience, such as the ICC, ON the happening of the hemagglutinin of influenza virus, His-6 or FLAG). In addition, protein or polynucleotide can be fused with other polypeptides which increase its function or determine its localization in the cell, such as a secretory sequence described here. Methods for obtaining recombinant fused protein, described above, are common in this area. Recombinant or protein may be isolated by methods well known in the field.

To analyze the efficacy of drugs and agents that inhibit or activate the function of VEGI, such as proteins of the owner or chemically derived agents or other proteins that interact with polynucleotide VEGI, buck adjusting or changing the expression of VEGI polypeptides or acting on their ability to inhibit angiogenesis, the use of transformed cell hosts. Method for testing efficacy of a drug or agent against VEGI or against angiogenesis may be, for example, blocking inhibitor of endothelial cell growth.

This invention encompasses compositions, including pharmaceutical compositions containing the polypeptides described herein. In some embodiments, these compositions contain polypeptide SEQ ID NO:4. This composition is applicable for inhibin is of angiogenesis. These compositions can optionally contain suitable fillers, such as pharmaceutically acceptable excipients, including buffers, which are well known in this field.

The invention also provides kits containing polypeptides described herein. In some embodiments, the composition contains a polypeptide SEQ ID NO:4. In some aspects, these sets can be used for the treatment of pathological angiogenesis, inhibition of angiogenesis or cancer treatment, such as reduction in tumor size. The kits of this invention are in suitable packaging and may optionally provide additional components such as buffers and instructions.

The invention also provides polypeptides described herein attached to a solid substrate. Methods of obtaining such attachments, such as attachment to the surface of the array is well known in this field. The polypeptides of the present invention attached to a solid substrate, such as particles of agarose, Sephadex, or the like, is applicable for screening molecules that selectively bind polypeptides described herein.

The invention also includes an array containing polypeptides VEGI isoforms of the present invention, as described herein. Thus, in one aspect, the invention provides mA is SIV, containing polypeptides VEGI isoforms encoded by polynucleotides of the present invention, as described herein. In another aspect, the invention provides an array containing the polypeptide containing the sequence shown in table 4 (SEQ ID NO:4), or its plot, where this plot has a length of at least about 5 contiguous amino acids or more, e.g., at least, 10, 15, 25, 50, 75, 100, 150 or more amino acids). In some embodiments, this site comprises amino acids 26 and 27 of the sequence shown in table 4 (SEQ ID NO:4). In other embodiments, this UCHASTOK is within amino acids 1-26 of the sequence shown in table 4 (SEQ ID NO:4).

In another aspect, the invention provides an array containing the polypeptide containing the sequence shown in table 5 (SEQ ID NO:5), or its plot, where this plot has a length of at least about 5 contiguous amino acids or more, e.g., at least, 10, 15, 25, 50, 75, 100, 150 or more amino acids). In some embodiments, this site comprises amino acids 26 and 27 of the sequence shown in table 5 (SEQ ID NO:5). In other embodiments, this site is located within amino acids 1-26 of the sequence shown in table 5 (SEQ ID NO:5).

The terms "polypeptide" and "protein" are used herein interchangeably, and as well from the local in this area, refer to polymers of amino acids of any length. In various embodiments, the polymer may be linear or branched, it may contain modified amino acids, it can be interrupted components that are not amino acids, and/or it can be assembled in a complex of more than one polypeptide chain. As is well understood specialists in this field, the polypeptide can be modified naturally or by intervention; for example, the formation of disulfide bonds, glycosylation, limitirovanie, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with the aiming component. In some embodiments, the polypeptides contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc), as well as other modifications known in this field.

The invention also includes functionally intact VEGI polypeptides described herein. Such variants can be obtained using methods standard in this area, such as conservative substitutions of amino acids. In various embodiments functionally intact variant contains (or, in some embodiments, consist of) any of one, two, three, four, five, six, seven, eight, nine, ten conservative substitutions is minislot.

Vectors and cells of the host

This invention also relates to vectors which include the selected polynucleotide of the present invention, the cells of the host, which are genetically engineered with the use of these recombinant vectors, or which are designed in another way to obtain polypeptides of the present invention, and to receive the polypeptides of the present invention recombinante ways.

The term "vector" refers to a plasmid, virus or other media known in this area, which was subjected to manipulation by the insertion or incorporation of genetic sequences VEGI-251, VEGI-192aor VEGI-192bor their fragments. Polynucleotide (generally, DNA) element, which makes the vector suitable for reproduction, may be the site of initiation of replication (origin of replication), which works in prokaryotic or eukaryotic cells. An example of the site of initiation of replication, which works in prokaryotic cells is colE1 ori. Recombinant vector requires additional marker selection to control the growth of organisms that carry this vector. Suitable selection markers include genes that protect organisms from antibiotics (resistance genes to antibiotics), such as ampicillin, streptomycin, chloramphenicol, liebespaar growth in conditions of deprivation of any connection (conditions auxotrophic growth) for expression as a protein in the cells. In the preferred embodiment of the present invention for propagation of the recombinant vector of these prokaryotic cells are bacteria. In particularly preferred versions of this invention, these bacteria are, in particular, the bacteria Escherichia coli or Bacillus sp. In the following preferred embodiment of the present invention for propagation of the recombinant vector eukaryotic cells are cells of the cell line or yeast cells. In particularly preferred versions of this invention the cells of the cell lines are cell lines Cho, COS, HeLa or T, and yeast cells are cells of Saccharomyces cerevisiae.

The invention includes various vectors (i.e. cloning and/or expressing the vectors, and vectors for cloning and/or replication), having cloned in them polynucleotide VEGI-192a, VEGI-192bor VEGI-251. These vectors can be used for expression of recombinant polypeptides, as well as a source of polynucleotides VEGI-192a, VEGI-192bor VEGI-251. Cloning vectors can be used to obtain a replica copies of polynucleotides VEGI-192a, VEGI-192bor VEGI-251they contain, or as a means of storing these polynucleotides in a Depository for future use. Expresser the matter of the vectors (and the cells of the host, containing those expressing vectors) can be used to produce polypeptides produced from polynucleotides they contain. They can also be used when it is desirable to Express the polypeptides VEGI-192a, VEGI-192bor VEGI-251in the individual, for example, for the induction of immune responses by polypeptide (polypeptide)encoded in expressing vector (vectors). Appropriate cloning and expressing vectors include any known in the field vectors, such as vectors for use in bacterial expression systems, expression systems mammalian, yeast and insects. Specific vectors and appropriate cell hosts known in this field and do not require detailed description here. See, for example, Gacesa and Ramji, Vectors, John Wiley and Sons (1994).

Cloning and expressing vectors usually contain a breeding marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed by this vector), although this marker gene may be transferred to another polynucleotide sequence, simultaneously introduced into the cell host. Only those cells are the masters, which was introduced in breeding gene will survive and/or grow under selective conditions. Typical breeding g is HN encode a protein (protein), which: (a) inform the resistance to antibiotics or other compounds, toxins, e.g. ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic failure or (C) provide critical nutrients not available from complex environments. The choice of a suitable marker gene will depend on the host cell, and appropriate genes for different hosts known in the field. Cloning and expressing vectors typically also contain a replication system, the focus master.

Suitable cloning vectors can be constructed in accordance with standard methods, or can be selected from a large number of cloning vectors are available in this area. Even though the cloning vector may vary depending on the host cell that you want to use, applicable cloning vectors usually reproduce or infect other programs, can have a single target for a specific restrictase and/or may carry genes for marker, which can be used in the selection of clones containing the vector. Suitable examples include plasmids and viruses bacteria, for example, pUC18, pUC19, BlueScript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR22, RMV, ColE1, pCR1, RP4, DNA phages and Shuttle vectors such as pSA3 and rat. These and many other cloning vectors are d is accessible from commercial suppliers, such as BioRad, Stratagene, and Invitrogen.

Expressing the vectors usually are replication competent polynucleotide constructs that contain polynucleotide encoding interest VEGI polypeptide. Polynucleotide encoding the VEGI polypeptide functionally linked to appropriate regulatory elements for transcription, such as promoters, enhancers and terminators. For expression (i.e. broadcast) usually also requires regulatory elements, such as the binding sites of ribosomes, the sites of translation initiation and stop codons. These regulatory elements (transcription and translation) can be obtained from polynucleotides VEGI (i.e. one of the genes VEGI isoforms) or they may be heterologous (i.e. derived from other genes and/or other organisms). Can also be included polynucleotide sequence encoding a signal peptide to the VEGI polypeptide could cross the cell membrane and/or to be disposed in the cell membrane or secretariats of cells. A number expressing vectors suitable for expression in eukaryotic cells, including yeast cells of birds and mammals, are known in this field.

Vectors containing interest polynucleotide, may be introduced into the cell host any number of suitable methods, including electroporation, Tr is Specchia using calcium chloride, the rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, the bombardment microkeratome, lipofectin and infection (where the vector is an infectious agent, such as vaccinia virus). The choice of ways of introducing vectors or polynucleotides will often depend on the host cell.

The invention also includes a cell-master, and a cell culture containing the cells of the host. This is a host cell containing at least one recombinant polynucleotide (generally, DNA) vector mentioned above. "Cells masters" are cells in which the vector can breed, and its DNA can be expressed. This cell can be prokaryotic or eukaryotic. This term includes any progeny of the considered host cell. It is clear that all progeny may not be identical to the parent cell, as can occur mutations that occur during replication. However, this offspring is enabled when you use the term "a host cell". How stable transfer, which means that foreign DNA is continuously maintained in the host, well-known in this field. When a host cell is selected from prokaryotic cells, it is preferably in a cell of bacteria, in particular Escherichia coli or Bacillus sp. When a host cell is eukaryotic the coy cell it preferably is a cell or cell line, in particular the cell line COS, Hela or T or cell yeast, in particular Saccharomyces cerevisiae cell.

Cells are the owners of this invention can be used, inter alia, as repositories of polynucleotides VEGI and/or carriers to obtain polynucleotides and/or VEGI polypeptides, as described herein. Cell owners may also serve as repositories of mutant polynucleotides VEGI-192a, VEGI-192bor VEGI-251as described hereinafter. These cells are the owners may be useful for screening a therapeutic protein or polypeptide, as described below.

Antibodies and their receipt

The invention also provides antibodies that selectively bind with the protein VEGI-192aand/or VEGI-192b(including fragments), as described here. The term "antibody" includes, but is not limited to, intact molecules, fragments thereof, such as Fab, F(ab')2, Fv, which are capable of binding the epitope determinants. These antibody fragments retain some ability to selectively contact the antigen or receptor and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigennegative fragment of the antibody molecules, can be obtained by cleavage of a whole antibody with the enzyme papain obtained with the eating of intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of the antibody molecules, which can be obtained by treating whole antibody with pepsin, followed by reduction with obtaining intact light chain and part of the heavy chain; receive two Fab'-fragment of the antibody molecule;

(3) F(ab')2fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent recovery; F(ab')2is a dimer of two Fab'fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable area light chain and the variable section of the heavy chain expressed as two chains; and

(5) single-chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of light chain, the variable region of the heavy chain associated with a suitable polypeptide linker as a genetically fused single-chain molecule.

In some embodiments the antibodies of this invention can be any of the following antibodies: polyclonal, monoclonal, single-chain (ScFv), mutants of these variants, fused protein containing part of the antibody (such as one or more CDR regions), humanized antibodies, chimeric antibodies, human antibodies, or any modified configuration of the molecule is of immunoglobulin, which contains the site of the recognition of the desired antigen specificity.

As specialists in this area, "monoclonal antibody" refers to a homogeneous population of antibodies, in which the monoclonal antibody consists of the amino acid (naturally occurring and not occurring in nature)that are involved in the selective binding of the antigen. Monoclonal antibodies are highly specific, directed against a single antigenic site. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain scFv, their mutants fused proteins containing antibody as part of a fused protein, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecules, which contains the site of the recognition of the desired antigen specificity and are able to bind to the antigen. Not assumed restrictions on the source of the antibody or the manner in which it is received (for example, using hybridoma, phage selection, recombinant expression, transgenic animals and so forth).

"Humanitarianism" antibody molecule called with antigennegative site, the cat is who, in essence, comes from the immunoglobulin of the species, which is not a person, and the rest of the structure of this immunoglobulin molecules based on the structure and/or sequence of a human immunoglobulin. This antigennegative site may contain either variable domains fused with the constant domain, or complementarity determining region (CDR)grafted on a suitable frame parts in the variable domains. Antigennegative sites may be sites of wild-type or modified by one or more amino acid substitutions, for example modified so that they had a closer resemblance to human immunoglobulin. Some forms of humanized antibodies retain all CDR sequences (for example, humanitariannet murine antibody, which contains all six CDRs of murine antibodies). Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six)which are altered with respect to the original antibody, which are also referred to as one or more CDR, "originating from" one or more CDRs of the antibody.

Methods for producing antibodies and fragments of antibodies known in the field. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated into the description by reference.)

This invention providing the em also antibody which selectively binds a polypeptide containing SEQ ID NO:4, SEQ ID NO:5 or a fragment, where the fragment is located within amino acids 1-26 SEQ ID NO:4 or SEQ ID NO:5, or the fragment comprises amino acids 26 and 27 of SEQ ID NO:4 or SEQ ID NO:5. The invention also provides antibodies that selectively bind VEGI-192aand VEGI-192bbut not other isoforms VEGI (not associated selectively with other VEGI isoforms, such as VEGI-251). The invention also provides antibodies that selectively bind VEGI-192aor VEGI-192b.

Further, this invention provides an antibody that selectively binds a polypeptide encoded by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a fragment.

In some embodiments, the invention provides an antibody that selectively binds to a polypeptide containing an area of at least 5, at least 10, at least 15, at least 20, at least 25, or more contiguous amino acids of SEQ ID NO:4 or SEQ ID NO:5, where this area is located within amino acid residues 1-26 shown in table 4 (SEQ ID NO:4) or table 5 (SEQ ID NO:5). In other embodiments, the invention provides an antibody that selectively binds to a polypeptide containing an area of at least 5, at least 10, at least 15, at least 20, at least 25 who do more contiguous amino acids of SEQ ID NO:4 or SEQ ID NO:5, where this plot includes amino acids 26 and 27 of SEQ ID NO:4 or SEQ ID NO:5.

In some embodiments, the antibody of the present invention inhibits the activity of VEGI; for example, such an antibody could stimulate angiogenesis. The methods of screening such antibodies are described below.

In some embodiments, the antibody of the present invention may be agonistic antibody in the sense that it stimulates the activity of VEGI. The methods of screening such antibodies are described below.

It is clear that in this context, when there are a variety of VEGI isoform selective binding means binding primarily (or even exclusively) with specific isoform in comparison with the other isoform (if they have no other indications). In some embodiments, the antibody selectively binds the VEGI polypeptide of the present invention in comparison with VEGI isoform is not from man. As an example, the antibody of the present invention could selectively bind human VEGI-192abut not mouse (not person) VEGI-192a.

Antibodies of the present invention may be linked (i.e. conjugated) with detektivami agent, or a hapten. This complex can be used to determine polypeptide (polypeptides) or fragments of the polypeptide)to which the antibody specifically binds to the sample, using standard immunochemical methods, such as the immunohistochemistry, as described by Harlow and Lane (1988), supra. Examples of types of immunoassays which can be used monoclonal antibodies of this invention are competitive and non-competitive immunoassays in a direct or indirect format. Examples of such immunoassays are the ELISA (solid phase) assay (ELISA), radioimmune assay (RIA) and sandwich(immunodeficiency)analysis. Detection using monoclonal antibodies of this invention can be performed using immunoassays that operate in forward, reverse or simultaneous modes, including immunohistochemical assays on physiological samples. Professionals in this field know or can easily choose other formats of immunoassays without undue experimentation.

Another way, which may give higher sensitivity, involves the binding of antibodies to low molecular weight haptens. Then these haptens can be specifically detected using the second reaction. For example, usually use such Galani as Biotin, which reacts with Avidya, or dinitrophenyl, pyridoxal, and fluorescein, which can react with specific antibodies against the hapten. Cm. Harlow and Lane (1988) supra.

Antibodies of the present invention may also be associated with different media. T is thus, this invention also provides compositions containing the antibodies and the media. The media can be active and/or inert. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. By its nature, this carrier can be either soluble or insoluble for the purposes of this invention. Specialists with expertise in this area will be known other suitable carriers for binding monoclonal antibodies, or they will be able to find them in the course of routine experimentation.

There are many different labels and methods of labeling known to specialists of ordinary skill in this field. Examples of the types of labels that can be used in this invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds and bioluminescent compounds. Specialists of ordinary skill in this field will be known other suitable labels for binding to monoclonal antibodies, or they will be able to find them using routine experimentation. In addition, the binding of these labels with a monoclonal antibody of this invention can be performed with use the of standard methods, normal for specialists of ordinary skill in this field.

For the purposes of this invention, the polypeptides of this invention can be detected by the antibodies of the present invention, if they are present in samples such as fluids and tissues. Is the use of antibodies is discussed in more detail below.

Compositions containing the antibodies, fragments or cell lines that produce these antibodies, are covered by the invention. When the pharmaceutical use of these compositions them together with a pharmaceutically acceptable excipient. The invention also includes arrays containing antibodies or fragments thereof. Antibodies can be immobilized on a surface, such as an array for use in detection and diagnostic assays, as described in more detail below. Antibodies can be immobilized on the carrier for purification described here polypeptides or their fragments.

Composition

In addition, this invention provides compositions, including pharmaceutical compositions containing the polypeptides, polynucleotides, antibodies, recombinant vectors and cells are the owners of this invention. These compositions may include a buffer, which is selected in accordance with the intended use of the polypeptide, antibody, polynucleotide, recombinant the th vector or host cell, and may also include other substances appropriate to the intended application. Specialists in this field can easily choose a suitable buffer, a great variety of which are known in this region, suitable for the intended purpose. In some cases, the composition is a pharmaceutical composition and may contain pharmaceutically acceptable excipients, which are known in this field and does not require detailed discussion here. Pharmaceutically acceptable excipients have been adequately described in numerous publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams and Wilkins.

Sets containing polynucleotide, polypeptides and/or antibodies of the present invention

As described herein, the invention also includes kits containing polynucleotide (polynucleotide), polypeptide (polypeptide) and/or antibodies of the present invention, such as diagnostic kits, for therapy. Sets the embodiment of the present invention include kits that allow you to test for the presence of polynucleotides, VEGI polypeptide and/or anti-VEGI-antibodies, such as any described herein, for detection and/or quantification of these molecules. Thus, the invention includes (a kit for detecting or quantifying polynucleotide VEGI in the sample, containing any of polynucleotides VEGI described herein; (b) a set containing any of the antibodies described herein, for the detection or quantitative determination of the VEGI polypeptide in the sample; (C) the set containing any of the polypeptides described herein, for the detection or quantitative determination of anti-VEGI-antibodies in the sample. The invention also provides kits containing polynucleotide or polypeptides of the present invention, for use in therapy.

The kits of this invention are in suitable packaging and may optionally contain additional components that can be used in this procedure. These optional components include, but are not limited to, buffers, exciting reagents, RealAudio reagents, labels, reacting surfaces, means for detection, control samples, instructions and explanatory information.

How to apply polynucleotides, polypeptides and antibodies: detection systems

The invention also provides methods of use of polynucleotides, polypeptides and/or antibodies VEGI-192aand VEGI-192bthe present invention for the detection of suitable targets in the sample. As this invention makes it clear that detection methods refer to any detection of the presence, absence, and quantity of the military definition. Procedures for conducting diagnostic tests (i.e. detection) using polynucleotides, polypeptides or antibodies are widely known in this field and are routine for the practice of ordinary skill in this field. Usually for carrying out the diagnostic method of the present invention from the compositions of the present invention is used as a reagent to detect a target with which it reacts in the sample. This target is provided by obtaining a suitable sample from the individual, which must be measured diagnostic parameter. Many types of samples are suitable for this purpose. If desired, the target may be partially purified from the sample or amplified prior to analysis.

This invention relates to a method for detecting the presence or absence or level of the polypeptide VEGI-192aor VEGI-192bin the sample, providing the contacting of the sample from a human or animal with antibodies that selectively bind to the one described here polypeptide, and detecting the presence, or absence, or quantity of complex formed between the polypeptide and these antibodies. Such detection is applicable for the purposes of diagnosis, prognosis and/or monitoring associated with angiogenesis diseases. In some the embodiments, the invention provides a method for the diagnosis of pathological angiogenesis, providing stage shielding the sample from a human or animal that is suspected of having pathological angiogenesis, with antibodies that recognize the polypeptide described herein, and detecting the presence or absence of a complex formed between the polypeptide and these antibodies.

Can be used for competitive analysis, in which antibodies that selectively bind to polypeptides VEGI-192aand/or VEGI-192battached to the solid carrier, and labeled VEGI-192aand/or VEGI-192band the sample obtained from the owner, skip over this solid carrier, and the number of labels, detective, for example, liquid scintillation chromatography, can be correlated with the number of VEGI-192aand/or VEGI-192bin this sample.

"Sandwich-analysis is similar to ELISA analysis. In sandwich analysis VEGI-192aand/or VEGI-192bskipped over solid media and binds to antibody attached to a solid carrier. Then the second antibody bind with this VEGI-192aand/or VEGI-192b. Then the third antibody that is labeled and specific second antibody is passed over a solid carrier and communicates with the second antibody, and then may be determined by the associated number.

Using standard who methodology, well known in this field can be constructed diagnostic analysis by application to the surface (i.e. a solid support), for example, microtiter plate or a membrane (e.g. nitrocellulose membrane), antibodies specific to the polypeptide VEGI-192aor VEGI-192bor both, or communicating with them, and contacting this covered the surface with serum, tissue or other biological or chemical sample obtained from a person, which suggest the presence associated with angiogenesis diseases. The presence or absence of the received complex formed between the polypeptide VEGI-192aor VEGI-192bin the sample and antibodies specific thereto can be detected by any of the known methods common in the field, such as spectroscopy or colorimetry of fluorescent antibodies. This method of detection can be used, for example, for the diagnosis or prediction of cancer.

Analysis of the levels of polypeptides VEGI-192aand/or VEGI-192bin the sample can use any of the methods based on antibodies, which are well known in this field. For example, expression of VEGI polypeptides-192aand/or VEGI-192bin tissues can be studied classical immunohistological methods (Jalkanen, M., et al., J.Cell.Biol. 101:976-985 (195); Jalkanen, M., et al., J.Cell.Biol. 105:3087-3096 (1987). Other methods based on antibodies that are applicable for the detection of gene expression of VEGI polypeptides-192aand/or VEGI-192binclude immunoassays, such as ELISA assay (ELISA) and radioimmunoassay analysis (RIA). Suitable labels for the analysis of antibody known in the field and include enzyme labels, such as glucose oxidase, and radioactive isotopes, such as iodine (125I121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and Biotin.

In addition to analyzing the levels of the VEGI polypeptide-192aand/or VEGI-192bin the sample obtained from the individual, the polypeptide VEGI-192aand/or VEGI-192bcan also be detected in vivo by imaging. Label antibodies or markers to obtain images of the in vivo polypeptide VEGI-192aor VEGI-192binclude labels, detected by x-radiography, NMR or ESR (electron spin resonance). For radiography suitable labels include radioisotopes, such as barium or cesium, which emit detective radiation, but are not obviously harmful to the subject. Suitable markers for NMR and ESR include markers with detektivami characteristic spin, such as deuterium, which can be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

The antibody or antibody fragment that selectively contact the VEGI polypeptide-192aand/or VEGI-192bthat have been marked with suitable, giving the image of a molecule, such as a radioisotope (for example,131I112In99mTc), impervious to radiation substance or material, the detected nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal, examined in relation to immune system disorders. Specialists in this field will be clear that the size of the subject and used the system for obtaining image will determine the number giving the image of molecules needed to obtain diagnostic images. In the case of radioactive molecules to the quantity of injectable radioactivity will typically be in the range of from about 5 to 20 millicurie99mTc. Then the labeled antibody or antibody fragment will mainly accumulate at the location of cells which contain the polypeptide VEGI-192aand/or VEGI-192b. Imaging of tumors in vivo is described Burchiel et al. (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S.W. and Rhodes, B.A., eds., Masson Publishing Inc. (1982)).

As it is clear to experts in the data is the second region, polypeptides VEGI-192aand/or VEGI-192bcan be detected using any agent that binds to these polypeptides.

In another embodiment this invention relates to a diagnostic kit containing antibodies that selectively bind to the polypeptides of the present invention, for example, antibodies that selectively bind VEGI-192aor VEGI-192bor both of the polypeptide, and auxiliary reagents for use in detecting the presence of these polypeptides in the sample. These reagents are well known in this field and is suitable for use in detecting the presence or absence of VEGI polypeptides in serum, tissue or other specimen. Considering the tissue samples can be obtained from monkeys, or humans, or other mammals. This kit may optionally include instructions for use, control and explanatory information.

This invention also provides a method for detecting the presence, or absence, or level of polynucleotides described here, involving the contacting of the sample from the individual, such as a person or an animal, with polynucleotides (in some embodiments the oligonucleotide), which selectively binds to the one described here polynucleotide, and detecting the presence or OTS is tste, or the amount of duplex formed between polynucleotides and polynucleotides in the sample. In some embodiments of the method of the present invention, which is applicable for the diagnosis of pathological angiogenesis, involves the stage of shielding the sample from a human or an animal, which suggest the presence of pathological angiogenesis, polynucleotides (such as an oligonucleotide)that is associated with described here polynucleotide, and detecting the presence or absence of a duplex formed between polynucleotides and polynucleotides in the sample. In another embodiment the present invention relates to RNA, DNA, or other nucleotide sequences for use in detecting the presence or absence of polynucleotides VEGI using polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR). Can be used other ways amplification-based primers. The DNA sequence of this invention shown in table 1 (SEQ ID NO:1) or table 2 (SEQ ID NO:2)may be used to design primers that are specifically associated with a polynucleotide sequence VEGI-192aor VEGI-192bin the case of PCR or cDNA VEGI-192aor VEGI-192bobtained by reverse transcription of RNA that encodes polypeptide VEGI- 192aor VEGI-192bfor the purpose of detecting the presence, absence, or quantification of polynucleotide VEGI-192aor VEGI-192bby comparing with the standard. These primers can be of any length, for example in the range of 7-40 nucleotides, preferably 10-15 nucleotides, most preferably 18 to 25 nucleotides. Reagents and controls necessary for the reactions of PCR or RT-PCR are well known in this field. Then amplificatoare products can be analyzed for the presence or absence of sequences polynucleotides VEGI-192aor VEGI-192bfor example, by fractionation in gels, hybridization with or without hybridization, the field of radiochemistry and immunochemical methods. This method is preferred because it requires only a small amount of sample to obtain sufficient quantities of DNA templates, which are conducting PCR or RT-PCR.

In some embodiments of the methods of detection involve the use of one or more primers for the amplification of interest sequence VEGI-192aand/or VEGI-192b. In other embodiments, the detection is performed by using specific probes (e.g., labeled probes), which detects the presence or absence (or can quantify) int representing the RES sequence VEGI- 192aand/or VEGI-192b. In some embodiments the probe contains a label.

In some embodiments, this method is used for level detection VEGI-192aor VEGI-192bby detecting the presence, or absence, or amount of cellular RNA that encodes a VEGI-192aor VEGI-192bor fragment described herein. Total cellular RNA can be isolated from the sample using any suitable method, such as one-step method using guanidine thiocyanate-phenol-chloroform, described by Chomczynski and Sacchi (Anal.Biochem. 162:156-159 (1987). Then the levels of mRNA encoding the VEGI polypeptide-192aand/or VEGI-192banalyze using any suitable method. These methods include Northern blot analysis, mapping with nuclease S1, polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) and reverse transcription in combination with the ligase chain reaction (- LCR).

In another embodiment this invention relates to a diagnostic kit which contains primers PCR or RT-PCR, one or more primers, such as primers, specific polynucleotide VEGI-192aor VEGI-192b; and/or primers specific for other VEGI isoforms, such as VEGI-251, VEGI-174, and support the additional reagents, which are well known in this field and which are suitable for use in detecting the presence or absence of polynucleotides VEGI isoforms or quantifying RNA, which encodes a polypeptide VEGI isoforms in the sample using PCR or RT-PCR, or one or more other methods of amplification. The considered samples can be obtained from humans or animals.

In another embodiment this invention relates to a diagnostic kit which contains probes, one or more probes, such as probes, specific polynucleotide VEGI-192aor VEGI-192band/or probes specific for other VEGI isoforms, such as VEGI-251, VEGI-174and ancillary reagents that are well known in the field and which are suitable for use in detecting the presence or absence of polynucleotides VEGI isoforms or quantifying RNA, which encodes a polypeptide VEGI isoforms in the sample, using techniques such as Northern blotting or one or more other ways. The considered samples can be obtained from humans or animals.

As is clear in this area, "sample" can be any sample obtained from the individual (often called "biological sample"): fluid body, cleto is the Naya line, tissue culture, or other source which contains or may contain the polypeptide or mRNA VEGI-192aand/or VEGI-192b. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid)which contain free VEGI polypeptide-192aand/or VEGI-192bthe fabric of the immune system and the circulatory system and other tissue sources, which have been detected, Express complete or Mature polypeptide VEGI-192aand/or VEGI-192band/or receptor VEGI-192aand/or VEGI-192b. Methods for obtaining tissue biopsies and body fluids from mammals are well known in this field. When the biological sample must contain mRNA, a preferred source is a tissue biopsy.

Under the "analysis of the expression level of the gene encoding the protein VEGI-192aand/or VEGI-192b"I mean qualitative or quantitative measurement or determination of the level of the VEGI polypeptide-192aand/or VEGI-192bor level of mRNA that encodes the polypeptide VEGI-192aand/or VEGI-192bin the first sample either directly (e.g., by determining or assessing the absolute level of the polypeptide or mRNA level)or relatively (e.g., by comparing the level of polypeptide VEGI-192aand/or VEGI-192bor level of MRR is in the second sample). Preferably, the level of the polypeptide or mRNA level VEGI-192aand/or VEGI-192bin the first sample is measured or evaluated and compared with a standard level of polypeptide or mRNA level VEGI-192aand/or VEGI-192band this standard is a second sample obtained from an individual not having problems, or is determined by averaging levels from a population of individuals not having a compromised immune system and circulatory system. As will be clear in this area, when the standard level of the polypeptide or mRNA level VEGI-192aand/or VEGI-192bis known, it can be used repeatedly as a standard for comparison.

As noted above, polynucleotide and polypeptides VEGI-192aand/or VEGI-192bapplicable for diagnosis of conditions involving abnormally high or low expression activities VEGI-192aand/or VEGI-192b. Given the cells and tissues, which is expressed VEGI-192aand/or VEGI-192b, as well as the activities modulated VEGI-192aand/or VEGI-192bobviously that significantly altered (increased or decreased) level of expression of VEGI-192aand/or VEGI-192bin individuo, in comparison with a standard or "normal" level, causing pathological conditions associated with system (systems) of the body in which VEGI 192aand/or VEGI-192bis expressed and/or is active.

The invention also provides methods that facilitate the diagnosis of VEGI-associated disorders or conditions. These methods help in performing clinical definitions relating to the classification, or nature of a pathologic angiogenesis or cancer prognosis, and may or may not be decisive in the final diagnosis. Thus, the method for facilitating the diagnosis of pathological angiogenesis, or prognosis of cancer or related diseases may include the stage of detecting the level of expression of VEGI isoforms (i.e. VEGI-192a, VEGI-192bin the sample from the individual. A method of facilitating diagnosis of angiogenesis-associated diseases may also include a stage of detecting altered levels of polynucleotide and/or polypeptide VEGI isoforms in the sample from the individual and/or detection of increased or decreased levels of polynucleotide and/or polypeptide VEGI isoforms in the sample from the individual.

This invention also provides a method of detecting the individual with an increased risk, which may or may not have detected the angiogenesis-associated disease and/or condition associated with abnormal levels of VEGI-192aand/or VEGI-192band maybe p is havlat or may not be detected signs of the disease before treatment ways as described here. The term "risk" means that the individual, as determined on the basis of the standard methods of risk assessment, has a greater likelihood of symptom or has one or more risk factors that correlate with the development of angiogenesis-associated diseases. An individual having one or more of these risk factors has a higher probability of developing angiogenesis-associated disease than an individual without these risk factors. Examples (i.e. categories) risk groups are well known in this area and are discussed here.

Polynucleotide VEGI-192aand/or VEGI-192bcan also be used as probes to detect the presence or absence of mutations or polymorphisms in the gene VEGI-192aand/or VEGI-192band any interest sequence VEGI-192aor VEGI-192bbeing or not a mutation. Mutant VEGI-192aand/or VEGI-192bcan be associated with angiogenesis or various disorders associated with the immune system or the circulatory system. Methods for detecting mutant polynucleotide sequences are well known in this field and include, for example, single-stranded conformational polymorphism (SSCP) and different methods based on amplification is posledovatelnosti for detection of mutations of the sequence, including point mutations, for example LCR, first NASBA, PCR, limited elongation of the primers, etc. Methods for detection of modified protein include Western blot analysis, capillary electrophoresis, mass spectroscopy and WAVE. Usually experiment on the detection performed in parallel with the control polynucleotide or VEGI polypeptide-192aand/or VEGI-192bor, in the case when determining the modified levels of expression, with a control sample having normal levels of polynucleotide or VEGI polypeptide-192aand/or VEGI-192b.

Sequences of this invention are valuable for chromosome identification. This sequence is specifically targeted at a particular location on an individual human chromosome and can gibridizatsiya with a particular location on the chromosome. In addition, currently there is a need to identify specific sites on the chromosome. Mapping of DNA relative to the chromosomes in accordance with this invention is an important first stage in the correlated these sequences.

Briefly, sequences can be mapped to chromosomes by obtaining PCR primers (preferably 15-25 BP) from the cDNA. Computer analysis of the 3'-untranslated region of this posledovatelno and used for quick selection of primers, which do not extend more than one exon in the genomic DNA that complicated as a process of amplification. Then the primers used for PCR screening of somatic cell hybrids containing individual chromosomes of man. Only hybrids containing the human gene corresponding to this primer will give amplificatory fragment.

PCR mapping of somatic cell hybrids is a quick procedure for attributing specific DNA to a specific chromosome. Using the present invention with the same oligonucleotide primers sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in the same way. Other mapping strategies that can be similarly used for mapping on chromosome include in situ hybridization, pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to construct libraries chromosome-specific cDNA.

Fluorescent in situ hybridization (FISH) cDNA clone with a stroke of metaphase chromosomes can be used to obtain a precise chromosomal location in one stage. This method can be used with such a short cDNA as 50 or 60 osnovaniya the review of this method see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once the sequence is mapped in the exact position on the chromosome, physical position of the sequence on the chromosome can be correlated with the data of genetic maps. Such data can be found, for example, in V, McKusick, Mendelian Inheritance in Man. The relationship between genes and diseases that have been mapped to the same chromosomal region, identify and then using the clutch (co-inheritance of physically adjacent genes).

You then need to determine the differences in the cDNA or genomic sequence between affected and unaffected subjects. If a mutation is observed in some or all of the affected individuals but not in any normal subjects, this mutation appears to be a causal factor of the disease; gene, localized in the chromosomal region associated with the disease could be one of 50-500 potential causal genes. This implies resolution mapping of 1 million base pairs and one gene per 20 TPN using the methods described above, it was determined that the chromosomal location of the VEGI was 9q32 with a very high confidence level. Previous studies on chromosomal mapping of communicating multiple defects once the development with the loci in this part of chromosome 9.

This invention is applicable also for the diagnosis or treatment of various disorders associated with the immune system and the circulatory system in mammals, preferably in humans. Such disorders include infections by bacteria, viruses and other parasites, immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmune diseases, graft versus host and any violation of the regulation of the function of cells of the immune and circulatory system, including, but not limited to, autoimmune disease, leukemias, lymphomas, immunosuppression, immune, inflammatory disease of the digestive tract, myelosuppression, etc.

For a number of violations substantially altered (increased or decreased) levels of gene expression VEGI-192aand/or VEGI-192bcan be detected in various tissues (e.g., tissue of the circulatory system) or other cells or bodily fluids (such as sera, plasma, urine, synovial fluid or spinal fluid)taken from an individual having such a violation, relative to the standard gene expression level VEGI - and/or VEGI-192b, i.e. the level of expression of VEGI-192aand/or VEGI-192bfrom individual do not have this violation. Thus, the invention provides a diagnostic method useful for diagnosis tie the frame with VEGI violations which involves measuring the expression level of the gene encoding the protein VEGI-192aand/or VEGI-192bin the sample from the individual and comparing the measured level of gene expression with a standard level of expression of VEGI-192aand/or VEGI-192bso that increase or decrease the level of expression of this gene compared to the standard indicates that the violation.

Thus, the invention provides a diagnostic method useful for diagnosis associated with VEGI violations involving measuring the expression level of the gene encoding the protein VEGI-192aand/or VEGI-192bin the sample from the individual and comparing the measured level of gene expression with a standard level of expression of VEGI-192aand/or VEGI-192bso that increase or decrease the level of expression of this gene compared to the standard indicates that the violation.

If the diagnosis of disorders has already been delivered in accordance with conventional methods, the present invention is applicable as a prognostic indicator and/or indicator when monitoring patients, detecting reduced expression of VEGI-192aand/or VEGI-192b, will experience a worse clinical outcome compared to patients expressing the gene at a level closer to standartenfuhrer.

How to apply polynucleotides, polypeptides and antibodies: screening tests

Polynucleotide and polypeptides of this invention can be used as research reagents and materials for the development of methods of treatment and diagnosis of human diseases.

This invention provides a method of identifying agents that modulate the activity of VEGI-192aand/or VEGI-192bthe methods for identifying agents that modulate the expression of VEGI-192aand/or VEGI-192bin the cage. In some embodiments, this assay is a cell-free analysis. In other embodiments, this analysis is an analysis based cells.

In this context, the term "modulate" includes "increase" and "decrease". In some embodiments of particular interest are agents that inhibit the activity of VEGI-192aand/or VEGI-192b. Such agents applicable to stimulate angiogenesis. In other embodiments of interest, the agents are agents that increase the activity of VEGI-192aand/or VEGI-192b. Such agents are of interest for the inhibition of angiogenesis and the treatment of angiogenesis-associated diseases.

Typically, the methods of screening or testing use agents or drugs, such as simple or complex the I organic or inorganic molecule, peptide, protein, oligonucleotide, polynucleotide, carbohydrate or lipoprotein. Can be synthesized many compounds, for example, oligomers, such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures, and they are also included in the term "agent". In addition, various natural sources can provide compounds for screening, such as extracts of plants or animal tissues, etc. Connections can be tested separately or in combination with each other.

This invention provides methods of identifying agents that modulate the activity of VEGI-192aand/or VEGI-192bafter bonding with VEGI-192aand/or VEGI-192b. This method typically involves contacting the test agent that selectively binds to VEGI-192aand/or VEGI-192bthe sample containing VEGI-192aand/or VEGI-192b; and the determination of the activity of VEGI-192aand/or VEGI-192bin the presence or in the absence of these agents. Increase or decrease in the activity of VEGI-192aand/or VEGI-192bin the presence of the agent compared to the absence of the agent indicates that the agent increases (an agonist) or decreases (is an antagonist) activity of VEGI-192aand/or VEGI-192b. Potential agonists and and antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of this invention and thereby increase or decrease its activity.

Analyses to test the ability of the agent to selectively contact the polypeptide of the present invention are known in this field. For example, the polypeptides of this invention can be attached to a solid carrier and an agent that selectively binds with the polypeptide may be identified using methods known in this field. Alternatively, agonists and/or antagonists VEGI-192aand/or VEGI-192bcan be detected by combining VEGI-192aor VEGI-192band a potential agonist and/or antagonist with membrane-bound receptors VEGI-192aor VEGI-192b(if such receptors are identified) or recombinant receptors in suitable conditions for the analysis of competitive inhibition. VEGI-192aor VEGI-192bcan be labeled, for example, with the use of radioactivity, so that the number of molecules of VEGI-192aor VEGI-192bassociated with the receptor can determine the effectiveness of potential agonist and/or antagonist.

Assays to test the activity of VEGI known in this field. Examples of such analyses based on the cells described in the examples in more detail, e.g. the trial of the action on the growth of vascular endothelial cells, education and organization of endothelial cells in capillaroscopy tubular structure or organization of endothelial cells into capillary vessels in the chorioallantoic membrane of chicken embryo.

Antibodies that selectively bind to polypeptides VEGI-192aand/or VEGI-192bcan be used as antagonists by binding with VEGI-192aand/or VEGI-192band prevent the performance of their activity.

The invention also provides methods for identifying agents that modulate the activity described here VEGI isoforms without linking with this VEGI isoform. Such agents include, but are not limited to, agents that are above (upstream) or below (downstream) the activity of VEGI in the regulatory cascade. These methods include analysis of the activity of VEGI in the presence or in the absence of the test agent.

The invention also provides methods of identifying agents that modulate the level of mRNA and/or VEGI polypeptide described herein.

Thus, this invention provides a method of identifying an agent that modulates the level of expression of VEGI in the cell, and the method comprises: contacting the test agent is a candidate cell that contains nucleic acid that encodes described C is followed by a session in the VEGI polypeptide, and the definition of actions of this agent on the expression of VEGI polypeptide. In some embodiments, this action is measured by detecting the level of mRNA encoding the VEGI polypeptide, using described here polynucleotides VEGI. In other embodiments, this action is measured by the level detection VEGI polypeptide using the antibodies described here.

Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA, or through the formation of a triple helix. Antisense methods are discussed in several studies (e.g., Okano, J.Neurochem. 56:560 (1991); "Oligodesoxynucleotides as Antisense Inhibitors of Gene Expression." CRC Press, Boca Raton, Fla. (1988)). The triple helix formation is discussed in some studies (e.g., Lee, et al., Nucleic Acids Research 6:3073 (1979); Cooney, et al.. Science 241:456 (1988); Dervan, et al.. Science 251:1369 (1991)). These methods are based on binding of polynucleotides with complementary DNA or RNA. For example, the 5'coding portion of polynucleotide, which encodes the Mature polypeptide of this invention may be used to design antisense RNA oligonucleotide with a length of about 10-40 BP DNA oligonucleotide design so that it was complementary to the gene involved in transcription, prevent the I hereby transcription and production of VEGI- 192aand/or VEGI-192b. Antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule to the polypeptide VEGI-192aand/or VEGI-192b. The above-described oligonucleotides can also be delivered into cells, so that the antisense RNA or DNA may expressroute in vivo to inhibit production of the polypeptide VEGI-192aand/or VEGI-192b.

The invention also provides methods of identifying such agents, as mutants or variants VEGI-192aand/or VEGI-192bwho can compete for binding to the target, which is associated VEGI-192aand/or VEGI-192b,and prevent the interaction of VEGI-192aand/or VEGI-192bwith their targets. Such mutants or variants can be agonists or antagonists VEGI-192aand/or VEGI-192b.

This invention provides a method of identifying agents that are associated with VEGI-192aand/or VEGI-192bproviding the contacting these agents with VEGI-192aand/or VEGI-192band then detecting the binding agents with VEGI-192aand/or VEGI-192b.

How to apply polynucleotides, polypeptides and antibodies: the treatment of diseases

This invention provides methods of inhibiting the growth of vascular endothelial cells, inhibition of angiogenesis, treatment or donkey the mix of diseases and processes which is mediated by uncontrolled angiogenesis for the treatment of cancer, such as suppression of tumor growth. In contrast to claims that VEGI-251is a membrane-bound protein, the examples show that VEGI-251is secretively protein, inhibits the growth of vascular endothelial cells and exerts an antiangiogenic effect in expression. In addition, the example also shows that VEGI-192ainhibits the growth of vascular endothelial cells.

Thus, compositions which can be used for this method of the present invention include, but are not limited to, described here polynucleotide, such as polynucleotide encoding the polypeptides SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; described here polypeptides, such as polypeptides SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a shortened form, which contains one or more amino acids from the region of amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6; and the agonist or antagonist described herein VEGI polypeptide such as an antibody that blocks the activity of VEGI polypeptide.

The invention also includes methods of delaying development of angiogenesis-associated diseases in the individual.

Polypeptides VEGI isoforms of the present invention (and polynucleotide encoding polypeptides VEGI isoforms) can the be used to reduce the formation of capillarity tubular structures, formed by endothelial cells in vitro. Polypeptides VEGI isoforms of the present invention can be used to inhibit the formation of endothelial cells, organized in capillaroscopy tubular structures in response to angiogenic factors such as FGF-2. In addition, described herein are isolated polypeptides VEGI isoforms of the present invention can also be used to inhibit the growth and organization of endothelial cells into capillary vessels in a modified chorioallantoic membrane of chicken embryo (CAM). As a result, the polypeptides VEGI isoforms of the present invention can be used to inhibit the formation of capillaries or capillarity structures from endothelial cells in vitro.

It should be clear that conditions caused by a decrease in the standard or normal level of activity of the polypeptides VEGI isoforms in individual, particularly disorders of the immune and circulatory systems, can be treated by the introduction of the polypeptides described herein VEGI isoforms (or polynucleotides encoding polypeptides isoforms VEGI). Thus, this invention also provides a method of treatment of an individual in need of an increased level of activity of VEGI isoforms, providing for the introduction to such an individual a pharmaceutical composition containing the number isolera the data polypeptide isoforms VEGI (or polynucleotide) of the present invention, such as the Mature form of the polypeptide VEGI isoforms of this invention, effective to increase the activity level of the polypeptide isoforms VEGI in this individual. This invention also provides a method of treatment of an individual in need of a reduced level of activity of VEGI isoform, providing for the introduction to such an individual a pharmaceutical composition comprising an amount of antagonist VEGI isoforms, such as an antibody specific against the VEGI isoforms, which blocks the activity of VEGI isoform, effective to reduce the level of activity of polypeptide isoforms VEGI, in this individual.

Shipping based polynucleotide

In some embodiments, the invention includes a method of inhibiting angiogenesis in a tissue or cell, which is to be called to an effective amount of the polypeptide having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, or SEQ ID NO:6, or a shortened form, which contains at least one or more amino acids from the amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6, came into contact with the tissue or cell or was located near the tissue or cells, so that angiogenesis is inhibited. In some embodiments, the invention includes a method of inhibiting angiogenesis, cover is a broad introduction to the individual, such as a person or animal, a composition containing a nucleic acid molecule encoding the polypeptide SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a shortened form, which contains at least one or more amino acids from the amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6 in a dose sufficient to inhibit angiogenesis. In some embodiments, the nucleic acid molecule is functionally linked to a regulatory sequence that controls gene expression. Such regulatory sequences are known in this field.

The individual is an animal, in some embodiments individual may be a mammal.

This invention also provides a method of treating or attenuating diseases and processes that are mediated by uncontrolled angiogenesis which the stages of introduction of the individual, such as a person or animal, a composition containing a nucleic acid molecule that encodes a polypeptide SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a shortened form, which contains at least one or more amino acids from the amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6 in a dose sufficient to regulation of angiogenesis. In some embodiments, the nucleic acid molecule is functionally linked to regulatory the sequence, which controls gene expression.

This invention also provides a method of treating cancer or suppression of tumor growth, providing the stage of introduction of the individual, such as a person or animal, a composition containing a nucleic acid molecule that encodes a polypeptide SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a shortened form, which contains at least one or more amino acids from the region of amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6 in a dose sufficient for suppression of tumor growth. In some embodiments, the nucleic acid molecule is functionally linked to a regulatory sequence that controls gene expression.

It is clear that, if they use a shortened form of VEGI and shortening takes place in the secretory signal sequence, this shortened form includes a secretory signal sequence, homologous or heterologous, to control the secretion of this protein. Heterologous secretory signal sequences known in the field.

Methods of delivery of polynucleotides for expression in the individual (as ex vivo and in vivo) are known in this field. Usually prepare and introduce a suitable polynucleotide vector design.

Can be used for targeted delivery of therapeutic compositions is s, containing polynucleotide expressing vector or sebenarnya polynucleotide. Ways receptor-mediated DNA delivery are described, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A.Wolff, ed.) (1994); Wu et al., J.Biol.Chem. (1994) 269-542; Zenke et al., Proc.Natl.Acad.Sci. USA (1990) 87:3655; Wu et al., J.Biol.Chem. (1991) 266:338. Therapeutic compositions containing polynucleotide can be in the range of about 100 ng - about 200 mg of DNA for local administration in the gene therapy Protocol. The concentration ranges of about 500 ng to about 50 mg, about 1 mg and about 2 mg, about 5 μg - 500 μg and about 20 μg - about 100 μg DNA can also be used in gene therapy Protocol. Therapeutic polynucleotide of the present invention can be delivered using vectors for gene delivery. Vector for gene delivery can be viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185 and Kaplitt, Nature Genetics (1994) 6:148). The expression of such coding sequences can coil using endogenous promoters for mammalian or heterologous promoters. The expression of the coding sequence can be either constitutive or regulated.

ectory-based viruses to deliver the desired polynucleotide and expression in a desired cell is well known in this field. Examples of vectors based on viruses include, but are not limited to, recombinant retroviruses (see, for example, PCT publication numbers WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/10218; WO 91/02805; U.S. patent numbers 5219740 and 4777127; UK patent No. 2200651 and EP 0345242), the vectors on the basis of alpha viruses (for example, vectors of the virus Sindbis virus, Semliki forest (ATSS VR-67; ATCC VR-1247), virus Ross river (ATSS VR-373; ATCC VR-1246) virus and Venezuelan equine encephalitis (renowned for VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and vectors based on adeno-associated virus (AAV) (see, for example, PCT publication numbers WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). You can also use the introduction of DNA related to killed adenovirus, as described in Curiel, Hum. Gene Ther. (1992) 3:147.

Can also be used non-viral vectors and methods, including, but not limited to, condensed with polycation-DNA related or not related only to killed adenovirus (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); the ligand-bound DNA (see, for example, Wu, J.Biol.Chem. (1989) 264:16985); delivery vectors based on eukaryotic cells (see, for example, U.S. patent No. 5814482; PCT publication numbers WO 95/07994; WO 96/17072; WO 95/30763 and WO 97/42338) and neutralization of the charge of the nucleic acid or fusion with cell membranes. Can also be used "naked" DNA. Examples of methods of introducing naked DNA described in public is of PCT No. WO 90/11092 and U.S. patent No. 5580859. Liposomes that can act as gene delivery vectors described in U.S. patent 5422120; PCT publications with numbers WO 95/13796; WO 94/23697; WO 91/14445 and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411 and in Woffendin, Proc.Natl.Acad.Sci. USA (1994) 91:1581.

So, for example, can be designed cells from patients with polynucleotides (DNA or RNA)encoding such polypeptide, as VEGI-251, ex vivo, and these engineered cells is administered to a patient should be treated this polynucleotides. Such methods are well known in this field and are obvious from these descriptions. For example, can be designed cells using a viral or retroviral particles containing DNA or RNA encoding the polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of the polypeptide in vivo, for example, by procedures known in the field. For example, the cell-producer for producing retroviral particle containing RNA encoding a polypeptide of the present invention may be administered to a patient for constructing cells in vivo and expression of the polypeptide in vivo. These and other routes of administration of the polypeptide of the present invention in this way should be obvious to specialists with expertise in this area from the descriptions given of the image is to be placed. For example, expressing the vector for constructing cells may be other than a retrovirus, such as adenovirus, which can be used to construct cells in vivo after combining with a suitable delivery vehicle.

Retroviruses from which can be produced retroviral plasmid vectors include, but are not limited to, virus murine leukemia, Malone, virus, spleen necrosis, retroviruses such as rous sarcoma virus, the virus of William Harvey sarcoma virus leukemia birds, the virus Gibbon leukemia, human immunodeficiency virus, adenovirus, virus, myeloproliferative sarcoma virus tumor of the breast. In one embodiment, the retroviral plasmid vector derived from a virus, murine leukemia, molony.

The vector typically includes one or more promoters. Suitable promoters that can be used include, but are not limited to, the retroviral LTR, the SV40 promoter and the promoter of the human cytomegalovirus (CMV), described by Miller and al. (Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellular promoters such as the promoters of eukaryotic cells, including, but not limited to, the promoters of histone, pol III, and β-actin). Other viral promoters that can be used include, but are not limited to, the promoters of adenovirus, etc the motors timedancing (TC) and the promoters of parvovirus B19. The choice of a suitable promoter will be obvious to specialists with expertise in this area contained herein explanation.

The sequence of the nucleic acid encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be used include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter or heterologous promoters, such as the promoter of the human cytomegalovirus (CMV)promoter respiratory syncytial virus (RSV), inducible promoters, such as the MMT promoter, the promoter metallothionein, the promoters of heat shock, the albumin promoter, the ApoAI promoter, promoters globin human, viral promoters timedancing, such as the promoter timedancing of herpes simplex virus, retroviral LTRS (including the modified retroviral LTR described here), promoter β-actin and promoters of human growth hormone. The promoter may also be the natural promoter, which controls the gene encoding this polypeptide.

When selecting the system retroviral vectors retroviral plasmid vector used for transduction of lines packaging cells to form a cell line-producers. Examples of packaging cells which may be transpire ovani, include, but are not limited to, cell line RE, RA, b-2, b-AM, PA12, kzt19-14, VT-19-17-H2, CRE, beta-CRIP, GP+E-86, GP+envAm12, and DAN, described in the article Miller, Human Gene Therapy 1:5-14 (1990), incorporated herein by reference in its entirety. This vector can transducible packaging cells through any means known in the field. Such methods include, but are not limited to, electroporation, the use of liposomes and deposition CaPO4. In one alternative, the retroviral plasmid vector may be encapsulated in a liposome or associated with the lipid and then introduced into the host.

Cell line-producers generates infectious retroviral vector particles which include the sequence (sequence) of the nucleic acids encoding these polypeptides. Such retroviral vector particles can then be used for transduction of eukaryotic cells in vitro or in vivo. Transduced eukaryotic cells will Express the sequence (sequence) of the nucleic acid that encodes the polypeptide. Eukaryotic cells, which can be transpulmonary include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, cultured myoblasts, keratinocytes, EN is teleline cells and bronchial epithelial cells.

These General principles are also applicable to other delivery systems based viruses such as AAV.

Delivery of polypeptides

This invention also provides a method of inhibiting angiogenesis, providing for the introduction to the individual, such as a human or an animal polypeptide described herein, such as the polypeptide SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a shortened form, which contains at least one or more amino acids from the region of amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6, in a dose sufficient to inhibit angiogenesis.

This invention also provides a method to treat or ameliorate diseases and processes that are mediated by uncontrolled angiogenesis which the stages of introduction of the individual, such as a person or animal, a composition comprising the polypeptide SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a shortened form, which contains at least one or more amino acids from the region of amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6, in a dose sufficient to control angiogenesis.

This invention also provides a method of treating cancer or suppression of tumor growth, providing the stage of introduction of the individual, such as a person or animal, a composition comprising the polypeptide SEQ ID NO:4, SEQ ID NO:5 or SEQ ID N:6, or a shortened form, which contains at least one or more amino acids from the region of amino acids 1-26 SEQ ID NO:4, amino acids 1-26 SEQ ID NO:5 or amino acids 1-85 SEQ ID NO:6, in a dose sufficient for suppression of tumor growth.

Methods for determining the activity of VEGI polypeptide (including a shortened form VEGI) is well known in this field and are described in detail in the examples, such as the trial of the action on the growth of vascular endothelial cells, formation capillaroscopy tubes, capillary growth in collagen gels placed on chorioallantoic membrane of chicken embryo, the growth of tumor xenografts.

In this context, the term "angiogenesis-associated disease" means a disease or condition associated with undesired and/or uncontrolled angiogenesis or a disease or condition for which is the best inhibition of angiogenesis. It includes diseases and processes mediated by undesired and/or uncontrolled angiogenesis. Examples of such angiogenesis-associated diseases, such as tumor growth, are described here.

Polypeptides VEGI isoforms described herein may be used to inhibit proliferation of endothelial cells, for example endothelial cells of the aorta. Thus, the VEGI polypeptide-192aand/or VEGI-19b can be used for the treatment of diseases and disorders, which is advantageous inhibition of endothelial cell growth.

The composition of the polypeptide isoforms VEGI will be prepared and implemented in accordance with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment only polypeptides isoforms VEGI), the place of delivery of the composition of the polypeptide isoforms VEGI, as a way of introduction, the scheme of administration and other factors known to practitioners. Thus, the "effective amount" of the polypeptide VEGI isoforms for these purposes is determined based on such considerations.

Polypeptides isoforms VEGI and agonists and antagonists of this invention can be used in combination with a suitable pharmaceutical carrier. Such compositions typically contain a therapeutically effective amount of this compound and a pharmaceutically acceptable carrier or excipient. Such media include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol and combinations thereof. This form should be suitable for the specific method (scheme) introduction.

This invention also provides a pharmaceutical package or kit containing one or more containers filled with one is m or more ingredients of the pharmaceutical compositions of the present invention. Such containers may be accompanied by a statement in the form prescribed by the governmental Agency regulating the manufacture, use or sale of pharmaceuticals or biological products which reflects approval by that office manufacture, use or sale for the introduction of man. In addition, the pharmaceutical compositions of this invention can be used in conjunction with other therapeutic compounds.

The pharmaceutical compositions can be administered in a suitable manner, for example local, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal ways. The pharmaceutical composition is administered in an amount that is effective for the treatment and/or prophylaxis of the specific indication. They are usually administered in amounts of at least about 10 g/kg body weight and in most cases they are administered in a quantity not in excess of about 8 mg/kg of body weight per day. In most cases, this dose is equivalent to approximately 10 g/kg to about 1 mg/kg of body weight per day taking into account the routes of administration, symptoms, etc.

Various delivery systems are known and can be used for introducing polypeptides VEGI isoforms of the present invention, for example, encapsulation in liposomes, microparticles, the microcapsule is, mediated receptor endocytosis (see, e.g., Wu and Wu, 1987, J.Biol.Chem. 262:4429-4432). Methods of introduction include, but are not limited to, local, intradermal, intramuscular, intraperitoneal, motiony, subcutaneous, intranasal, epidural, ophthalmic, and oral ways. These compounds can be administered in any conventional manner, for example by infusion or bolus injection, by absorption through epithelial or Muco-cutaneous mucosa (for example, the mucous membrane of the mouth, rectal mucosa and intestinal mucous membrane, and so on), and can be administered together with other biologically active agents.

In a specific embodiment, it may be desirable introduction pharmaceutical compositions of the invention locally to the area that needs treatment. This may be done, for example, and without limitation, local infusion during surgery, by local application, for example when holding the bandage on the wound, by injection, by means of a catheter, by means of a suppository, or by means of the graft, with a specified transplant is a porous, non-porous, or gelatinous material, including membranes, such as membranes or fibers of the elastomer Silastic.

The evaluation of disease is carried out using standard methods known in the data is the second region, such as methods of image acquisition and monitoring of suitable markers.

Specialist with expertise in this area should be clear that, because the polypeptides VEGI isoforms of this invention are members of the TNF-family, Mature secretiruema form of this protein may be released in soluble form from the cells which Express polypeptides described herein VEGI isoforms by proteolytic cleavage. Thus, when this Mature form or soluble extracellular domain of the polypeptide isoforms VEGI is added from an exogenous source to cells, tissues or the body of the individual, the polypeptide will exhibit its physiological activities on its target cells of that individual. Cells expressing this transmembrane polypeptide type II can also be added to cells, tissues or the body of the individual, and these added cells will be contacted with cells expressing the receptor polypeptides described herein VEGI isoforms, resulting in cells expressing the polypeptides VEGI isoforms, can have an effect (for example, regulation of growth and regulation of endothelial cells) bearing receptors of the target cells.

As mentioned above, it is known that VEGI has a strong anti-proliferative effects on endothelial cell growth. So about what atom, VEGI can also be used to regulate the development of endothelial cells from circulating hematopoietic and endothelial progenitor cells.

Thus, this invention provides methods of enhancing angiogenesis, providing for the introduction of inhibitor VEGI-192aand/or VEGI-192bso that angiogenesis is enhanced. Such amplification may be desirable, for example, in the context of States associated with obstruction of a blood vessel, for example, in ischemic conditions or heart attacks. These forms may be administered locally or systemically using known in the field of methods.

The antibody of the present invention, which blocks or inhibits the activity of VEGI polypeptide, can be used to stimulate or enhance angiogenesis.

Examples

Below are examples of the present invention, which are given for illustrative purposes only and not to limit the present invention. In light of this description, numerous embodiments within the appended claims will be obvious to a person of ordinary skill in this field.

The invention will be further described with reference to the following examples, however, it should be clear that the invention is not limited to such examples.

It was recently reported about the opening specific for endothelial cells gene product, growth inhibitor of vascular endothelial cells (VEGI) (Zhai Y, et al., FASEB J., 13:181-189, 1999; Zhai Y, et al., Int. J. Cancer, 82:131-136, 1999). This protein consists of 174 amino acids, i.e. VEGI-174with 20-30% homology sequence relative to the members of the superfamily of TNF. Northern-blot analysis of a wide range of cell lines and primary cell cultures shows that the gene VEGI-174is expressed predominantly in endothelial cells. In addition, mRNA VEGI-174detected in many organs of the adult, suggesting a physiological role of this gene in normal vascular network. Function VEGI-174investigated for a number of cellular and animal models. Recombinant truncated form of VEGI-174inhibited proliferation of endothelial cells with high efficiency, but not acted on the growth of any other types of investigated cells. Shortened form of this protein inhibited the formation capillarity structures by endothelial cells in collagen gels and the sprouting of capillaries in collagen gels placed on chorioallantoic membrane of chicken embryo. Overexpression of Sekretareva form VEGI-174in cancer cells of the colon of a mouse (MC-38) almost completely prevented tumor growth of these cells in syngeneic mice C57/BL. In addition, the joint inoculation of cancer cells Molo is Noah cancer man with ovary cells Chinese hamster, sverkhekspressiya secreterial form VEGI-174resulted in significant inhibition of tumor growth of xenografts of breast cancer in naked mice.

Example 1. ELISA analysis of human sera

Normal human serum of healthy adult individuals, male and female, received from the Bank of sera Lombardi Cancer Center. For measuring the content of VEGI in sera used a sandwich ELISA. Serum samples (100 μl) or varying amounts of recombinant protein VEGI in 3% BSA was added in 96-well plates, coated with a polyclonal anti-VEGI-antibody and blocked with 3% BSA. Added monoclonal antibody (100 μl, 2 μg/ml) against VEGI (3-12D). Added biotinylated antibody against mouse IgG (2 μg/ml Vector laboratories, Burlingame, CA) followed by the addition of a complex of avidin-horseradish peroxidase with 3,3',5,5'-tetramethylbenzidine (Vector laboratories) as a substrate. These samples were incubated at room temperature for 10 minutes, the reaction was stopped by 50 μl of 1M H2SO4and then analyzed at 450 nm using a spectrophotometric tablet reader using the standard curve y=-0,72+0,409·log(x). Used a range of standard protein 0,32-1000 ng/ml.

Example 2. Northern-blotting

Northern blots of multiple tissues and dot-blot panels of many tissues (Clontech, Palo Alto, CA) and hybridized vrestore ExpressHyb (Clontech), double-stranded cDNA probes. Full size used VEGI-174the probe was a cDNA fragment HindIII-BamHI (GenBank access number AF03990) in pCDNA3.1 (Invitrogen, Carlsbad, CA). For isoform-specific probes using PCR amplification was obtained matrix VEGI-251297 BP encoding a 99 N-terminal amino acids, and noted32P-dCTP using the method of random priming (Life Technologies, Invitrogen, CA). VEGI-174-specific probe corresponding to the 22 N-terminal amino acids, was obtained by end-labeling of PCR product 66 P.N. These blots hybridized at 42°during the night and washed in wash buffer 1 (2 X SSC, 0.1% lauryl sulfate) and wash buffer 2 (1 X SSC, 1% LTOs) at 42°With subsequent radioautography at -70°With intensifying screen.

Example 3. 5'RACE and cloning isoforms VEGI

5'-sequence VEGI amplified from panel RACE many tissues (ORIgene, Rockville, MD) according to the manufacturer's instructions. This panel contains the cDNA samples obtained from 24 individual human tissue, with adapter, legirovannym with 5'-ends of the cDNA. Two rounds of "nesting" (nested) PCR was performed using two pairs of oligonucleotide primers. In the first round of PCR used adaptery primer ADP1, 5' CGGAATTCGT CACTCAGCG 3' (SEQ ID NO:8) and VEGI-gene-specific primer GSP1, 5' CCCGGATCCT ATAGTAAGAA GGCTCC 3' (SEQ ID NO:9). Then the reaction products were diluted 1:10 with water. Then dilute the HRC the samples used for the second round of PCR with other adapternum primer, ADP2 5' AGCGCGTGAA TCAGATCG 3' (SEQ ID NO:10) and VEGI-gene-specific primer GSP2, 5' CGGTGGATCC CGAGTTTGTC TCACAACTG 3' (SEQ ID NO:11). PCR products were separated in agarose gel, purified and sequenced on an automated synthesizer ABI.

Example 4. Selection VEGI-251and VEGI-192a

Gene-specific primers designed in accordance with the results of the sequencing of the RACE products were used to repeat the second round of PCR to confirm the identities of the sequences. Then gel purified RACE products were cloned in the plasmid pCR3.1 (Invitrogen, Frederick, MD) and sequenced. Based on these sequences were designed specific for isoforms of the primers. Common reverse primer Vg161 (161), 5' GTGTAATCCA CCAAAGAG 3' (SEQ ID NO:12) were used to direct the primers listed in table 8.

Example 5. Cell culture and treatment TNFα

Endothelial cells of the umbilical vein of a person (HUVE) and endothelial cells of fetal bovine heart (FBHE) were obtained from Clonetics (Walkersville, MD) and were grown in EGM-2 (Clonetics). Dermal microvascular endothelial cells (HMVE), endothelial cells of the human coronary artery (NSE) and NIH3T3 cells were obtained from American type culture Collection (ATSS) and were grown in EGM2-MV (Clonetics). Endothelial cells of the aorta of adult cows (AVE) and endothelioma brain of mice bEND.3, which were the kind gift of Dr. who and Peter Bohlen from ImClone Inc. New York, NY. The smooth muscle cells of the human coronary artery (HCASM) (Clonetics) and cells of AWE cultured in full (Biofluids, Biosource International, Camarillo, CA), 10% FCS, 1 ng/ml of factor 2 fibroblast growth (Promega, Madison, WI). EAO, cell line, derived from human vascular endothelial cells, was a gift from Dr. Cora-Jean Edgell, University of North Carolina. These cells together with bEND.3 brain mice and cell lines endothelioma H5V supported in full with 10% FCS. Subconfluent cells grown in the cups of 100 mm, were treated with different doses of factor and tumor necrosis (TNFα) (Biosource International, Camarillo, CA) before analysis of RNA.

Example 6. Analyses of the protection of ribonuclease

For probes that are specific with respect to specific isopom, was obtained using PCR cDNA fragments from VEGI-174(862-1062 rn), VEGI-251(1-160 P.N.), VEGI-192a(277-656 P.N.) person and was built between sites EcoRI and NotI pcDNA3 (Invitrogen) in the antisense direction. Probe for mouse gene β-actin (824-942) cloned into pSP72 (Promega) between sites HindIII and BamHI. VEGI - and β-actin-matrix was linearizable using HindIII and EcoRI, respectively. Antisense run-off probes synthesized using RNA polymerase SP6 with a set for transcription Maxiscript (Ambion, Woodward TX). For protection against nucleases using a set of RPAIII (Ambion, TX) 15-20 µg of total RNA hybridized overnight with 1-3×105pulse/min each probe at 52° C. Cleavage by RNase was performed with a dilution of 1:100 mixture of RNase A/T1 (Ambion) for 30 minutes at 37°C. cleavage Products were besieged, were separated in 6% polyacrylamide gel and subjected to autoradiography at -70°C.

Example 7. Analysis of the structure of the gene

Organization of the gene VEGI person analyzed using PCR using clone bacterial artificial chromosome (BAC) (Genome Systems, Inc. St. Louis, MO). Designed PCR primers from exon sequences, which gave an overlapping PCR products. These PCR products sequenced to determine their relative positions. The primers for intron region were designed based on the GenBank entry for the reference NT_017569 chromosome 9, which correspond to the sequences between the bases 2643881 and 2694724. They are listed in table 9. Using DNA human placenta as a matrix carried out extra-long PCR using PCR set rTth XL from Perkin Elmer (Foster City, CA) and the following conditions for extra-long PCR: 95°sec, 1 min, 97°sec, 15 sec, 60,5°C, 10 min, 17 cycles; 97°sec, 15 sec, 60,5°C, 10 min plus 15 sec elongation, 13 cycles with subsequent final elongation at 72°C for 11 minutes These PCR fragments were obtained using the primer pairs shown in table 9. These PCR products are sequenced.

The PCR product 2b also sequenced with the use the of primers 61, 57, 51 and 53.

Example 8. Expression plasmids and transient transfection

Open reading frames VEGI-H4 and VEGI-251 was insertional in pcDNA3.1-myc (Invitrogen, MD) to obtain peptides bearing a C-terminal myc-tag. The resulting plasmid was transfusional in cells AWE and HUVE for studies of cellular localization. For transfection of cells 3×104cells were sown on a cover glass (Lab-Tek chambered coverglass (Nalge, Naperville, IL) and maintained throughout the night. Plasmid DNA (400 ng) in 25 µl of serum-free full mixed with reagent PLUS (4 μl) (Gibco-BRL). The reagent lipofectamine (Gibco) (1 μl) was 200-fold, and it was mixed with the DNA solution within 15 minutes. Then the complex DNA-lipofectamine was added to the cells with 200 ál of full and incubated at 37°C for 3 hours. These cells were allowed to recover in containing serum culture medium for 36 hours before immunoablative and subsequent fluorescence microscopy. Full coding region VEGI-174or VEGI-251also built between sites EcoRI and BamHI pEGFP-C2 (Clontech) to obtain the fused proteins GFP-VEGI. Merged constructs GFP-VEGI was transfusional in cells AWE, as described above. After 48 hours after transfection cellular localization of these fused proteins was investigated using fluorescence microscopy.

Example 9. Immunoablative for subcellular localization

T is inficirovannye cells in the aorta of adult cows (AVE) and HUVE cells were washed SFR and were fixed with a mixture of 3.7% paraformaldehyde/0.1% Triton X-100 in STR for 10 minutes, was permeabilities 0.5% Triton X-100 in STR for 5 minutes and then incubated with a dilution of 1:300 anti-myc-monoclonal antibodies 9-10TH (Sigma) for 1 hour. Cells were washed SFR and incubated with a monoclonal antibody against mouse IgG, conjugated with the dye Texas Red at a dilution of 1:60 (Jackson ImmunoResearch Labs, Inc. West Grove, PA), then washed three times SFR. Cells were visualized confocal fluorescent microscopy (Olympus IX70-SIF).

Example 10. Obtaining monoclonal antibodies

Six-week female mice Balb/C (Charles River Laboratories, Wilmington, MA) were subcutaneously injected with purified recombinant protein VEGI (residues 20-174) in an amount of 50 μg per mouse in 0.1 ml complete adjuvant's adjuvant (Life Technologies). After intraperitoneal booster injection of mice with higher titers were obtained final intraperitoneal injection of antigen 30 μg/mouse. The spleen cells were isolated and merged with cells of the mouse myeloma SP2/0 using polyethylene glycol 1500 (Boehringer Mannheim). Hybridoma were selected using HAT and environment were subjected to screening using ELISA. Positive hybridoma cloned, and the subclass of the monoclonal antibodies was determined using set mouse isotypes (SIGMA, MO). Hybridoma were cultured in the medium INTEGRA CL 350 (INTEGRA Biosciences, Inc., liamsville, MD), supernatant collected and monoclonal antibodies were purified using b the LCA A-agarose AffiGel (BioRad).

Example 11. Obtaining polyclonal antibodies

Four-six new Zealand white SPF rabbits (Charles River) were inoculable subcutaneously with 100 µg expressed in E. coli recombinant VEGI (as described above)was mixed with complete adjuvant's adjuvant (Life Technologies). After intramuscular booster injection, serum was collected from rabbits that detect a significant immune response. Serum was purified by absorption on the lysate of E. coli cells (transformed with empty expression vector), then the lysate of the cells of smooth muscles of the human coronary artery.

Example 12. Analysis of VEGI in mammalian cells and conditioned media

Encoding district full-VEGI-251built between sites HindIII and BamHI pcDNA3 (Invitrogen). These plasmids pcDNA3, including vector, transfusional in breast cancer cells MDA-MB231 by electroporation. Stable transfectants were selected with 2 mg/ml G418 sulfate (Gibco). Conditioned medium was concentrated using the column Method (limit bandwidth based on the molecular weight of 10000). Both cell lysates and conditioned medium were immunoprecipitated using protein A/G-agarose (Oncogene, Boston, MA) and polyclonal antibodies against VEGI. Samples were analyzed by Western blotting. Detection was performed with a dilution of 1:1000 mouse monoclonal antibody 1-8F and visualized the antibodies is om against mouse IgG, conjugated with horseradish peroxidase (Set ECL, Amersham).

Example 13. Lentiviral transfer gene transfer

Lentiviral transfer vectors containing VEGI-174, sVEGI and VEGI-251were obtained using previously described methods (Dull et al. J.Virol 72, 8463-8471, 1998; Naldini, et al. Proc.Natl.Acad.Sci. USA 93, 11382-11388, 1996; Naldini, et al. Science 272, 263-267, 1996; Stewart, et al. Proc.Natl.Acad.Sci. USA 96. 12039-12043, 1999). Briefly, lentiviral transfer vector of received cells T with 3 plasmids: transducible plasmids pHR'CMV-VEGI packing plasmids pCMV ΔR8.2 Δvpr, and plasmids shell pCMV-VSV-g Viral supernatant collected every 24 hours for two days after transfection, purified by filtration through a 0.45 µm filter and the titer was determined using analysis of P24 (Dull et al. J.Virol 72, 8463-8471, 1998; Naldini, et al. Proc.Natl.Acad.Sci. USA 93, 11382-11388, 1996; Naldini, et al. Science 272, 263-267, 1996; Stewart, et al. Proc.Natl.Acad.Sci. USA 96,12039-12043, 1999). One of GHGs amount of P24 was determined as one infectious dose for tissue culture (TCID). For analyses at the cellular toxicity of HUVE cells were sown at a density of 2×104per well in 24-hole tablet for 20 hours before infection with viral supernatant. It was expected that the number of cells will double after 20 hours after seeding. Increasing the dose of viral vector was added to HUVE cells. The multiplicity of infection (MOI) was evaluated as TCID per cell at the time of infection. The number of attached cells, the left is already in the culture 24 hours after viral infection, was determined using the counter of Coulter.

Example 14. Analysis of oncogenic potential in vivo

Stable transfetsirovannyh cells MDA-MB-231, containing an empty vector pcDNA3, VEGI-174, VEGI-251or sVEGI, were injected with in the fatty tissue of the mammary glands of female Nude naked mice (1×106cells per injection). There were 2 sites of injection on the animal and 15 animals in each group who received the transfectants VEGI-174, VEGI-251and sVEGI. A group of 5 animals that were injected with the vector pcDNA, was used as control. The dimensions of the resulting tumor xenografts were registered blind. Determination of the density of microvessels was performed as described (Weidner, et al. N Engi J Med 324, 1-8, 1991). Briefly, intratumoral microvessels immunoablative mouse monoclonal antibody against mouse CD31 (RESOURCES-1) (clone MEC, Pharmingen International, San Diego, CA). The antibody was diluted 1:100 in SFR, incubated overnight, fixed in paraffin sections of tumors with a thickness of 5 μm and visualized biotinylated antibody against rat IgG (Vector Laboratories) according to the method Vectastain ABC (Vector Laboratories). Each sample was examined at low magnification (X10 objective lens and X10 eyepiece lens) to identify areas with the highest vascularization of the tumor ("hot spots", see reference (Weidner, et al. N Engi J Med 324, 1-8, 1991)). In these zones examined m ximala 10 fields of view with increasing h (X40 objective lens and X10 eyepiece lens; 0.16 mm2on one field) and the expected average values. Large vessels with muscular walls excluded. Internal clearance was not required to identify the vessel. Any positively stained endothelial cells or clusters of cells, clearly separated from adjacent MicroSCADA, tumor cells and components of the connective tissue, considered as a separate subject counting microvessels. All measurements were performed blind. The results were analyzed by one-factor variance analysis (ANOVA). The level of significance a priori was set at P value less than 0.05.

Example 15. Detection of protein VEGI in human sera

As an initial screening to determine whether there is a VEGI in a soluble form, the inventors analyzed the Bank of human sera of healthy adults using monoclonal antibodies. The inventors were able to detect by using ELISA with antibody against C-end concentration VEGI in the range between 1 and 10 ng/ml (figure 1). This testified to the fact that, in addition to the previously characterized VEGI, which is believed to be membrane-bound, soluble form of VEGI could also have important physiological significance. Because previous studies have shown adaptations activity of recombinant C-terminal peptide, and overexpression of Polner Smirnova VEGI- 174was neeffektivnoi on tumor xenografts formed transfitsirovannykh cancer mice, the inventors reasonable way decided that contains the end of the soluble peptide found in human sera, hardly came from a VEGI-174. This observation led the authors to suggest that VEGI gene can Express alternative forms in human tissues.

Example 16. Detection of multiple transcripts VEGI and cloning of new isoforms VEGI

Using the full-size cDNA originally discovered VEGI person as a probe Northern blots of tissues of a healthy person reproducibly detected multiple bands in the following sizes: 7,5 TPN, 2,0 TPN and 1.5 TPN (figure 2). These multiple transcripts VEGI found to some extent overlapping tissue distribution and demonstrated the existence of a family of VEGI. To clarify the structure of the VEGI-related transcripts, the inventors undertook the selection VEGI isoforms using PCR. Using VEGI-specific 3'-primer, 5'RACE was used for amplification of 5'-ends of mRNA VEGI from a number of human tissues. Then the RACE products were cloned into pcR3.1 and sequenced (figure 3). Analysis of the sequence revealed two new sequence VEGI, VEGI-251and VEGI-192a(table 8 and figure 3). At the core is ve of these 5'sequences were designed isoform-specific primers and were allocated a full-sized cDNA made from an array of cDNA panels. Thus, there were three VEGI isoforms from fetal brain, adult uterus and lung (figure 3A and b). These new cDNA contains an open reading frame of 251 and 192 amino acids (figure 3B) with the calculated molecular masses 28086 and 21052 daltons, respectively. These two new peptide VEGI, VEGI-251and VEGI-192ahave a total of 151 amino acid residue from the source VEGI (Zhai, et al. hit J Cancer 82, 131-136, 1999), now called VEGI-174. The hydrophobicity profile of these proteins showed hydrophobic region of 20 amino acid residues in the VEGI-251near its N-Terminus (figure 3B), which was absent in the VEGI-174and VEGI-192a. It was predicted that this sequence contains a signal peptide. Another vysokopetrovsky may transmembrane region was previously identified at the N-end VEGI-174(figure 3B).

Example 17. Expression of isoforms VEGI

Individual profiles the expression of these isoforms additionally explored Northern analysis of blots of many tissues. Transcript VEGI-2517,5 TPN was detected in the placenta, kidney, lung and liver, whereas the transcript VEGI-1742 TPN observed in the liver, kidney, skeletal muscle and heart (figure 4). When using the same probes on the dot-band RNA from multiple tissues was observed that, in addition to overlapping expression between VEGI-251and VEGI-174in the prostate W is Lesa, the salivary glands and the placenta, VEGI-251was more abundant than VEGI-174in fetal kidney and fetal lung, whereas VEGI-174was more abundant in heart, skeletal muscle, pancreas, adrenal gland and liver (table 10). The value of such expression profiles at the present time is not quite obvious to the authors of the present invention. mRNA VEGI-192acould not easily be detected using Northern blot due to its low content.

The expression of VEGI in vitro was also investigated by way of protection from RNase. In accordance with previous observations for VEGI-174, VEGI-251and VEGI-192awere found in the same types of cells that VEGI-174being present in endothelial cells, including endothelial cells of the coronary artery (NSE), endothelial cells of the umbilical vein (HUVE) and endothelial cells of microvessels person (HMVE), and not detected in the smooth muscle of the human coronary artery (HCASM), AVE and mouse endothelioma bEND.3. It should also be noted that more than one isoform, expressives in the same type of cells, and the VEGI isoform-251was the most abundant. This suggests that the expression of these isoforms plays a regulatory role in the function of VEGI.

Table 10.
Expression of RNA VEGI-174and VEGI-251in human tissues*
ClothVEGI-174VEGI-251
The brain++-
Heart++++-
The aorta+-
Skeletal muscle+++-
The colon++±
Bladder+±
Uterus+-
The prostate gland+++++
Stomach+++++
Egg+-
Ovary+-
Pancreas++++±
The pituitary gland++-
The adrenal gland+++±
Thyroid gland+-
Salivary gland++++++++
Mammary gland+±
Kidney+++++
Liver ++++-
The small intestine+++++
Spleen++±
The thymus++±
Peripheral leukocytes+-
Lymph node+±
Bone marrow+-
Appendix++
Easy++++
Trachea+++
Placenta+++++++
Fetal brain+-
The rest of the heart+-
Fetal kidney+++++
The rest of the liver++-
Fetal spleen++
Fetal thymus+-
Fetal lung+++++

* Comparison of expression of RNA VEGI-174and VEGI-251using dot-blot of poly a + RNA of many human tissues. -: not detected; ±: insignificant above the background level; +: is the low tier; ++++: abundant. The expression of VEGI-192anot detected.

Example 18. Organization of the gene VEGI person

To determine the structural relationship of the three transcripts VEGI analyzed the organization of the gene VEGI person. This was performed using the clone YOU, and genomic DNA extracted from samples of human placenta. It was found that the gene VEGI man stretches for 17 TPN with 4 exons and 3 introns (figure 6). The intron-exon boundaries are governed by the rule GT-AG. On the basis of fragment size 2 intron 1 was estimated at 13-15 TPN, although the sequence information could not be obtained from this PCR product. All three isoforms have a common area of 438 BP, encoding residues 29-174 VEGI-174(table 12)encoded by exon IVb (figure 6), but their 5'-regions are formed from the alternative use of exons. Interestingly, VEGI-251and VEGI-192ause the acceptor splicing sites of exons to generate their respective products (table 11). Studies with protection from ribonuclease and 5'RACE using genomic probes and RNA HUVE showed that the presumed site of transcription initiation for VEGI-251localized approximately 100 BP against the direction of transcription from its ATG (unpublished data. Chew LJ), but these sites for VEGI-174and VEGI-192ahave not yet been mapped. Due to the very low content of RNA VEGI- 192asubsequent studies have focused on VEGI-251. Although it is currently unclear whether all these isoforms are initiated from the same promoter, the authors of this invention, however, conclude that the value of education multiple transcripts could be differential regulation of synthesis, which, in turn, may indicate the relative importance of one particular VEGI isoforms.

Example 9. Transcripts VEGI isoforms are induced in parallel by TNFα

In order to check the possibility of differential regulation of transcription VEGI isoforms, analyzed the regulation of genes VEGI using the example antiangiogenic actions when processing TNFα. Although many studies describe the antiproliferative action of proinflammatory cytokines such as TNFαon endothelial cells, these cytokines may also be angiogenic depending on the dose and the system used (see discussion). Such modulatory effects on endothelial cells may serve to regulate levels specific for endothelial cells of the cytokine, such as VEGI. The authors of this invention have found that the concentration of 15 ng/ml TNFα or higher concentrations may induce increasing levels of RNA VEGI in endothelial cells as large soudo the (umbilically Vienna), and small vessels (dermal MicroSCADA) (figure 7), and that all isoforms were induced in both of these types of endothelial cells. It is also clear that VEGI-251remains the most represented of these isoforms. This parallel increase regulation of transcripts VEGI by TNF not only indicates that mediated VEGI activity is potentially a target of action of TNFαbut also that the control functions VEGI through the synthesis of multiple peptides, most likely, lies at the post transcriptional level.

Example 20. Subcellular localization

Because TNF-like peptides are often present in the form of membrane-bound and water-soluble forms, this possibility was investigated using transfected with the recombinant VEGI cells AWE.

Experiments on localization was performed with constructs bearing a myc tag at the C-ends of the coding regions VEGI (figure 8). Expression design VEGI isoforms in pcDNA3.1-myc was transfusional in cells AWE and analyzed the distribution of VEGI-myc-product by means of immunocytochemistry with anti-myc-antibody. VEGI-174were detected in the cell distribution in the endoplasmic reticulum/Golgi apparatus (figure 8A). However, VEGI-251found more limited perinuclear granular staining (figure 8B). In both cases, the localization of glue on the internal surface was not detected by confocal fluorescence microscopy. In HUVE cells was found that VEGI-251-myc-containing granules are not calves Weibel-Palade endothelial cells, the signal Tue didn't colocalizes with staining for factor von Willebrand's disease (vWF) (figure 8C). Thus, it is possible that, in contrast to vWF, processing VEGI-251does not include regulated secretory path in endothelial cells.

To determine whether these isoforms to be determined N-end differences in subcellular localization, got chimeric expression constructs GFP-VEGI as with polnorazmernyi VEGI, and with their respective unique N-terminal sequences. These constructs with GFP-tagged at their N-ends (figure 8) transtorno was transfusional in cells AVE. As shown in figures 8F-8J, except 8G, the distribution of GFP-VEGI differed from the distribution of GFP. Full-VEGI-174showed localization to the endoplasmic reticulum/Golgi apparatus (ER/Golgi), as observed previously with myc-labeled VEGI-174while the first 22 balance VEGI-174were insufficient to target the distribution of GFP in specific intracellular organelle (figure 8G). In contrast, the first 99 amino acids VEGI-251were sufficient to GFP was localized in vesicles, which bordered on the plasma membrane. This distribution shall be observed as fluorescent bands, which delineates the border cells (figure 8H-J). Observations authors suggest that VEGI-251can be localized in secretory vesicles that undergo constitutive exocytosis.

In contrast to GFP-VEGI-251the absence of localization in the plasma membrane VEGI-251-myc (figure 8B) strongly suggests that the C-terminal myc tag was lost in this cell, possibly due to the cleavage of the secretory signal and the secretion of soluble C-terminal fragment. Know that the authentic signal sequence located downstream of transcription (downstream) of N-ends, such as in GFP-VEGI-251and GFP-VEGI-1-99, not tsapralis during synthesis in the endoplasmic reticulum (ER). This difference in the distribution profiles VEGI-251with the C-terminal and N-terminal tags were in agreement with secretory mechanism, which involves the splitting of VEGI-251on or within its N-terminal sequence before release into the extracellular environment.

Example 21. Demonstration of VEGI in conditioned cell medium

Taking into account the hydrophobic residues in the N-end VEGI-251and the observed subcellular distribution, it seems likely that VEGI-251is secretively protein. To test this hypothesis received stable transfectants VEGI-251in breast cancer cells is EleSy MDA-MB-231. As a negative control were also transfectants vector pcDNA3. The expression of these constructs in MDA-MB-231 was confirmed by analysis of the protection from RNase. It should be noted that neither the transfection vector or transfection of VEGI did not affect the survival and proliferation of these cells in vitro. The air-conditioned environment transfectants were collected, concentrated and immunoprecipitates polyclonal antibody to VEGI and subjected to Western analysis with monoclonal anti-VEGI-antibody 3-12D. The results of the inventors discovered a protein with a molecular mass of approximately 25 kDa (figure 9A). The appearance of the doublet cannot be easily explained, but may be the result of alternative glycosylation or other posttranslational modifications of recombinant peptide in transfected cells MW. Protein VEGI was not detected in media from nitrostilbene cells or cells carrying an empty vector pcDNA (figure 9A). VEGI-174could not be detected in the conditioned medium under the same experimental conditions (not shown). In a separate experiment, Western analysis of concentrated conditioned HUVE cells environment also revealed a band of similar molecular weight, in comparison with the band obtained with transfectants VEGI-251and air-conditioned HUVE cells also were immunoprecipitates Policl the national antibody (figure 9B). These observations indicate that VEGI-251is not membrane-bound protein, and is, on the contrary, secretively protein.

Example 22. Overexpression of VEGI-251in endothelial cells causes cell death

To study the biological activity of VEGI-251on endothelial cells was selected antivirusa the gene delivery system for transfection of cells HUVE expression constructs VEGI. Antivirusnuu delivery of genes were first examined with GFP-design and confirmed that more than 90% of HUVE cells could be transpulmonary (not shown). Observations of the authors of the invention with VEGI-174, VEGI-251and Sekretareva form of VEGI with the signal peptide of IL-6, sVEGI showed that only the secreted form of VEGI, including VEGI-251and sVEGI were cytotoxic against HUVE cells (figure 10A), whereas VEGI-174had no such action.

These results indicate that HUVE cells have membrane receptors for VEGI, which can be activated through an autocrine mechanism.

Example 23. Antitumor activity of VEGI-251

Previously it was shown that recombinant form of VEGI-174carrying secretory signal peptide, sVEGI, was effective at inhibiting the growth of tumors carcinoma of the colon MS in vivo (Zhai, et al. hit J Cancer 82, 131-136, 1999). Because natural VGI- 251is secretively protein, the inventors have determined whether he could also inhibit the growth of tumor xenografts in vivo. Selected fifteen lines stably transfected clones of MDA-MB-231 for each design with five lines transfected with the vector controls. Cells from each group were combined and were injected with in the fatty tissue of the breast females Nude naked mice. The volume of tumors was determined in dependence on the time after injection. Compared in this way cell culture VEGI-174-transfected and sVEGI-transfected clones. United transfetsirovannyh vector clones were used as controls. Nitrostilbene the original cells were also analyzed and it was found that they are identical with transfitsirovannykh vector clones.

The results of the inventors show that overexpression of full-VEGI-174cancer cells had little effect on tumor growth of xenografts (figure 10B). However, overexpression of intact VEGI-251and also fused protein sVEGI was significantly delayed tumor growth. These observations are in full agreement with effect lentiviruses transfection in vitro (figure 10A), confirming the biological activity of natural VEGI-251. Then, the inventors determined the effect is AirExpress full-VEGI- 251cancer cells for neovascularization of tumors. It was found that associated with tumors of the density of microvessels significantly reduced when the expression of VEGI-251. The degree of reduction was comparable with the degree of reduction in sverkhekspressiya sVEGI tumors. Because protein sVEGI consists of a secretory signal peptide of IL-6 and residues 23-174 VEGI-174these results show that the residues 23-174 contain biological equivalent native VEGI-251. Taken together, these findings demonstrate that the secretion of VEGI-251in the extracellular matrix is essential for its antitumor activity. In addition, like sVEGI, this antitumor activity of VEGI-251not due to a direct action on tumor cells, but rather with the fact that it prevents the development associated with tumor vascular network.

Example 24. Identification of potential isoforms VEGI

It is established that in the analysis of the Northern blot membrane of human tissues using cDNA probe VEGI numerous number of bands mRNA appears in various tissues (figure 11). Because the experimental conditions used in these experiments, is not conducive to unspecific binding of these probes, according to an estimate based on the approximate dimensions of mRNA molecules were present in at least three isoforms, soo is relevant to 7.5 TPN, 2,0 TPN and 1.5 TPN respectively. The different distribution of these isoforms in different tissues suggested that they play different physiological roles.

Example 25. Confirmation of new isoforms VEGI

Using the method of rapid amplification of cDNA ends (RACE) (commercially available from OriGene Technology, Rockville, MD) to search for the VEGI isoforms used a panel of cDNA libraries representing different human tissues. This panel contains the cDNA samples obtained from 24 individual human tissue, with adapter, legirovannym with the 5'-end of each cDNA molecule. For polymerase chain reaction (PCR) used gene-specific oligonucleotide primer (GSP), the corresponding part of the cDNA VEGI, and adultery primer (ADP) (figure 12). Two rounds of "nesting" (nested) PCR was performed using two pairs of oligonucleotide primers. After the first round of PCR (94°3 min, 4 cycle 94°C for 30 sec, 65°C for 30 sec, 72°C for 2 min; then 72°C for 6 min) adapternum primer (ADP1, 5'-CGGAATTCGT CACTCAGCG-3') (SEQ ID NO:8) and VEGI-gene-specific primer (GSP1, 5'-CCCGGATCCT ATAGTAAGAA GGCTCC-3') (SEQ ID NO:9), the reaction products were diluted 1:10 with water. Diluted PCR samples used in the second round of PCR (94°C for 3 min; 35 cycles of 94°C for 30 sec; 54°C for 30 sec; 72°C for 2 min; then 72°in the course is 6 min) with another adapternum primer (ADP2, 5'-AGCGCGTGAA TCAGATCG-3') (SEQ ID NO:10) and VEGI-gene-specific primer (GSP2, 5'-CGGTGGATCC CGAGTTTGTC TCACAACTG-3')(SEQ ID NO:11). The PCR products were fractionally agarose gel. Positive DNA fragments were cut from the gel, purified for sequence analysis. From various tissues received four PCR product with different lengths (figure 13). These PCR products were subjected to DNA sequencing, confirming that the nucleotide sequences of these PCR products differed from each other. These isoforms are named now VEGI-174, VEGI-192a, VEGI-192band VEGI-251in accordance with the number of amino acid residues in the proteins encoded by these cDNA molecules.

Example 26. Cloning of the full-size cDNA of these isoforms

Gene-specific primers designed in accordance with the results of sequencing of PCR products obtained from experiments with RACE, described above, used to repeat the second round of PCR to confirm the specificity of their sequences. Then peeled the RACE products were cloned in the plasmid pCR3.1 Invitrogen (San Diego, CA) to obtain samples of high-quality DNA for sequencing. Based on the presence of a stop codon and the initiating codon at the 5'-sequence VEGI-192aand VEGI-192bin the frame read-designed full-size cDNA molecule VEGI-192ausing two pairs great the Mer for "nesting" (nested) PCR: Vg3A: 5'-AATCTCACCT GTCTCTGCCT G-3' (SEQ ID NO:43) and Vg-3'-1: 5'-CTAAACCGTT GTCCCTGTGG-3' (SEQ ID NO:44); Vg3B: 5'-CCTGTAAAAA TGGTTATAGT AG-3' (SEQ ID NO:45) and VG-3'-2: 5'-GGTGGCAGAG GACTTTC-3' (SEQ ID NO:46). Full-size cDNA molecule VEGI-192bdesigned using primer VG4B 5'-CTCTACTTAC GCCAAGG-3' (SEQ ID NO:47) and primer JY2 5'-CCCGGATCCT ATAGTAAGAA GGCTCC-3' (SEQ ID NO:48). cDNA libraries from which these were identified isoforms, was used for PCR. As VEGI-192aand VEGI-192bcloned into pCR3.1 from Invitrogen (San Diego, CA). Because it failed to detect the initiating codon in reading frame in the 5'-sequence VEGI-251used a pair of gene-specific primers vg5B: 5'-CACCACATAC CTGCTTG-3' (SEQ ID NO:49) and vg161: 5'-GTGTAATCCA CCAAAGAG-3' (SEQ ID NO:12) to select full-size cDNA clone of VEGI-251 from running in the form of array panel of cDNA libraries from OriGene (Rockville, MD). cDNA sequence VEGI-192ashown in table 13.

Example 27. Antiangiogenic and anticancer activity of VEGI-251

Recombinant protein derived from a shortened form of VEGI-174consisting of residues 29-174, is a strong inhibitor of carcinogenesis (Zhai Y, et al., FASEB J., 13:181-189, 1999; Zhai Y, et al. Int.J.Cancer, 82:131-136, 1999). It is shown that VEGI no direct effect on the growth of cancer cells and the mechanism of action of VEGI in the inhibition of tumor growth is the inhibition of the formation of blood vessels in these tumors. Thus, the full-size cDNA VEGI-251was transfusional the cell line of breast W is Lesa human MDA-MB-231, then implanted in the fatty tissue of the breast females Nude naked mice to show that the product of this gene could inhibit tumor growth by these cells. To inhibit the growth of endothelial cells in the immediate vicinity of the cancer cells need to VEGI gene product was released to the outside of the transfected cells. It was shown that the gene product could be detected in conditioned media of transfected cells in culture and b) that these transfetsirovannyh cells could grow into tumors in naked mice.

The breast cancer cells MDA-MB-231 was transfusional either empty vector or cDNA full-VEGI-174or cDNA full-VEGI-251or fused gene, which protein VEGI was associated with a secretory signal peptide derived from interleukin-6 (TL-6/VEGI). In the analysis of Western blotting using monoclonal antibodies (13-2D) against VEGI VEGI gene product is251was detected in the conditioned media of transfected cells (figure 14). In contrast, the gene product VEGI-174were not detected in air-conditioned environments.

Stable transfetsirovannyh clones of MDA-MB-231 were injected with in the fatty tissue of the breast Nude females naked mice (1×106per injection). The sizes of tumors metering is whether as a function of time. Analyzed the density of microvessels in the tumor. Speakspace full-VEGI-174cancer cells had little effect on the growth of tumor xenografts, and overexpression of Sekretareva forms hypothetical extracellular domain VEGI and a full-length VEGI-251significantly inhibited tumor growth (figure 15). In addition, it was shown by carrying out immunohistochemical analysis of sections of tumors using monoclonal antibodies against VEGI that protein VEGI was available in tumors. Some of the tumors (G9-1R) had cancer cells which have produced a significant amount of VEGI-251while others had cancer cells which have produced little VEGI-251(G9-2R) compared with tumors formed by cells (G10-2R), transfitsirovannykh vector (figure 16). The more VEGI-251was producirovanie these tumors, the slower was the rate of growth of these tumors (figure 17). These results show that VEGI-251secreted transfitsirovannykh cancer cells and that the secretory VEGI-251is a strong inhibitor of tumor growth by inhibiting the growth of endothelial cells surrounded by tumors. In contrast, VEGI-m not secretarials these cells and, therefore, could not inhibit the growth of endothelial to ATOC.

Example 28. Tissue distribution of expression of VEGI

Unlike other members of the TNF family VEGI-174specifically expressed in endothelial cells. Northern-blot analysis of the total RNA preparations of the 23 cell lines and primary cell cultures showed that VEGI detected only in HUVE and venous endothelial cells (figure 18). VEGI-174was not observed in arterial endothelial cells. Using Northern blots of multiple tissues transcript VEGI-174was found in many adult tissues, including placenta, lung, skeletal muscle, kidney, pancreas, spleen, prostate, small intestine and colon (figure 18), which implies that the product of this gene may play a role in the normal functioning of the vascular network.

Example 29. Specific inhibition of endothelial cell growth by VEGI

A shortened version of VEGI-174received in accordance with the estimated extracellular domain consisting of residues 39-174. This protein is expressed in E. coli, purified to apparent homogeneity assessed by electrophoresis in LTO-polyacrylamide gel, and experienced in a variety of cellular assays. This truncated protein was able to specifically inhibit the proliferation of aortic endothelial the data cells (AVE) adult cows (figure 19). Almost complete inhibition of endothelial cell growth was observed when 10 μg/ml with the concentration value of premaxillae inhibition (IC50approximately 1 µg/ml (approximately 70 nm). In contrast, this protein did not affect the growth of cancer cells of human breast (MDA-MB-231 or MDA-MB-435) under the same experimental conditions (figure 19). VEGI not also inhibited cell proliferation T-cell human leukemia (Jurkat), lymphoma cells, human Burkitt's lymphoma (Raji)cells monocytic leukemia person (TPR-1) or cells of human promyelocytic leukemia (HL60), i.e. cells that are often sensitive to the cytotoxic activity of TNF. The effectiveness of VEGI was lower than the activity that would be expected from cytokine; however, it is comparable with the activity of known protein inhibitors of angiogenesis such as angiostatin, endostatin, and CD40 ligand (another member of the TNF family). The relatively low observed activity shortened VEGI may also be partly due to suboptimal website shortening or absence of post-translational modifications, such as glycosylation, as this recombinant protein expressed in E. coli.

Example 30. Increasing regulation (up-regulation) gene VEGI in the confluent endothelially cells

Specific against the AI endothelial cell inhibiting activity VEGI suggests, this protein acts as a negative regulator of angiogenesis. To show that the gene VEGI buck regulated (down-regulated) in proliferating endothelial cells, but polysoude regulated when these cells are at rest, investigated the expression of VEGI in cultured cells HUVE using Northern blotting (figure 20). Low levels of mRNA VEGI observed in svezhevyslannyh HUVE cells; however, the increase in the number of cells in these cultures the level of mRNA VEGI respectively increases and reaches a plateau after reaching confluently these cultures.

Example 31. Inhibition of education capillaroscopy tubes by endothelial cells on collagen gels

Antiangiogenic activity of shortened VEGI investigated using a model of angiogenesis in vitro (Montesano, R. and OOrci, L., Cell 42:469, 1985). Endothelial cells cultured on the surface of three-dimensional collagen gel form resting monolayer when the culture reaches confluently. However, after stimulation of the cells of the monolayer angiogenic factor, such as FGF-2, these cells invaziruyut gel and form capillaroscopy tubular structure in the gel. This formation of tubules may also be inhibited angiogenic factors. The degree of education of the tubes can be determined quantitatively using to the computer image analysis (Li, L. Y., Biochemistry 33:10999, 1994). Shortened VEGI-174was able to inhibit the formation of capillarity tubules cells AVAE (figure 21). The value of the IC50for inhibition was approximately 1 μg/ml, which is similar to the value observed for the inhibition of endothelial cell growth.

Example 32. Inhibition of growth of capillaries in collagen gels placed on chorioallantoic membrane of chicken embryo

Antiangiogenic activity of VEGI-174additionally demonstrated using a modified method on the chorioallantoic membrane of chicken embryo (CAM) (Nguyen, M. et al., Microvasc. Res. 42:31, 1994). This method is based on the growth of new capillary blood vessels in the tablet of the collagen gel, placed on HIMSELF. Blood vessels are stimulated to vertical growth polymerized collagen gel in the presence of angiogenic factors such as FGF-2 or VEGF, drenched in this gel. The inhibitor was included in these gels to determine its effect on the growth of capillaries. The degree of angiogenesis in the gel was evaluated using FITC-dextran, injectable into the bloodstream ITSELF. Recombinant VEGI-174(5,0 µg/ml) inhibited approximately 50% of the growth of new capillaries in collagen gels induced by FGF-2 (2.0 µg/ml) (figure 22).

Example 33. Inhibition of growth of tumor xenografts conheceram the cells SNO, sverkhekspressiya VEGI

Anticancer activity of VEGI is demonstrated using a model of tumor xenograft implantation in the fatty tissue of the breast females Nude naked mice. Secreterial form VEGI-174design by replacing the N-terminal and hypothetical transmembrane segments VEGI-174(residues 1-25) secretory signal peptide derived from interleukin-6 person. This secreterial design VEGI-174cloned in eukaryotic expressing vector, which was then transfusional in the cells of the Chinese hamster ovary (Cho). The expression of the corresponding construct was confirmed by Northern blot analysis. The secretion of the modified VEGI transfitsirovannykh cells was confirmed by the ability of the conditioned medium to inhibit the growth of cells in AWE. Then transfetsirovannyh cells SNO mixed with cancer cells human breast (MDA-MB-231 or MDA-MB-435), and these cell mixture were injected with in the fatty tissue of the breast naked mice. The growth of tumor xenografts were recorded after injection. Despite the high carcinogenesis used lines of breast cancer cells, was observed a marked inhibition of tumor growth of xenografts formed or cells MDA-MB-231 (figure 23A)or cells MDA-MB-435 (Fig whom RA 23C). When we repeat this experiment using cells MDA-MB-435 this coinjected has also led to a complete inhibition of tumor development. Transfetsirovannyh vector cells SNO had no effect on tumor growth in any case. Because VEGI not inhibited the growth of breast cancer cells in culture, this anticancer activity of this protein was the result of its antiangiogenic activity.

Example 34. Inhibition of cell proliferation in AWE VEGI-192a

Full-VEGI-192aexpressed in E. coli and was re-laid and purified using the method described in PCT application WO 01/55174. Specifically designed expression vector (PET11, Novagen)containing the polynucleotide insert, which encodes a full-sized polypeptide VEGI-192a. This expression vector was transformed into E. coli and transformed E. coli were grown for expression of the polypeptide VEGI-192a. The cells were collected and destroyed cells were isolated calf inclusions. OD280the solution containing these calf inclusions, brought to 5.0 to 8.0 M urea solution. The final solution contained the following restorative components: 10 mm beta-mercaptoethanol, 10 mm DTT (dithiothreitol), 1 mm restored glutathione (GSH), 0.1 mm oxidized glutathione (GSSG). The final pH of this solution was to 10.0. This solution was rapidly diluted in the 20 volumes of 20 mm Tris™ base, the pH is brought to 9.0 and then slowly brought up to 8.0 1 M HCl by bringing the pH to 8.8 for 24 hours, then to 8.6 within 24 hours, etc. until the pH became an 8.0. Alternatively, proteins can be re-laid using dialysis. OD2808 M urea solution was brought to 0.5 and were dialyzed against 20 volumes of Tris™-base. the pH of this solution was slowly brought again to 8.0. Then re-laid material was concentrated by ultrafiltration and separated by gel-filtration, for example, on a column with Sephacryl™ S-300 using 20 mm Tris™, HCl, 0.4 M urea, pH 8.0. Fractions with S-300 could be checked by electrophoresis in non-ordinator-PAG. Misfolded protein migrated with a very high molecular weight, whereas a properly laid proteins migrated with normal molecular weight.

Protein VEGI-192aof fractions with S-300 was tested for their ability to inhibit the growth of endothelial cells. Aortic endothelial cells of adult cows (AVE) were sown in triplicate at a concentration of 8000 cells per well in 24-well plates in full (Gibco, Gaithersburg, MD), 10% fetal calf serum. The environments were added FGF-2 (1 ng/ml). These cultures are maintained at 37°C, 5% CO2within 6 days. Then the cells were trypsinization and the number of cells was determined with the use of the cation counter of Coulter (Hialeah, FL).

As shown in figure 24, correctly laid 12A, but not misfolded VEGI-192aof fractions with S-300 was able to inhibit the growth of cells AVE. Almost complete inhibition of the growth of endothelial cells was achieved at 1000 ng/ml, and the concentration value of premaxillae inhibition (IC50) was approximately 10 ng/ml.

1. Polynucleotide encoding a polypeptide that inhibits the growth of vascular endothelial cells, with the specified polynucleotide contains (a) the nucleotide sequence of SEQ ID NO: 1; (b) the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4; or the complement of the specified polynucleotide, and with the specified polynucleotide optionally contains noncoding and/or coding nucleotide sequence for the expression, purification or detection of the polypeptide.

3. Polynucleotide according to claim 1, which essentially consists of the nucleotide sequence of SEQ ID NO: 1 or its complement.

4. Polynucleotide according to claim 1, which includes the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, or its complement.

5. Polynucleotide according to claim 1, which essentially consists of a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 4, or its complement.

6. Polynucleotide according to claim 1, which further comprises detektiruya tag.

7. Polynucleotide according to claim 1, which is immobilized on the surface.

8. Polynucleotide according to claim 1, which encodes a polypeptide having antiangiogenic biological activity.

9. The expression vector containing polynucleotide according to any one of claims 1 to 8.

10. A host cell E. coli containing the vector of claim 9.

11. The polypeptide that inhibits the growth of vascular endothelial cells, containing the amino acid sequence of SEQ ID NO: 4, and optionally the amino acid sequence for expression, purification or detection of the polypeptide.

12. The polypeptide according to item 11, which essentially consists of the sequence SEQ ID NO: 4.

13. The polypeptide according to item 11, which has the inhibitory angioprotective.

14. A protein that exhibits growth inhibitory vascular endothelial cells activity containing the amino acid sequence of SEQ ID NO: 4, fused with the amino acid sequence, which increases its function, determines its localization in the cell increases immunological reactivity, facilitates its binding to the substrate or carrier, or for easy cleaning.

15. Protein at 14, in which the amino acid sequence of SEQ ID NO: 4 is fused with the amino-acid sequence that encodes an epitope selected from the group consisting of the ICC, ON the happening of the hemagglutinin of influenza virus, His-6 and FLAG.

16. Polynucleotide encoding a protein according to any one of p and 15.

17. The expression vector containing polynucleotide on item 16.

18. Pharmaceutical composition for inhibiting angiogenesis, containing an effective amount of polynucleotide of claim 8 or polypeptide according to item 13 and a pharmaceutically acceptable filler.

19. The pharmaceutical composition according p containing an effective amount of the polypeptide according to item 13.

20. An antibody that selectively binds to the polypeptide according to claim 11, obtained by immunization of a mammal the polypeptide according to item 11.

21. The antibody according to claim 20, where the polypeptide essentially consists of the sequence SEQ ID NO: 4.

22. Therapeutic method ing is berbania angiogenesis, introducing the individual a composition containing a polypeptide essentially consisting of the amino acid sequence of SEQ ID NO: 4 in a pharmaceutically acceptable carrier in a dosage sufficient to inhibit angiogenesis.

23. therapeutic method according to item 22, in which the polypeptide consists of the amino acid sequence of SEQ ID NO: 4.

24. therapeutic method according to item 22, in which the individual is a person.

25. therapeutic method of suppressing tumor growth, providing an introduction to the individual a composition containing a polypeptide essentially consisting of the amino acid sequence of SEQ ID NO: 4 in a pharmaceutically acceptable carrier at a dose sufficient for suppression of tumor growth.

26. therapeutic method according A.25, in which the polypeptide consists of the amino acid sequence of SEQ ID NO: 4.

27. therapeutic method according A.25, in which the individual is a person.



 

Same patents:

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention relates to a plasmid vector able to autonomic replication in the strain Escherichia coli K-12 at any temperature. Plasmid comprises region or with mutation, gene rep from pMW119 and DNA fragment. DNA fragment can be integrated into chromosome of microorganism belonging to Escherichia coli W or Escherichia coli B as result of homologous recombination. Plasmid is fused into a microorganism belonging to Escherichia coli W or Escherichia coli B followed by preparing amino acid. Also, invention relates to strains Escherichia coli DH5α/pMTS11910 (FERM BP-6904) and Escherichia coli DH5α/pMTS11914 BP-6905) designated for storage the plasmid vector. Strains Escherichia coli WLA-131 (FERM BP-6902) and Escherichia coli WL-1133 (FERM BP-6903) are producers of amino acid leucine. Invention provides carrying out superexpression of gene and to obtain stable plasmids.

EFFECT: valuable biological properties of plasmids and strains.

13 cl, 2 dwg, 1 tbl, 5 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, immunology, molecular biology, pharmacy.

SUBSTANCE: invention describes variants of MCP-1-binding molecules. One of MCP-1-binding molecule comprises at least one variable region of immunoglobulin (VH) heavy chain comprising of hypervariable sites CDR1, CDR2 and CDR3 while other molecules comprises both light and heavy chains. Invention proposes DNA constructs encoding indicated MCP-1-binding molecules and expressing vector carrying at least one of these DNA constructs. Invention describes a method for preparing MCP-1-binding molecule. Invention discloses a method for treatment of disease or disorder mediated by MCP-1 or eotaxine-1 based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode. Invention describes a pharmaceutical composition based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode and used in treatment of disease or disorder mediated by MCP-1 or eotaxine-1 in a patient. MCP-1-binding molecules inhibit binding MCP-1 with its receptor. The full immobilized antibody is highly specific as far as it binds human recombinant MCP-1 with value KD = (43 ± 2.9) x 1012 and can be used in medicine.

EFFECT: valuable medicinal properties of antibodies, improved method of treatment.

13 cl, 5 dwg, 4 tbl, 2 ex

FIELD: biotechnology, genetic engineering, medicinal microbiology.

SUBSTANCE: invention proposes recombinant plasmid pAce90 expressing Vibrio cholerae cloned ace gene (accessory cholera enterotoxin) and E. coli strain M15[pREP4]pAce90 (KM 194) as a producer of accessory cholera enterotoxin carrying indicated plasmid. Plasmid is prepared by incorporation of Vibrio cholerae ace gene by sites of Bam HI - Hind III in polylinker of vector plasmid pQE30 in orientation providing the transcription direction under control of T5 promoter. The strain E. coli M15[pREP4]pAce90 (KM 194) is a producer of recombinant protein ace wherein this strain is prepared by transformation of the strain E. coli M15[pREP4] with recombinant plasmid pAce90. Production of ace protein occurs by induction with isopropyl-β-O-thiogalactoside with the yield 7-10% of cellular proteins. Using plasmid pAce90 and the strain E. coli (KM 194) provides accelerating the process for preparing Vibrio cholerae ace protein.

EFFECT: improved preparing method of protein ace, valuable properties of plasmid and strain.

3 cl, 2 dwg, 3 ex

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: invention describes variants of monoclonal antibodies showing specificity to TRAIL-receptor DR4. By one of variant antibodies are produced by hybridoma 2E12 recorded in ATCC at number PTA-3798. Each of antibody variants possesses apoptosis-inducing activity both in vivo - in the concentrations less 10 mg/kg in target cells expressing DR4 and in vitro - in the presence of a cross-linking agent in the concentrations less 1 mcg/ml in target cells. Invention discloses variants of methods for selective induction of apoptosis in cells expressing DR4, and variants of methods for inhibition of DR4-expressing cells based on using antibodies. Invention describes variants of compositions, methods for treatment of a patient suffering from inflammatory or autoimmune disease and methods in treatment of a patient suffering from malignant tumor wherein these compositions are based on antibody to DR4 for inducing apoptosis in cells expressing DR4. Also, invention discloses variants of nucleic acids, purified polypeptides, expression vectors and host-cells used in preparing antibody. Using the invention provides delaying tumor growth and decreasing case of its regression that can be used in tumor therapy.

EFFECT: improved and valuable properties of antibody.

103 cl, 169 dwg, 6 tbl, 30 ex

FIELD: biotechnology, immunology, biochemistry, medicine.

SUBSTANCE: invention proposes peptide concatemer inducing production of antibodies against apolipoprotein B-100 that inhibit lipase effect and inhibit binding LDL with LDL receptors. This concatemer consists of amino acid sequence of peptide repeating four times. Amino acid sequence is given in the invention description. Also, invention describes a concatemer-base vaccine used in treatment and prophylaxis of obesity and a method for preparing concatemer in E. coli cells using a vector. Invention discloses a polynucleotide encoding concatemer and expressing vector comprising the indicated polynucleotide. Using the invention provides inhibition of obesity.

EFFECT: valuable medicinal properties of concatemer and vaccine.

7 cl, 16 dwg, 1 tbl, 6 ex

FIELD: medicine, virology, molecular biology, genetic engineering.

SUBSTANCE: invention relates to nucleic acid constructions and to vaccine preparations containing such construction and to using such preparations in medicine. Nucleotide sequence that encodes HIV-1 gag protein or its fragment containing gag epitope and HIV-1 Nef protein or its fragment containing nef epitope, and RT protein or its fragment containing RT epitope is coupled operatively with heterologous promoter wherein gag sequence is optimized by cordons. This sequence is a component of pharmaceutical composition and used in treatment and/or prophylaxis of HIV-infection and AIDS.

EFFECT: valuable medicinal properties of constructions, protein and pharmaceutical composition.

15 cl, 50 dwg, 22 ex

FIELD: biotechnology, immunology, genetic engineering.

SUBSTANCE: invention relates to a variant of protein subtilysine from Bacillus amyloliquiefaciens with change Y217L and comprising T-cellular epitope. This T-cellular epitope of indicated variant comprises one or some amino acid changes chosen from group consisting of residues corresponding to positions: 76, 79 and 122 wherein indicated subtilysine variant has optionally change at one or some following positions: 3, 31, 40, 41, 46, 47, 48, 50, 76, 101, 104, 107, 111, 128, 147, 154, 181, 182, 183, 185, 206, 215, 218, 238, 247, 248, 250, 254, 258 and 262. Also, invention relates to DNA molecules that encode subtilysine variants, host-cells containing this DNA, and to compositions used in skin care and containing indicated subtilysine variants. Invention provides preparing subtilysine variants that elicit reduced immunogenic response in human as compared with the parent subtilysine.

EFFECT: improved and valuable immune properties of protein, improved preparing methods.

31 cl, 25 dwg, 3 tbl, 14 ex

FIELD: virology, immunology, medicine.

SUBSTANCE: invention relates to genomic RNA of JEV Korea isolate with nucleotide sequence of SEQ ID NO:15, as well as to infective cDNA obtained from genomic RNA useful in therapy, vaccination, and diagnosis. Also described are recombinant vector containing infective cDNA, method for production thereof, method for production of JEV infective RNA-transcript, synthetic JEV and vaccine against JEV.

EFFECT: new methods for medicine applications.

26 cl, 40 dwg, 5 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to polynucleotides encoding polypeptide modulating cancer cell proliferation, monoclonal antibody, expression vector as well as expression vector, host cell, pharmaceutical composition and pharmaceutical agent for modulating of cancer cell proliferation. Present invention is useful in determination of abnormal cell proliferation malignancy.

EFFECT: agent and method for effective cancer disease treatment.

12 cl, 6 dwg, 2 ex

FIELD: chemical and pharmaceutical industry.

SUBSTANCE: invention relates to lyophilized pharmaceutical immunocytokine composition including immunocytokine and containing as cytokine component interleukin-2 (IL-2), sugar or aminosugar, amino acid and surfactant, wherein said composition contains: immunocytokines from 0.1 to 25 mg/ml; sugar or aminosugar 1-200 mg/ml; amino acid 1-200 mMol/l; and surfactant 0.001-1 mass %.

EFFECT: composition for parantheral administering with prolonged storage time even at increased temperatures.

13 cl, 8 ex, 4 tbl, 2 dwg

FIELD: biotechnology, molecular biology, genetic engineering.

SUBSTANCE: method involves transformation of the strain yeast Pichia pastoris PS99 (his4 pep4::PHO85) with the constructed plasmid pPIC9HabIL-2 and preparing the strain Pichia pastoris PS107(pPIC9HabIL-2) that represents a producer of hybrid protein consisting of human plasma blood albumin and human IL-2. Invention provides preparing highly stable hybrid protein consisting of human plasma blood albumin and human IL-2 by simplified technology Invention can be used in preparing hybrid proteins preparations.

EFFECT: improved preparing method, valuable properties of strain and hybrid protein.

4 cl, 2 dwg, 5 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, immunology, biochemistry, medicine.

SUBSTANCE: invention proposes peptide concatemer inducing production of antibodies against apolipoprotein B-100 that inhibit lipase effect and inhibit binding LDL with LDL receptors. This concatemer consists of amino acid sequence of peptide repeating four times. Amino acid sequence is given in the invention description. Also, invention describes a concatemer-base vaccine used in treatment and prophylaxis of obesity and a method for preparing concatemer in E. coli cells using a vector. Invention discloses a polynucleotide encoding concatemer and expressing vector comprising the indicated polynucleotide. Using the invention provides inhibition of obesity.

EFFECT: valuable medicinal properties of concatemer and vaccine.

7 cl, 16 dwg, 1 tbl, 6 ex

FIELD: chemical-pharmaceutical industry, proteins.

SUBSTANCE: invention concerns to cytokine-containing fused proteins showing the enhanced therapeutic index, and methods for enhancing the therapeutic index of these fused proteins. Fused proteins are able for binding with cytokine receptors of more one type expressed on cells and to bind with cells of more one type also. Except for, fused proteins possess a half-time value in blood stream of a patient as compared with that of corresponding natural cytokines.

EFFECT: improved and valuable properties of cytokines.

21 cl, 3 tbl, 11 ex

FIELD: molecular biology, genetic engineering, biochemistry.

SUBSTANCE: invention relates to polynucleotides optimized for expression in plants and encoding processing enzymes. Polynucleotides encode mesophilic, thermophilic or hyperthermophilic enzymes that are activated under conditions suitable for interaction with a necessary substrate. Incorporation of these polynucleotides into the plant genome results to preparing "self-processing" transgenic plants wherein their parts, for example, grain, fruit or seed express one or more indicated enzymes and have the varied composition. Autoprocessing plants can be used, for example, for preparing foodstuffs eliciting improved taste.

EFFECT: improved and valuable biological properties of plants.

29 cl, 23 dwg, 6 tbl, 41 ex

FIELD: biology, in particular gene engineering, microbiological industry, production of new lactam antibiotics.

SUBSTANCE: constructed is recombinant plasmid of pVR1 expression containing DNA fragment which encodes functionally active hybrid protein (DAOcbd), comprising D-amino acid oxydase of Trigonopsis variabilis BKM Y-2601 strain and chitin-coupling domain of A1 Bacillus circulans domain. As a result of transformation of E.coli strain with disclosed recombinant plasmid and selection of transformed clones new Escherichia coli strain C 41 (DE3)/pVR1 as producer of DAOcbd providing high yield of reconbinant enzyme is obtained. Said active DAO in combination with chitin-coupling domain enables purification of target protein wherein in purification process immobilized enzyme form is obtained.

EFFECT: improved method for purification of target proteins.

3 cl, 3 dwg, 2 tbl, 5 ex

FIELD: medicine, biotechnology, pharmacy.

SUBSTANCE: invention relates to exchangers of ligand/receptor specificity delivering antibodies to receptors on pathogen. In particular, invention describes variants related to manufacturing and using exchangers of ligand/receptor specificity. Exchangers comprise at least one specificity domain containing ligand for receptor wherein ligand is not antibody or its part, and at least one antigenic domain combined with abovementioned specificity domain wherein antigenic domain comprises epitope of pathogen or toxin. Advantage of the invention involves enhanced specificity in delivery of drug.

EFFECT: improved and valuable properties of exchangers.

30 cl, 5 tbl, 6 ex

FIELD: medicine, oncology, peptides.

SUBSTANCE: invention relates to a chimeric protein used in treatment of malignant lymphomas. Invention proposes chimeric protein consisting of two peptides representing inhibitor of cyclin kinases as an active fragment p16INK4a with amino acid sequence 84-13 or 84-106 as a therapeutic agent and the second peptide VP22 with sequence 140-301 of virus herpes simplex as a transporting agent. Advantage of the invention involves the development of preparation of high effectiveness of penetration into cells and possessing cytostatic and cytotoxic effects.

EFFECT: valuable medicinal properties of chimeric protein.

1 dwg, 2 ex

FIELD: biotechnology, medicine.

SUBSTANCE: Pcup1-Aβ-SUS35MC hybrid gene is produced on the base of CUP1 promoter, sequence encoding of full-length human amyloid beta (Aβ) peptide, sequence encoding of M and C domains of Sup35 protein of Saccharomyces cerevisiae, and translation terminating sequence.

EFFECT: method for scale screening of Aβ-peptide aggregation regulating agents.

2 dwg

FIELD: biotechnology, gene engineering, medicine.

SUBSTANCE: claimed composition contains inert carrier, diluent, and antigen component in effective amount. As antigen component it contains hybrid proteins such as PEPHER2/neu.1-HSP70, PEPHER2/neu.2-HSP70, PEPHER2/neu.3-HSP70 or PERErbB1vIII-HSP70, or mixture thereof. Also disclosed are recombinant plasmid DNA encoding biosynthesis of hybrid proteins comprising in vaccine composition and method for production thereof by their gene expression in composition of recombinant proteins in E.coli followed by purification of target product.

EFFECT: new anti-tumor preparation.

6 cl, 1 dwg, 9 ex

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