The selected nucleic acid molecule encoding a tie-2 ligand of tie-2 ligand and method of production, plasmid (options) , antibody, conjugate, ligand body, pharmaceutical composition, method of stimulating neovascularization, the method of maintaining a cell, a method of identifying an antagonist of tie-2 receptor

 

The invention relates to biotechnology, in particular genetic engineering, and can be used for TIE-2 ligand. TIE-2 ligand is produced by culturing cells transformed by a vector containing a nucleotide sequence encoding a TIE-2 ligand. TIE-2 ligand or ligand of the body, including additional plot of immunoglobulin, in the form of pharmaceutical composition used to stimulate neovascularization in a mammal, as well as to maintain cells expressing TIE-2 receptor. The antibody specifically binding to TIE-2 ligand, obtained by immunization of an animal TIE-2 ligand. The invention allows the development of tools for the diagnosis and treatment of diseases affecting the endothelial cells. 12 N. and 7 C.p. f-crystals, 11 ill.

This international application claims the priority placed on the simultaneous consideration of applications U.S. No. 418595 filed April 6, 1995, No. 373579, filed January 17, 1995, No. 353503, filed December 9, 1994, No. 348492, filed December 2, 1994, no 330261, filed October 27, 1994, No. 319932, filed October 7, 1994, the contents of each of which are included here by reference. In the description there are links to various publications. Content the present invention relates to the field of genetic engineering and more specifically, the gene of the receptor tyrosinekinase and their cognate ligands, their embedding in recombinant DNA vectors, and to receive the encoded proteins in recipient strains of microorganisms and recipient eukaryotic cells. More specifically, the present invention relates to new ligands, known as TIE-2 ligands that bind the TIE-2 receptor, as well as to methods of producing these TIE-2 ligands. The present invention also proposed that the nucleic acid sequences encoding the TIE-2 ligands, and methods of obtaining nucleic acids encoding these TIE-2 ligands and their gene products. TIE-2 ligands, as well as encoding their nucleic acids may be useful in the diagnosis and treatment of certain diseases affecting the endothelial cells and the corresponding TIE receptors, such as neoplastic diseases, including tumor angiogenesis, treatment of wounds, thromboembolic diseases, atherosclerosis and inflammatory diseases. More generally, the biologically active TIE-2 ligands can be used for promotion of growth, survival and/or differentiation of cells expressing TIE-2 receptor. Biologically active TIE-2 ligand can be used for in vitro podderjanie, endothelial cells of the heart and blood vessels, epithelium of the crystalline lens and the epicardium of the heart. In another embodiment, such a ligand can be used to support cells that are designed for the expression of TIE-2 receptor. Next, TIE-2 ligands and their corresponding receptors can be used in analytical systems to identify agonists or antagonists of the TIE-2 receptor.

Background of the invention

The behavior of the cells responsible for the development, preservation and restoration of the differentiated cells and tissues is regulated largely at the expense of intercellular signals transmitted by growth factors and other ligands and their receptors. These receptors are located on the surface of the respective cells, and they bind peptides or polypeptides, known as growth factors, and other hormone-like ligands. The result of such interactions are rapid biochemical changes in the respective cells, as well as quick and long-term restructuring of the expression of cellular genes. Some receptors are associated with different cell surfaces, can associate specific growth factors.

Phosphorylation of tyrosines in proteins due to tyrosinekinase represents the present protein genes encode tyrosine kinase transmembrane receptor polypeptide growth factors and hormones, such as epidermal growth factor (EGF), insulin, insulin-like growth factor (IGF-1), growth factors derived from platelets (PDGF-A u-B) and fibroblast growth factors (FGFs). (Heldin et al., Cell Regulation, 1: 555-566 (1990); Ullrich et al., Cell, 61: 243-54 (1990)).

In each case, these growth factors appear to act by binding to the extracellular part of the corresponding receptors, leading to activation of the internal tyrosine kinase, which is present on cytoplasmically part of the receptor. The receptors of growth factors, endothelial cells are of particular interest due to the possible involvement of growth factors in a number of important physiological and pathological processes, such as vasculogenesis, angiogenesis, atherosclerosis, and inflammatory diseases (Folkman et al., Science, 235: 442-447 (1987)). In addition, some receptors of hematopoietic growth factors are tyrosine kinases; these include c-fms, which is the factor receptor 1, colony stimulating, Sherr et al., Cell 41: 665-676 (1985), u c-kit, the receptor for primitive hematopoietic growth factor, reported by Huang et al., Cell 63: 225-33 (1990).

The receptors tyrosinekinase divided into evolutionary subfamilies on the basis of the characteristic structures of their ectodomains (Ullrich. et al., Cell, 61: 243-54 (1990(division II), each of which contains repeated homologous sequences with a high content of cysteine in their extracellular domains. A separate area with a high content of cysteine is also found in the extracellular domains of eph-like kinase. Hirai et al., Science 238: 1717-1720 (1987); Lindberg et al., Mol. Cell. Biol., 10: 6316-24 (1990); Lhotak et al., Mol. Cell. Biol. 11: 2496-2502 (1991). PDGF receptors, and c-fms and c-kit receptors tyrosinekinase can be grouped in division III, while FGF receptors form a subclass IV. Typical members of both subclasses are extracellular folding fragments, stable nutrizyme disulfide bonds. Such so-called immunoglobulin (Ig)-like folds found in the proteins of immunoglobulin superfamily, which includes a wide range of other receptors on cell surfaces, with or associated with cells or soluble ligands. Williams et al., Ann. Rev. Immunol., 6: 381-405 (1988).

The receptors tyrosinekinase differ in their specificity and epinasty. Typically, the receptor tyrosinekinase are glycoproteins, which consist of (1) an extracellular domain capable of binding a specific factor (factors) growth; (2) a transmembrane domain, which is usually the alpha-helical cha is of the protein; (4) the tyrosine kinase domain, which is the enzymatic component of the receptor; and (5) carboxyterminal tail, which in many receptors involved in recognition and binding of substrates for tyrosine kinase.

It is reported that processes such as alternative exon splicing and alternative selection gene promoter or polyadenylation sites can lead to the formation of several different polypeptides from a single gene. Such polypeptides may contain (or may not) contain various domains listed above. As a consequence, some extracellular domains can be expressed as a separate secreted proteins, and some forms of the receptor may not contain a tyrosine kinase domain, and contains only the extracellular domain embedded in the plasma membrane through the transmembrane domain plus short carboxyterminal tail.

The gene encoding endothelial cell transmembrane tyrosinekinase, initially identified through RT-PCR as an unknown homologous tyrosinekinase cDNA fragment from cell leukemia person described Partanen et al., Proc. Natl Acad. Sci. USA, 87: 8913-8917 (1990). This gene and encoded them a protein called "tie" that is what camping, that tie mRNA is present in all tissues of the human fetus and mouse embryo. After studying tie mediator was localized in the cells of the heart and vascular endothelial cells. tie mRNA were localized in the endothelium of blood vessels and the endocardium of mouse embryos 9.5 to 18.5 day of their existence. Was shown reinforced tie expression during neovascularization associated with the development of the ovarian follicle and granulation tissue in skin wounds. Korhonen et al., Blood 80: 2548-2555 (1992). Thus, it is assumed that the tie plays a role in angiogenesis, which is important for the development of therapies for solid tumors and other diseases dependent on angiogenesis, such as diabetic retinopathy, psoriasis, atherosclerosis and arthritis.

Two structurally related protein TIE receptor in rats, as reported, are encoded by different genes with similar expression profiles. One gene, called tie-1 is a mouse homologue tie man. Maisonpierre et al., Oncogene 8: 1631-1637 (1993). Another gene, tie-2, may be the mouse homologue of murine tek gene, which, like a tie, as was reported to be expressed in mice exclusively in endothelial cells and their presumptive precursors. Dumout et al., Oncogene 8: 1293-1301 (1993).

Ccana. Significant levels of tie-2 transcripts are also present in other populations of embryonic cells, including the epithelium of the crystalline lens, the epicardium of the heart and parts of the mesenchyme. Maisonpierre et al., Oncogene 8: 1631-1637 (1993).

Preferential expression of the TIE receptor in the vascular endothelium suggests that TIE plays a role in the development and preservation of the vascular system. This includes roles in determination, differentiation and proliferation of endothelial cells, migration of cells and copy elements vessels. It was reported that analysis of mouse embryos with a deficit of TIE-2 showed that the TIE-2 are important for angiogenesis, especially for the formation of a network of vessels in the endothelial cells. Sato, T. N. et al., Nature 376: 70-74 (1994). In the Mature vascular system TIE can function as a survival factor, preservation of endothelial cells and reactions on the impact of pathogens.

Summary of invention

In the present invention proposed a composition comprising a TIE-2 ligand, practically free of other proteins. The present invention also proposed an isolated nucleic acid molecule encoding a TIE-2 ligand. The selected nucleic acid may be DNA, cDNA or RNA. In the present invention is a vector comprising videoready in suitable host cells of polypeptide, with the activity of TIE-2 ligand. Cells are suitable hosts can be bacterial, yeast, insect cells or mammalian. In the present invention proposed a method of obtaining a polypeptide having the biological activity of TIE-2 ligand, which comprises growing cells of the host system is a vector in the conditions for production of the polypeptide and the allocation thus obtained polypeptide.

The present invention, which describes an isolated nucleic acid molecule encoding a TIE-2 ligand, further proposed for the development of ligand fragment, or derivative of it, or another molecule that is an agonist or antagonist of the receptor as a therapeutic agent for treating patients suffering from disorders, including cells, tissues or organs that Express a TIE receptor. The present invention also proposed an antibody that specifically binds to such a therapeutic molecule. This antibody can be monoclonal or polyclonal. In the present invention a method of use of such monoclonal or polyclonal antibodies to determine the number of therapeutic molecules in the image is about, which specifically binds TIE-2 ligand. This antibody can be monoclonal or polyclonal. Thus, the present invention also provides therapeutic compositions comprising an antibody that specifically binds TIE-2 ligand in a pharmaceutically acceptable carrier. In the present invention a method of blocking blood vessel growth in a mammal by introducing an effective amount of a therapeutic composition comprising an antibody that specifically binds TIE-2 ligand in a pharmaceutically acceptable carrier.

In the present invention the following therapeutic composition that includes TIE-2 ligand in a pharmaceutically acceptable carrier. In the present invention a method of promotion of neovascularization in a patient by introducing him an effective amount of a therapeutic composition comprising a TIE-2 ligand in a pharmaceutically acceptable carrier. In one embodiment, the method can be used for promotion of wound healing. In another embodiment, the method can be used to treat ischemia.

In another embodiment, the present invention proposes, in which the TIE-2 ligand can be anywhereman with CIT is sobienie also proposed that the receptor body, which specifically binds TIE-2 ligand. In the present invention proposed a therapeutic composition comprising the receptor body which specifically binds TIE-2 ligand in a pharmaceutically acceptable carrier. A method of blocking blood vessel growth in a mammal by introducing an effective amount of a therapeutic composition comprising the receptor body which specifically binds TIE-2 ligand in a pharmaceutically acceptable carrier.

The present invention also proposed antagonist of TIE-2 receptor, as well as a way of inhibiting the biological activity of TIE-2 ligand in a mammal, comprising administration to the mammal of an effective amount of a TIE-2 antagonist. In accordance with the invention, the antagonist may be an antibody or other molecule capable of specific binding or TIE-2 ligand, or TIE-2 receptor. For example, the antagonist can be TIE-2 receptor body.

Brief description of drawings

Fig.1A and 1B: TIE-2 receptor body (TIE-2 PB) inhibits the development of blood vessels in chorioallantoic membrane chicken (HIMSELF). A separate piece of absorbing gel foam (Gelfoam) dipped 6 µg PBS, injected immediately under HIMSELF dnout and explore. Fig.1A: the embryos treated ENK-1 RB /Genk-1 ecto/h IgG1 Fc/ was viable and had normal developed blood vessels in surrounding HIMSELF. Fig.1B: all embryos treated TIE-2 RB (r, TIE-2 ecto/h IgG1 Fc) died, a decrease in size, and were almost completely devoid of surrounding blood vessels.

Fig.2: Vector pJFE14.

Fig.3: Restriction mapgt10.

Fig.4: nucleic acid Sequence and the selected amino acid (single letter code) of human TIE-2 ligand from clonegt10 encoding htie-2 ligand 1.

Fig.5: nucleic acid Sequence and the selected amino acid (single letter code) of human TIE-2 ligand from T98G clone.

Fig.6: nucleic acid Sequence and the selected amino acid (single letter code) of human TIE-2 ligand from the clone pBluescript KS encoding human TIE-2 ligand 2.

Fig.7: Results of the Western blot showing activation of TIE-2 receptor, due to the TIE-2 ligand 1 (L1 band), but not at the expense of TIE-2 ligand 2 (lane 2), control (Mosk.) activation is also absent.

Fig.8: Results of Western blotting showing that pre-treatment of NAES cells excess TIE-2 ligand 2 (paavani with the what happens when pre-processing NAES cells MOCK medium (lane 1).

Fig.9: Histogram representation of binding to the immobilized surface rat TIE2 IgG TIE2 ligand in CS ras, Rat2 ras, SHEP and T98G concentrated (10x) air-conditioned environment. Specific binding of rat TIE2 (GTE) demonstrates a significant reduction in binding activity in the presence of 25 μg/ml of soluble TIE2 RB compared to a smaller decrease in the presence of soluble trkB RB.

Fig.10: the Binding of recombinant human TIE-2 ligand 1 (hTL1) and human TIE-2 ligand 2 (hTL2) in cos cell supernatant fluids with immobilized surface of human TIE-2 RB. Specific binding of human TIE-2 define incubare samples at 25 µg/ml) or soluble human TIE2 RB or trkB RB; a significant reduction in binding activity is observed not only for the samples incubated with TIE2 RB man.

Fig.11: Results of the Western blot demonstrating that the TIE-2 receptor body (indicated by TIE-2 RB, or as here, TIE2-Fc) blocks the activation of TIE-2 receptors due to the TIE-2 ligand 1 (TL1) in HUVEC cells, whereas an unrelated receptor body (TRKB-Fc) does not block this activation.

Detailed opinione new ligand, which binds TIE-2 receptor. The present invention includes a TIE-2 ligand, and its amino acid sequence and also functionally equivalent molecules in which amino acid residues substituted for residues within the sequence resulting in a silent change. For example, one or more of amino acid residues in the sequence can be replaced with another amino acid (amino acids) is similar polarity, which acts as a functional equivalent, and get silent replacement. The substituents of the amino acids in the sequence can be selected from other members of the class to which belongs this amino acid. For example, the class of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, Proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include aspartic acid and glutamic acid. In the scope of the present invention also includes proteins, or fragments thereof, or derivatives that exhibit the same or similar biological activity, and derivatives, which difference is deplane, binding with the antibody molecule or other cellular ligand, etc.,

The present invention also includes a nucleotide sequence that encodes the protein described here as a TIE-2 ligand 1, and cells that are genetically engineered to produce the protein, for example, by transfection, transduction, infection, electroporation, or microinjection of nucleic acid encoding a TIE-2 ligand 1, described herein, in a suitable expression vector.

Further, the present invention covers nucleotide sequence encoding the protein described here as a TIE-2 ligand 2, as well as cells that are genetically engineered to produce the protein, for example, by transfection, transduction, infection, electroporation, or microinjection of nucleic acid encoding a TIE-2 ligand 2, described here, in a suitable expression vector.

The specialist should be clear that the present invention encompasses DNA and RNA sequences that hybridize to the selected sequence encoding TIE-2 ligand, under conditions of moderate stringency, as defined, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ed., vol.1, pp. 101-104, C molecule, having a sequence selected from the amino acid sequence of TIE-2 ligand, obtained as described previously, and the molecule having the sequence of nucleic acids, which hybridizes with such a sequence of nucleic acids, as well as the sequence of nucleic acid which is degenerate in respect of the above sequences as a result of the genetic code, but which encodes a ligand that binds TIE-2 receptor.

Any of the methods known in the art for embedding the DNA fragments into a vector may be used to construct an expression vector encoding a TIE-2 ligand through the use of appropriate control signals to the transcription/translation and protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination). The expression of the nucleic acid sequences encoding a TIE-2 ligand or peptide fragment may be regulated by a second nucleic acid sequences such that the protein or peptide is expressed in a host transformed by a recombinant DNA moluccanum element, well-known specialists. Promoters that can be used to control expression of the ligand include (but are not limited to) long terminal repeat as described y Squinto et al. /Cell 65: 1-20 (1991)/; SV40 early promoter site (Bernoist and Chambon, Nature 290: 304-310), the CMV promoter, the M-MuLV 5’ terminal repeat the promoter contained in the 3’ long terminal repeat sarcoma virus Payca (Yamamoto et al., Cell 22: 787-797 (1980)), the promoter of the gene timedancing herpes virus (Wagner et al., Proc. Natl Acad. Sci. USA 78: 144-1445 (1981)), the promoter of adenovirus, the regulatory sequences of the gene of metallothionein (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression vectors, as a promoter-lactamases (Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)); see also "Useful proteins from recombinant bacteria" in Scientific American, 242: 74-94 (1980); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADH (alcoholdehydrogenase) promoter, RDK (fosfoglitserin-kinata) promoter, alkaline phosphatase promoter, and the following sections transcriptional control of animals that exhibit tissue specificity and have been utilized in transgenic animals: control plot elastase gene 1, which is active in acinar to the 1987); control plot of the insulin gene, which is active in the beta cells of the pancreas (Hanahan, Nature 315: 115-122 (1985)), the control section immunoglobulin gene, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), the control section virus mouse mammary tumor that is active in cells of the ovaries, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45: 485-495), the control section albumin gene, which is active in liver (Pinkert et al., 1987, Genes and Devel 1: 268-276), the control portion of a gene of alpha-fetoprotein, which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); control plot of the gene alpha 1-antitrypsin deficiency, which is active in liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), control plot beta-globin gene, which is active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); the control portion of a gene of the basic protein of myelin, which is active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48: 703-712); control gene light chain 2 myosin, which is active in skeletal muscle (Shani, 1985, Nature 314: 283-286), and the control portion of a gene hormone releasing hormone that is active in the hypothalamus (Mason et al., 198 the ski to gibridizatsiya with RNA sequence encoding TIE-2 ligand to modulate its expression (Ecker, U.S. patent No. 5166195, issued November 24, 1992).

Thus, in accordance with the present invention, the expression vectors capable of replication in a bacterial or eukaryotic host containing a nucleic acid encoding a TIE-2 ligand, as he is here disclosed, used for transfection of the host and due to this direct expression of such nucleic acid to produce a TIE-2 ligand, which can then be identified in a biologically active form.

In the sense, as used herein, biologically active form includes a form that can be contacted with the TIE-2 receptor and cause such biological response as differencirovannoe operation or effect on the phenotype of cells expressing this receptor. Such biologically active form, for example, include the phosphorylation of the tyrosine kinase domain of the receptor TIE-2.

The expression vectors containing the gene insert can be identified in the four General approaches: (a) DNA-DNA hybridization, (b) in the presence or absence of "marker" gene functions, and (C) on the expression of the built-in sequences and (d) determining, using R. In the first approach, the presence of alien the surrounding sequence, which homologous gene, codereuse built-in TIE-2 ligand. In the second approach, the system of the recombinant vector/host can be identified and selected on the basis of the presence or absence of certain "marker" gene functions (e.g., activity timedancing, resistance to antibiotics, according to phenotype transformation, the formation of occlusion bodies in baculoviruses and so on) caused by embedding in a vector of foreign genes. For example, if encoding TIE-2 ligand nucleic acid is integrated into the sequence of the marker gene in the vector, recombinants containing the insert can be identified by the absence of the function of the marker gene. In the third approach, recombinant expression vectors can be identified by analyzing the foreign gene product expressed by the recombinant. Such analysis can be based, for example, on the physical or functional properties of the gene TIE-2 product, for example, by binding of the ligand to the TIE-2 receptor, or part thereof, which may be associated, for example, with detektivami antibody, or part thereof, or by binding with antibodies generated against a protein TIE-2 ligand or its parts.

Cells of the present invention is described. In the fourth approach, you can get a DNA nucleotide primers corresponding to the tie-2-specific DNA sequence. These primers can then be used in PCR tie-2 gene fragments (PCR Protocols: A Guide To Methods and Applications, Edited by Michael A. Innis et al., Academic Press (1990)).

Recombinant ligand can be distinguished in any way that provides the subsequent formation of a stable, biologically active protein. So, for example (but not from the point of view of constraints), the ligand can be isolated from cells or as soluble proteins or as the body of inclusion, of which the ligands can be quantitatively extracted with 8M of guanidinylation and dialysis. For further purification of the ligand can use conventional ion-exchange chromatography, chromatography hydrophobic interaction chromatography with reversed phase or gelfiltration.

In additional embodiments of the invention recombinant TIE-2 ligand encoding gene can be used to inactivate or "knockout" endogenous genes through homologous recombination, and thereby create cells with deficiency of TIE-2 ligand, tissue or animal. So, for example, (and not for limitation, recombinant TIE-2 ligand encoding gene can be constructed so that it th gene. This design is under the control of a suitable promoter can be embedded in these cells as stem cells of the embryo, through methods such as transfection, transduction or injections. Cells containing this construct can then be selected by resistance to G418. Cells lacking an intact gene encoding TIE-2 ligand can then be identified, for example by southern blotting, PCR determination, Northern blot or analysis of gene. Cells lacking an intact gene encoding TIE-2 ligand can then be drained from the cells of the embryo at an early stage to generate transgenic animals with deficiency of such a ligand. Such animals can be used to determine specific in vivo processes, which are usually dependent on the ligand.

The present invention also provides antibodies against TIE-2 ligands described herein that can be used for detection of ligands, for example, for diagnostic purposes. To obtain monoclonal antibodies directed to the TIE-2 ligand, it is possible to use any techniques that can provide antibody molecules with continuous cultivation of cell lines. For example, in the scope of the invention includes a method of spacecraft using a hybrid In-human cells (Kozbor et al., 1983, Immunology Today 4: 72), and methods of EBV-hybrid to obtain monoclonal human antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp.77-96), and the like.

Monoclonal antibodies can be monoclonal antibodies, human or chimeric human-mouse (or other species) monoclonal antibodies. Monoclonal human antibodies can be obtained by any of numerous methods known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80: 7308-7312; Kozbor et al., 1983, Immunology Today 4: 72-79; Olsson et al., 1982, Meth. Enzymol. 92: 3-16). Chimeric antibody molecules can be obtained so that they contained the murine antigen-binding domain with constant human areas (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 6851; Takeda et al., 1985, Nature 314: 452).

Various well-known specialists procedures can be used to obtain polyclonal antibodies to epitopes described herein TIE-2 ligands. To obtain antibodies can be immunizati different host animals by injecting the TIE-2 ligand or its fragment or derivative, such as (but not limited to rabbits, mice and rats.

To enhance the immunological response, you can use various adjuvants, depending on the host species, which include (but are not granberget-active substances, as lysolecithin, planovye polyols, polyanion, peptides, oil emulsions, hemocyanine lymph snails, dinitrophenol, and such potentially useful human adjuvants, as BCG (Bacille Calmette-Guerin) u Corynebacterium paryum.

Molecular clones of antibodies to selected epitopes TIE-2 ligand can be obtained by known methods. Recombinant methods (see, for example, Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Sprind Harbor Laboratory, Cold Sprind Harbor, New York) can be used to construct sequences of nucleic acids that encode molecules of monoclonal antibodies or their antigen binding sites.

In the present invention proposed molecule antibodies, as well as fragments of such antibody molecules. Antibody fragments which contain the idiotype of the molecule, can be created by known methods. For example, such fragments include, but are not limited to F(AB’)2the fragment, which can be obtained by pepsin digestion of the antibody molecules; the Fab’fragments which can be obtained by restoring the disulfide bridges of F(ab’)2fragment; and Fab fragments, which can be obtained by treating the antibody molecules with papain and regenerating agent. The antibody molecules can be distinguished by known methods, HPLC (high performance liquid chromatography), or their combinations.

The present invention also includes an immunoassay to determine the amount of TIE-2 ligand in a biological sample by:

a) implementation of contacting a biological sample with at least one antibody which specifically binds TIE-2 ligand so that this antibody form a complex with any TIE-2 ligand present in the sample;

b) determining the amount of the complex, thereby determining the number of TIE-2 ligand in a biological sample.

Further, the present invention encompasses the analysis to determine the number of TIE-2 receptors in biological samples by:

a) implementation of contacting a biological sample with at least one ligand of the invention so that this ligand to form a complex with the TIE-2 receptor,

b) determining the number of complexes and thereby determining the number of TIE-2 receptor in a biological sample.

In the present invention proposed the use of TIE-2 ligand to maintain survival and/or growth and/or differentiation of cells expressing TIE-2 receptor. Thus, the ligand can be used as a Supplement to maintain, for example, culture endotine systems analysis, suitable for identification of agonists or antagonists of the TIE-2 receptor. Such analytical systems may be useful in identifying molecules that can promote the inhibition of angiogenesis. For example, in one embodiment, antagonists of the TIE-2 receptor can be identified as a test molecule, which is able to interfere in the interaction of TIE-2 receptor with a biologically active TIE-2 ligand. Such antagonists are identified by their ability to 1) block the binding of biologically active TIE-2 ligand to the receptor, when measuring, for example, using BlAcore biosensor technology (BlAcore; Pharmacia Biosensor, Piscataway, NY), or 2) block the ability of biologically active TIE-2 ligand to cause a biological reaction. These biological responses include, but are not limited to) the phosphorylation of TIE-2 receptor, or that are located in the forward direction of the circuit components transduction TIE-2 signal, or the survival, growth or differentiatio containing TIE-2 cells.

In one embodiment, cells engineered to Express the TIE-2 receptor, could depend on the growth of adding TIE-2 ligand. These cells provide a suitable analytical system for the identification of additional am version autocrine cells, designed so that they are able to joint expression and TIE-2 ligand and receptor may represent a suitable system for the analysis of potential agonists or antagonists.

Therefore, the present invention can be used for the introduction of the TIE-2 receptor in cells that do not normally Express this receptor, thereby allowing these cells to demonstrate a strong and easily visible reaction to the ligand that binds the receptor. The type of reaction depends on the used cells, and not on a specific receptor entered in these cells. Appropriate cell lines can be chosen in such a way as to obtain a reaction representing the value of the tests and to detect molecules that can act on a receptor tyrosine kinase. These molecules may be molecules of any type, including (but not limited to) peptides ones and molecules that will act in the described systems, the receptor-specific manner.

One of the most suitable to use systems is a system, which includes the introduction of the TIE-2 receptor in a cell line of fibroblasts (for example, NIH3T3 cells) so that this receptor, which is not usually the and various well-known methods to quantify effects of fibroblast growth factors (for example, the incorporation of thymidine or other types proliferation analyses; see van Zoelen, 1990, "The Use of Biological Assays For Detection Of Polypeptide Growth Factors" in Progress Factor Research, Vol.2, pp.131-152; Zhan and M. Goldfarb, 1986, Mol. Cell. Biol., Vol.6, pp.3541-3544). These analyses differ in the additional advantage that any products can be analyzed on a cell line that has entered the receptor, and as a parent cell line in which this receptor is absent; only specific effects on cell lines with the receptor should be considered as managed due to the launch of the receptor. Such cells can be constructed further so that they are expressed TIE-2 ligand, thus creating an autocrine system suitable for the analysis of molecules that act as antagonists/agonists of this interaction.

Thus, in the present invention proposed cell hosts containing nucleic acid encoding a TIE-2 ligand and a nucleic acid encoding a TIE-2 receptor.

The interaction of TIE-2 receptor/TIE-2 ligand is also useful system for identifying small molecule agonists or antagonists of the TIE-2 receptor. So, for example, fragments, mutants or derivatives of TIE-2 ligand can be identified, which Svyazinvest to define the active portion of the molecule. Further, identification of the ligand allows you to define a crystalline structure (using x-rays) of the complex of receptor/ligand, thereby allowing to determine the binding site on the receptor. Knowledge of the binding site to allow rational design of new agonists and antagonists.

Specific binding of the test molecule with the TIE-2 receptor can be determined in several ways. For example, the actual binding of the test molecule with cells expressing tie-2, it is possible to define or measure, determining or measuring (i) the test molecule associated with the surface of intact cells, (ii) the test molecule, made with TIE-2 protein in cell lysates, or (iii) the test molecules associated with TIE-2 in vitro. Specific interaction between the test molecule and TIE-2 can be estimated using reagents that exhibit unique properties such interaction.

As a specific, limitiruyuschie example methods of the present invention can be used in the following way. Consider the case in which you want to define the content of the TIE-2 ligand in the sample. Different dilutions of the sample (test molecules) in parallel with a negative control (NC), not containing otati cells, which Express tie-2 in the presence of detected labeled TIE-2 ligand (in this example radioiodinated ligand). The number of TIE-2 ligand in the test sample can be estimated by defining a number of125I-labeled TIE-2 ligand, which binds to the controls, and in each of the dilutions, and then compare the obtained values for samples from the calibration curve. The more TIE-2 ligand contained in the sample, the less125I-ligand is associated with the TIE-2.

The number of125I-ligand associated with TIE-2, can be determined by measuring the amount of radioactivity per cell, or by binding TIE-2 ligand from the cell surface proteins using DSS, by way Meakin and Shooter, 1991, Neuron 6: 153-163, and determining the amount of labeled protein in cell extracts, using, for example, polyacrylamide gel electrophoresis, which helps to identify the labeled protein with a size corresponding TIE-2 ligand/TIE-2 receptor. The specific interaction between the test molecule/TIE-2 can be further tested by analysis of various diluting its control ligand that does not bind the TIE-2 receptor, and therefore no significant impact on competition between labeled TIE-2 ligand and testowanie TIE-2 ligand/TIE-2, for example, such as (but not limited to, anti-TIE-2 antibody, or TIE-2 receptor body, as they are described here, as you might expect, will participate in the competition between125I-TIE-2 ligand and a test molecule for binding the TIE-2 receptor.

Detektirano labeled TIE-2 ligands include, but are not limited to, TIE-2 ligand, covalently linked or ecovalence associated with a radioactive substance, a fluorescent substance, a substance which has an enzymatic activity, a substance that can serve as a substrate for the enzyme (preferred enzymes and substrates associated with colorimetrically detectivesyme reactions), or a substance that can be recognized by the antibody molecules, i.e. detektirano labeled molecules antibodies.

In another embodiment, the specific binding of the test molecule with TIE-2 can be measured by evaluating the secondary biological effects bind the TIE-2 ligand/TIE-2 receptor, including (but not limited to, cell growth, and/or differentiatio, or immediate early gene expression or phosphorylation of TIE-2. For example, the ability of test molecules to induce differentiatio can be tested in cells in which there is no e in comparative cells, which do not contain tie-2, will serve as an indicator of the specific interaction of test molecules with TIE-2. Similar analyses can be performed by detecting induce immediate early genes (e.g., fos and jun) ti-2-minus and tie-2-plus cells or due to the detection of the phosphorylation of TIE-2, using standard analysis of phosphorylation, known to specialists. Such analyses can be useful for the identification of agonists or antagonists that competitive contact TIE-2.

Similarly, in the present invention, a method of identifying molecules that have biological activity of TIE-2 ligand comprising (i) exposing cells which Express tie-2, test molecules, and (ii) detecting specific binding of the test molecule with the TIE-2 receptor, where the specific binding to TIE-2 positively correlated with the TIE-2 activity. Specific binding can be determined, either directly analyzing the binding or secondary biological effects of binding, as discussed previously. This method may be particularly useful in identifying new members of the family TIE ligands or in the pharmaceutical industry for skinlogics activity. In a preferred, specific, limitiruyushchem variant of the invention it is possible to prepare a large plate culture of cells that contain alternating rows RS (or fibroblasts) cells, which are either tie-2 or less, or designed to be tie-2-plus. Then you can add a test molecule so that each column of the plate or part would contain a test molecule. Then each to assess the presence or absence of growth and/or differentiation. So you can skanirovat a very large number of test molecules for their activity.

In additional embodiments, the present invention proposed methods for determining or measuring the TIE-like activity, or identification of molecules with such activity, including: (i) exposure of the test molecule to the protein TIE-2 receptor in vitro under conditions that allow binding; and (ii) detecting binding of the test molecule with TIE-2 protein when binding of the test molecule with TIE-2 correlates with TIE-like activity. In accordance with such methods TIE-2 may or may not be substantially purified, may be associated with a solid substrate (e.g., affinity column or as the be any known in the art. In preferred embodiments, the binding of the test molecule can be detected or measured by evaluating its ability to compete with detektirano labeled known TIE-2 ligands for TIE-2 receptor binding.

The present invention also provides a method for detecting ability of test molecules to function as an antagonist of TIE-like activity, comprising detecting the ability of a molecule to inhibit the effect of the binding TIE ligand with TIE-2 cells, which Express tie-2. Such antagonist may (or may not) interfere with the binding to TIE-2 ligand with TIE-2 receptor. The effects of binding of the TIE-2 ligand with TIE-2 receptor are preferably biological or biochemical effects, including (but not limited to survival or cell proliferation, transformation of cells, the induction of immediate early gene, or the proliferation of TIE-2.

Further, the present invention offers a method for identifying antibodies or other molecules that can neutralize the ligand or to block its binding to the receptor, as well as molecules identified in this way. As a non-limiting example of this method can be carried out in the analysis, which is an example with multiple-cell plastic plate. The control can then be entered in the cell known amount of a TIE ligand, which was Myc-tagged, and then any labeled TIE ligand that binds the receptor body, can be identified using a reporter antibody directed against the Myc-tag. Then this analytical system can be used for screening of test samples for molecules that can: (i) contacting the labeled ligand or (ii) contacting the receptor body, and block binding of the receptor body at the expense of the labeled ligand. For example, a test sample containing presumably representing the interest of the molecule, together with a known amount of labeled ligand can be introduced into the cell and to determine the amount of labeled ligand that binds with the receptor body. By comparing the amount of bound labeled ligand in the test sample with the amount in the control, you can define samples containing molecules that can block the binding of the ligand to the receptor. Interest molecules, defined this way, it is possible to allocate, using the well-known specialists ways.

Once you have defined the blocker binding ligand, special the p receptor, or it binds with the ligand, as well as how to carry out analysis to determine whether a molecule blocker to neutralize the biological activity of the ligand. So, for example, using the analysis of binding due to the BIAcore biosensor technology (or its equivalent) in which either TIE the body of the receptor, or TIE ligand covalently linked to a solid substrate (for example, carboxymethylcysteine or gold surface), the specialist can determine whether the associated molecule blocker specifically with the ligand or with the body of the receptor. To determine whether a molecule blocker to neutralize the biological activity of the ligand, the specialist can carry out the analysis using phosphorylation (see example 5), or, in another embodiment, due to a functional bioanalysis, as survival analysis, at the expense of primary cultures, such as endothelial cells. In another embodiment, the molecule blocker, which contact the body of the receptor, can be agonists, and the specialist should be clear how to determine this through the implementation of appropriate analysis to identify additional agonists TIE-2 receptor.

As TIE-2 receptor was identified in connection with endothelial cells and, as in the estuaries is that TIE-2 ligand may be useful to induce vascularization in diseases or conditions, when this vascularization is shown. Such diseases or disorders include wound healing, ischemia, and diabetes. On the other hand, antagonists of the TIE-2 receptor, such as the receptor body, as disclosed in examples 2 and 3, and TIE-2 ligand 2, as disclosed in example 9, can be useful for preventing or reducing vascularization, thereby preventing or attenuating, for example, tumor growth.

As the cells containing the TIE-2 receptor, apparently, should be adjusted in the direction of increasing vascularization, TIE-2 ligand of the body can also be useful for preventing or reducing, for example, tumor growth. TIE-2 ligands may be useful for delivery of toxins it contains the receptor cells. In another embodiment, such other molecules like growth factors, cytokines or nutrients can be delivered to cells containing the TIE-2 receptor, due to the TIE-2 ligands. TIE-2 ligands can also be used as a diagnostic reagent for TIE-2 receptor, to determine the receptor in vivo or in vitro. Where the TIE-2 receptor is associated with a disease state, TIE-2 ligand of the body can be used as diagnostic reagents for the determination of the disease, for example, when coloring danioninae, includes TIE-2 ligands or ligand of the body, here described, peptide fragments or derivatives in a pharmaceutically acceptable carrier. Proteins TIE-2 ligand, peptide fragments or derivatives thereof can be entered systemically or locally. Can be any suitable types of injection, known in the art, including (but not limited to intravenous, vnutrishkolnoe, intraarterial, vnutripuzarnoe, oral, subcutaneous, intraperitoneal, or by local injection or surgical implants. You can also prepare compositions with delayed release.

The present invention also proposed that the isolated and purified nucleic acid molecule containing the nucleic acid sequences encoding TIE-2 ligand, where the sequence of nucleic acids selected from the group consisting of:

(a) sequences of nucleic acids that contain the plot, encoding TIE-2 ligand person, as shown in Fig.4, Fig.5 or Fig.6,

(C) sequences of nucleic acids that hybridize under conditions of moderate stringency to the sequence of the nucleic acid in (a), and which encode a TIE-2 ligand that binds TIE-2 the definition of the code with respect to sequences of nucleic acids (a) or (b) and which encode a TIE-2 ligand, which binds TIE-2 receptor.

In another embodiment, the invention proposed by the selected nucleic acid molecule which encodes a TIE-2 ligand, where the nucleic acid sequence is:

(a) a nucleic acid sequence comprising the site, encoding TIE-2 ligand person, as shown in Fig.4, Fig.5 or Fig.6,

(C) a nucleic acid sequence that hybridizes in conditions of moderate stringency to the sequence of the nucleic acid (a) and which encodes a TIE-2 ligand that binds TIE-2 receptor, or

(C) a nucleic acid sequence, which, if not for the degeneracy of the genetic code, hybridizes would with a sequence of nucleic acids (a) or (b), and which encodes a TIE-2 ligand that binds TIE-2 receptor.

The present invention also proposed isolated and purified TIE-2 ligands encoded by the selected nucleic acid molecule of the present invention. In the present invention is a vector that includes the allocation of a nucleic acid molecule comprising a nucleic acid sequence encoding the ligand of TIE-2 people. In one embodiment, the constructed vector, oppressive, comprising a DNA molecule encoding the ligand of TIE-2, where the DNA molecule is operatively linked to a sequence controlling the expression. The present invention also proposed a system host-vector for production of the polypeptide having biological activity of the ligand of TIE-2, which includes the expression vector of the present invention in cells of a suitable host. In one embodiment, suitable host cells can be bacterial cells, yeast cells, insect cells or mammalian cells. In the present invention proposed a method of obtaining a polypeptide having the activity of biologically active TIE-2 ligand, which comprises growing cells of the host system is the vector of the present invention, the conditions for production of the polypeptide and isolating the thus obtained polypeptide.

The present invention describes the selected nucleic acid molecule encoding a TIE-2 ligand, and proposed the creation of a ligand, its fragment or derivative, or other molecule that is an agonist or antagonist of the receptor as a therapeutic agent to treat patients suffering from diseases nastojashem the proposed invention the body of the ligand, which specifically binds to TIE-2 receptor, or TIE-2. In an alternative embodiment, the present invention proposed the body of the ligand, which includes TIE-2 ligand associated with the permanent site of the immunoglobulin. In one embodiment, the body of the ligand includes TIE-2 ligand 1 or TIE-2 ligand 2 and FC portion of human IgG1. The body of the ligand can be used in a method of treatment of humans or animals or for diagnostic purposes.

The present invention also proposed an antibody that specifically binds to such therapeutic molecules. This antibody can be monoclonal or polyclonal. In the present invention a method of using such monoclonal or polyclonal antibodies to determine the number of therapeutic molecules in the sample taken from the patient for the purpose of monitoring the progress of treatment.

Further, in the present invention proposed a therapeutic composition comprising a ligand of TIE-2 person or body of ligand, and conjugated with him cytotoxic agent. In one embodiment, the cytotoxic agent may be a radioisotope or a toxin.

The present invention also proposed an antibody that specifically binds TIE-2 ligand person. This and the division TIE-2 ligand person, including:

a) connecting at least one TIE-2 binding of the substrate with the solid matrix,

C) incubation of the substrate with the cell lysate for this substrate has formed a complex with any TIE-2 ligand in the cell lysate,

c) washing the solid matrix and

(d) elution TIE-2 ligand person from the United substrate. As the substrate may be used any substance that specifically binds TIE-2 ligand person. In one embodiment, the substrate is chosen from the group consisting of anti-TIE-2 ligand antibodies, TIE-2 receptor, and TIE-2 receptor body. The present invention also proposed that the receptor body which specifically binds TIE-2 ligand, and a therapeutic composition comprising the receptor body in a pharmaceutically acceptable carrier, and a method of blocking blood vessel growth in humans, including the introduction of an effective amount of therapeutic composition.

The present invention also proposed a therapeutic composition that includes TIE-2 ligand or person or body of ligand in a pharmaceutically acceptable carrier, and a method of promotion of neovascularization in a patient, comprising the administration to a patient an effective IRS cells, which Express TIE-2 receptor, which comprises contacting cells with detektirano labeled TIE-2 ligand or the body of the ligand, under conditions providing linking detektirano labeled ligand with TIE-2 receptor, and determining whether associated detektirano labeled ligand with the TIE-2 receptor, thereby identifying cells as cells expressing TIE-2 receptor. The present invention also proposed a therapeutic composition that includes TIE-2 ligand or the body of the ligand, and conjugated with him cytotoxic agent. The cytotoxic agent may be a radioisotope or a toxin.

In the present invention, a method for determining the expression of TIE-2 ligand by cells, which includes obtaining mRNA from cells containing mRNA with labeled nucleic acid molecules encoding TIE-2 ligand, under conditions of hybridization, the detection of mRNA hybridized with labeled molecules and thereby determining the expression of TIE-2 ligand in the cell.

In the present invention a method for determining TIE-2 ligand in tissue sections which comprises effecting contact of these tissue sections with labeled nucleic acid molecules encoding TIE-2 ligand, under conditions of hybridization, the and in tissue sections.

EXAMPLE 1. Identification of the cell line AVE as reporter cells for TIE-2 receptor

Adults BAE cells registered in the European Depository cell cultures under registration number ESAS No. 92010601 (see PNAS 75: 2621 (1978)). Northern (RNA) analysis revealed moderate levels of tie-2 transcripts in AWE (adult bovine arterial endothelial =Adult Bovine Arterial Endothelial) cell lines, consistent with in situ-hybridization results, which demonstrates the almost exclusive localization of tie-2 RNA in the vascular endothelial cells. Why were investigated AVE cell lysates on the subject the presence of TIE-2 protein, as well as from the point of view of the degree to which this TIE-2 protein is tyrosine-phosphorylated in normal growth conditions. AVE cell lysates collected and immunoassays with subsequent Western blotting immunogenic proteins due to the TIE-2-specific and phosphotyrosine-specific antisera. The omission or inclusion of TIE-2 peptides as specific blocking molecules during TIE-2 immunopositive allow us to determine TIE-2 as moderately detected a protein of approximately 150 KD, phosphotyrosine which in steady state is reduced to almost redaktiruem levels of C is as follows. AVE cells with a low number of passages are sown in the form of a monolayer with a density of 2106cells/150 mm plastic Petri dishes (Falcon) and cultured in Dulbecco modified environment Needle (DMEM) containing 10% calf serum (10% BCS), 2 mM L-glutamine (9) and 1% each of penicillin and streptomycin (P-S) in an atmosphere of 5% CO2. Before collecting the cell lysate of the cells subjected to serum starvation for 24 hours in DMEM(Q)P-S, followed by aspiration of the medium and washing the plates of ice buferizovannyiy phosphate saline (PB S), supplemented by orthovanadate sodium, sodium fluoride and benzamidine sodium. Cells subjected to lysis in a small volume of this wash buffer, which was supplemented with 1% NP40 detergent and protease inhibitors, PMSF and Aprotinin. Insoluble debris removed from the cell lysates by centrifugation at 14000 g for 10 minutes at 4C, and the supernatant immunoassay anticorodal-specific TIE-2 receptor, by adding (or not adding) blocking peptides to about 20 μg/ml of lysate. Immunosurgery proteins share at the expense of the PAGE (to 7.5% Laemmli gel), and then perform elektroprenos on PVDF membrane the shape by incubation of membranes with NKR-related secondary anticorodal with subsequent treatment of the ECL reagent (Amersham).

EXAMPLE 2. Cloning and expression of TIE-2 receptor bodies for based on epinasty investigations of interactions of TIE-2 ligand

Create expression design, which can secrete the protein consisting of the full extracellular part of the rat TIE-2 receptor, sratoga with the permanent site of the gamma-2 immunoglobulin (IgGl Fc). This fusion protein is called TIE-2 "receptor body (RB) and you can usually expect that he exists as a dimer in solution, due to the formation of disulfide bonds between the individual IgGl Fc tails. The Fc portion of the TIE-2 SB obtained as follows. The DNA fragment encoding the Fc portion of human IgGl, which extends from the hinge area to the carboxy end of the protein was amplified from cDNA of human placenta due to PCR with oligonucleotides corresponding to the sequence of human IgG1, the resulting DNA fragment of clone in plasmid vector. The corresponding DNA restriction fragments of the plasmid encoding the full-size TIE-2 receptor, and plasmids IgG1 Fc man, are ligated with each of the short sides PCR-derived fragment, which was designed to drain in a frame TIE-2 and human IgG1 Fc protein-coding sequences. Thus brandy and cytoplasmically domains. An alternative way of obtaining CENTURIES disclosed y Goodwin et al., Cell 73: 447-456 (1993).

Milligramme number of TIE-2 RB produced by cloning TIE-2 S DNA fragment into the pVL1393 baculovirus vector and subsequent infection of Spodoptera frugiperda SF-21AE cell lines insects. In another embodiment, it is possible to use the cell line SF-9 (ATS registration N CRL-1711) or cell line BTI-TN-5b1-4. DNA encoding a TIE-2 RB, clone as Eco RI-Notl fragment in baculovirus plasmid transfer pVL 1393. Plasmid DNA purified by centrifugation with a density gradient of cesium chloride recombine with the viral DNA, mixing 3 μg of plasmid DNA with 0.5 μg Baculo-Gold DNA (Pharminigen), with subsequent introduction into liposomes using 30 µg lipofectin (Lipofectin, GIBCO-BRL). A mixture of DNA-liposome add to F-21AE cells (2106cells/60 mm Cup) in TMN-FH modified Grace's medium for insect cells (GIBCO-BRL) for 5 hours at 27With in 5 days in TMN-FH medium supplemented with 5% fetal calf serum. Wednesday tissue cultures are harvested for blastophaga purification of recombinant viruses that carry out the previously described method (O'reilly, D. R., L. K. Miller and V. A. Luckow, Baculovirus Expression Vectors - A Laboratory Manual. 1992, New York: W. H. Freeman) .gif">-D-galactopyranoside, GIBCO-BRL). After 5 days incubation at 27With non-recombinant plaques assessed by positive chromogenic reaction on X-gal substrate and mark their position. Then visualize recombinant plaques, adding the second surface layer containing 100 µg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, Sigma). Putative recombinant viral plaques are selected due to aspiration of plaques and purified through multiple cycles of selection plaques to ensure homogeneity. Stock viruses create through serial low-multiplicity passage of the purified plaques virus. Get stock of low passage of a single viral clone (vTIE-2 receptor body).

SF-21AE cells cultured in the medium containing no serum (SF-900 II, Gibco BRL), containing a solution of IX antibiotic/antimycotic (Gibco BRL) and 25 mg/l gentamicin (Gibco BRL). As the surface active agent added Pluronic F-68 to a final concentration of 1 g/l of Culture (4 l) grown in the bioreactor (Artisan Cell Station System) for at least three days before infection. Cells grown at 27With, Hazira up to 50% dissolved oxygen at a gas flow rate of 80 ml/min (sobiraut in the middle of the logarithmic growth phase (approximately 2106cells per ml), concentrated by centrifugation and infect 5 plaque-forming units vTIE-2 receptor body cells. Cells and AMF inoculum was adjusted to 400 ml of freshly environment, and the virus absorb for 2 hours at 27With the spinner. Then the culture again suspended to a final volume of 8 liters of fresh, not containing serum, the medium and cells incubated in the bioreactor, using previously described conditions.

Culture medium SF21AE cells infected with TIE-2 receptor body, collected by centrifugation (500 g, 10 min) at 72 hours after infection. The supernatant was adjusted to pH 8 by NaOH. Add EDTA to a final concentration of 10 mm and the pH of the supernatant liquid again lead to 8. The supernatant was filtered (0.45 μm, Millipore) and exposed to the protein And the column (protein And sepharose 4 fast flow or HiTrapp protein And, both from Pharmacia). The column was washed with PBS containing 0.5 M NaCl, up until the absorption at 280 nm drops to baseline. The column was washed with PBS and elute with 0.5 M acetic acid. The column fractions are immediately neutralized elwira in ampoules containing 1 M Tris pH 9. Peak fractions containing the TIE-2 PB, collect and cialiswhat against PBS.

P is of known ligand for TIE-2 receptor, it would be reasonable to assume that this could provide an opportunity to clarify the functions of TIE-2 by introducing "an excess of soluble TIE-2 receptor bodies (TIE-2 R) in the developing system. Potential TIE-2 RB to bind and thereby to neutralize the available TIE-2 ligands may lead to the observed disturbances in the normal development of blood vessels and characterization of the ligand. To study whether the use of TIE-2 RB for developmental disorders of the blood vessels in the early embryo chick, small pieces of biologically absorbing foam moisten TIE-2 R and immediately placed under chorioallantoic the membrane in position directly on the side of the primitive embryo.

Early chicken embryo develops on top of the yolk of a small disk of cells that are covered with chorioallantoic membrane (CAM). Endothelial cells, which should line vasculature in the embryo, formed as from the outside-and inside-embryonic cells. Extraembryonal derived endothelial cells, which provide the main source of endothelial cells in the embryo occurs from areas of the mesenchyme, which is located on the sides around the actual embryo, directly under HIMSELF. As these mesenchymal cells is to called hemangioblastoma. In turn, hemangioblast serve as a source of mixed populations of angioblasts (offspring of endothelial cells) and hemocytoblasts (prepotentials hematopoietic precursor). The formation of the rudiments of the circulatory system begins when the seed endothelial cells divided by the education bubble thickness of one cell, which surrounds the primitive blood cell. Proliferation and migration of these cell components leads to the formation of the network is filled with blood microvessels under HIMSELF, which ultimately covers the embryo and connects with disabilities, intraembryonic received vascular elements.

The newly fertilized eggs obtained from Spafas, Inc. (Boston, MA), incubated at 99,5F (37,5C, 55% RH. After about 24 hours of development of the egg shell is washed with 70% ethanol and a dental drill to make the holes of 1.5 cm in the blunt end of the egg. The shell membrane is removed to create an air space directly above the embryo. Cut a small rectangular pieces of Gelfoam sterile (decision Upjohn) with a scalpel and moisten equal concentrations or TIE-2, or Enony domain instead of TIE-2 extracellular domain (Maisonpierre et al., Oncogene 8: 3277-3288 (1993). Each piece of foam gel absorbs approximately 6 μg of protein in 30 µl. Sterile tweezers watchmaker used to create small holes in HIMSELF in the position of a few mm to the side of the primitive embryo. A large part of the piece R-soaked Gelfoam is placed under HIMSELF and egg shells covered with pieces of masking tape. Other eggs of the same stage of development process in parallel RB unrelated, neural downregulation of receptor receptor, ENK-1 (Maisonpierre et al., Oncogene 8: 3277-3288 (1993). Development provide an opportunity to proceed for 4 days, and then the embryo research on the subject of visual research. The embryos are removed, gently breaking the shell in a Cup with warm RB, and carefully cut out the embryo from the surrounding ITSELF. Out of 12 eggs, processed each RB, 6 TIE-2 RB and 5 ENK-1 R treated embryos developed after phase observations at the beginning of the experiment. A striking difference is observed between the developed embryos, as seen in Fig.1A and 1B. Those that have been processed ENK-1 RB, apparently, developed relatively normally. Four of the five ENK-1 embryos were viable, which can be judged from the beating of the heart. Moreover, extraembryonal vascular network, which visually bulabog HIMSELF. On the contrary, those that were treated TIE-2 RB, were severely limited, had only 2-5 mm in diameter compared with the diameter of 10 mm for ENK-1 RB embryos. All embryos treated TIE-2 RB, died, and he HIMSELF had been deprived of blood vessels. The ability of TIE-2 RB to block the development of blood vessels in chickens demonstrates that TIE-2 ligand necessary for the development of the vascular system.

EXAMPLE 4. Identification of the activity of specific binding TIE-2 in the conditioned medium of ras oncogene-transformed cell lines SS mouse cultured myoblasts

Screening 10 times concentrated cell-conditioned medium (10 x SMS) from different cell lines on the presence of soluble TIE-2-specific binding activity (Visage; hrmi iosensor, iscataway, NY) reveals binding activity in not containing serum environment from oncogene-ras-transformed SS cells (SS-ras), R 2-ras (which is a ras transformed fibroblast cell line), glioblastomas 98G person and neuroblastoma cell line, known as S-1.

SS-ras 10X SMS, source derived from stable-transfectional line SS of cultured myoblasts, which were oncogene transformed by transfection of T-24 mutants basis SV40 was physically associated with gas the expression plasmid for to make possible the selection transfection clones. Obtained G418 resistant ras SS cells in the usual way support in the form of a monolayer on plastic cups in DMEM/glutamine/penicillin-streptomycin medium, supplemented with 10% fetal calf serum (FS). Not containing serum SS-ras 10X receive SMS sowing cells at 60% of confluently not containing serum specific environment for 12 hours. (Zhan and Goldfarb, Japan. ll. il. 6: 3541-3544 (1986)); Zhan, et al. Opodepe 1: 369-376 (1987)). Wednesday is drained and replaced with fresh D/O/P-S for 24 hours. The medium was collected, and the cells are again fed fresh D/O/P-S, which also collected after another 24 hours. These SMS complement protease inhibitors SF (1 mm) and Aprotinin (10 μg/ml), and concentrated 10-fold in sterile membranes exclusion size (Amicon). TIE-2 binding activity can be neutralized by incubation environment with an excess of TIE-2 RB, but not by incubation with ENK-1 RB, before BIAcore analysis.

Binding activity of 10x define the SMS using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ), which allows you to monitor biomolecular interactions in real time due to the plasmon resonance surface. Peeled TIE-2 CENTURIES covalently bound for the notes (Pharmacia Biosensor, Piscataway, NJ). The surface of sensor chip activate using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide (EDC), followed by immobilization of TIE-2 RB (25 μg/ml, pH 4.5) and deactivation of unreacted sites at the expense of 1.0 M ethanolamine (pH 8.5). The surface of the negative control ENK-1 receptor body prepared in the same way.

Wash buffer in this system is HBS (10 mM Hepes 3,4 mm EDTA, 150 mm NaCl, of 0.005% surfactant P20, pH 7.4). 10x SMS samples centrifuged for 15 minutes at 4To further clarify, using a sterile connecting low protein filter 0.45 µm Millipore, Bedford, MA). To each sample SMS add dextran (2 mg/ml) and P20 surface active agent (0,005%). Aliquots of 40 ál injection through immobilized surface (either TIE-2, or ENK-1) at a flow rate of 5 μl/min for 8 minutes register receptor binding. Activity binding (% of resonance, RU) is defined as the difference between the base value determined for 30 seconds before injection of the sample, and the value obtained after 30 sec after injection. Regeneration of the surface by one 12-µl pulse 3M MgCl2.

The instrument noise level is the a journey to the receptor. For C2C12-ras air-conditioned environment binding activity are in the range of 60-90 EN for TIE-2 RB immobilized surface. For the same samples analyzed on ENK-1 RB immobilized surface, the measured activity was found to be less than 35 EN. Specific binding to TIE-2 receptor body appreciate incubare samples with an excess of either soluble TIE-2, or ENK-1 R before the analysis of activity linking. Adding soluble ENK-1 RB has no effect on TIE-2 binding activity for any sample, whereas in the presence of soluble TIE-2 binding to the surface is in 2-30 times smaller than the value measured in the absence of TIE-2. Re-analysis using more than 50 times concentrated C2C12-ras CCM leads to four-fold amplification compared to the background value for a signal of specific binding TIE-2.

EXAMPLE 5. C2C12-ras SMS contains activity that induces tyrosine phosphorylation of TIE-2 receptor C2C12-ras 10x SMS examined for the ability to induce trainferrovie TIE-2 in the cells of AWE.AVE cells after starvation in serum briefly incubated with C2C12-ras CCM, is subjected to lysis and immunoassays, and then performed Western BCI C2C12-ras 10x SMS, as follows. Wednesday AWE cells subjected to starvation, as described previously, remove and replace the particular environment or 10x CCM, which was preheated to 37C. After 10 minutes, the medium was removed and cells washed twice in ice excess of chilled PBS, supplemented orthovanadate (NaF) benzamidine. Cell lysis and TIE-2-specific immunoassay carry out the previously described method.

AVE cells inkubirovanie for 10 minutes with the specified environment lead to induction of TIE-2 TRAINFORTRADE, whereas incubation with C2C12-ras CCM stimulates at least a hundred-fold amplification of TIE-2 phosphorylation. This activity is almost completely disappears when the preliminary incubation of C2C12-ras 10x CCM for 90 minutes at room temperature with 13 μg TIE-2 RB-fixed protein 6-separatrix balls. Environment during incubation with only one protein 6-separate not lose this activity phosphorylation.

EXAMPLE 6. Expression cloning of a TIE-2 ligand COS-7 cells were cultured in modified Dulbecco environment Needle (DMEM) containing 10% fetal bovine serum (FBS), 1% each of penicillin and streptomycin (P/S) and 2 mm glutamine in AE Needle (EMEM) with 10% FBS, (P/S) and 2 mm glutamine. Get the full length cDNA clones of mouse TIE-2 ligands by screening libraries SS ras cDNA in pJFE14 vector, expressed in COS cells. This vector, as shown in Fig.2, is a modified version of the vector pSR(Takebe, et al. 1988, Mol. Cell. Biol. 8: 466-472). This library created using two BSTX1 restriction site in pJFE14 vector.

COS-7 cells short transferout or pJFE14 library, or a control vector through DEAE-dextran transfection scheme. Briefly, COS-7 cells were seeded with a density of 1.0106cells in 100 mm plates 24 hours before transfection. For transfection cells were cultured in not containing serum DMEM containing 400 μg/ml DEAE-dextran, 1 μm of chloroquine and 2 mm glutamine and 1 μg of the appropriate DNA for 3-4 hours at 37C in an atmosphere of 5% CO2. Environment for transfection sucked off and replace buferizovannyiy phosphate saline with 10% DMSO in 2-3 minutes. After such a DMSO shocked COS-7 cells placed in DMEM with 10% FBS, 1% each of penicillin and streptomycin and 2 mm glutamine for 48 hours.

As a TIE-2 ligand is secreted, it is necessary to make cells pronitsayemyye PBS, a then incubated with PBS containing 1.8% formaldehyde, for 15-30 minutes at room temperature. The cells are then washed XXX PBS and incubated for 15 minutes with PBS containing 0.1% Triton X-100 and 10% calf serum in order to make the cells permeable to block nonspecific binding sites. Screening carried out by direct localization of staining, using the TIE-2 receptor body, which consists of the extracellular domain of TIE-2, merged with the permanent plot IgG1. This receptor body get as presented earlier in example 2. 100 mm plates with transfectional, fixed and made permeable cells COS probe by incubating them for 30 minutes with TIE-2-RB. The cells are then washed twice PBS and incubated for an additional 30 minutes with PS (10% calf serum) conjugate IgG anti-human alkaline phosphatase. After three PBS washes, the cells incubated in the substrate of alkaline phosphatase within 30-60 minutes. Then Cup examined under a microscope for the presence of stained cells. For each of the stained cells a small area of cells that includes this painted cage, cut out from a Cup, using the tip of a plastic pipette, and then allocate plasmid DN electroporation, selected and obtained from these colonies plasmid DNA used for transfection of COS-7 cells, which probe on the subject of TIE-2 ligand expresii as evidence of binding to TIE-2 receptor bodies. This allows you to identify individual colonies encoding TIE-2 ligand. Confirmation of expression of TIE-2 ligand receive through the phosphorylation of TIE-2 receptor, using previously described in example 5 method. Plasmid clone encoding TIE-2 ligand deposited in ATSS 7 October 1994. and marked as "pJFE14 encoding TIE-2 ligand" under the registration number of ATSS No. 75910.

EXAMPLE 7. Isolation and sequencing of full length cDNA clone encoding a TIE-2 ligand man

cDNA library of fetal lung man in lambda gt 10 (see Fig.3) are obtained from Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were seeded with a density of 1.25106/2020 cm plates and filters to remove fingerprints in accordance with standard procedures (Sambrok et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

Selection of clones tie-2 ligand person is as follows. the 2.2 kb Xhol fragment from the deposited clone of tie-2 ligand (ATSS No. 75910 - see example 6) mark at the expense of the statistics is desalu carried out at 65C in hybridization solution containing 0.5 mg/ml DNA salmon sperm. The filters are washed at 65With 2SSC, 0,1% SDS and exhibit on film Kodak XAR-5 overnight at -70C. Positive phages clean. Lysates of phage with high titers of pure phage used for DNA extraction on a Qiagen column, using standard methods (Qiagen, Inc., Chatsworth, CA, 1995 catalog, page 36). Phage DNA digested by EcoRl to highlight the cloned cDNA fragment for subsequent sublimirovanny. The lambda phage vector containing DNA tie-2 ligand person, they accumulate in ATSC 26 October 1994. under the namegt10 encoding htie-2 ligand 1 (ATSS registration No. 75928). Phage DNA can be directly subjected to DNA sequencing by the method using dideoxyribose termination (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA. 74: 5463-5467).

Subclavian TIE-2 ligand of human rights in the expression vector mammalian

Clonegt10 encoding htie-2 ligand 1 contains EcoRl site, located at 490 base pairs in the forward direction from the start of the coding sequence for tie-2 ligand person. Encoding section can be cut out using the unique is emer, Spel-site located 70 BP 5’ from the initiation codon, and WRI (also known as ll) site, located at 265 b in the direction of the 3’ stop codon, can be used to cut the full coding of the site. Then it can be subclinical in pJFE14 cloning vector using Xbal (compatible with Spel ledge) and Pstl sites (Pstl and Vre sites both form blunt ends).

Sequencing of TIE-2 ligand man

Encoding portion of clonegr10 encoding tie-2 ligand 1, is sequenced using ABl 373A DNA sequencing machine and a set of Taq Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). Nucleotide and amino acid sequence selected TIE-2 ligand person from clonegt10 encoding a tie-2 ligand 1 shown in Fig.4.

In addition, the full-size cDNA clones TIE-2 ligand person get through the screening of a cDNA library of human glioblastoma T98G in pJFE14 vector. Clones encoding tie-2 ligand man, identified through DNA hybridization using a 2.2 kb Xhol fragment from the deposited clone of tie-2 ligand (ATSS No. 75910) as a probe (see example 6). Encoding the plot is sequenced using AB1 373A DNA sequencing machine and a set of Taq Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foste tie-2 ligand 1. As is shown in Fig.4, clonegt10 encoding tie-2 ligand 1 contains an additional glycine residue, which is encoded by nucleotides 1114-1116. The coding sequence of T98G clone does not contain this glycine residue, but on the other hand, is identical to coding sequence of clonegt10 encoding tie-2 ligand 1. In Fig.5 shows the nucleotide and deduced amino acid sequence and TIE-2 ligand man from T98G clone.

EXAMPLE 8. Isolation and sequencing of a second full-size cDNA clone encoding a TIE-2 ligand man

A cDNA library of fetal lung man in lambda gt-10 (see Fig.3) are obtained from Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were seeded with a density of 1.25106/2020 cm plate and get the replica filters in accordance with standard procedures (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Duplicate filters sceneroot under mild conditions (2x SSC, 55(C) probes obtained for tie-2 L-1 sequence of human rights. One of the duplicate filters probe 5’ probe, encoding amino acids 25-265 tie-2 L-1 person, as predstavlennosti person (see Fig.4). Both probe hybridized when 55C in hybridization solution containing 0.5 mg/ml DNA salmon sperm. The filters are washed in 2x SSC at 55And during the night exhibit at the x-ray film. In addition, duplicate filters hybridized under normal conditions of stringency (2x SSC, 65C), with full length coded probe murine tie-2L (F3-15, Xhol insert). Select three positive clone, which satisfy the following criteria: (i) hybridization was not observed with full-length mouse) probe in the normal stiffness, and (ii) hybridization occurs under mild conditions with both 5’ - and 3’probes. EcoRl digestion ragovoy DNA obtained from these clones, indicating two independent clones with the size of the inserts of approximately 2.2 KB and approximately 1.8 KB. Insert a 2.2 KB EcoRl subcloning in EcoRl sites as pBluescript KS (Stratagene) and the expression vector mammal, suitable for use WITH cells. Define the two orientation for expression vector of the mammal. Insert a 2.2 KB in pBluescript KS deposited in ATSC 9 December 1994 and identified as pBluescript KS encoding TIE-2 ligand 2 people. Starter website coding sequence TIE-2 ligand 2 contains approximately 355 is a PR expression, any vector cloning scheme of DEAE-dextran-transfection. In short, COS-7 cells were seeded at a density of 1.0106cells per 100 mm plate 24 hours before transfection. For transfection cells were cultured in not containing serum DMEM containing 400 μg/ml DEAE-dextran, 1 μm of chloroquine and 2 mm glutamine, and 1 μg of the appropriate DNA for 3-4 hours at 37C in an atmosphere of 5% CO2. Transfection medium is sucked off and replace buferizovannyiy phosphate saline with 10% DMSO in 2-3 minutes. After such a DMSO shocked COS-7 cells placed in DMEM with 10% FBS, 1% each of penicillin and streptomycin, and 2 mm glutamine for 48 hours.

As a TIE-2 ligand is secreted, it is necessary to make the cells permeable to determine the binding of the receptor probe body with the ligand. Transfection COS-7 cells were seeded with a density of 1.0106cells per 100 mm plate. These cells ml PBS, and then incubated with PBS containing 1.8% formaldehyde for 15-30 minutes at room temperature. The cells are then washed with PBS and incubated for 15 minutes in PBS containing 0.1% Triton X-100 and 10% calf serum to give a cell Prony the military localization of staining, using the TIE-2 receptor body, which consists of the extracellular domain of TIE-2, merged with the permanent plot IgG1. This receptor body receive, as described in example 2. Transfection COS cells probe, incubare them for 30 minutes with TIE-2 RB. The cells are then washed twice PBS, fixed with methanol, and then incubated for an additional 30 minutes with PBS/10% calf serum conjugate anti-IgG-human alkaline phosphatase. After three washes with PBS, cells incubated in alkaline-phosphatase the substrate within 30-60 minutes. Then Cup examined under a microscope to determine the presence of stained cells. Cells expressing one orientation of the clone, but not a different orientation, as can be seen, bind the TIE-2 receptor body.

The specialist should be obvious that the described methods can be used to further identify other related members of the family TIE ligands.

Sequencing of the second TIE-2 ligand man

Encoding portion of clone pBluescript KS encoding TIE-2 ligand 2 people, is sequenced using AV A DNA sequencing machine and a set of Taq Dyedeoxy Terminator Cycle Sequencing Kit (Applid Biosystems, Inc., Foster City, CA). Nucleotide and deduced amino acid sequence of TIE-2 ligand person from clone pBluesript KS, to what place

Air-conditioned environment of OS cells expressing either TIE-2 ligand 2 (TI2), or TIE-2 ligand 1 (TL1) is compared in relation to their ability to activate TIE-2 receptors, naturally present in the endothelial cell line human.

Lipofectamine reagent (GIBCO-BRL, Inc.)and recommended circuits used for transfection of COS-7 cells either one pJFE14 expression vector, pJFE14 vector containing cDNA TIE-2 ligand 1 person or RMT expression vector (Kaufman R. J., 1985, Proc. Natl. Acad. Sci. USA 82: 689-693) containing cDNA TIE-2 ligand 2 people. COS the medium containing secreted ligands, collected three days later and concentrated 20 times by diafiltration (DIAFLO ultrafiltration membrane, Amicon, Inc.). The number of active TIE-2 ligand 1 and TIE-2 ligand 2, present in these environments, identify and expressed as the amount (in resonance units, R. U.) TIE-2 receptor activity specific binding, determined in the BlAcore analysis of binding.

Northern-blotting (RNA) analysis revealed significant levels of TIE-2 transcripts in NAAS (endothelial cells of the aorta person) primary endothelial cells (Clonetics, Inc.), therefore, these cells were used to determine is whether the TIE-2 Rotz in complete nutrient medium for cultivation of endothelial cells (Clonetics, Inc.), containing 5% fetal calf serum, soluble extract of bovine brain, 10 ng/ml GF man, 1 mg/ml hydrocortisone, 50 μg/ml gentamicin and 50 ng/ml amphotericin-Century

Determining which of T L1 and TL 2 can activate the TIE-2 receptor in NAAS cells, as follows. Polyethlyene cells NAES forced to starve in the serum for two hours in Dulbecco environment MEM high glucose with added L-glutamine and penicillin-streptomycin at 37With the subsequent replacement of the environment of fast-containing ligand air-conditioned COS environment for 7 minutes at 37C in an incubator with 5% CO2. The cells are then subjected to lysis, and TIE-2 receptor protein produce by immunosurgery lysates TIE-2 peptide anticorodal, with subsequent Western blotting with antiphosphotyrosine anticorodal, exactly according to the method of example 1. The results obtained are shown in Fig.7. Levels of phosphotyrosine on TIE-2 receptor (TIE-2-S) induce processing of NEAS cells TIE-2 ligand 1 (L1 band), and not TIE-2 ligand 2 (lane L2) air-conditioned COS environment. MOCK represents the conditioned medium from COS, transfected JFE14 empty the Vano by using BlAcore analysis TIE-2 receptor specific activities in transtitional COS environment and by immunostaining L1 and L2-expressing OS cells TIE-2 receptor bodies.

As L2 activates TIE-2 receptor, the applicants decided to determine whether the TIE-2 to serve as an antagonist TL1 activity. Analyze NAES phosphorylation, in which cells are first incubated with the "excess" TL2 followed by the addition of dilute TL1. Was reasonable that prior to the TIE-2 receptor, due to the high levels L2 can prevent subsequent stimulation of the receptor with subsequent exposure to L1 present in limited concentrations.

Polyethlyene NAES cells subjected to starvation in serum as described previously, and then incubated for 3 minutes at 37With 1-2 ml of 20 x COS /JF14-L2 air-conditioned environment. The control plate only handle 20 x COS medium (MOCK). Plates are removed from the incubator, and then add different dilution COS/JFE14-TL1 environment, followed by an additional incubation of the plates for 5-7 minutes at 37C. Cells are then washed, subjected to lysis and explore TIE-2-specific trainferrovie in the lysates by immunosurgery receptors and Western blotting, as described previously. L1 dilution is carried out, using 20 x COS/JFE14-TL1 Wednesday, diluted to 2, 0.5×, 0.1 X or 0.02 X is Oh technology shows they contain a similar number of TIE-2-specific binding activity, i.e., 445 R. U. and 511 R. U. for L1 and TL2, respectively. The results antiphosphotyrosine the Western blot shown in Fig.8, show that, compared with pre-treatment of NAES cells MOCK medium (lane 1), pre-processing NAES cells excess TIE-2 ligand 2 (lane 2) counteracts the corresponding ability diluted TIE-2 ligand 1 to activate the TIE-2 receptor (TIE-2-R).

These data indicate that unlike TL1 TL2 is not able to stimulate the TIE-2 receptor kinase activity in NAAS cells. Moreover, pre-incubation of endothelial cells with high concentrations of TL2 and then adding TL1 blocks the ability TL1 to stimulate the TIE-2 receptor, proving that TL2 is an antagonist of TIE-2 receptor.

EXAMPLE 10. Identification of TIE-2-specific binding activity in kondicionirovanie environment and the supernatant fluids COS cells

Binding activity of 10x SMS from cell lines CS ras, Rat2 ras. SHEP and T98G or supernatant fluids COS cells after transfection with either TIE-2 ligand 1 person (hTL1) or TIE-2 ligand 2 (hTL2) is determined using a biosensor technolo surface plasma resonance (SPR). Peeled TIE-2 RB human or rat covalently connects at the expense of primary amines with a layer of carboxymethylcysteine SM touch chip research purity (Pharmacia Biosensor, Piscataway, NJ).

The surface of sensor chip activate using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N’-(3-dimethylamino-propyl)carbodiimide (EDC), followed by immobilization of TIE-2 RB (25 μg/ml, pH 4.5) and deactivation of unreacted parts of 1.0 M ethanolamine (pH 8.5).

Usually 9000-10000 EN each receptor bodies connected with touch chip.

As running buffer in the system use S (10 mm Hepes, 150 mm NaC1, 0,005% RO surface-active agent, pH 7.4). The samples are centrifuged for 15 minutes at 4C, and then purified using a sterile 0.45 μm filter, connecting little protein (Millipore; Bedford, MA). To each sample add dextran (2 mg/ml) and surface-active agent P20 (0,005%). Aliquots of 40 ml is injected over immobilized surface (or THE human or rat) at a flow rate of 5 μl/min, and register binding receptors within 8 minutes. Binding activity (resonance units, RU) is defined as the difference between the baseline determined for 30 seconds to ineterests for each 15 μl of 3M input gl2.

SMS samples (SS ras, Rat2 ras, SHEP, T98G) testing immobilized on the surface THE RB rats, whereas recombinant hTL 1 and hTL 2 testing immobilized on the surface THE RB person. In each case, specific binding to TIE-2 receptor body assessed by incubation of samples 25 μg/ml of either soluble TIE-2 (rat or human) RB, or trkB RB before binding activity. As is shown in Fig.9 and 10, the addition of soluble trkB RB causes a small reduction in TIE-2 binding activity, whereas the addition of soluble TIE-2 RB greatly reduces binding activity compared with the results in the absence of TIE-2 RB.

EXAMPLE 11. TIE-2 specifically blocks the activation of TIE-2 receptor, due to the TIE-2 ligand 1

The applicants wanted to determine whether soluble TIE-2 RB to serve as a competitive inhibitor by blocking the activation of TIE-2 receptor, due to the TIE-2 ligand 1 (TL1). To implement this L1-containing COS medium pre-incubated with either TIE-2, or DVT-R, and then compare their ability to activate TIE-2 receptors, naturally present in the endothelial cell line human.

Air-conditioned COS the environment is obtained from COS cells, transfi the TL1), and collect, as shown in example 9, except that the environment is filtered under sterile conditions, but do not concentrate. The amount of TL 1 determined and expressed as the amount (in resonance units, R. U.) TIE-2 receptor-specific binding activity, as defined in the BlAcore analysis of binding.

Northern-blotting (RNA) reveals significant levels of TIE-2 transcriptof in HUVEC (endothelial cells of the umbilical vein of a person), primary endothelial cells (Clonetics, Inc.). Therefore, these cells are used to determine whether the TIE-2 receptor tyrosine-fosforilirovanii during exposure in the presence of TIE-2 or Thcv-RB COS medium containing L1. HUVEC cells support at 37C, 5% CO2in a complete environment for the cultivation of endothelial cells (Clonetics, Inc.), containing 5% fetal calf serum soluble extract of bovine brain with 10 µg/ml heparin, 10 ng/ml human EGF, 1 μg/ml hydrocortisone, 50 μg/ml gentamicin and 50 ng/ml amphotericin-B. Determining whether TL1 activate TIE-2 receptor in HUV EC cells, as follows. Confluently Cup HUV EC cells subjected to starvation in serum within two to four hours in Dulbecco MEM with low content is logania, which contains 0.1 mm of orthovanadate sodium, potential inhibitor phosphoribosyltransferase. Meanwhile, air-conditioned COS medium, pre-incubated for 30 minutes at room temperature with either TIE-2, or TrkB-RB add up to 50 µg/ml Medium for starvation removed from HUVEC cups and incubated with RB-containing COS environment for 7 minutes at 37C. HUV EC cells are then subjected to lysis, and protein TIE-2 receptor allocate by immunosurgery TIE-2 peptide anticorodal, with subsequent Western blotting with antiphosphotyrosine antibody as disclosed in example 1. The results obtained are shown in Fig.11. Levels of phosphotyrosine on TIE-2 receptor induce processing of HUVEC cells TIE-2 ligand 1 (TL1) relative to those observed for the control medium (MOCK), and this induction is specifically blocked by pre-incubation with TIE2-RB (TIE2-Fc), but not by incubation with TrkB-RB (TrkB-Fc). These data indicate that soluble TIE-2 RB can serve as a selective inhibitor to block the activation of TIE-2 receptor, due to the TIE-2 ligand 1.

EXAMPLE 12. Design of TIE-2 ligand tel

Expression design create thus, in order to ensure the of ne (TL 1) or TIE-2 ligand 2 (T L2), merged with a constant area gamma-1 immunoglobulin (IgG1 Fc). These fusion proteins called TIE-2 "ligand of the body" (TL1 or TL2-). Fc part TL1-Fc and TL2-Fc was prepared as follows. The DNA fragment encoding the Fc portion of human IgG1, which extends from the hinge area to the carboxy end of the protein, amplified from cDNA of the human placenta in the PCR with oligonucleotides corresponding to the published sequence of human IgGI, the resulting DNA fragment of clone in plasmid vector. The corresponding DNA restriction fragments of the plasmid encoding full TL 1 or TL2 and Fc plasmids human IgG1, are ligated to each side of the short obtained PCR fragment, which was designed to drain in a frame TL1 or TL2 with the protein coding sequence of the IgG1 Fc man.

Milligramme number TL2-Fc produced by cloning DNA fragment TL2-Fc in pVL 1393-baculovirus vector and subsequent culturing the infected cell line of the insect Spodoptera Frugiperda SF-21AE. In another embodiment, it is possible to use the cell line SF-9 (ATS registration number CRL-1711), or cell line BP1-TN-5b1-4. DNA encoding TL2-Fc clone as EcoR1-Not1 fragment in baculovirus is 0.5 mg Baculo-Gold DNA (Pharminigen), with the subsequent introduction of liposomes using 30 mg lipofectin (GIBCO-BRL). DNA liposomal mixture is added to SF-21AE cells (2 106 cells) 60 mm Cup (in TMN-FH medium). A modified medium for insect cells Fraces (GIBCO-BRL) for 5 hours at 27C, followed by incubation at 27C for 5 days in TMN-FH medium supplemented with 5% fetal calf serum. The culture medium of the tissues collected for purification plaques of recombinant viruses that carry out using previously described methods (O'reilly, D. R., L. K. Miller and V. A. Luckow, Baculovirus Expression Vectors - A Laboratory Manual. 1992, New York: W. H. Freeman), except that the agarose surface layer contains 125 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-b-D galactopyranoside, GIBCO-BRL). After 5 days of incubation at 27°C calculate the non-recombinant plaques positive chromogenic reaction with X-gal substrate, and mark their position. Then recombinant plaques visualize, adding additional second surface layer containing 100 mg/ml MTT (3-/4,5-dimethylthiazol-2-yl/-2,5-diphenyltetrazolium, Sigma).

Plaques alleged recombinant viruses are selected, sucking, and clear in repeated cycles of selection plaques to ensure homogeneity. Sapat stock with a small number of passages of a single viral clone (vTL2-Fc clone No. 7).

F-21AE cells cultivated in not containing serum medium (SF-900 II, Gibco BRL) containing 1x antibiotic/intimidatingly solution (Gibco BRL) and 25 mg/l gentamicin (Gibco BRL). As surface-active agent added Pluronic F-68 to a final concentration of 1 g/l of Culture (4 l) grown in the bioreactor (Artisan Cell Station System) for at least three days prior to infection. Cells grown at 27With, Hazira up to 50% of the dissolved oxygen in the gas flow rate of 80 ml/min (for aeration spray on the ring). Stirring is carried out by the impeller at a speed of 100 rpm, the Cells are harvested in mid-logarithmic growth phase (approximately 2106cells per ml), concentrated by centrifugation and infect 5 plaque-forming units vTL2-Fc to the cell. Cells and AMF inoculum was adjusted to 400 ml by adding fresh medium, and the virus to adsorb for 3 hours at 27With the spinner. Then the culture is again suspended in a final volume of 8 l not containing fresh serum medium, and cells incubated in the bioreactor, using previously described conditions.

Culture medium from vTL2-Fc infected SF21AE cells are harvested by centrifugation (500 about 10 minutes) at 72 h of th is of 10 mm and a pH of supernatant was adjusted to 8. The supernatant was filtered (0.45 mm, Millipore) and injected into a column of protein A (protein a, sepharose 4, the flow velocity or HiTrap protein A, both from Pharmacia). The column was washed with PBS containing 0.5 M NaCl until then, until the absorption at 280 nm drops to baseline. The column was washed with PBS and elute with 0.5 M acetic acid. Fractions from the column immediately neutralized elwira in ampoules containing 1 M Tris, pH 9. Peak fractions containing TL2-FC gather and cialiswhat against PBS.

Deposits

The following have been deposited in the American type culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, in accordance with the Budapest Treaty. Plasmid clone encoding TIE-2 ligand, was deposited with ATSC October 7, 1994, and marked as "pJFE14 encoding TIE-2 ligand" under ATS registration no. 75910. Recombinant baculovirus Autographa californica encoding TIE-2 receptor body, was deposited with ATSC October 7, 1994 and designated as "vTIE-2 receptor body" under ATS registration No. VR2484. The lambda phage vector containing DNA tie-2 ligand person was deposited with ATSC October 26, 1994 and designated asfgt10 encoding tie-2 ligand 1 under ATS registration No. 75928. Plasmid clone that encodes a second TIE-2 ligand, was deponirovano 75963.

The present invention is not limited to those disclosed here specific options. Naturally, various modifications of the invention in addition to the disclosed here will be apparent to experts from the preceding description and accompanying drawings. Such modifications should be included in the scope of the attached claims.

Claims

1. The selected nucleic acid molecule encoding a TIE-2 ligand having the amino acid sequence shown in Fig.4 or Fig.5.

2. Dedicated TIE-2 ligand, which is a TIE-2 ligand having the amino acid sequence shown in FIG.4 or FIG.5, essentially free from other proteins.

3. A plasmid, designated as pJFE14 encoding a TIE-2 ligand (ATS registration no. 75910).

4. Plasmid under item 3, used for transfection line COS-cells, for the production of TIE-2 ligand under item 2.

5. Vector, denoted asgt10 encoding hTIE-2 ligand 1 (ATSS registration No. 75928).

6. Vector for p. 5, characterized in that it is used for transfection line COS-cells for the production of TIE-2 ligand under item 2.

7. A method of obtaining a TIE-2 ligand under item 2, including Viridian the conditions ensuring the production of the ligand, and the allocation thus obtained ligand.

8. The antibody that specifically binds to TIE-2 ligand on p. 2, obtained using the TIE-2 ligand under item 2.

9. The antibody under item 8, characterized in that it is a monoclonal antibody.

10. Conjugate that includes TIE-2 ligand on p. 2 and conjugated with him cytotoxic agent.

11. Conjugate under item 10, wherein the cytotoxic agent is a radioisotope or a toxin.

12. Ligand body, which specifically binds to TIE-2 receptor, and which includes a TIE-2 ligand on p. 2, merged with a constant area of immunoglobulin.

13. Ligand body under item 12, characterized in that a constant site of an antibody is the Fc part of human IgG1.

14. Pharmaceutical composition for stimulating neovascularization in a mammal, comprising an effective amount of TIE-2 ligand on p. 2 or ligand of the body under item 12, and a pharmaceutically acceptable carrier.

15. Way to stimulate neovascularization in a mammal, comprising the administration to a mammal the pharmaceutical composition under item 14.

16. The method according to p. 15, wherein the mammal is man.

17. Way PoA TIE-2 receptor, in the presence of an effective amount of TIE-2 ligand under item 2.

18. The method according to p. 17, wherein the cell expressing TIE-2 receptor, is an endothelial cell.

19. A method of identifying an antagonist of TIE-2 receptor, comprising contacting cells expressing TIE-2 receptor, with (a) a test compound; and (b) TIE-2 ligand under item 2, the conditions for binding of the ligand to the receptor and determining whether the test compound to inhibit the binding of ligand to the receptor.

 

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