The selected nucleic acid molecule encoding a chimeric tie-2 ligand (options), chimeric or modified tie-2 ligand and a method thereof, a vector, a method of obtaining a system of vector-host, conjugate, the pharmaceutical composition

 

The invention relates to biotechnology, in particular genetic engineering, and can be used to produce chimeric or modified TIE-2 ligand. Chimeric TIE-2 ligand that binds and activates TIE-2 receptor, contains N-terminal domain, helically twisted domain and a fibrinogen-like domain, which is selected from TIE-2 ligand 1, TIE-2 ligand 2, TIE-3 ligand, TIE-ligand 4. The modified ligand can have a nucleotide change 1102-1104 sequence in Fig.4, encoding cysteine, or nucleotides 555-557 sequence in Fig.6, encoding arginine. Chimeric TIE-2 ligand is produced by culturing cells transformed by a vector containing a nucleic acid encoding a chimeric TIE-2 ligand. Pharmaceutical composition for the induction of hematopoiesis or neovascularization contains chimeric or modified ligand. The invention allows the development of tools that enhance hematomas. 8 N., and 15 C.p. f-crystals, 44 ill., 1 PL.

This application claims priority to U. S. Serial No. 08/740223 filed October 25, 1996, and U.S. Provisional application 60/022999 filed August 2, 1996. Throughout the application, reference is made to various publications. The description of these publications in their entirety are included in kachele specifically, to the genes for 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, this invention relates to a new modified TIE-2 ligand that binds TIE-2 receptor, as well as methods of producing and applying the modified ligand. Further, the invention provides a nucleic acid sequence encoding a modified ligand, and methods of generating nucleic acid that encodes a modified ligand, and the product of the gene. Modified TIE-2 ligand, and encodes its nucleic acid can be applied in the diagnosis and treatment of certain diseases involving endothelial cells and associated TIE-receptors, such as neoplastic diseases involving the tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis and inflammatory diseases. In addition, the modified ligand can be used to enhance proliferation and/or differentiation of hematopoietic stem cells. More generally, activating the project, migration and/or differentiation and/or stabilization or destabilization of cells expressing TIE-receptor. Biologically active modified TIE-2 ligand can be used to support in vitro expressing TIE-receptor cells in culture. Cells and tissues expressing TIE-receptor include, for example, heart and vascular endothelial cells, epithelial lens cells of the epicardium of the heart and early hematopoietic cells. The alternative of such a ligand may be used to maintain cells which are designed genetic engineering for the expression of the TIE receptor. In addition, a modified TIE-2 ligand and related cognate receptor can be used in test systems to identify additional agonists or antagonists of this receptor.

Background of the invention

Cellular behavior is responsible for the development, maintenance and reparation of differentiated cells and tissues is regulated, in large part, intercellular signals sent through growth factors and similar ligands and their receptors. Receptors localized on the cell surface of cells of the respondents and they bind peptides and polypeptides, known as factors rotisserie cells respondents as well as quick and long-term adjustment in the expression of cellular genes. Some receptors are associated with different cell surfaces, can associate specific growth factors.

Phosphorylation of tyrosine residues in proteins by tyrosine kinases is one of the key ways in which signals transducers (transferred) through the cytoplasmic membrane. Some currently known genes proteincontaining encode transmembrane receptors for polypeptide growth factors and hormones such as epidermal growth factor (EGF), insulin, insulin-like growth factor 1 (IGF-1), platelet-derived growth factors (PDGF-A and-b) and fibroblast growth factors (FGF) (Heldin et al., Cell Regulation, 1:555-566 (1990); Ullrich, et al., Cell 61:243-254 (1990)). In this case, these growth factors have shown their activity by binding to the extracellular part of the related receptors, leading to activation of the internal tyrosine kinase that is present in the cytoplasmic part of the receptor. The receptors of growth factors, endothelial cells are of particular interest because of the possible involvement of growth factors in several important physiological and pathological processes, such as education and raspatory hematopoietic growth factors are tyrosine kinases; they include c-fms, which is the receptor for colony-stimulating growth factor, Sherr, et al., Cell, 41:665-676 (1985), and c-kit, the receptor undifferentiated hemopoietic growth factor, reported in Huang, et al., Cell 63:225-233 (1990).

Receptor tyrosine kinase have been subdivided on the evolution of the subfamily based on the characteristic structure of their ectodomains (Ullrich, et al., Cell 61:243-254 (1990)). These subfamilies include EGF-receptor-like kinase (division I) and insulin receptor-like kinase (division II), each of which contains a repeating homologous enriched with cysteine sequences in their extracellular domains. One enriched in cysteine region was also detected in the extracellular domains of eph-like kinase. Hirai, et al., Science 238:1717-1720 (1987); Lindberg, et al., Mol. Cell. Biol., 10:6316-6324 (1990); Lhotak, et al., Mol. Cell. Biol., 11:2496-2502 (1991). PDGF receptors as well as c-fms and c-kit-receptor tyrosine kinase, can be grouped in division III; whereas FGF-receptors form a subclass IV. Typical members of both subclasses are extracellular unit stacking, stabilized by interchain disulfide bonds. These so-called immunoglobulin(Ig)-like styling found in proteins of the superfamily ietosnovamy or soluble ligands. Williams, et al., Ann. Rev. Immunol., 6:381-405 (1988).

Receptor tyrosine kinase differ in their specificity and affinity. Typically, the receptor tyrosine kinase are glycoproteins, which consist of (1) an extracellular domain capable of binding a specific factor (factors) growth; (2) a transmembrane domain, which is usually located in the alpha-helical portion of this protein; (3) juxtamembrane (kolomanbrunnen) domain, where the receptor may be regulated, for example, phosphorylation of a protein; (4) tyrosinekinase domain, which is the enzymatic component of the receptor; and (5) carboxykinase tail, which in many receptors involved in recognition and binding of substrates for tyrosine kinase.

It was reported that processes such as alternative splicing of exons and alternative selection gene promoter or polyadenylation sites, capable of producing several different polypeptides from a single gene. These polypeptides can contain or not to contain various domains listed above. As a result, some extracellular domains can be expressed in the form of individual secreted proteins and some forms of the receptor may not be tyrosinekinase domain is plus the short carboxykinase tail.

The gene encoding the transmembrane tyrosinekinase endothelial cells, initially identified using RT-PCR as homologous to the unknown tyrosinekinase cDNA fragment from leukemic human cells, has been described Partanen, et al., Proc. Natl. Acad. Sci. USA, 87:8913-8917 (1990). This gene and encoded them protein has been called a "TIE" that is an abbreviation for "tyrosine kinase domain homology with Ig and EGF". Partanen, et al., Mol. Cell. Biol., 12:1698-1707 (1992).

It was reported that mRNApresent in all fetal tissues and embryonic tissues of mice. In the study of mRNAwas localized in cardiac and vascular endothelial cells. Specifically, mRNAwas localized in endothelia blood vessels and the endocardium of 9.5 to 18.5-day mouse embryos. Enhanced expressionwas discovered during the formation of new blood vessels associated with the development of ovarian follicles and granulation (granular) tissue in skin wounds. Korhonen, et al., Blood, 80:2548-2555 (1992). Thus, it was assumed that the TIE play a role in angiogenesis, which is important for the development of therapies for solid tumors and some other angiogene is that two structurally related rat TIE-receptor protein encoded by different genes with similar expression profiles. One gene, called-1 is the mouse homologueman. Maisonpierre, et al., Oncogene, 8:1631-1637 (1993). Another gene-2 may be the mouse homologue of the mouse gene tek, which, as reported, like, is expressed in mice exclusively in endothelial cells and their alleged cell precursors. Dumont, et al., Oncogene, 8:1293-1301 (1993). The human homologue-2 described in Ziegler, U. S. Patent No. 5447860, issued September 5, 1995 (where he referred to as the "ork"), which is included in its entirety here by reference.

It was found that both genes on a large scale is expressed in the endothelial cells of the embryonic and postnatal tissues. Significant levels-2 transcripts were also present in other populations of embryonic cells, including in the epithelium of the crystalline lens, the epicardium of the heart and parts of the mesenchyme. Maisonpierre, et al., Oncogene, 8:1631-1637 (1993).

The predominant expression of the TIE receptor in vascular endothelium suggests that TIE plays a role in the development and maintenance of the vascular system. This could play a role in determining endothelial cells, proliferation for TIE-2, illustrate its importance in angiogenesis, in particular for the formation of a network of blood vessels in the endothelial cells. Sato, T. N., et al., Nature 376:70-74 (1995). In the Mature vascular system TIE could function in the survival of endothelial cells, maintaining and reciprocal reactions on the pathogenic effect.

TIE-receptors expressed in immature hematopoietic stem cells, b cells and sub-megakaryocytes, suggesting, thus, the role of ligands that bind these receptors, in early hematopoiesis, in the differentiation and/or proliferation of b-cells and in the path of differentiation of megakaryocytes. Iwama, et al., Biochem. Biophys. Research Communications 195:301-309 (1993); Hashiyama, et al., Blood 87:93-101 (1996); Batard, et al., Blood 87:2212-2220 (1996).

The invention

This invention provides a composition comprising a modified TIE-2 ligand, essentially not containing other proteins. In the application here, the modified ligand of TIE-2 is called the TIE ligand-family ligands, typical representatives of which include ligands TL1, TL2, TL3 and TL4, described here, which have been modified by addition, deletion or replacement of one or several amino acids, or by marking, for example, the Fc part of human IgG-1, but to the RNA TIE-2 ligand, containing at least a portion of the first TIE-2 ligand and part of the second TlE-2-ligand, which is different from the first. As a non-restrictive example, the first TIE-2 ligand is TL1, and the second TIE-2 ligand is TL2. This invention considers other combinations with additional family members ligands of TIE-2. For example, other combinations possible to create chimeric TIE-2 ligand, including, but not limited to, combinations in which the first ligand is selected from the group consisting of TL1, TL2, TL3 and TL4, and the second ligand different from the first ligand selected from the group consisting of TL1, TL2, TL3 and TL4.

This invention also provides an isolated nucleic acid molecule encoding a modified TIE-2 ligand. In one embodiment, the selected nucleic acid molecule encodes a TIE-2 ligand TIE-family ligands, typical representatives of which include ligands TL1, TL2, TL3 and TL4, described here, which have been modified by addition, deletion or replacement of one or several amino acids, or by marking, for example, the Fc part of human IgG-1, but which retain their ability to bind the TIE-2 receptor. In another embodiment, the selected nucleic acid molecule encodes Bodiroga TIE-2 ligand and part of the second TIE-2 ligand, which is different from the first. As a non-restrictive example, the first TIE-2 ligand is TL1, and the second TIE-2 ligand is TL2. This invention considers other combinations with additional family members ligands of TIE-2. For example, other combinations possible, including, but not limited to, combinations in which the selected nucleic acid molecule encodes a modified TIE-2 ligand, which is a chimeric TIE-2 ligand containing a portion of the first ligand selected from the group consisting of TL1, TL2, TL3 and TL4, and part of the second ligand different from the first ligand selected from the group consisting of TL1, TL2, TL3 and TL4.

The selected nucleic acid molecule can be a DNA, cDNA or RNA. This invention also provides a vector containing an isolated nucleic acid molecule encoding a modified TIE-2 ligand. This invention also provides a system of vector - host for the production in a suitable cell host polypeptide having the biological activity of the modified TIE-2 ligand. Resembling the host-cell can be cells of bacteria, insect or mammal. This invention also provides a method matrial growing cells of the host system - the vector under conditions that allow production of the polypeptide and the extract thus obtained polypeptide.

This invention related to a selected nucleic acid molecule that encodes a modified TIE-2 ligand, provides further development of this ligand as a therapeutic agent for treating patients suffering from disorders involving cells, tissues or organs expressing TIE-2 receptor. This invention also provides an antibody that specifically binds to such a therapeutic molecule. This antibody can be monoclonal or polyclonal. This invention also provides a method of using such monoclonal or polyclonal antibody to measure the amount of therapeutic molecules in a sample taken from a patient, for the purpose of monitoring the progress of therapy.

This invention also provides an antibody that binds a modified TIE-2 ligand described above. This antibody can be monoclonal or polyclonal. Thus, this invention also provides a therapeutic composition comprising an antibody that specifically binds to a modified TIE-2-lohankristen vessels in mammals by introduction of an effective amount of a therapeutic composition, containing an antibody that specifically binds to a modified TIE-2 ligand described herein, in a pharmaceutically acceptable carrier.

This invention provides further a therapeutic composition containing a modified TIE-2 ligand described herein, in a pharmaceutically acceptable carrier. This invention also provides a method of enhancing the formation of new blood vessels in the patient by introducing an effective amount of a therapeutic composition containing an activating receptor of a modified TIE-2 ligand described herein, in a pharmaceutically acceptable carrier. In one embodiment, this method can be applied for the treatment of ischemia. Another option activates the receptor modified TIE-2 ligand described herein, is used alone or in combination with other hematopoietic factors to stimulate proliferation or differentiation of hematopoietic stem cells, b-cells or megakaryocytes.

Alternative, the invention provides a conjugation of a modified TIE-2 ligand with a cytotoxic agent and a therapeutic composition, prepared on the basis of this conjugate. Further, the invention provides a protein of receptarea (receptorbody)the invention also provides a method of inhibiting the growth of blood vessels in a mammal by introducing an effective amount of receptical, which specifically binds to a modified TIE-2 ligand in a pharmaceutically acceptable carrier.

This invention also provides an antagonist of TIE-2 receptor, as well as a way of inhibiting the biological activity of TIE-2 in a mammal, involving the introduction of this mammal an effective amount of an antagonist of TIE-2. According to this invention, the antagonist may be modified TIE-2 ligand described herein that binds to TIE-2 receptor but does not activate it.

Brief description of drawings

Fig.1A and 1B TIE - 2-receptarea (TIE-2-RB) inhibits the development of blood vessels in the embryonic chicken chorioallantoic membrane (Cam). One piece rezorbiruetsa gelatin (Gelfoam), soaked 6 ug RB inserted directly into 1-day-old chick embryos. After 3 days incubation 4-day embryos and surrounding HIMSELF was removed and examined. Fig.1A: the embryos treated EHK-1-RB (rEHK-1 ecto/hIgG1 Fc), were viable and had normal developed blood vessels in surrounding HIMSELF. Fig.1B: all embryos treated TIE-2-RB (r, TIE-2-RB ecto/hIgG1 Fc), were dead, reduced in size and almost completely devoid of surrounding blood vessels.

Fig. - Christ.new acid and decoded amino acid sequence (one letter code) of human TIE-2 ligand 1 from clonegt10 encoding htie-2 ligand 1.

Fig.5 is the nucleic acid sequence and the decoded amino acid sequence (one letter code) of human TIE-2 ligand 1 from T98G clone.

Fig.6 is the nucleic acid sequence and the decoded amino acid sequence (one letter code) of human TIE-2 ligand 2 of the clone pBluescript KS encoding human TIE-2 ligand 2.

Fig.7 is a Western blot showing activation of TIE-2 receptor TIE-2 ligand 1 (Lane L1), but not TIE-2 ligand 2 (Lane 2) or control.

Fig.8 is a Western blot showing that pre-treatment of cells NAES excess TIE-2 ligand 2 (Lane 2) impairing the ability of divorced TIE-2 ligand 1 to activate the TIE-2 receptor (TIE2-R) compared with pre-treatment of cells NAES environment MOCK (Lane 1).

Fig.9 is a Western blot demonstrating the ability TL2 competitive inhibition of activation TL1 TIE-2 receptor, using a line of hybrid human cells, EA.hy926.

Fig.10 (a, b, c, d) representation in the form of a histogram of the binding surface with immobilized TIE-2-IgG TIE-2 ligand in air-conditioned SS ras, Rat2 ras, SHEP and T98G concentrated (10x) environment. Specific binding krysi the TIE-2-RB in comparison with a small reduction in the presence of soluble trkB-RB.

Fig.11 a, b is the binding of recombinant human TIE-2 ligand 1 (hTL1) and human TIE-2 ligand 2 (hTL2) supernatant COS cells, with a surface with immobilized human TIE-2-"receptaculum" (RB). Specific to human TIE-2 binding was determined by incubation of the samples with 25 μg/ml soluble or hTIE-2-RB, or trk-RB; a significant decrease in the activity of binding was observed only for samples incubated with hTIE-2-RB.

Fig.12 is a Western blot showing that TIE-2-receptarea (denoted TIE-2-RB, or, as here, the TIE-2-Fc) inhibits the activation of TIE-2 receptor TIE-2 ligand 1 (TL1) in HUVEC cells, whereas foreign receptarea (TRKB-Fc) did not inhibit this activation.

Fig.13 - agarose gels showing the serial cultivation [undiluted (1) - 10-4] products of RT-PCR TL1 and TL2, derived from mouse embryonic liver E14,5 (Tracks 1 - General Track 3 - stromal enriched Track 4 - hematopoietic precursor cells c-kit+TER119) and murine fetal thymus (Lanes 2).

Fig.14 - agarose gels showing the serial cultivation [undiluted (1) - 10-3] products RT=PCR TL1 and TL2, derived from murine fetal cortical stromal cells of the thymus (Lanes 1 - CDR1+/A2B5-) and medullary straw is in angiogenesis. TL1 presents (), TL2 presents (*), TIE-2 presents (T), VEGF presents ([]) and flk-1 (VEGF receptor) presents (V).

Fig.16 - slides in situ hybridization, showing the character of the temporal expression of TIE-2, TL1, TL2 and VEGF during angiogenesis associated with follicular development and formation of the corpus luteum (yellow body) in the ovary of rats that received the serum of pregnant mares. Column 1: the early pre-ovulatory follicle; Column 2: pre-ovulatory follicle; Column 3: the early corpus luteum; and Column 4: athleticly follicle; the Number And glossy area; Series: VEGF; Number: TL2; Series D: TL1 and Row E: TIE-2 receptor.

Fig.17 - comparison of amino acid sequences of the Mature protein TL1 and Mature protein TL2. The sequence TL1 is the same, which is shown in Fig.4, except that the putative leader sequence was removed. Similarly, the sequence TL2 is the same, which is shown in Fig.6, except that the putative leader sequence was removed. Arrows indicate residues Arg49, Cys245 and AGD TL1, which correspond to residues in positions of amino acids 69, 265 and 284, respectively TL1 shown in Fig.4.

Fig.18 is a Western blot covalent multimeric binding curve TIE-2-IgG with immobilized TL1 in quantitative cell-free test link.

Fig.20 is a typical curve showing the binding "legendtina" TIE-2 ligand 1, containing the fibrinogen-like domain of the ligand associated with the Fc domain of IgG (TL1-fFc), with immobilized ectodomain TIE-2, quantitative cell-free test link.

Fig.21 - nucleotide and decrypted (single-letter code) amino acid sequence of the TIE ligand-3. The coding sequence starts at position 47. The fibrinogen-like domain starts at position 929.

Fig.22 - comparison of amino acid sequences of members of the family TIE-ligands. mTL3 = mouse TIE ligand-3; hTL1 = human TIE-2 ligand 1; chTL1 = chicken TIE-2 ligand 1; mTL1 = mouse TIE-2 ligand 1; mTL2 = mouse TIE-2 ligand 2; hTL2 = human TIE-2 ligand 2. Prisoners in blocks areas indicate conservative regions of homology among the members of this family.

Fig.23 - nucleotide and decoded amino acid sequence (one letter code) TIE ligand-4. The arrow shows the position of the nucleotide 569.

Fig.24 - nucleotide and decoded amino acid (single letter code) sequences of chimeric TIE-ligand labeled 1N1C2F (Chimera 1). The putative leader sequence is encoded by nucleotides 1-60.

Fig.25 nucleotides 2N2C1F (Chimera 2). The putative leader sequence is encoded by nucleotides 1-48.

Fig.26 - nucleotide and decoded amino acid (single letter code) sequences of chimeric TIE-ligand labeled 1N2C2F (Chimera 3). The putative leader sequence is encoded by nucleotides 1-60.

Fig.27 - nucleotide and decoded amino acid (single letter code) sequences of chimeric TIE-ligand labeled 2N1C1F (Chimera 4). The putative leader sequence is encoded by nucleotides 1-48.

Detailed description of the invention

As described in more detail below, applicants have created a new modified TIE-2 ligands that bind the TIE-2 receptor. This invention provides a composition comprising a modified TIE-2 ligand, essentially not containing other proteins. In the application here, TIE-2 ligand is called a TIE ligand-family ligands, typical representatives of which include ligands L1, TL2, TL3 and TL4, described here, which have been modified by addition, deletion or replacement of one or several amino acids or tagging, for example, the Fc part of human IgG-1, but which retain their ability to bind the TIE-2 receptor. Modified TIE-2 ligand includes the English, which is different from the first. As a non-restrictive example, the first TIE-2 ligand is L1 and the second ligand is TL2. The invention also considers other combinations with additional members of the family TIE-2 ligands. For example, other combinations possible to create chimeric TIE-2 ligand, including, but not limited to, combinations in which the first ligand is selected from the group consisting of L1, TL2, TL3 and TL4, and the second ligand different from the first ligand selected from the group consisting of TL1, TL2, TL3 and TL4.

This invention also provides an isolated nucleic acid molecule encoding a modified TIE-2 ligand. In one embodiment, the selected nucleic acid molecule encodes a TIE-2 ligand TIE-family ligands, typical representatives of which include ligands TL1, TL2, TL3 and TL4, described here, which have been modified by addition, deletion or replacement of one or several amino acids, or by marking, for example, the Fc part of human IgG-1, but which retain their ability to bind the TIE-2 receptor. In another embodiment, the selected nucleic acid molecule encodes a modified TIE-2 ligand, which is a chimeric TIE-2 ligand, soderjaschie non-restrictive example, the first TIE-2 ligand is TL1, and the second TIE-2 ligand is TL2. This invention considers other combinations with additional family members ligands of TIE-2. For example, other combinations possible, including, but not limited to, combinations in which the selected nucleic acid molecule encodes a modified TIE-2 ligand, which is a chimeric TIE-2 ligand containing a portion of the first ligand selected from the group consisting of TL1, TL2, TL3 and TL4, and part of the second ligand different from the first ligand selected from the group consisting of TL1, TL2, TL3 and TL4.

The invention includes a modified TIE-2 ligands and their amino acid sequences and their functionally equivalent variants, as well as proteins or peptides, including mutants with substitutions, deletion or insertion mutants described sequences that bind the TIE-2 receptor and act as agonists or antagonists. Such variants include variants in which amino acid residues substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues in this sequence can be replaced by the NT, resulting in a silent change. Replacement of amino acids in this 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, series, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In the scope of the present invention also includes proteins or their fragments or derivatives that exhibit the same or similar activity as modified TIE-2 ligands described herein, and derivatives which are differentially modified during or after translation, for example by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc., Functionally equivalent molecules include molecules that contain modifications, including N-terminal modifications that occur, wsle the second takes place in some bacterial expression systems (e.g., E. Li).

The invention also includes nucleotide sequences that encode proteins described herein as a modified TIE-2 ligands, and cell-hosts, including yeast, bacteria, viruses, and mammalian cells that are genetically engineered to obtain these proteins, for example, transfection, transduction, infection, electroporation, or microinjection of nucleic acid that encodes a modified TIE-2 ligands described herein, in a suitable expressing vector. The invention also includes the introduction of a nucleic acid that encodes a modified TIE-2 ligands, using methods of gene therapy, as described, for example, in Finkel and Epstein FASEB J. 9:843-851 (1995); Guzman, et al., PNAS (USA) 91:10732-10736 (1994).

The person skilled in the art will also understand that the invention includes DNA sequences and RNA which hybridize with a nucleotide sequence encoding a TIE-2 ligand, under conditions of moderate stringency, as defined, for example, in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 Ed., Vol.1, pp. 101-104, Cold Spring Harbor Laboratory Press (1989). Thus, the molecule of nucleic acid covered by this invention includes a molecule having nucleotide, obtained as described above, and the molecule having a nucleotide sequence that hybridizes with such a nucleotide sequence, and a nucleotide sequence that is a degenerate sequence of the above sequence as a result of the degeneracy of the genetic code, but which encodes a ligand that binds TIE-2 receptor and having the amino acid sequence and other primary, secondary and tertiary characteristics, which are quite duplicate the modified TIE-2 ligand described herein, so that this molecule has the same biological activity as the activity of a modified TIE-2 ligand described herein.

This invention provides an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds and activates TIE-2 receptor, containing the nucleotide sequence encoding a TIE-2 ligand 1, in which a part of the nucleotide sequence that encodes the N-terminal domain of TIE-2 ligand 1, replaced by a nucleotide sequence that encodes the N-terminal domain of TIE-2 ligand 2. This invention also provides such a nucleic acid molecule with dodman TIE-2 ligand 1, replaced by a nucleotide sequence that encodes a twisted helical domain of TIE-2 ligand 2.

This invention also provides an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds and activates TIE-2 receptor, comprising the nucleotide sequence encoding a TIE-2 ligand 1, in which a portion of the nucleotide sequence encoding the N-terminal domain of TIE-2 ligand 1, replaced by a nucleotide sequence that encodes a N-terminal domain of TIE-2 ligand 2, and further modified so that it encodes a different amino acid instead of a cysteine residue encoded by nucleotides 784-787, as shown in Fig.27. The cysteine residue is preferably substituted by a serine residue. In another embodiment, the nucleic acid molecule additionally modified to encode other amino acids instead of the arginine residue encoded by nucleotides 199-201, as shown in Fig.27. The arginine residue is preferably substituted by a serine residue.

This invention also provides an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds and activates TIE-2 receptor, containing the nucleotide is th amino acid instead of a cysteine residue at amino acid position 245. The cysteine residue is preferably substituted by a serine residue.

This invention provides further an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds but does not activate the TIE-2 receptor, containing the nucleotide sequence encoding a TIE-2 ligand 1, in which a portion of the nucleotide sequence that encodes a N-terminal domain of TIE-2 ligand 1, demeterova. This invention also provides such a nucleic acid molecule, optionally modified in such a way that a part of the nucleotide sequence encoding a twisted helical domain of TIE-2 ligand 1, demeterova and part encoding a fibrinogen-like domain fused in reading frame with a nucleotide sequence that encodes a constant region of human immunoglobulin gamma 1 (IgG1 Fc).

This invention provides further an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds but does not activate, the TIE-2 receptor, containing the nucleotide sequence encoding a TIE-2 ligand 2, in which part of the nucleotide sequence that encodes a N-terminal domain of TIE-2 ligand 2, demeterova. The invention of the th nucleotide sequence, coding twisted helical domain of TIE-2 ligand 2, demeterova and part encoding a fibrinogen-like domain fused in reading frame with a nucleotide sequence that encodes a constant region of human immunoglobulin gamma 1 (IgG1 Fc).

This invention provides further an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds but does not activate, the TIE-2 receptor, containing the nucleotide sequence encoding a TIE-2 ligand 1, in which a portion of the nucleotide sequence encoding a fibrinogen-like domain of TIE-2 ligand 1, replaced by a nucleotide sequence that encodes a fibrinogen-like domain of TIE-2 ligand 2. This invention also provides such a nucleic acid molecule, optionally modified in such a way that a part of the nucleotide sequence encoding a twisted helical domain of TIE-2 ligand 1, replaced by a nucleotide sequence that encodes a twisted helical domain of TIE-2 ligand 2.

This invention further provides a modified TIE-2 ligand encoded by any nucleic acid molecule of the present invention.

This invention provides Anda, different from the first, and the first and second TIE-2 ligands selected from the group consisting of TIE-2 ligand-1, TIE-2 ligand-2, TIE ligand-3 or TIE ligand-4. Preferably chimeric TIE-ligand contains at least part of the TIE-2 ligand 1 and part TIE-2 ligand-2.

This invention also provides a nucleic acid molecule that encodes a chimeric TIE-ligand shown in Fig.24, 25, 26 or 27. The invention also provides chimeric TIE-ligand shown in Fig.24, 25, 26 or 27. Additionally, the invention provides chimeric TIE-ligand shown in Fig.27, modified so that it has a different amino acid instead of a cysteine residue encoded by nucleotides 784-787.

Any of the methods known to the person skilled in the art, the introduction of DNA fragments into a vector may be used to construct expressing vectors encoding a modified TIE-2 ligand, using suitable signal regulation of transcription/translation and encoding this protein sequences. These methods may include methods for the synthesis of recombinant DNA in vitro and recombination in vivo (genetic recombination). Expression of nucleic acid sequence that encodes a mod is inovas acid, which is operatively linked to a sequence encoding a modified TIE-2 ligand, so that the modified protein or peptide TIE-2 ligand is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a modified TIE-2 ligand described herein can be controlled by any promoter/enhancer element known in this field. Promoters that can be used for the regulation of expression of the ligand include, but are not limited to) long terminal repeat as described in Squinto et al. (Cell 65:1-20 (1991)); the early promoter region SV40 (Bernoist and Chambon, Nature 290:304-310), the promoter, CMV, 5'-terminal repeat M-MuLV, the promoter contained in the 3'long terminal repeat of rous sarcoma virus (Yamamoto, et al., Cell 22:787-797 (1980)), the promoter timedancing herpes (Wagner et al., Proc. Natl. Acad. Sci. USA 18: 144-145 (1981)), the promoter of adenovirus, the regulatory sequences of the gene of metallothionein (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expressing vector, such as a promoter-lactamases (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)) or the 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); the promoter of apostardinerointe), the alkaline phosphatase promoter, and the following regulatory region transcription animals that exhibit tissue specificity and have been utilized in transgenic animals; the regulatory region of the gene elastase I, which is active in the acinar cells of the pancreas (Swift et al., 38:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987); the regulatory region of the gene of insulin that is active in the beta cells of the pancreas (Hanagan, Nature 315:115-122 (1985)); the regulatory region of the gene of the immunoglobulin, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), the regulatory region of the virus tumors in the mammary gland of mice, which is active in testicular cells, the cells of the breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), the regulatory region of the gene of albumin, which is active in liver (Pinkert et al., 1987, Genes and Devel., 1:268-276), the regulatory region of the gene 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); regulatory region of the gene alpha-1-antitrypsin, which is active in liver (Kelsey et al., 1987, Genes and Devel., 1:161-171); regulatory area of the beta-globin gene, which is active in myeloid cells (Mogram et al., Nature 315:338-340; Kollias et al., 1986, Cell 16:89-Cell 18:703-712); the regulatory region of the gene light chain 2 myosin, which is active in skeletal muscle (Shani, 1985, Nature 315:283-286), and the regulatory region of the gene gonadotropin releasing hormone that is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378). In addition, the invention includes receiving antisense compounds that can specifically gibridizatsiya with a RNA sequence encoding a modified TIE-2 ligand to modulate its expression. Ecker, U. S. Patent No. 5166195, issued November 24, 1992.

Thus, according to this invention expressing vectors that can replicate in a bacterial or eukaryotic host containing a nucleic acid encoding a modified TIE-2 ligand described above, are used for transfection of the host and thereby to direct the expression of such nucleic acid to produce a modified TIE-2 ligand, which can be allocated in a biologically active form. In the application here, the biologically active form includes a form capable of binding TIE-receptor and cause a biological response, such as a function of differentiation, or to influence the phenotype of cells expressing this receptor. Such biologically active form of the economic activity can actually be antagonistic in relation to TIE-receptor. In alternative embodiments, the active form of the modified TIE-2 ligand is a form that can learn TIE-receptor and thereby to act as a targeting agent for the receptor for use as diagnostic and therapeutic tools. In accordance with such variants of this active form should not cause any change in the phenotype expressing any TIE cells.

Expressing the vectors containing the gene insertions can be identified using four main approaches, such as: (a) DNA-DNA hybridization, (b) the presence or absence of the function of "marker" gene (s) expression of the built-in sequences and (d) PCR detection. In the first approach, the presence of a foreign gene integrated in expressing vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous built, codereuse modified TIE-2 ligand gene. In the second approach, the recombinant system vector/host can be detected and selected on the basis of the presence or absence of certain functions of "marker" gene (e.g., activity timedancing, resistance to antibiotics, transformation of genes in the vector. For example, if a nucleic acid encoding a modified TIE-2 ligand, is embedded in 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 expressing vectors can be identified by analysis of foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the gene product of a modified TIE-2 ligand, for example, by binding of the ligand with TIE-receptor, or part thereof, which may be marked, for example, detektivami antibody or part thereof, or the binding of the antibody produced against the protein of the modified TIE-2 ligand or part thereof. Cells of this invention may temporarily or preferably a constitutive and permanent to Express the modified TIE-2 ligand, as described herein. In the fourth approach can be cooked nucleotide primers DNA corresponding to specific DNA sequences. These primers could then be used for education using PCS fragment genethe n any way, which makes possible the subsequent formation of a stable, biologically active protein. Preferably the ligand is secreted into the culture medium, from which it is extracted. Alternative ligand can be isolated from cells or in the form of soluble proteins, either in the form of inclusion bodies, from which it can be extracted quantitatively 8M chloride guanidine and dialysis in accordance with well known methods. For further purification of the ligand can be used in affinity chromatography, conventional ion-exchange chromatography, hydrophobic chromatography, chromatography with reversed phase or gel filtration.

In additional embodiments of the present invention, as described in detail in the Examples, the gene encoding a modified TIE-2 ligand, can be used for inactivation of a "knockout" endogenous gene by homologous recombination, and thereby create a TIE-ligandprotein cell, tissue or animal. For example, but not limitation, may be constructed in the gene encoding recombinant human TIE ligand-4, containing insertional mutation, for example, genethat iactiveaware would native gene encoding TIE ligand-4. This design apart from the way transfection, transduction or injection. Then cells containing this construct can be selected by resistance to G418. Cells that do not have an intact gene encoding TIE ligand-4, can then be identified, for example, a blot on the Southern, R-detection, Northern blot testing or analysis of expression. Cells that do not contain the intact gene encoding TIE ligand-4, can then be merged with early embryonic cells for producing transgenic animals, the lack of such ligand. Such an animal can be used to identify specific processes in vivo, the rate depends on the ligand.

The invention also provides antibodies to the modified TIE-2 ligand described herein, which are applicable for the detection of the ligand, for example in the diagnosis. To obtain monoclonal antibodies directed against a modified TIE-2 ligand, can be applied to any method that provides the formation of molecules of antibody continuous cell lines in culture. For example, in the scope of this invention are the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as Trinny way, the way of using the antibodies of the person (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp.77-96), etc.

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 this field (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U. S. A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16). Can be derived molecules chimeric antibodies containing the mouse antigennegative domain with constant regions of human (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851; Takeda et al., 1985, Nature 314: 452).

Various procedures known in this field can be used to produce polyclonal antibodies to epitopes described here is a modified TIE-2 ligand. To obtain antibodies of different animal hosts, including, but not limited to, rabbits, mice and rats can be immunized by injection of a modified TIE-2 ligand or a fragment or derivative. To increase the immune response can be used a variety of adjuvants depending on the type of owner, including, but not limited to, adjuvants's adjuvant (complete and incomplete), mineral gels such as GI is Lena and polyoxypropylene (pluronic), polyanion, peptides, oil emulsions, hemocyanine fissurella, dinitrophenol, and potentially applicable adjuvants for humans, such as BCG (Bacille Calmette-Guerin) and Corinebacterium parvum.

A molecular clone of an antibody to a selected modified epitope of TIE-2 ligand can be obtained by known methods. The methodology of recombinant DNA (see, for example, Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) can be used to construct sequences of nucleic acids that encode the antibody molecule or its antigennegative district.

This invention provides a molecule antibodies, as well as fragments of such antibody molecules. Antibody fragments which contain the idiotype of this molecule can be obtained by known methods. For example, such fragments include, but are not limited to: the F(ab')2-the fragment that can be obtained by pepsin cleavage of the molecule antibodies; Fab'fragments which can be obtained by reduction of disulfide bridges of F(ab')2fragment, and Fab fragments, which can be obtained by treating the antibody molecules with papain and regenerating agent. Molecules antibodies can be purified by known methods, in the liquid chromatography high resolution) or their combination.

The invention also includes an immunoassay for measuring the amount of a modified TIE-2 ligand in a biological sample

a) contacting the biological sample with at least one antibody that specifically binds to a modified TIE-2 ligand in such a way that the antibody forms a complex with any modified TIE-2 ligand present in the sample; and

b) measuring the amount of this complex and, thus, the measurement of the amount of the modified TIE-2 ligand in a biological sample.

Further, the invention includes an analysis to measure the amount of TIE-receptor in a biological sample

a) contacting the biological sample with at least one ligand of the present invention in such a way that the ligand forms a complex with TIE-receptor; and

b) measuring the amount of this complex and, thus, by measuring the number of TIE-receptor in a biological sample.

This invention also provides the use of a modified TIE-2 ligand that activates TIE-2 receptor, as described herein, to maintain the survival and/or growth and/or migration and/or differentiation expressing TIE-2 receptor cells. Thus, this ligand can be used in the e applicants modified TIE-2 ligand for TIE-2 receptor makes it possible to use test systems, applicable for the identification of agonists or antagonists of the TIE-2 receptor. Such test systems would be useful in identifying molecules capable of stimulating or inhibiting angiogenesis. For example, in one embodiment, antagonists of the TIE-2 receptor can be identified as molecules that are capable of resisting the interaction of TIE-2 receptor with a modified TIE-2 ligand that binds TIE-2 receptor. Such antagonists are identified by their ability 1) to inhibit the binding of biologically active modified TIE-2 ligand to the receptor, as measured, for example, using BIAcore biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ); or 2) to inhibit the ability of the biologically active modified TIE-2 ligand to cause a biological response. These biological responses include, but are not limited to, phosphorylation of TIE-receptor or located along transduction path components transduction TIE-signal or the survival, growth or differentiation of bearing TIE-receptor cells.

In one embodiment, the cells are designed for the expression of the TIE receptor, can be dependent on the growth from the addition of a modified TIE-2 ligand. Tagonists, can counteract the activity of the modified TIE-2 ligand on these cells. Alternative autocrine cells, designed to ensure the ability of these cells to coexpression as a modified TIE-2 ligand and the receptor, can be a valuable system for the analysis of potential agonists and antagonists.

Thus, this invention provides for the introduction of the TIE-2 receptor in cells that do not normally Express this receptor, which allows these cells to be deep and easily distinguishable response to a ligand that binds the receptor. The callable type of response depends on the cells and does not depend on the specific receptor entered in this cell. Can be selected suitable cell line, giving the answer that is most applicable for the analysis and for the detection of molecules that can act on tyrosinekinase receptors. These molecules can be a molecule of any type, including, but not limited to, peptide and ones molecules that will act in the described systems receptorpositive way.

One of the more applicable systems for such use involves the introduction of TIE-receptor (or chem is nutriclean domain TIE-receptor) in line fibroblastic cells (for example, cells NIH3T3), so that the receptor, which is usually not Mediaset proliferative or other responses may, however, after the introduction in fibroblasts analyzed various well-established methods to quantify the effects of fibroblast growth factors (e.g., thymidine incorporation, or other types of tests proliferation; 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 tests have the additional benefit that any drug can be tested on cell lines that have entered the receptor and the source cell lines that do not have receptor; only specific effects on cell lines with the receptor could be considered as mediasuite entered by the receptor. Such cells can be, in addition, designed for expression of a modified TIE-2 ligand that creates autocrine system, applicable for testing molecules that act as antagonists/agonists of this interaction. Thus, the invention provides cell host containing a nucleic acid encoding a modified TIE-2 ligand, and civet also a valuable system for the identification of agonists or antagonists with molecules of small size TIE-receptor. For example, can be identified fragments, mutants or derivatives of the modified TIE-2 ligand that bind the TIE-receptor, but do not enter any other biological activity. An alternative characteristic of the modified TIE-2 ligand makes possible an additional characteristic of the active parts of this molecule. In addition, identification of the ligand allows you to define the x-ray structure of the complex of receptor/ligand, which allows to identify the binding site on the receptor. Knowledge of the binding site will provide important insights into the rational design of novel agonists and antagonists.

Specific binding of the test molecule with TIE-receptor can be measured in different ways. For example, the true binding of the test molecule with cells expressing TIE can be detected or measured by detecting or measuring (i) the test molecule associated with the surface of intact cells; (ii) test molecules cross-linked to protein a TIE, in cell lysates; or (iii) test molecules associated with TIE, in vitro. Specific interaction between the test molecule and TIE can be measured using reagents that demonstrate the unique properties of this interaction.

In their way. Consider the case in which should be measured using a modified TIE-2 ligand in the sample. Different dilution (test molecules) in parallel with a negative control (NC), not containing activity of the modified TIE-2 ligand, and a positive control (PC) containing a known amount of a modified TIE-2 ligand may be exhibited by cells expressing TIE, in the presence of detektirano labeled modified TIE-2 ligand (in this example, radioiodinated ligand). The amount of the modified TIE-2 ligand in the test sample can be assessed by determining the number of125I-labeled modified TIE-2 ligand that binds to the controls and in each of the dilutions, followed by comparison of the sample with a standard curve. More than a modified TIE-2 ligand in the sample, the less125I-ligand will be contacted with a TIE.

The amount of bound125I ligand can be determined by measuring the amount of radioactivity per cell or crosslinking of the modified TIE-2 ligand protein on the cell surface using LTOs, as described in Meakin and Shooter, 1991, Neuron 6:153-163, and detecting the amount of labeled protein in cell extracts using, for example the plans for the complex TIE-receptor/modified TIE-2 ligand. Specific interaction of the test molecule and TIE can be further tested by adding to the tests of various dilutions of unlabeled control ligand that does not bind the TIE-receptor and, therefore, does not have significant effects on the competition between the modified TIE-2 ligand and a test molecule for TIE-linking. Alternatively, you can expect the molecule, which, as you know, destroys the binding TIE-receptor/modified TIE-2 ligand, such as, but not limited to, antibody against TIE or antibody against TIE-receptor described here will be to counteract the competition between125I modified TIE-2 ligand 2 and the test molecule for binding to TIE-receptor.

Detektirano labeled modified TIE-2 ligand includes, but is not limited to, a modified TIE-2 ligand, covalently linked or ecovalence with a radioactive substance, a fluorescent substance, a substance having 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 antibody molecule, which preferably ablate can be measured by the assessment of secondary biological effects of binding of a modified TIE-2 ligand/TIE-receptor, including, but not limited to, cell growth and/or differentiation or expression of immediate early genes or phosphorylation TIE. For example, the ability of the test molecule to induce differentiation can be tested in cells that do not haveand in comparable cells expressing; differentiation in expressingcells, but not in comparable withoutthe cells would indicate a specific interaction of the test molecule/TIE. A similar analysis could be carried out by detecting the induction of immediate early gene (for example,or) inminus andplus the cells or detection of phosphorylation TIE using the standard tests phosphorylation, known in this area. Such analysis can be used to identify agonists or antagonists, which are not tied competitive with TIE.

Similarly, the invention provides a method of identifying a molecule that has a biological activity of a modified TIE-2 ligand that includes all the specific binding of the test molecule with the TIE receptor, in which specific binding TIE is positively correlated with TIE-like activity. Specific binding can be detected either by analysis of the direct binding or by secondary biological effects of binding discussed above. This method may be particularly applicable in the identification of new members of the family TIE-ligands or, in the pharmaceutical industry, in screening a large number of peptide and ones of molecules (e.g., peptidomimetics) associated with TIE biological activity. In the preferred characteristic non-restrictive embodiment, the present invention can be prepared with a big net culture wells that contain alternating rows of cells RS (or fibroblasts, see), who are either cellsminus or cells-plus. Can then be added to various test molecules so that each column of the grid or its part contained different test molecule. Then each well can be evaluated for the presence or absence of growth and/or differentiation. Thus, it may be subjected to screening for this activity on a large number of test maligant-like activity or identification of the molecule, with this activity, comprising (i) exposing the test molecule TIE-receptor protein in vitro in conditions that may lead to the binding, and (ii) detecting binding of the test molecule with TIE-receptor protein, in which the binding of the test molecule with TIE-receptor correlates with TIE-2 ligand-like activity. According to such methods TIE-receptor may or may not be essentially cleaned, can be attached to a solid carrier (such as affinity column or ELISA test) or can be incorporated into an artificial membrane. Binding of the test molecule with TIE-receptor can be assessed by any method known in this field. In preferred embodiments, binding of a test molecule can be detected or measured by assessing its ability to compete with detektirano labeled known TIE-ligands for binding to TIE-receptor.

This invention also provides a method for detecting ability of test molecules to function as an antagonist of TIE-ligand-like activity, which includes the detection ability of this molecule to inhibit the effect of the binding TIE-TIE ligand with a receptor on the cell expressing the receptor. Such Anta is D. The effects of binding of the TIE-2 ligand with TIE-receptor are preferably biological or biochemical effects, including, but not limited to, survival or cell proliferation, transformation of cells, induction of immediate early genes or phosphorylation TIE.

This invention provides a further as a way of identifying antibodies or other molecules that can neutralize the ligand or block the binding of receptors and molecules identified in this way. As a non-restrictive example, the method can be performed by means of analysis, which is conceptually similar to ELISA analysis. For example, the antibody against TIE-receptor may be associated with a solid carrier, such as plastic advance the tablet. Then as the control of a known amount of a modified TIE-2 ligand, which was the ICC are labeled ICC), may be introduced into the hole and then any labeled modified TIE-2 ligand that binds with the antibody against the receptor can be identified using a reporter antibodies directed against the ICC-label. This test system can then be used for screening of test samples for molecules that are capable of i) svjazyvaites with the antibody against the receptor and labeled ligand. For example, the hole may be a test sample containing a potential interest in the molecule with a known amount of the labeled ligand, and may be measured by the amount of labeled ligand that binds with the antibody against the receptor. By comparing the amount of bound labeled ligand in the test sample with the amount of control can be identified molecules that can block the binding of ligand to the receptor.

Interest molecules identified thereby, can be isolated using methods well known to specialists in this field.

Upon detection of the antagonist (inhibitor) binding of the ligand to the person skilled in the art will be able to perform a second test to determine whether the associated blocker with the receptor or ligand, as well as tests to determine whether a molecule blocker to neutralize the biological activity of the ligand. For example, by applying the test of binding using a biosensor BIAcore technology (or equivalent test), in which the TIE-"receptarea or modified TIE-2 ligand or logandale" covalently associated with the solid carrier (e.g., from carboxymethylate (inhibitor) specifically with the ligand, "legendtina" or "receptaculum". To determine whether a molecule blocker to neutralize the biological activity of the ligand, the person skilled in the art could perform a test of phosphorylation (see Example 5) or, alternatively, functional Biotest, such as the survival test, using primary cultures, such as endothelial cells. Alternative molecule blocker, which binds with the antibody to the receptor, could be an agonist, and the person skilled in the art could determine this by conducting a suitable test to identify additional agonists TIE-receptor.

In addition, this invention considers further compositions, in which the TIE-ligand is a receptor-binding domain described herein TIE-2 ligand. For example, TIE-2 ligand 1 contains helically twisted domain (starting at the 5'end and extending to nucleotide at position ~1160, Fig.4, and the provisions of ~1157, Fig.5) and a fibrinogen-like domain (encoded by the nucleotide sequence of Fig.4, starting at position ~1161, and when the position -1158, Fig.5). Believe that the fibrinogen-like domain of TIE-2 ligand 2 starts at or near the same amino acid sequence as in ligand IE-2-ligand-3 begins at or near amino acid sequence, which is encoded by the nucleotides shown in Fig.21. Multimerization helically twisted domains during the formation of the ligand makes it difficult to clean. However, as described in Example 19, the applicants have found that the fibrinogen-like domain contains a domain bind the TIE-2 receptor. However, Monomeric forms of the fibrinogen-like domain, apparently, are not intended to bind the receptor. Studies using myc-labeled fibrinogen-like domain, which was assembled in "clusters" using antibodies against myc, showed that they really bind the TIE-2 receptor. [Methods of obtaining "arranged in clusters of ligands and liganded" described in Davis et al., Science 266:816-819 (1994)]. On the basis of this discovery, applicants have received "legendtina" that contain fibrinogen-like domain of TIE-2 ligands associated with the Fc domain of IgG ("fFc"). These logandale, which form dimers, effectively bind the TIE-2 receptor. In accordance with this invention considers that the provision of the modified TIE-liganded that can be used as targeting agents for tumor and/or associated vasculature, where necessary antagonist TIE.

Further, this invention provides the preparation of the ligand, its frste therapeutic tools for treatment of patients suffering from disorders involving cells, tissues or organs expressing TIE-receptor. Such molecules can be used in the method of treatment of the human or animal or in a diagnostic method.

Because TIE-receptor was identified in connection with endothelial cells and, as shown here, the locking TIE-2 ligand 1, apparently prevents the formation of new blood vessels, applicants expect that a modified TIE-2 ligand described herein can be applied to induce the formation of new vessels in case of diseases or disorders, when shown this induction. Such diseases or disorders could include wound healing, ischemia, and diabetes. These ligands can be tested in animal models and can be used therapeutically, as described for other agents, such as growth factor vascular endothelial (VEGF), the other specific for endothelial cells factor, which is angiogenic. Ferrara, et al., U. S. Patent No. 5332671, issued July 26, 1994. Link Ferrara, and also other papers describe research in vitro and in vivo, which can be used to demonstrate antigenome factor in the acceleration of blood flow to ischemic myocardium, acceleration of healing p what do et al., European Patent Application 0550296 A2, published July 7, 1993; Banai et al., Circulation 89:2183-2189 (1994); Unger, et al., Am.J. Physiol. 266:H1588-H1595 (1994); Lazarous, et al., Circulation 91:145-153 (1995)]. In accordance with this invention, a modified TIE-2 ligand may be used alone or in combination with one or more additional pharmaceutically active compounds, such as, for example, VEGF or basic fibroblast growth factor (bFGF), as well as cytokines, neurotrophins, and so on,

Conversely, antagonists of the TIE-receptor, such as a modified TIE-2 ligands that bind to the receptor but do not activate it as described here, "receptical" described herein in Examples 2 and 3, and TIE-2 ligand 2, described in Example 9, could be used for preventing or reducing the formation of new vessels and, therefore, prevent or ameliorate, e.g., tumor growth. These agents can be used alone or in combination with other compositions, such as antibodies against VEGF, which has been shown to be applicable in the treatment of conditions in which the goal of therapy is blocking (inhibiting) angiogenesis. Applicants expect that a modified TIE-2 ligand described herein can also be used in combination with such agents as entitely determined that TIE-ligands expressroute in cells in tumors or in cells that are closely associated with tumors. For example, TIE-2 ligand 2 is likely to be strongly associated with tumor endothelial cells. Therefore, he and other TIE-antagonists may also be applicable in preventing or weakening of, for example, tumor growth. In addition, the TIE-ligands or legendtina" (ligandbodies) can be used to deliver toxins to the bearing the receptor cell. Alternative other molecules, such as growth factors, cytokines or nutrients can be delivered in a carrier TIE-receptor cell through TIE-ligands or legendtina. TIE-ligands or logandale, such as a modified TIE-2 ligand described herein can also be used as diagnostic reagents for TIE-receptor for the detection of this receptor in vivo or in vitro. When TIE-receptor associated with a pathological condition, the TIE-receptors or logandale, such as a modified TIE-2 ligand, can be used as diagnostic reagents to detect diseases, such as staining of tissue or imaging of the whole body. Such reagents include radioisotopes, fluorochromes, dyes, enzymes and Biotin. Such diagnostic or Ter(USA) 90:8996-9000 (1993), included here as a complete reference.

In other embodiments, TIE-ligand that activates the receptor modified TIE-2 ligand described herein, is used as hematopoietic factors. Various hematopoietic factors and their receptors are involved in proliferation and/or differentiation and/or migration of different cell types contained in the blood. Because TIE-receptors expressed in early hematopoietic cells, it is expected that the TIE-ligands play a comparable role in proliferation, or differentiation, or migration of these cells. So, for example, contains TIE composition can be obtained, analyzed and tested in biological systems in vitro and in vivo and is used therapeutically as described in any of the following links: Sousa, U. S. Patent No. 4810643; Lee, et al., Proc. Nail. Acad. Sci. USA 82:4360-4364 (1985); Wong, et al., Science, 228:810-814 (1984); Yokota, et al., Proc. Natl. Acad. Sci. USA 81:1070 (1984); Bosselman, et al., WO 9105795, published May 2, 1991, entitled "Stem Cell Factor" and Kirkness, et al., WO 95/19985, published July 27, 1995 entitled "Haemopoietic Maturation Factor. Thus, activating the receptor modified TIE-2 ligand can be used to diagnose or treat conditions in which the normal hematopoi depressed, including, but not limited to, anemia, thrombocytopenia, leukopenia and granulotsitopenii. In polachi differentiation of progenitor cells of the blood in situations when the patient has the disease, such as acquired immunodeficiency syndrome (AIDS), which caused a decrease of the normal levels of blood cells, or in a clinical setting in which it is desirable increase in hematopoietic populations, such as the condition that occurs when bone marrow transplantation or for the treatment of aplasia or mielosupression under the action of irradiation, chemical treatment or chemotherapy.

Activating the receptor modified TIE-2 ligand of the present invention can be used alone or in combination with another pharmaceutically active agent such as, for example, cytokines, neurotrophins, interleukins, etc., In the preferred embodiment, these ligands can be used in conjunction with any of the aforementioned factors, which are known to induce the proliferation of stem cells or other hematopoietic precursors, or factors acting on these cells in the path of hematopoesis, including, but not limited to, together with the factor of hematopoietic maturation, thrombopoietin, stem cell factor, erythropoietin, G-CSF, GM-CSF, etc.

In an alternative embodiment, the antagonists of the TIE-receptor used for diagnosis or treatment of patients whose jeetendra proliferative disorders forming blood bodies, such as thrombocythemia, polycythemia and leukemia. In such embodiments, the treatment may include the use of a therapeutically effective amount of a modified TIE-2 ligand, TIE-antibodies, TIE-receptical, conjugate of a modified TIE-2 ligand or legendtina or fFc described above.

This invention also provides pharmaceutical compositions containing a modified TIE-2 ligand or legendtina described herein, peptide fragments or derivatives pharmacologically acceptable carrier. Modified TIE-2-ligand proteins, peptide fragments or derivatives may be administered systemically or topically. Can be used any suitable route of administration known in this field, including, but not limited to, intravenous, vnutrispinalnaya, intraarterial, through the nose, oral, subcutaneous, intraperitoneal or local injection or surgical transplant. Provided compositions with prolonged action.

This invention also provides an antibody that specifically binds to such a therapeutic molecule. This antibody can be monoclonal or polyclonal. The invention also provides a method of using such may of the patient, for the purpose of monitoring the progress of therapy.

Further, this invention provides a therapeutic composition comprising a modified TIE-2 ligand or the protein logandale and conjugated them with a cytotoxic agent. In one embodiment, the cytotoxic agent may be a radioisotope or a toxin.

This invention also provides an antibody that specifically binds to a modified TIE-2 ligand. This antibody can be monoclonal or polyclonal.

Further, this invention provides a method of purification of a modified TIE-2 ligand, comprising the following:

a) binding at least one TIE-linking of the substrate with the solid matrix;

b) incubation of this substrate with the cell lysate so that the substrate forms a complex with any modified TIE-2 ligand in the cell lysate;

c) washing the solid matrix; and

(d) elution of the modified TIE-2 ligand from the bound substrate.

The substrate may be any substance that specifically binds to a modified TIE-2 ligand. In one embodiment, the substrate is selected from the group consisting of antibodies against a modified TIE-2 ligand, TIE-and receptor fused protein TIE-receptorligand TIE-2 ligand, as well as a therapeutic composition comprising the protein recepticle in a pharmaceutically acceptable carrier, and a method of blocking blood vessel growth in man, introducing an effective amount of therapeutic composition.

This invention also provides a therapeutic composition comprising an activating receptor of a modified TIE-2 ligand or the protein logandale in a pharmaceutically acceptable carrier, as well as a way to enhance the formation of new blood vessels in the patient, providing for the introduction to the patient an effective amount of therapeutic composition.

In addition, this invention provides a method of identifying cells expressing TIE-receptor, which involves contacting cells with detektirano labeled modified TIE-2 ligand or legendtina under conditions that allow binding detektirano labeled TIE ligand with the receptor, and determining whether associated detektirano labeled TIE ligand with the receptor, and identification, thus, this cell as cells expressing TIE-receptor. This invention also provides a therapeutic composition comprising a modified TIE-2-Li gang is to be a radioisotope or a toxin.

This invention also provides a method of detecting expression of a modified TIE-2 ligand cell, which includes obtaining mRNA from the cell, contacting the mRNA so obtained with a labelled nucleic acid molecule that encodes a modified TIE-2 ligand, under conditions of hybridization, the presence of mRNA, hybridizing with labeled molecule, and thereby detecting the expression of a modified TIE-2 ligand in the cell.

The invention provides further a method of detecting expression of a modified TIE-2 ligand in the tissue section, which provides for the contacting of the tissue section with labeled nucleic acid molecule that encodes a modified TIE-2 ligand, under conditions of hybridization, the presence of mRNA, hybridizing with labeled molecule, and thereby detecting the expression of a modified TIE-2 ligand in the tissue section.

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

Adult cells BAE registered in the European Cell Culture Repository under ESAS#92010601 (see PNAS 75:2621 (1978)). Northern analyses (RNA) revealed moderate levels-2 transcripts in cell lines AB is th localization RNA-2 in vascular endothelial cells. Therefore, we investigated the lysates of the cells AWE the presence of protein TIE-2, as well as the extent to which this protein is TIE-2 fosfauriliruetsa on tyrosine in normal conditions versus serum-free growth conditions. Lysates AVE cells were collected and subjected to thus with subsequent Western blotting immunoprecipitating proteins with TIE-2-specific and phosphotyrosine-specific antisera. The exclusion or inclusion of TIE-2 peptides as specific blocking molecules during thus TIE-2 has made possible an unambiguous identification of TIE-2 in the form of moderately detected a protein of ~150 kDa, phosphotyrosine stationary States which are reduced almost to redetection levels in the pre-treatment of cells in serum-free medium (starvation).

The cultivation of AVE cells and collecting cell lysates was carried out as follows. Cells AWAE with a low number of passages were sown in the form of a monolayer at a density of 2106cells/150-mm plastic Petri dish (Falcon) and cultured in modified according to the method of Dulbecco environment Needle (DMEM) containing 10% fetal whom b>. Before collecting cell lysates cells maintained in serum-free medium (starvation) for 24 hours in DMEM/Q/P-S, followed by aspiration of the medium and washing of cups chilled on ice phosphate buffered saline (PBS) with added ortho-Vanadate sodium, sodium fluoride and benzamidine sodium. Cells were literally in a small volume of this washing buffer, to which was added 1% detergent NP40 and protease inhibitors PMSF and Aprotinin. Insoluble residues cells were removed from the cell lysates by centrifugation at 14000g for 10 minutes at 4And supernatant subjected thus by antisera specific for TIE-2 receptor, in the presence or in the absence of the blocking peptides added to ~20 μg/ml of lysate. Immunoprecipitated proteins were separated by polyacrylamide gel electrophoresis (7.5% Laemmli gel) and then transferred by electroblotting on a PVDF-membrane and incubated either with different TIE-2-specific or phosphotyrosine-specific antisera. Protein TIE-2 was visualized by incubation of this membrane with the secondary connected with horseradish peroxidase antisera, followed by processing really TIE-2 ligand

Was created in expression design, which could give the secretory protein consisting of the extracellular part of the rat TIE-2 receptor, fused to the constant region of human immunoglobulin gamma 1 (IgG1 Fc). This protein was named TIE-2-receptarea" (receptorbody) (RB), and you would expect that he in norm exists as a dimer in solution by the formation of disulfide bonds between the individual tails IgGl Fc. Fc-part TIE-2-RB was prepared as follows. The DNA fragment encoding the Fc-portion of human IgGl, which extends from the hinge region to carboxilic of this protein, amplified from placental cDNA person using PCR with oligonucleotides corresponding to the published sequence of the human IgGl; the resulting DNA fragment was cloned in the plasmid vector. Appropriate restriction fragments of DNA from a plasmid that encodes a full-sized TIE-2 receptor, and containing Fc human IgGl plasmids ligated on each side of the short obtained using a PCR fragment, which was designed in such a way that it could be drained in the frame readout sequence encoding a TIE-2 and Fc protein of human IgGl. Thus, the obtained slidemenu TIE-2. An alternative way of obtaining RB described in Goodwin, et al., Cell 73:447-456 (1993).

The number of the order of milligrams TIE-2-RB was obtained by cloning a DNA fragment TIE-2-RB in baculovirus vector pVL1393 and subsequent infection of the cell line of the insect Spodoptera frugiperda SF-21AE. Alternative you can use the cell line SF-9 (ATCC Accession No. CRL-1711) or cell line BTI-TN-5bl-4. DNA encoding a TIE-2-RB, cloned in the form of Eco RI-NotI fragment in baculovirus transport plasmid pVL1393. Plasmid DNA purified by centrifugation in a density gradient of cesium chloride, recombinable into viral DNA by mixing 3 μg of plasmid DNA with 0.5 μg Baculo-Gold DNA (Pharminigen) with subsequent introduction into liposomes using 30 µg Lipofectin (GIBCO BRL). A mixture of DNA-liposomes were added to the cells SF-21AE (2106cells on 60 mm Cup) in TMN-FH (Modified atmosphere Grace for insect cells (GIBCO BRL) for 5 hours at 27C, followed by incubation at 27C for 5 days with medium TMN-FH, supplemented with 5% fetal calf serum. Environment for tissue culture were collected for purification plaques of recombinant viruses, which was performed using the methods described previously, the Loy agarose contained 125 μg/ml X-da (5-bromo-4-chloro-3-indolyl--D-galactopyranoside; GIBCO-BRL). After 5 days incubation at 27With non-recombinant plaques were evaluated in points using positive chromogenic reaction with X-gal is a substrate and their positions marked. Then recombinant plaques were visualized by adding a second covering layer containing 100 µg/ml MTT (bromide 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; Sigma). Estimated plaques of recombinant virus was removed by suction tubes and cleaned numerous cycles of selection plaques to ensure homogeneity. The baseline of the virus was obtained by serial passage with a low multiplicity of infection cleaned from virus plaques. Received line from a low number of passages of a single viral clone (vTIE-2-receptarea).

Cells SF-21AE were cultured in serum-free medium (SF-900 II, Gibco DRL) containing 1a solution of the antibiotic/fungicide (Gibco BRL) and 25 mg/ml Gentamicin (Gibco BRL). Pluronic A-68 (block copolymers of polyoxyethylene and polyoxypropylene) was added as a surfactant to a final concentration of 1 g/l of Culture (4 l) were grown in the bioreactor (Artisan Cell Station System) for at least three days before initsiirovaniem. Cells were grown at 27106cells/ml), concentrated by centrifugation, and infected with 5 plaque-forming units fused protein vTIE-2-receptical on the cell. Cells and the inoculum was brought to 400 ml of fresh medium and the virus was adsorbing for 2 hours at 27With in a rotating flask. Then this culture resuspendable in the final volume of 8 liters of fresh serum-free medium and cells were incubated in the bioreactor using the above-described conditions.

Culture medium from infected fused protein vTIE-2-receptarea SF21AE cells were collected by centrifugation (500g, 10 minutes) at 72 hours after infection. Cellular supernatant brought to pH 8 with NaOH. Added EDTA to a final concentration of 10 mm and the pH of the supernatant is re-conveyed to 8. Supernatant was filtered (0.45 μm, Millipore) and applied to a protein A-column (protein A sepharose 4 fast flow or HiTrap protein A, both from Pharmacia). The column was washed in PBS containing 0.5 M NaCl until the absorbance at 280 nm was reduced to background. The column was washed in PBS and were suirable 0.5 M acetic acid. Faction, erwerbende with speakers, Portela, United and were dialyzed against PBS.

EXAMPLE 3. The demonstration that TIE-2 plays a crucial role in the development of the vascular network

Elucidation of the functions of TIE-2 was achieved by introducing an excess of soluble TIE-2-receptical (TIE-2-RB) in the developing system. Potential TIE-2-RB to bind and thereby to neutralize the available TIE-2 ligand leads to the observed disruption of the normal development of the vasculature and normal properties of this ligand. To determine whether you can use TIE-2-RB for developmental disorders of the blood vessels in early chicken embryos, small pieces of biologically reformiruemoi foam impregnated TIE-2-RB and injected immediately under chorioallantois the membrane provisions directly on the side of undifferentiated embryo.

Early chicken embryos on top of the yolk of a small disc cells, which covered chorioallantoic membrane (CAM). Endothelial cells that will line the blood vessels in the embryo arise from sources as unembryonated and vnutribronhialnah cells. Unembryonated endothelial cells, which provide the main source of endothelial cells in the embryo come from the intergrowth of the mesenchyme, which raibaut, they give rise to a common ancestor as endothelial and hematopoietic lines of differentiation, called extent. In turn, hemangioblast gives rise to a mixed population of angioblasts (precursors of endothelial cells) and hepatoblastoma (poly potent hematopoietic precursors). The formation of the embryonic circulatory system begins when the precursors of endothelial cells are split with the formation of the bubble thickness of one cell, which surrounds the undifferentiated blood cells. Proliferation and migration of these cell components over time produces a network filled with blood microvessels under ITSELF, which will eventually penetrate the embryo to connect with limited formed vnutribronhialno vascular elements.

Sizeofloatarray chicken eggs obtained from Spatas Inc. (Boston, MA), incubated at 99,5F and 55% relative humidity. At approximately 24 hours of development of the egg shell was wiped with 70% ethanol and dental drill was to cut a hole 1.5 cm in blunt top of each egg. The shell membrane was removed to open air space directly above AMB the concentrations TIE-2 or ENK-1-receptical. ENK-1-receptarea was prepared as described in Example 2, using the extracellular domain ENK-1 instead of the extracellular domain of TIE-2 (Maisonpierre et al., Oncogene 8:3277-3288 (1993)). Every piece of Gelfoam was absorbed approximately 6 μg of protein in 30 µl. Sterile tweezers for observations used for making a small incision in HIMSELF in position at a distance of a few millimeters to the side of undifferentiated germ. Larger piece impregnated RB Gelfoam was introduced under HIMSELF, and egg shells were closed tightly on top of a piece of tape. Other similarly processed eggs processed RB outsider, expressed by neurons receptor tyrosine kinase, ENK-1 (Maisonpierre et al., Oncogene 8:3277-3288 (1993)). Development was allowed to run for 4 days and then the embryos were subjected to visual examination. Embryos were removed by careful breaking-shell cups with warmed PBS and careful cutting of the embryo with the environment ITSELF. Out of 12 eggs, processed, each of RB, 6 treated TIE-2-RB and 5 treated EHK-1-RB embryos developed further stage observed at the beginning of the experiment. A dramatic difference was observed between the developed embryos, as shown in Fig.1A and 1B. Embryos treated EHK-1-RB, apparently, fu is rdca. In addition, unambivalent vascular network, visually detectable due to the presence of erythrocytes, was plentiful and stretched a few inches under lateral HIMSELF. In contrast, embryos treated with TIE-2-RB, were strongly retarded in growth, with 2-5 mm in diameter compared with the diameter of >10 mm for treated EHK-1-RB embryos. All treated TIE-2-RB embryos were dead and did not contain blood vessels. The ability to TIE-2-RB to block the development of blood vessels in chick embryos showed that the TIE-2 ligand is necessary for the development of the vascular network.

EXAMPLE 4. Identification of TIE-2-specific binding activity in conditioned medium from mouse line myoblastic cells SS transformed rac-oncogene

Screening of 10-fold concentrated cell-conditioned media (10SMS) from various cell lines in the presence of soluble TIE-2-specific binding activity (BIAcore; Pharmacia Biosensor, Pitcataway, NJ) showed binding activity in serum-free medium from cells SS transformed by oncogenic ras (C2C12-), cells RAT 2-rac (which are ras-transformed line fibroblast the p>

10SMS C2C12-came from a stable transtitional line of cultured myoblasts SS that oncogene transformed by transfection of a mutant T-24 H-ras standard calcium phosphate methods. Expressing plasmid resistance to neomycin based on SV40 physically connected with expressing ras plasmid for selection of transfected clones. Obtained G418 resistant cells ras-C2C12 routine was maintained in the form of a monolayer on plastic cups in DMEM/glutamine/penicillin-streptomycin, supplemented with 10% fetal calf serum (FCS). Serum-free medium 10SMS with C2C12-were prepared by seeding cells at 60% of confluently in serum-free defined medium and cultivation within 12 hours [Zhan and Goldfarb, Mol. Cell. Biol. 6:3541-3544 (1986); Zhan, et al., Oncogene 1:369-376 (1987)]. This medium was discarded and replaced with fresh DMEM/Q/P-S and were cultured for 24 hours. This medium was collected and cells were re-filed fresh DMEM/Q/P-S, which also collected after another 24 hours. These SMM supplemented with protease inhibitors PMSF (1 mm) and Aprotinin (10 μg/ml) and then concentrated 10-fold in sterile gel-filtration membranes (Amicon). TIE-cation with EHK-1-RB, before BIAcore analysis.

Binding activity of 10The SMS was measured using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ), which exposes monitoring biomolecular interactions in real time through the surface plutonomy resonance. Purified TIE-2-RB covalently linked via primary amines with a layer of carboxymethylcysteine touch crystal TM research grade (Pharmacia Biosensor, Piscataway, NJ). The surface of the touch crystal activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC), followed by immobilization TIE-2-RB (25 μg/ml, pH 4.5) and deactivation of unreacted sites 1,0 M ethanolamine (pH 8.5). Negative control surface ENK-1-receptor-antibodies was obtained in a similar manner.

Used in this system, the buffer of the mobile phase was HBS (10 mm Hepes, 3.4 mm EDTA, 150 mm NaCl, of 0.005% surfactant P20, pH 7.4). Sample 10 x SMS centrifuged for 15 minutes at 4And then osvetleni using sterile connecting low-molecular-weight protein of 0.45 µm filter (Millipore; Bedford, MA). To each sample SMM added dextran (2 mg/ml) and surfactant P20 (0,005%). Aliquot 40 ál of the W receptor was observed within 8 minutes. Activity binding (resonance units, D) was measured as the difference between the background value determined for 30 s before the injection of the sample, and the measurement performed within 30 seconds after injection. Regeneration of the surface was performed one 12 ál-pulse 3 M MgCl2.

The noise level of the device is equal to 20 in D; thus, any activity of binding to a signal above 20 D can be interpreted as a real interaction with the receptor. For SC-air-conditioned environments binding activity were in the range of 60-90 D for the surface with immobilized TIE-2-RB. For the same samples tested on the surface with immobilized EHK-1-RB measured activity were less than 35 RE. Specific binding to TIE-2-receptaculum was assessed by incubation of the samples with an excess of either soluble TIE-2-, or EHK-1-RB before testing activity linking. Adding soluble EHK-1-RB did not affect the activity of the binding TIE-2 any of the samples, whereas in the presence of soluble TIE-2 binding to the surface is 2/3 smaller than when measured in the absence of TIE-2. Re-test with the use of >50concentrated C2C12-CMM gave a 4-fold increase over fonolibro tyrosine TIE-2 receptor

C2C12-10CMM was tested for its ability to induce phosphorylation of tyrosine TIE-2 in the cells of AWE. Starving in serum-free medium cells AWE briefly incubated with C2C12-CMM, literally and subjected to thus and Western analysis as described above. Stimulation of starving cells of AWE serum-free medium C2C12-10CMM was performed as follows. The environment of the cells AWE, starving as described above, were removed and replaced or specific environment, or 10CMM, which was preheated to 37C. After 10 minutes the medium was removed and cells washed twice in ice excess of chilled PBS, supplemented by orthovanadate/NF/benzamidine. Lysis of cells and TIE-2-specific immunoprecipitation performed as described above.

Cells AWE, incubated for 10 minutes with a specific environment, found no induction of phosphorylation of tyrosine TIE-2, whereas incubation with C2C12-CMM stimulated at least 100 times phosphorylation of TIE-2. This activity is almost entirely East and room temperature with 13 μg TIE-2-RB, associated with the pellet protein G-Sepharose. Wednesday, incubated with protein G-Separate, did not make this fosforiliruyusciye activity.

EXAMPLE 6. Expression cloning of a TIE-2 ligand

Cells COS-7 were cultured in modified according to the method of Dulbecco environment Needle (DMEM) containing 10% fetal calf serum (FCS), 1% each of penicillin and streptomycin (P/S) and 2 mm glutamine in an atmosphere of 5% CO2. Murine cell line cultured myoblasts SSwere cultured in minimum essential medium Needle (EMEM) with 10% FCS, P/S) and 2 mm glutamine. The full-size cDNA clones of mouse TIE-2 ligand was obtained by screening the cDNA library SSin the vector pJFE14 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). The library was obtained using two BSTX1 restriction enzymes cut sites in the vector pJFE14.

Cells COS-7 was temporarily transfusional or library pJFE14, or a control vector according to the Protocol of DEAE-dextran-transfection. Briefly, cells COS-7 were sown at a density of 1.0106cells/100-mm Cup for 24 hours before transformation μm 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 was aspirated and replaced with PBS with 10% DMSO for 2-3 minutes. After that, DMSO-shocked cells COS-7 were placed in DMEM with 10% FCS, 1% each of penicillin and streptomycin and 2 mm glutamine and were cultured for 48 hours.

As a TIE-2 ligand is secreted, it was necessary to make the cells permeable for detecting binding of the probe receptarea with ligand. 2 days after transfection cells were washed in PBS and then incubated with PBS containing 1.8% formaldehyde, for 15-30 minutes at room temperature. Then cells were washed in 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 was performed by direct localization of staining with the use of TIE-2-receptical (RB), which consists of the extracellular domain of TIE-2, fused with the constant region of IgG1. This receptarea was obtained as described in Example 2. A Cup with a diameter of 100 mm, with transfitsirovannykh, fixed and permeable cells COS - probed by incubating them for 30 minutes with TIE-2-RB. Then tile three washes with PBS cells were incubated in the substrate for alkaline phosphatase within 30-60 minutes. The cap is then viewed under a microscope to detect the presence of stained cells. For each stained cells a small area of cells, including stained cells were viscerale from a Cup with the tip of a plastic pipette and then was isolated plasmid DNA, and used for electroporation of bacterial cells. Individual bacterial colonies obtained by electroporation, viscerale, and plasmid DNA obtained from these colonies were used for transfection of cells COS-7, which was probed for the expression of TIE-2 ligand to be detected by binding to TIE-2-receptorrelated. This allowed us to identify individual clones encoding TIE-2 ligand. Confirmation of expressions TIE-2 ligand was obtained by phosphorylation of TIE-2 receptor using the method described in Example 5. Plasmid clone encoding TIE-2 ligand, was deposited in ATSC October 7, 1994 and designated as "pJFE14 encoding TIE-2 ligand", under the access number of ATSS Accession No. 75910.

EXAMPLE 7. The selection and sequencing of the full-size cDNA clone encoding a TIE-2 LIGAND man

A cDNA library of the lung of a human embryo in lambda gt-10 (see Fig.3) was obtained from Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were sown at a density of 1.25

The allocation of human clones-2-ligand was carried out as follows. The XhoI fragment of 2.2, etc., N. from the deposited clone-2-ligand (ATSS No. 75910, see Example 6 above) were labeled by random praimirovanie to a specific activity of approximately 5108pulse/min/ng. Hybridization was carried out at 65With the solution for hybridization, containing 0.5 mg/ml DNA salmon sperm. Filters were washed at 65With 2SSC, 0.1% of LTOs and exhibited on film Kodak XAR-5 overnight at -70C. Positive phage was purified by titration of the plaques. Lysates of pure phage with high titer phage was used for DNA extraction using Qiagen columns using standard methods (Qiagen, Inc., Chatsworth, CA, 1995 catalog, page 36). The phage DNA was digested EcoRI to release the cloned cDNA fragment for subsequent sublimirovanny. Vector-based phage lambda containing human DNA-2-ligand, were deposited in ATSC October 26, 1994, entitled "gt10, cognosci DNA method dideoxyadenosine chain (Sanger, et al., 1977, Proc. Natl. Acad. Sci. USA, 74: 5463-5467).

Subclavian human DNA-2-ligand in expressing vector mammal can be performed as follows. Clonegt10 encoding htie-2 ligand 1 contains the EcoRI site located at a distance of 490 p. N. during transcription from the start of the coding sequence of human TIE-2 ligand. Coding region can be carved using unique restriction sites against the course and in the course of transcription from the initiation codon and the stop codon of transcription, respectively. For example, to clip the full coding region can be used SpeI site, located at 70 p. N. 5' relative to the initiation codon, and the website Bpu1102i (also known as VR), located at 265 p. N. 3' relative to the stop codon. Then the area can be subcloned into the cloning vector pJFE14 on sigam XbaI (complementary SpeI) and > PST websites > PST and Bpu1102i previously a small mistake).

Coding region htie-2 ligand 1 from clonegt10 sequenced using DNA sequencing machine (ABI A and sequencing Taq Dideoxy Terminator Cycle (Applied Biosystems, Inc., Foster City, CA). Nucleotide and corresponding amino acid placenta is shown in Fig.4.

In addition, the full-size cDNA clones htie-2 ligand 1 were obtained by screening the cDNA library glioblastoma T98G human vector pJFE14. Clones encoding TIE-2 ligand man, identified DNA hybridization using XhoI fragment of 2.2, etc., N. from the deposited clone tie-ligand 2 (ATSS No 75910) as a probe (see Example 6 above). Coding region sequenced using DNA sequencing machine (ABI A and sequencing Taq Dideoxy Terminator Cycle (Applied Biosystems, Inc., Foster City, CA). This sequence was almost identical sequence of clonegt10 encoding htie-2 ligand 1. As shown in Fig.4, clonegt10 encoding htie-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 residue of glycine, but otherwise identical to coding sequence of clonegt10 encoding htie-2 ligand 1. Fig.5 shows the nucleotide and corresponding amino acid sequence of human TIE-2 ligand from T98G clone.

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

The cDNA library easy (EME) src="https://img.russianpatents.com/chr/215.gif">106/Cup 2020 cm and filter replicas were obtained according to standard procedures (Sambrook et al.; (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The filters in the two surfaces were subjected to screening in a relatively mild conditions (2SSC, 55C) using probes prepared for the sequence TIE-2 ligand person. Some of the filter-duplicates were analyzed using the 5'probe, encoding amino acids 2-265 TIE-2 ligand person, as shown in Fig.4. The second filter is a duplicate was analyzed using the 3'probe, encoding amino acids 282-498 sequence TIE-2 ligand (see Fig.4). Both probes hybridized at 55With the solution for hybridization, containing 0.5 mg/ml DNA salmon sperm. Filters were washed in 2SSC at 55With and exposed overnight to x-ray film. In addition, filters duplicate hybridized also with normal severity (2SSC, 65C) with full coded probe of mouse TIE-2 ligand 1 (F3-15, XhoI insert). Was viscerale three positive clones that met SL is (i) hybridization was observed at low stringency with probes 5' and 3'. The splitting of the EcoRI DNA phage obtained from these clones showed two independent clones with insert sizes of approximately 2,2 T. p. N. and approximately 1.8 T. p. N. Inserting EcoRI 2,2, etc. ad was subcloned into the EcoRI sites as pBluescript KS (Stratagene), and expressing the vector mammal suitable for use in COS cells. For expressing the vector of mammal were discovered two orientations. Insert 2,2, etc. ad in pBluescript KS was deposited in ATSC 9 December 1994 and named pBluescript KS encoding a TIE-2 ligand 2 people. Starter website coding sequence TIE-2 ligand 2 is approximately 355 p. N. during transcription from the EcoRI site pBluescript.

Cells COS-7 transfusional or expressing vector or a control vector according to the Protocol of DEAE-dextran-transfection. Briefly, cells COS-7 were sown at a density of 1.0106cells/100-mm Cup for 24 hours before transfection. For transfection, these cells were cultured in serum-free DMM containing 400 μg/ml DEAE-dextran, 1 μm 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 was aspirated and replaced with PBS 10% is of CIN and 2 mm glutamine and were cultured for 48 hours.

As a TIE-2 ligand is secreted, it was necessary to make the cells permeable for detecting binding of the probe receptarea with ligand. Transfetsirovannyh cells COS-7 were sown at a density of 1.0106cells/Cup 100 mm Cells were washed in PBS and then incubated with PBS containing 1.8% formaldehyde, for 15-30 minutes at room temperature. Then cells were washed in 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 was performed by direct localization of staining with the use of TIE-2-receptical, which consisted of the extracellular domain of TIE-2, fused with the constant region of IgGl. This receptarea was obtained as described in Example 2. Transfetsirovannyh cells COS-7 was probed by incubating them for 30 minutes with TIE-2-receptaculum. Then cells were washed twice in PBS, fixed with methanol and then incubated for another 30 minutes with PBS/10% FCS/conjugate human IgG with alkaline phosphatase. After three washes with PBS cells were incubated in the substrate for alkaline phosphatase within 30-60 minutes. The cap is then viewed under a microscope for detecting the current orientation of the clone, bind the TIE-2-recepticle.

The person skilled in the art will understand that the described methods can be used to further identify other related members of the family TIE-ligands.

Coding region of the clone pBluescript KS encoding TIE-2 ligand 2 people, sequenced using DNA sequencing machine AS A and sequencing Tag Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc. Foster City, CA).

Nucleotide and corresponding amino acid sequence of human TIE-2 ligand from the clone pBluescript KS encoding TIE-2 ligand 2 persons, shown in Fig.6.

EXAMPLE 9. TIE-2 ligand 2 is a receptor antagonist

Conditioned medium from COS cells expressing either TIE-2 ligand 2 (TL2) or TIE-2 ligand 1 (TL1), were compared for their ability to activate TIE-2 receptors, present in natural endothelial cell lines human.

Lipofectamine reagent (GIBCO BRL, Inc.) and recommended protocols used for transfection of cells COS-7 or one expressing vector pJFE14, vector pJFE14 containing cDNA TIE-2 ligand 1 person, or expressing vector RMT (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693) containing cDNA TIE-2 ligand 2 people. COS-containing media of secretarian the DIAFLO, Amicon, Inc.). The number of active TIE-2 ligand 1 and TIE-2 ligand 2 present in these environments was determined and expressed as the number (in resonance units, RE) activity of TIE-2 receptor-specific binding measured in the test BIAcore binding.

Northern (RNA) analysis revealed significant levels of TIE-2 transcripts in endothelial cells, NES (Human Aortic Endothelial Cell) (Clontech, Inc). Thus, these cells were used to study whether the TIE-2 receptor phosphorylated at tyrosine in the exposure environments COS containing TIE-2 ligands. Cells NAES maintained in complete medium for cultivation of endothelial cells (Clontech, Inc.), which contained 5% fetal calf serum, soluble extract of bovine brain, 10 ng/ml human EGF, 1 mg/ml hydrocortisone, 50 mg/ml gentamicin and 50 ng/ml amphotericin-Century Assessment of whether TL1 and TL2 to activate TIE-2 receptor in the cells of NAES performed as follows. Polyethlyene cells NAES maintained in serum-free medium (starvation) for 2 hours in an environment of Dulbecco MEM high glucose with added L-glutamine and penicillin-streptomycin, at 37Since then replace medium to fast landamerica condie the entrances were literally and TIE-2 receptor protein was extracted by immunoprecipitating lysates by anticorodal against TIE-2-peptide, exactly as described in Example 1. The results are presented in Fig.7. Levels of phosphotyrosine on TIE-2 receptor (TIE-2-R) were induced by treating the cells with COS environments, air-conditioned TIE-2 ligand 1 (Lane L1), but not TIE-2 ligand 2 (Lane L2). MOCK represents the conditioned medium from cells COS-7, transfected with the empty vector pJFE14.

Proof that as TL1 and TL2 specifically associated with the TIE-2 receptor, has been demonstrated using BIAcore to determine the activities of the TIE-2 receptor-specific binding in transfected COS-environments and immunoablative expressing TL1 and TL2 cells COS TIE-2-receptorrelated.

Since TL2 does not activate the TIE-2 receptor, the applicants decided to determine whether TL2 to be able to serve antagonist activity TL1. Performed tests of phosphorylation cells NAES, in which cells are first incubated with the "excess" TL2, and then added divorced TL1. This is justified by the fact that the primary occupation of the TIE-2 receptor due to high levels TL2 may prevent the subsequent stimulation of the receptor after exposure in TL1 present in limiting concentration.

Polyethlyene cells NAES were in serum-free medium (halcyonidae 20COS/pJFE14-TL2-environment. Control cups were processed 20COS/pJFE14 - only medium (MOCK). Cups were removed from thermostat and then added different cultivation environments COS/pJFE14-TLl, followed by further incubation cups for 5-7 minutes at 37C. the cells are Then washed, literally and TIE-2-specific tyrosine phosphorylation in lysates was determined by immunoprecipitating receptor and Western blotting as described above. Breeding TL1 performed by using COS/pJFE14-TLl, diluted to 2, 0.5×, 0.1 or 0.02 x x adding environment 20COS/pJFE14 - only. The analysis of the original 20x TL1 and 20x TL2 COS-environments using a biosensor BIAcore technology showed that they contained the same number of activities TIE-2-specific binding, i.e., 445 D and 511 D for TL1 and TL2, respectively. Results antibodies against phosphotyrosine Western-blotting, as shown in Fig.8 shows that when compared with pre-treatment of cells NAES environment MOCK (lane 1) pre-treatment of cells NAES excess TIE-2 ligand 2 (lane 2) counteracts (has antagonistic action) the subsequent ability diluted TIE-2 ligand 1 to activate the TIE-2 receptor (TIE is of a hybrid cell line human EA.Hy926 (see Example 21 for a detailed description of this cell line and its maintenance). Conducted experiments in which non-concentrated environment for COS cells containing L1, was mixed with various dilutions or MOCK environment, or the environment, air-conditioned TL2, and was placed on monolayers starving (in serum-free medium) cells EA.hy926 for 5 minutes at 37C. Then the medium was removed, cells were harvested by lysis and TIE-2-specific tyrosine phosphorylation was investigated using Westernblot, as described above. Fig.9 shows an experiment that contains three groups of treatments, when considering from left to right. As shown in the four tracks on the left, the cell treatment with EA.hy926 one environment lx COS-TL1 strongly activated by endogenous TIE-2-R in these cells, whereas lx TL2 COS-Wednesday was inactive. However, a mixture of TL1 either MOCK or TL2 showed that TL2 may block the activity TL1 dependent on dose. In the Central of the three pairs of tracks relation TL2 (or MOCK) were decreased, while the number of TL1 in the mixture, respectively, was increased from 0.1 x 0.3 X. If any of these relations mixtures track TL1:TL2 showed a reduced level of phosphorylation of TIE-2-R compared with the level of phospho what about TL2 (or MOCK) decreased (shown in three pairs of tracks to the right), has reached the point at which the TL2 in the sample was too diluted for effective inhibition activity TL1. The relative amount of each ligand present in these air-conditioned COS environments, could be estimated from their units binding, measured by BIAcore analysis, or Western blots of COS environments with ligand-specific antibodies. Thus, we can conclude that for effective blocking activity L1 in vitro requires only a small molar excess (several times) TL2. This is important because we observed some examples of in vivo (see Example 17 and Fig.16), in which mRNA TL2 reach significant amounts relative to the amounts of TL1. Thus, TL2 may play an important physiological role in the effective blocking of signal transmission TIE-2-R in those sites.

Taken together, these data confirm that in contrast to TL1, TL2 unable to stimulate endogenous expressed TIE-2-R on endothelial cells. In addition, when a small (very small number of times) molar excess TL2 may block the stimulation TL1 TIE-2 receptor, which suggests that TL2 is a naturally occurring antagonist of TIE-2 receptor.

EXAMPLE 10. Identification of TIE-2-specific binding of the asset is/img> SMS from cell lines C2C12-, Rat2, SHEP and T98G or supernatants COS cells after transfection with either TIE-2 ligand 1 person (hTL1) or TIE-2 ligand 2 (hTL2) was measured using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ), which carries out monitoring of biomolecular interactions in real time through the surface plutonomy resonance (SPR). Purified rat or human TIE-2-RB covalently linked via primary amines with a layer of carboxymethylcysteine touch crystal TM research grade (Pharmacia Biosensor, Piscataway, NJ). The surface of the touch crystal activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethyl-aminopropyl)carbodiimide (EDC), followed by immobilization TIE-2-RB (25 μg/ml, pH 4.5) and deactivation of unreacted sites 1,0 M ethanolamine (pH 8.5). As a rule, 9000-10000 RE each receptor antibodies was associated with touch crystal.

Used in this system, the buffer of the mobile phase was HBS (10 mm Hepes, 150 mm NaCl, of 0.005% surfactant P20, pH 7.4). Samples were centrifuged for 15 minutes at 4And then osvetleni using sterile connecting low-molecular shall esto P20 (0,005%). Aliquots of 40 μl were injected with through the surface with immobilized proteins (either mouse or human TIE-2) when the speed of the current 5 ál/min and receptor binding was observed within 8 minutes. Activity binding (resonance units, D) was measured as the difference between the background value determined for 30 s before the injection of the sample, and the measurement performed within 30 seconds after injection. Regeneration of the surface was performed one 15 µl-pulse 3 M MgCl2.

SMS-samples (C2C12-, Rat2, SHEP and T98G) were tested on the surface with immobilized mouse TIE-2-RB, whereas recombinant hTLl and hTL2 tested on the surface with immobilized human TIE-2-RB. In each case, specific binding to TIE-2-receptaculum was assessed by incubation of the samples with 25 μg/ml of either soluble TIE-2-RB (rat or human) or trkB-RB before testing activity linking. As shown in Fig.10 and 11, the addition of soluble trkB-RB causes a small reduction in TIE-2-binding activity, whereas the addition of soluble TIE-2-RB significantly reduces binding activity compared to the activity measured in the absence of TIE-2-RB.

EXAMPLE 11. TIE-2-RB specifically b is este competitive inhibitor to block the TIE-2 receptor TIE-2 ligand 1 (TL1). To do this, TLl-containing COS-environment was preincubator or TIE-2-RB, or TrkB-RB and then compared their ability to activate TIE-2 receptors, present in the natural line of human vascular endothelial cells.

Air-conditioned COS-environment received from the cells COS-7, transfected or one expressing vector pJFE14 (MOCK) or vector pJFE14 containing cDNA TIE-2 ligand 1 (TL1) person, and collected as described in Example 9 above, except that the medium was sterile filtered, but not focused. The number of TL1 was determined and expressed in the form (in resonance units, D) TIE-2 receptor-specific binding activity, measured test BIAcore binding.

Northern (RNA) analysis revealed significant levels-2-l transcripts in undifferentiated endothelial cells HUVEC (Human Umbilical Vein Endothelial Cell, endothelial cells, umbilical vein (Clontech, Inc.). Thus, these cells were used to study whether the TIE-2 receptor can be phosphorylated on tyrosine upon exposure in the presence of TIE-2-RB or TrkB-RB with environments COS containing TL1. The HUVEC cells were maintained at 37C, 5% CO2in a complete environment for the cultivation of endothelial bleeprin, 10 ng/ml human EGF, 1 μg/ml hydrocortisone, 50 μg/ml gentamicin and 50 ng/ml amphotericin-Century Assessment of whether TL1 activate TIE-2 receptor in HUVEC cells was performed as follows. Confluently Cup HUVEC were starving for 2-4 hours in the environment of Dulbecco MEM high glucose at 37With subsequent 10-minute incubation in the medium for fasting, containing 0.1 M ortho-Vanadate sodium, a potent inhibitor phosphotyrosine. Meanwhile, air-conditioned COS-environment was preincubator 30 minutes at room temperature with either TIE-2-RB, or Trkb-RB added to 50 µg/ml Then the environment for starvation removed from HUVEC cups and cups were incubated with RB-aderasa COS environment for 7 minutes at 37C. Then cells HUVEC were literally and TIE-2 receptor protein was extracted by immunoprecipitating anticorodal against TIE-2-peptide with subsequent Western blotting with antibody against phosphotyrosine as described in Example 1. The results are presented in Fig.12. Levels of phosphotyrosine on TIE-2 receptor were induced by treating the cells with HUVEC TIE-2 ligand 1 (TL1) relative to the levels observed with the control medium (MOCK), and this induction is specifically blocked predicability as a selective inhibitor to block the activation of TIE-2 receptor TIE-2 ligand 1.

EXAMPLE 12. Design of TIE-2-liganded

Was created in expression design, which can give the secretory protein consisting of the full coding sequence TIE-2 ligand 1 (TL1) of a person or TIE-2 ligand 2 (TL2) person, fused with the constant region of immunoglobulin gamma-1 man (IgG-Fc). These fused proteins called TIE-2-"legendele" (TLl-Fc or TL2-Fc). Fc-part TLl-Fc and TL2-Fc was prepared as follows. The DNA fragment encoding the Fc-portion of human IgG1, which extends from the hinge region to carboxilic of this protein, amplified from placental cDNA person using PCR with oligonucleotides corresponding to the published sequence of human IgG1; the resulting DNA fragment was cloned in the plasmid vector. Appropriate restriction fragments of DNA from a plasmid that encodes a full TL1 or TL2, and containing the human IgG1 Fc plasmids ligated on each side of the short received by means of the PCR fragment, which was designed in such a way that it could be drained in reading frame sequence encoding TL1 or TL2, protein-Fc of human IgG1.

The number of milligrams order TL2-2-FC was obtained by cloning a DNA fragment TL2-Fc in Bacelona to use the cell line SF-9 (ATCC Accession No. CRL-1711) or cell line BTI-TN-5bl-4. DNA encoding TL2-2-Fc, cloned in the form of Eco RI-NotI fragment in baculovirus transport plasmid pVL1393. Plasmid DNA recombinable into viral DNA by mixing 3 μg of plasmid DNA with 0.5 μg Baculo-Gold DNA (Pharminigen) with subsequent introduction into liposomes using 30 µg Lipofectin (GIBCO BRL). A mixture of DNA-liposomes were added to the cells SF-21AE (2106cells on 60 mm Cup) in TMN-FH (Modified atmosphere Grace for insect cells (GIBCO BRL) for 5 hours at 27C, followed by incubation at 27C for 5 days with medium TMN-FH, supplemented with 5% fetal calf serum. Environment for tissue culture were collected for purification plaques of recombinant viruses, which was performed using methods previously described (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 covering layer of agarose contained 125 μg/ml X-gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside; GIBCO-BRL). After 5 days incubation at 27With non-recombinant plaques were evaluated in points using positive chromogenic reaction with X-gal is a substrate and powergamer 100 µg/ml MTT (bromide 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; Sigma). Estimated plaques of recombinant virus was removed by suction tubes and cleaned numerous cycles of selection plaques to ensure homogeneity. The baseline of the virus was obtained by serial passage with a low multiplicity of infection cleaned from virus plaques. Received source line with a low number of passages of a single viral clone (vTL2-Fc Clone # 7).

Cells SF-21AE were cultured in serum-free medium (SF-900 II, Gibco BRL) containing 1a solution of the antibiotic/fungicide (Gibco BRL) and 25 mg/ml Gentamicin (Gibco BRL). Pluronic A-68 (block copolymers of polyoxyethylene and polyoxypropylene) was added as a surfactant to a final concentration of 1 g/l of Culture (4 l) were grown in the bioreactor (Artisan Cell Station System) for at least three days before infection. Cells were grown at 27With passing of gas up to 50% dissolved oxygen at the speed of current gas 80 ml/min (for aeration in bubbling fluidised bed ring). The stirring was conducted marine stirrer at a speed of 100 rpm, the Cells were collected in the phase of mid-logarithmic growth (~2106cells/ml), concentrated by centrifugation, and infected with 5 plaque-forming edinienie 2 hours at 27With in a rotating flask. Then this culture resuspendable in the final volume of 8 liters of fresh serum-free medium and cells were incubated in the bioreactor using the above-described conditions.

Culture medium from infected fused protein vTL2-Fc SF21AE cells were collected by centrifugation (500g, 10 minutes) at 72 hours after infection. Cellular supernatant brought to pH 8 with NaOH. Added EDTA to a final concentration of 10 mm and the pH of the supernatant is re-conveyed to 8. Supernatant was filtered (0.45 μm, Millipore) and applied to a protein A-column (protein A sepharose 4 fast flow or HiTrap protein A, both from Pharmacia). The column was washed in PBS containing 0.5 M NaCl until the absorbance at 280 nm was reduced to background. The column was washed in PBS and were suirable 0.5 M acetic acid. Fractions after elution from the column was collected in tubes containing 1 M Tris pH 9 for neutralization. The fraction of peaks containing protein TL2-FC United and were dialyzed against PBS.

EXAMPLE 13. Expression of TIE-1, TIE-2, L1 AND TL2 in kidney cancer

Experiments on in situ hybridization was performed on tumor tissue of kidney cancer man using cDNA probes TIE-1, TIE-2, TL1 And TL2. Expression of TIE-1, TIE-2, TL1 And TL2 negatively regulated in the vascular network of apogolize from endothelial cells of blood vessels in mesenchyme (TL1). It has been shown that VEGF dramatic negatively regulated in this tumor tissue. Brown, et al., Am. J. Pathol. 143:1255-1262 (1993).

EXAMPLE 14. Expression of TIE-1, TIE-2, YOU AND TL2 in the healing of wounds

Experiments on in situ hybridization was performed on cross sections of tissue obtained from a rat model of skin wound, using cDNA probes TIE-1, TIE-2, TL1 and TL2. Model of wound healing involves pressing a small mirror drills to the skin of rats and removing a small cylindrical tube of skin. As healing begins at the base of the wound, is a vertical section through the fabric and is used for in situ hybridization. In the test tissue sample TL1 and TL2, apparently, have a small negative regulation at 4 days after their imperfections. In contrast to this small negative regulation of expression of TL1 and TL2 in this tissue, the expression of VEGF, which may precede the expression of TL1 and TL2, dramatic negatively regulated.

EXAMPLE 15. Expression of TIE-ligands in the liver and thymus of the embryo

PCR using reverse transcriptase (RT-CR) was performed on the liver of the mouse embryo E14.5 and thymus of E17 mouse embryos.5. The agarose gel electrophoresis of the products of RT-PCR found that in the liver of the mouse embryo district Strom is the same tissue stromal cells enriched RNA TIE-2 ligand 2 (TL2), but TL2 is not in hematopoietic cells precursors (Fig.13). In the thymus of the mouse embryo stromal cells enriched TL2 (Fig.14).

EXAMPLE 16. System TIE-receptor/ligand angiogenesis

Although the system TIE-2/TIE-ligand, apparently, plays an important role in the biology of endothelial cells, has not been shown that it plays a significant active role in the early-intermediate stages of the formation of new blood vessels (for example, proliferation and migration of angioblasts or endothelial cells, the formation of tubules and other events early stage of the formation of blood vessels). In contrast to the receptors and factors known what they mediashout these aspects of the development of blood vessels, the later expression of TIE-2 and L1 during the formation of new blood vessels suggests that this system plays a special role in later stages of development of the blood vessels, including structural and functional differentiation and stabilization of new blood vessels. The nature of the expression of TIE-2/TL1 is also consistent with the ongoing role in maintaining the structural integrity and/or physiological characteristics of a developed vascular network.

TIE-ligand 2 (TL2), apparently, is a competitive inhibitor of TL1. Protrans the t to play multiple roles, essential for the appropriate development or re-modeling (correction) vessels (for example, destabilization/dedifferentiate Mature endothelial cells, making possible the formation of new blood vessels from an existing vascular network, inhibiting unwanted formation of blood vessels and regression (reverse development)/involution Mature blood vessels).

Fig.15 is a schematic depiction of a hypothetical role of TIE-2/TIE-ligands in angiogenesis. In this drawing TL1 represented as (), TL2 represented as (*), TIE-2 is represented as (T), VEGF is represented as ([])and flk-1 (VEGF receptor) is represented as (Y).

EXAMPLE 17. Expression of TIE-ligands in the female reproductive system: expression in the ovary

Preliminary observations made in experiments investigating the expression of the TIE receptor and TIE-ligands in the female reproductive system, consistent with the hypothesis that TL1 plays a role in the formation of new blood vessels, which over time should be for VEGF. The expression pattern TL2 is also consistent with antagonism of action TL1 and specific role in the reverse development of blood vessels. To confirm this can be investigated expression of relevant mRNA after experimental interslice aspects of the formation of new blood vessels/reverse development of blood vessels (for example, together with staining of endothelial cells, vascular stocks). Angiogenesis associated with the development of the follicle and formation of the corpus luteum (yellow body) in the ovaries certain stage of adult female rats or after induced ovulation in prepubertal animals was monitored using in situ hybridization. Fig.16 contains pictures of the slides in situ hybridization showing the temporal expression of TIE-2, TL1, TL2 and VEGF during the ovarian cycle [Column 1: early pre-ovulatory follicle; Column 2: pre-ovulatory follicle; Column 3: early yellow body; and Column 4: atleticheskii follicle; the Number And glossy area; Series: VEGF; Number: TL2; Series D: TL1; and the Number E: TIE-2 receptor]. These studies found that VEGF, TL1 and TL2 are expressed in spatial and temporal coordinates in accordance with the development and reverse development of the vascular network, specifically regarding the establishment of a vascular system, which is generated during the transformation of the ovarian follicle into the corpus luteum (CL).

Briefly, expression of VEGF is increased in the granulation layer of the follicle before it vascularization during luteinization (education yellow body). During the process of formation of CL higher levels of VEGF expression are seen in costaude moderately high and diffusely distributed in the developing yellow body. In contrast, significantly increased expression of TIE-2 ligand 1 occurs only late in the process of formation of the yellow body, after the formation of the primary vascular plexus. Later, expression TL1 noticeable around the yellow body is clear of the capillary network of the yellow body.

TL2 exhibits a more complex expression than VEGF or TL1. In the developing yellow body TL2 is expressed at higher levels ahead of the developing capillary plexus - between Central, not having vessels, district CL, where the expression of VEGF is the highest, and the most peripheral part of the CL, where the dominant is the expression of the TL1 and where the process of luteinization completed and the vascular system is the most Mature. TL2 is also expressed, apparently, at high levels in the follicular layer of large follicles that undergo atresia. While TL1 also noticeable in atleticheskij follicles, VEGF is not expressed in them.

The distribution of expression, described above, is most consistent with the role of VEGF in the initiation of angiogenesis, TL1, applicable late in the process - for example, in modeling and/or final stabilization of the vascular network.

In proiti (during the formation of the yellow body), and in areas that are not able to develop a new vascular network in the future have reverse development of blood vessels (atrioticheskie follicles). This suggests a more dynamic and complex role TL2, which will probably include the destabilization of the existing vascular network (re-development) or developing vascular network (required for dynamic simulation of forming new blood vessels).

EXAMPLE 18. Test bind receptical and test binding and competition ligands

Was developed quantitative cell-free test binding with two different forms for the detection of binding of the TIE-2-receptical or binding and competition ligands. In a variant binding receptical this test, the TIE-2 ligands (purified or partially purified; TL1 or TL2) is applied to the tablet ELISA. Then add receptarea in various concentrations, which binds to the immobilized ligand dependent on dose. At the end of 2 hours the excess receptical wash, then the number associated with the tablet, is detected using specific antibodies against human Fc, which is labelled alkaline phosphatase. Excess reporter antibody wash, then spend the reaction is of odometre. Fig.19 shows a typical curve of binding TIE-2-IgG. This test is used to assess the integrity of the TIE-2-IgG after injection in rats and mice. The test can also be used in the form of competitive analysis ligands, in which purified or partially purified TIE-ligands compete with the immobilized ligand for recepticle. TIE-2-ectodomain put on a tablet ELISA. Then Fc-labeled fragments of the fibrinogen-like domain of TIE-ligands (TLl-fFc and TL2-fFc) associated with this ectodomain and can be detected using the same antibody against human Fc as described above. Fig.20 shows an example of binding TLl-fFc with TIE-2-ectodomains. This version of the test can also be used to quantify levels TLl-fFc in serum or other samples. If simultaneously with TLl-fFc add unlabeled ligand (again, either peeled or unpeeled), then there is a competition between the labeled fragment of the ligand and a full-sized ligand. Full sized ligand may displace the Fc-labeled fragment and get the curve of competition.

EXAMPLE 19. Cell line EA.hy926 can be used as a reporter cell line for the activity of the ligand

EA.hy926 is a cell GI. atl. Acad. Sci. USA 80, 3734-3737 (1983)]. It was found that cells EA.hy926 Express significant levels of TIE-2 receptor protein with low basal levels of phosphotyrosine. The density at which cells EA.hy926 passedout before using them for receptor tests, and the degree of their confluences at the time of analysis can affect the abundance of TIE-2 receptor and relative indutsiruemoi in response to the processing of the ligand. By selecting the next mode for the cultivation of these cells cell line EA.hy926 can be used as a reliable system for the analysis of the activities of the TIE-2 ligands.

Cells EA.hy926 seeded at 1.5106cells in flasks T-75 (Falconware) and every second day served Wednesday, Dulbecco MEM high glucose, 10% fetal calf serum, L-glutamine, penicillin-streptomycin, and 1x gipoksantin/aminopterin/thymidine (HAT, Gibco/BRL). After 3-4 days of growth the cells passedout once again at 1.5106cells per flask T-75 and additionally cultivate within 3-4 days. For tests phosphorylation cells obtained as described above were subjected to serum starvation by replacing the culture medium DMEM with high content glucosteroids media or purified ligand was added to the flask in a total volume of 1.5 ml, followed by incubation at 37C for 5 minutes. The flask was removed from thermostat and put on a layer of ice. The medium was removed and replaced with 1.25 ml of buffer for lysis containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% of LTOs 6 20 mm Tris, pH of 7.6, 150 mm NaCl, 50 mm NaF, 1 mm ortho-Vanadate sodium, 5 mm benzamidine and 1 mm EDTA containing protease inhibitors PMSF, Aprotinin and leupeptin. After 10 minutes on ice to solubilize membranes Cup was viscerale and cell lysates were osvetleni microcentrifuge at high speed for 10 minutes at 4C. TIE-2 receptor was immunoprecipitated from the clarified supernatant by incubation in the cold with polyclonal anticorodal against TIE-2 and pellet Protein G-conjugated to Sepharose. Pellets were washed three times with cold buffer for lysis of cells and boiled for 5 minutes in sample buffer Laemmli and then were loaded on a 7.5% LTOs-polyacrylamide gels. Separated proteins transferred by electroblotting on a PVDF-membrane (Lamblia-P) and then subjected to Western blot analysis using antibodies against phosphotyrosine and ECL reagent. Subsequent comparison of the total levels of TIE-2 protein on the same blots were performed by detaching the antibodies against phosphotyrosine ("strip") and re-incubation of full-size cDNA clone encoding TIE-2 ligand-3 mammal

TIE-2 ligand-3 (TL3) cloned from the genomic library mice YOU (Research Genetics) by hybridization of duplicate library with mouse TLl-probe or mouse L2-probe corresponding to the complete coding sequence of these genes. Each copy of this library hybridized with the use of phosphate buffer at 55With during the night. After hybridization the filters were washed with 2SSC, 0.1% of LTOs at 60With subsequent exposure of the filters to x-ray film. Were found a strong hybridization signals corresponding to mouse TL1 and mouse TL2. In addition, the identified signals that are slightly hybridities as mouse TL1 and mouse TL2. DNA corresponding to these clones were purified, then digested with restrictase and two fragments, which were hybridisable with the original probe was subcloned into the bacterial plasmid and sequenced. The sequence of these fragments contained two exons with homology relative to the mouse TL1 and mouse TL2. To identify tissue containing transcripts corresponding to TL3, the primers used were specific for this sequence the mouse uterus in lambda gt-11. (Clontech Laboratories, Inc., Palo Alto, CA).

Plaques were sown at a density of 1.25106/Cup 2020 cm and filter replicas were obtained according to standard procedures (Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Filters in two replicates were subjected to screening at "normal" severity (2SSC, 65C) 200 p. N. radioactive PCR probe, prepared for the sequence TL3 mouse. Hybridization was carried out at 65C in a solution containing 0.5 mg/ml DNA salmon sperm. Filters were washed in 2SSC at 65With and exposed for 6 hours on x-ray film. Two positive clones, which were hybridities in two replications, visceralis. The splitting of the EcoRI DNA phage obtained from these clones showed two independent clones with the size of the inserts of approximately 1.2 T. p. N. and approximately 2.2, etc., ad Insertion EcoRI 2,2, etc. ad was subcloned into the EcoRI sites pBluescript KS (Stratagene). The sequence analysis showed that this longer the clone had the initiator methionine codon and signal peptide, but otherwise it encodes the probe, homologous as m is agtttgttagg

US1: ccagctggcagatatcagg

The following PCR reaction was performed using gene-expression libraries derived from murine cell lines C2C12ras and MG87. In the primary PCR reactions specific primer US2 used together with the vector-specific oligonucleotides to create opportunities amplification in any orientation. PCR was performed in a total volume of 100 ml using 35 cycles of 94C, 1 min; 42With or 48C for 1 min; 72C, 1 min. the Second PCR reaction included a second specific primer, US1, which was contained in the product of the primary PCR, together with the same oligonucleotides. Secondary reactions were 30 cycles, using the same temperature and time periods mentioned earlier. On the basis of sequences obtained from a total of four independent PCR reactions using two different libraries cDNA was transcribed 5'-end sequence TL3. Northern analysis revealed moderate to low levels of transcript mouse TL3 in the rat placenta. Expression of mouse TL3 gave a transcript of ~3 M. p. H. the coding sequence of Full-TL3 shown in Fig.21.

Then the mouse follow alnost human TL3, by hybridization of either genomic or cDNA library of a man with a probe corresponding to the mouse TL3, as described earlier, for example, in Example 8, supra.

EXAMPLE 21. The allocation of full-genoino clone encoding TIE-2 ligand-4 man

TIE-2 ligand-4 (TL4) cloned from the genomic library mice YOU (YOU are HUMAN (II), Genome Systems Inc.) by hybridization of duplicate libraries or radioactive probe TL1 person corresponding to the complete coding fibrinogen sequence TL1 (nucleotides 1153-1806 Fig.4) or with radioactive probe TL3 mouse, the corresponding segment of the 186 nucleotides from the region of the fibrinogen mouse TL3 (nucleotides 1307-1492 Fig.21). Each probe was labeled using PCR using specific oligonucleotides and standard PCR conditions, except that the dCTP was replaced by32P-dCTP. Then, the PCR mixture was passed through a gel filtration column to separate the probe from the free32P-dCTP. Each copy of this library hybridized with the use of phosphate buffer and radioactive probe at 55With overnight using standard hybridization conditions. After hybridization the filters were washed with 2SSC, 0.1% of LTOs at 55

The full sequence of the human TL4 can be obtained by sequencing a complete clone of YOU that is contained in the deposited bacterial cells. The exons can be identified by homology with known members of the family TIE-ligands, such: as TL1, TL2 and TL3. Can then be determined the complete coding sequence TL4 splicing together of exons from genomic clone TL4, which, in turn, can be used to obtain protein TL4. Alternative these exons can be used in I TL4 protein. The exons can be identified from the clone YOU homology with protein domains, such as domains of fibrinogen, twisted helical domains or protein signals, such as signal peptide sequence. Missing exons from the clone, YOU can be received by the close identification of clones of YOU, for example by using all of the deposited clone YOU as probes for screening genomic libraries of a person, such as used here, by using a sequence of exon contained in this clone of YOU, and for screening cDNA libraries, or by the 5'- or 3'-RACE procedure using oligonucleotide primers based on the sequences of exons TL4.

Identification of additional members of the family TIE-ligands

A new sequence of TTE-ligand-4 can be used in the rational search for additional members of the family TIE-ligands using approaches that take advantage of the existence of conservative segments of strong homology between known members of this family. For example, comparison (alignment) amino acid sequence TIE-ligands shows a few conservative areas of th is these blocks, in combination with segments of homology to previously known or new TIE-ligands can be used to identify new TIE-ligands.

Highly conserved regions among TL1, TL2 and TL3 can be used in the design of degenerate oligonucleotide primers that can be preimenovati PCR reactions using cDNA. cDNA-matrix can be formed by reverse transcription of tissue RNA using oligo d(T) or other suitable primers. Then aliquots of PCR reactions can be subjected to electrophoresis on agarose gel. Received amplificatoare DNA fragments can be cloned insert in the plasmid, sequenced and the obtained DNA sequences can be compared with sequences of all known TIE-ligands.

Selected size amplificatoare DNA fragments from these PCR reactions can be cloned into the plasmid to enter in E. Li by electroporation and the resulting transformants can be spread on selective agar. Bacterial colonies from PCR of transformants can be analyzed by sequencing of plasmid DNA to purify standard used for plasmid procedures.

The cloned fragments containing the segment of the new TIE-l is from the cDNA library. For example, genomic sequence TL4 person can be used to obtain a cDNA clone containing the complete coding sequence TL4 person, hybridization cDNA library of a man with a probe corresponding to TL4 person, as described earlier.

EXAMPLE 22. Cloning of the complete coding sequence hTL4

Both 5' - and 3'-coding sequence of the genomic clone TL4 human encoding TIE ligand-4 (hTL-4 ATCC Accession No.98095), was obtained by splitting restrictase, blotting the and Southern hybridization hTL-4-clone with coding sequences of mouse TL3 subsequent sublimemovies and sequencing hybridization fragments. Coding sequences corresponding to the N-terminal and C-terminal amino acids hTL-4, was used to design PCR primers (shown below), which, in turn, were used for PCR amplification TL4 from cDNA of human ovarian. Strip PCR identified as appropriate TL4 human DNA sequencing using the sequencing machine. DNA AS A and sequencing Taq Dideoxy Terminator Cycle (Applied Biosystems, Inc. Foster City, CA). PCR strip then was subcloned into the vector pCR-script and several plasmid clones analysera Fig.23. In another embodiment of this invention, the nucleotide at position 569 replaced And G, leading to the replacement of amino acids with Q in C.

Used here, the PCR primers described above were constructed in the following way:

hTL4atg 5'-gcatgctatctcgagccaccATGCTCTCCCAGctagccatgctgcag-3'

hTL4not 5'-gtgtcgacgcggccgctctagatcagacTTAGatgtccaaaggccgtacccgtatcatcat-3'.

Small letters indicate the sequence of "tails" added to PCR the primers to facilitate cloning of the amplified PCR fragments.

EXAMPLE 23. Design and characterization of modified TIE-ligands

For a better understanding of a number of observable properties was undertaken a genetic analysis of TIE-2 ligand-1 and TIE-2 ligand-2 (TL1 and TL2). Although TL1 and TL2 have the same structural homology, they exhibit different physical and biological properties. The most outstanding characteristic that distinguishes these two ligand, is that they are both associated with the TIE-2 receptor, TL1 is agonist, while TL2 is an antagonist. When non conditions of electrophoresis both protein covalent find a multimeric structure. TL1 is produced in the form of a mixture of crosslinked by disulfide bonds of multimers, primarily trimers, and molecular particles higher boradcasting particles. Also, while TL2 is produced well in most expression systems, TL1 is expressed poorly and it is difficult to obtain in large quantities. Finally, the conditions of obtaining and purification also, apparently predispose TL1 to inactivate proteolytic cleavage site located near aminocore.

To study these differences, several modified ligands were constructed as follows.

23.1. Replacement of cysteine

Research what factors can contribute to various physical and biological properties of these two molecules revealed the presence TL1 residue cysteine (CYS 265 in Fig.4; CYS 245 in Fig.17) prior to the fibrinogen-like domain in TL1, but not in TL2 - i.e. in TL2 does not have a corresponding cysteine residue. The remainder CYS265 in TL1 is encoded TGC and is located at nucleotides ~1102-1104 (see Fig.4) when the approximate location of the connection between twisted spirally and fibrinogen-like domains. Since cysteine residues are involved in the formation of disulfide bonds, the presence of which contributes in tertiary structure and biological properties of the molecule, believed that perhaps the presence of remnant CYS265 in TL1 may be, at konstruirovanie expression plasmid, which contained a mutation in TL1, in which CYS (remainder 265 in Fig.4; the remainder 245 in Fig.17) was replaced by the amino acid (serine), which does not form disulfide bonds. In addition, mutant L1/CYS-, was constructed in the second expression plasmid, which is mutated in approximately corresponding position in TL2 (Met247 in Fig.17), so that the balance was now a cysteine. As neutropenia and mutant expression plasmids TL1 and TL2 were transliterowany cells COS-7, cell supernatant containing recombinant proteins, was collected and the samples were subjected to both regenerative and non electrophoresis LTO-NAAG and subsequent Western-blot.

Fig.18 shows Western blots with non conditions as neutropenia and mutated proteins, TL1 and TL2, showing that the mutant TL1/CYS-moves in the form of a dimer, which is more like TL2, and that the mutant TL2/CYS+able to form a trimer, and multimer higher order, more like TL1. When testing these two mutant proteins for their ability to induce phosphorylation in expressing TIE-2 cells mutant TL1/CYS-was able to activate the TIE-2 receptor, whereas the mutant TL is istein (residue 265 in Fig.4; residue 245 in Fig.17) TL1 in the series it was found that covalent structure TL1 is similar to the structure of TL2, i.e. first of all is dimeric. Modified molecule TL1 still behaves as an agonist, therefore, trimers and/or multimeric structure of a higher order is not the determining factor, giving TL1 ability to activate the receptor. Although the destruction of cysteine produced a molecule with more desirable properties, it did not improve the level of production TL1.

23.2. Deletions of domains

The nucleotide sequence encoding TL1 and TL2, have a common genetic structure, which can be subdivided into three domains based on the amino acid sequences of the Mature proteins. Approximately 215 last amino acid residues of each of the Mature protein contains six cysteines and have strong similarities with the domain of fibrinogen. Therefore, this area was named "fibrinogen-like domain, or "F-domain. The Central region of the Mature protein containing approximately 205 residues, had a high probability assumptions "of helically twisted structure and was named "spirally twisted" domain or C-domain. Approximately 55 aminobenzene residue Mature protein stevem" domain or the N-domain. Modified ligands described herein, are identified using the terminology in which N = N-terminal domain, C = spirally twisted domain, F = fibrinogen-like domain, and the numbers 1 and 2 refer to TL1 and TL2, respectively. Thus, 1N indicates the N-terminal domain of TL1, 2F indicates the fibrinogen-like domain TL2, etc.

To test, whether contained fibrinogen-like domain (F-domain) TIE-2 ligands of TIE-2-activating activity, were constructed expression plasmids, which had deletions of helically twisted and N-terminal domains and had only part of the DNA sequence, encoding an F-domain (for TL1, beginning with Fig.4 if ~the nucleotide 1159, amino acid residue ARG284; for TL2, corresponding to ~ 1200 nucleotide in Fig.6, the amino acid residue 282). Then this mutant construct was transfusional in COS cells. The supernatant containing the recombinant protein was collected. Mutant TLl/F-domain was tested for its ability to bind the TIE-2 receptor. The results showed that the monomer mutant TL1/F-domain was not able to bind the TIE-2 in detektiruya level.

But when the monomer TLl/F-domain was myc-tagged and then subjected to the formation of clusters antibody against the ICC-label, he found detecting phosphorylation in expressing TIE-2 cell line.

Thus, it was determined that the F-domain of TIE-2 ligands involved in binding of the receptor, but truncated protein consisting of only one F-domain is not sufficient for binding of the receptor. It put forward the possibility that the helically twisted domain was responsible for holding together several fibrinogen-like domain that may be important for receptor binding. In an attempt to prove this hypothesis, the F-domain was merged with the Fc part of human IgG1 antibodies. Since Fc-plots timeresults when the expression of mammalian cells, these recombinant proteins that mimic theoretical configuration of the F-domain, were native ligands for dimerization. This construction of the F-domain-Fc bound, but does not activate the receptor. Obviously, multimerization driven by other parts of these ligands is necessary to allow the ligands to bind the TIE-2 receptor. In addition, some other factor outside of the F-domain should contribute to the phosphorylation of the receptor.

Then designed mutants that had no fibrinogen-like domain and, therefore, contained only the N-terminal and helically twisted domains. They were not able to contact the receptor. For and labelled FLAG-tag at N-end with getting designs, named FLAG-1C1F and FLAG-2C2F. Although these molecules were strongly stained cells COS-7, transfected for the expression of TIE-2 receptor, they did not give a positive response in the test phosphorylation. Thus, the N-domain does not contain a significant factor for the activation of the receptor, although, as described infra, the ability of the chimeric molecule 2N2C1F activate the receptor shows that even the N-domain of inactive ligand can perform this role.

Differences in behavior between the ICC marked shortened F-domain and the Fc-labeled shortened F-domain, as described earlier, suggest that TIE-ligands may contact only in dimeric form or in multimeric, with higher order multimerization forms. Indeed, non electrophoresis in LTO-SDS page showed that the TIE-ligands exist in nature in dimeric, trimeric and multimeric forms. That shortened proteins FLAG-1C1F and FLAG-2C2F can contact the TIE-2 receptor without dimerization synthetic label (such as Fc), whereas the truncated F can not, suggests that the area is at least partially responsible for the aggregation of F-domain.

23.3. Sharing designs (chimeras)

The applicants noted that the level of production TL1 in the cell is ke to explain this difference and also to characterize agonistic activity TL1 in comparison with antagonistic activity TL2.

Were constructed four chimeras, in which the N-terminal domain or the fibrinogen-like domain was subjected exchange between TL1 and TL2, and which were named using the terminology described earlier, so that, for example, 1N1C2F refers to a Chimera, having N-terminal and helically twisted domains TL1, together with the fibrinogen-like domain of TL2. Were designed following 4 chimeras:

Chimera 1 - 1N1C2F

Chimera 2 - 2N2C1F

Chimera 3 - 1N2C2F

Chimera 4 - 2N1C1F

Nucleotide and amino acid sequence of the Chimera 1-4 shown in Fig.24-27, respectively.

Each Chimera was embedded in the individual expressing vector pJFE14.

Then chimeras were transfusional cells COS-7, together with an empty vector pJFE14, native TL1 and native TL2 as controls and collected cultural supernatant.

To determine the influence on the expression of these ligands, dilution 1:5 dilution 1:50, COS-7-supernatants dot-bottiroli on nitrocellulose. Three ligand, which contained the N-domain TL1 (i.e., native TL1, 1N2C2F and 1N1C2F), then probed rabbit antibody specific for the N-terminal TL1. Three ligand containing N-domain TL2 (i.e., native TL2, 2N1C1F and 2N2C1F), probed rabbit antibody specific for the N-end TL2. The cut is high, than the level of any molecule containing N-domain TL1, regardless of the design of the rest of the protein. It was concluded that the N-domain has mainly to regulate the level of expression of the ligand.

The next question was related to the ability or inability of these chimeras to activate TIE-2 receptor. Cells EA.hy926 processed four chimeras, and TL1 as a positive control for phosphorylation and TL2 or supernatant pJFE14-transfected cells COS-7 as a negative control for phosphorylation. These cells were literally and TL2 was immunoprecipitated from the lysate of cells and subjected to electrophoresis in LTO-PAG. The samples were subjected to Western-blotting and probed with antibody against phosphotyrosine to detect any phosphorylated receptors. Unexpectedly, only constructs containing the fibrinogen-like domain TL1 (2N1C1F and 2N2C1F), could fosforilirovanii TIE-2 receptor. Thus, although the N-terminal region TL1 is essential for activation, it can be replaced by N-terminal region TL2, i.e., information that determines the ligand is an agonist or antagonist, in fact, contained in the fibrinogen-like domain.

Thus, it was determined that the F-domain, in addition to tie the th molecule, changed by replacing its F-domain (F-domain TL1, modified TL2 acted as an agonist.

However, the design 2N1C1F was somewhat more potent. The signal produced by Chimera 2N1C1F, apparently, is somewhat stronger than the signal chimeras 2N2C1F, which leads to the assumption that the C-domain TL1, although not crucial for phosphorylation may increase the strength TL1. However, because the sample used to test the phosphorylation were normalized against the concentration of ligand, does not exclude the possibility that a stronger signal phosphorylation only shows a larger content of the ligand. Therefore, the test of phosphorylation was repeated with valerauko quantities of ligand for determination, are active chimeras of the action of various forces. The ligand concentration in OS-7 supernatant ligand transpency determined by biosensor BIAcore technology according to previously described methods (Stitt, T. N., et al., (1995) Cell 80:661-670). The method was measured by BIAcore binding activity of the supernatant with TIE-2 receptor in relative units, called resonance units (PE). Watched as a rule, it is a good correlation between D and the concentration of ligand, and 400 D activity corresponded to ~1 helirovanie. The results showed that the Chimera 2N2C1F was clearly more active than native TL1 or Chimera 1N1C2F, at the same concentration.

Another interesting aspect of these exchange structures are their levels of expression. Each of the four chimeras were tested on the level of production in COS cells, the ability to communicate with TL2 and the ability to fosforilirovanii TIE-2. The results of these experiments showed that chimeras 1 and 3 were producirovanie at levels comparable to TL1, whereas chimeras 2 and 4 were producirovanie at levels comparable to TL2. Thus, a high level of education of the protein correlated with the N-terminal domain TL2. In addition, when testing endothelial cells EA.hy926 chimeras 2 and 4 were active, whereas chimeras 1 and 3 were inactive. Thus, this activity (phosphorylation of the receptor correlates with fibrinogen-like domain TL1. Therefore, chimeras 2 and 4, each had the desirable properties of high levels of production, as well as agonistic activity.

23.4. Proteoliticeski resistant design

Based on the observation that a large fraction of drugs TL1 often Proteoliticeski cleaved near the N-Terminus, an assumption was made that the arginine residue located in position the activity of this protein in vivo, and that replacement of the arginine-serine (R49S) can increase the stability of this protein without binding effect on its activity. Was designed such mutant TL1 and it was discovered that he is as active as native TL1, but he did not show resistance to proteolytic cleavage.

23.5 Raman mutants

The most active of the chimeric structures, 2N1C1F, has been further modified so that the cysteine is encoded by nucleotides 784-787, as shown in Fig.27, was converted into serine. This molecule (called 2N1C1F (C246S)) expressives well, strongly activated the receptor, was resistant to proteolytic cleavage and was mainly dimeric and not more multimeric. Thus, 2N-domain, apparently confers resistance to proteases of this molecule. Finally, this molecule was further modified to eliminate potentially sensitive to protease site encoded by nucleotides 199-201, as shown in Fig.27, with the receipt of molecules (called 2N1C1F (R51S C246S)), which, as expected, is activating, well expressed, dimeric and resistant to proteases.

The table summarizes Modific is the competitiveness bind the TIE-2 receptor, the ability to activate the TIE-2 receptor, type formed structure (monomer, dimer, and so on) and their relative levels of production. Unmodified TL1 (simple, without the dashes) and TL2 (shaded) is shown with three domains in the form of blocks. Thus, the shaded blocks show the domains of TL1. The cysteine located at position 245 of the Mature protein TL1, listed as "With". "X"-"C" indicate that the cysteine residue has been replaced by another amino acid, such as mutant TL1 CYS-. Similarly, "X","R" in the latest designs indicate replacement of AGD in position 49 of the Mature protein TL1. "C" is present in one modified structure TL2, indicating the mutant TL2 CYS+. Design with tails Fc or FLAG-marking, is also shown.

On the basis of expressed here claims the person skilled in the art will easily see what can be done for more designs to create additional modified and chimeric TIE-2 ligands, which have modified properties. For example, you can create a structure consisting of an N-terminal domain of TL2 and F-domain TL1, fused with the Fc part of human IgG antibodies. One would expect that this design will bind and activate the TIE-2 receptor. This way they are all in the scope of this invention.

23.6. Materials and methods

Construction of chimeras

Exchanging structure was built in the vector pJFE14 in which the site bI was replaced by the AscI site. Then this vector was digested AscI and NotI to obtain AscI-NotI-patterns. DNA fragments for these chimeras were obtained by means of PCR using suitable oligonucleotides.

Insert FLAG-1C1F AND FLAG-2C2F was subcloned into the structure of the vector RMT, which was split EcoRI and NotI. Shortened "CF" was obtained by using PCR, and the label FLAG and the previous signal sequence of trypsin was designed by annealing synthetic oligonucleotides.

Transfection

All designs were transfusional cells COS-7 using DEAE-Dextran or Lipofectamine in accordance with standard protocols. Cell culture was collected 3 days after transfection and centrifuged at 1000 rpm for 1 minute, and supernatant was transferred into a fresh tube and kept at -20C.

Staining of FLAG-lClF-transfected and FLG-22F-transfected cells

6-hole tablets cells COS-7 transfusional TIE-2 receptor. COS-7 supernatant from different ligand transpency incubated these cells for 30 minutes with subsequent "https://img.russianpatents.com/chr/176.gif">With methanol for 3 minutes, washed once with PBS, and incubated with antibody against FLAG M2 (IBI; dilution 1:3000 in PBS/10% calf serum (TC) for 30 minutes. Cells were washed once with PBS, and incubated with conjugated antibody goat antibodies against mouse IgG/alkaline phosphatase (AP) (Promega; 1:1000) in SFR/10% CU. Cells were washed twice in PBS, and incubated with phosphate substrate, BCIP/NBT, with 1 mm levamisole.

Tests phosphorylation

Cultivation COS-supernatant to study the dependence of the dose-response was performed in supernatant cells COS-7, transfected with the empty vector pJFE14. Cells EA, which in nature Express TIE-2 receptor, starved in serum-free medium for >2 hours followed by the introduction of appropriate COS-supernatant for 10 minutes at 37C in an atmosphere of 5% CO2. Then cells were washed in chilled PBS on ice and literally 1% NP40-buffer for lysis, containing protease inhibitors (10 μg/ml leupeptin, 10 μg/ml Aprotinin, 1 mm PMSF) with subsequent immunoprecipitating antibody specific for TIE-2 receptor. Then the samples were subjected to Western blot turns using antibodies against phosphotyrosine (pTyr).

Dot-blots

Samples were applied to nitrocellulose IU cells SNO and infected with baculovirus insect cells

Obtaining virus

The gene for the chimeric ligand (called 2N1C1F (C246S)) was built in baculovirus expressing plasmid and recombinable with viral DNA for the preparation of recombinant baculovirus, amplified and collected using methods described previously (O'reilly, D. R., L. K. Miller and V. A. Luckow, Baculovirus Expression Vectors-A Laboratory Manual. 1992, New York: W. H. Freeman). The cells of the insect SF21 (Spodoptera frugiperda), obtained from Invitrogen, adapted and were propagated at 27C in serum-free medium Gibco SF900 II. Uninfected cells were grown at a density of 1106cells/ml cell Density was determined by the expense of viable cells using hemocytometer. The original viral material for ligand was added to the bioreactor at low multiplicity of infection of 0.01-0.1 plaque-forming units (PFU) per cell for the start of the infection. The infection process is continued for 3-4 days for maximal virus replication without ravlecheniya significant lysis of the cells. From the cell suspension were taken sterile aliquots in sterile centrifuge bottles and the cells were removed by centrifugation (1600 rpm, 30 minutes). Cell-free supernatant was collected in sterile vials and stored at 4106cells. Serial dilution of the initial viral material was added to the attached cells and the mixture incubated with swing, to allow the virus to adsorb to the individual cells. Added a covering layer of agar and cups were incubated for 5 days at 27C. Viable cells were stained with neutral red, revealing the annular plaques, which are believed to obtain a titer of virus expressed in plaque-forming units per milliliter (PFU/ml PFU/ml).

Exposure of the cells to obtain protein

Uninfected SF21 cells were grown in the dish for tissue culture and virus containing the gene of the chimeric ligand was added at a multiplicity of infection of 1 to 10 PFU/cell. The virus was allowed to adsorb for 90 minutes at 27With a slow swing, after which the cells were given fresh quantity of serum-free medium Sf-900 II. After 3 days of growth at 27With the culture fluid was collected and the ligand were detected by Western blot turns.

Expression of cell SNO chimeras TIE-2 ligands

Chimeras TIE-2 ligands to the pcDNA3, pMT21, pED or other.

Plasmids were transfusional cells Cho DG44 (Urlaub, G. and Chasin, L. A. 1980. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77:4216-4220; Urlaub, G., Kas, E., Carothers, A. M. and Chasin, L. A. 1983. Deletion of the diploid dihydrofolate locus from cultured mammalian cells. Cell 33:405-412) precipitation with calcium phosphate or cationic liposomes. In the case of vectors, not having dhfr breeding marker plasmid pSV2.dhfr was cotranslationally at 20% molar with respect to the plasmid containing the chimeric TIE-ligand. DHFR+cells were selected by growth in selective medium (medium not containing nucleosides and nucleotides containing 10% fetal calf serum), and clones were screened for the production of chimeric TIE-ligands using Western blot turns with TIE-2-receptaculum. Clones expressing the desired protein was subjected to several cycles of gene amplification using graded concentrations of methotrexate in selective medium. Clones with high expression of identified after amplification of genes such methods Western blot turns.

Cell lines expressing chimeric TIE-ligands, were cultured in monolayers, suspension flasks, roller bottles and bioreactors in selective medium or in medium without selection, and they can vyrashivat is 12301 Parklawn Drive, Rockville, Maryland 20852, in accordance with the Budapest Treaty. Plasmid clone encoding TIE-2 ligand, was deposited in ATSS 7 October 1994 and was named "pJFE14 encoding TIE-2 ligand" under access number 75910. Recombinant baculovirus Autographa californica encoding TIE-2-receptarea, was deposited in ATSS 7 October 1994 and was named "vTIE-2-receptarea" under access number VR24484. The lambda phage vector containing DNA tie-2 ligand was deposited in ATSC October 26, 1994 and was named "lgtIO encoding htie-2 ligand 1" under access number 75928. Plasmid clone that encodes a second TIE-2 ligand, was deposited in ATSC 9 December 1994 and was named "pBluescript KS encoding TIE-2 ligand 2 person" under access number 75963. The strain of E. coli DHIOB containing plasmid pBeLoBacll with the insertion of a gene TL-4, encoding TIE ligand-4, was deposited in ATS 2 July 1996 and was named "hTL-4" under access number 98095.

This invention is not limited in its scope described here are typical options. Indeed, various modifications of the present invention, also described herein will be obvious to specialists in this field from the preceding description and accompanying drawings. It is assumed that such modifications are within the scope of prila inovas acid, encoding a chimeric TIE-2 ligand that binds and activates TIE-2 receptor, chimeric where the ligand contains an N-terminal domain, helically twisted domain and a fibrinogen-like domain, with at least two of these domains derived from different TIE-2 ligands selected from the TIE-2 ligand 1, having amino acid sequence corresponding to the amino acid sequence shown in Fig.4 and 5, the TIE-2 ligand 2 having amino acid sequence corresponding to the amino acid sequence shown in Fig.6, TIE-3 ligand having the amino acid sequence corresponding to the amino acid sequence shown in Fig.21, and TIE-ligand 4 having the amino acid sequence corresponding to the amino acid sequence shown in Fig.23, the ligand may be modified such that the nucleotide corresponding to the nucleotide 1102-1104 Fig.4, encode another amino acid instead of a cysteine, and optionally, the nucleotide corresponding to the nucleotide 555-557 Fig.6, encode another amino acid instead of arginine.

2. The nucleic acid molecule under item 1, encoding a chimeric TIE-2 ligand, where the N-terminal domain, Speer is kleinova acid on p. 2, containing the nucleotide sequence encoding a TIE-2 ligand 1, where a part of the nucleotide sequence that encodes the N-terminal domain of TIE-2 ligand 1, replaced by a nucleotide sequence that encodes the N-terminal domain of TIE-2 ligand 2.

4. The nucleic acid molecule under item 3, characterized in that the part of the nucleotide sequence which encodes a helically twisted domain of TIE-2 ligand 1, replaced by a nucleotide sequence which encodes a helically twisted domain of TIE-2 ligand 2.

5. The nucleic acid molecule under item 3 or 4, characterized in that the modified such that encodes a different amino acid instead of a cysteine residue encoded by nucleotides 784-786, as shown in Fig.27.

6. The nucleic acid molecule under item 5, wherein said modified so that encodes a serine residue instead of a cysteine residue.

7. The nucleic acid molecule under item 5 or 6, characterized in that it further modified so that encodes a different amino acid instead of the arginine residue encoded by nucleotides 199-201, as shown in Fig.27.

8. The nucleic acid molecule under item 7, characterized in that it is modified in such oppositionalist, presented on Fig.27.

10. The molecule of nucleic acid on p. 4 having the sequence shown in Fig.2 5.

11. The selected nucleic acid molecule encoding a modified TIE-2 ligand that binds and activates TIE-2 receptor, comprising the nucleotide sequence encoding a TIE-2 ligand 1, having amino acid sequence corresponding to the amino acid sequence shown in Fig.4 or Fig.5, which is modified so that encodes a different amino acid instead of a cysteine residue at amino acid position 265.

12. The nucleic acid molecule according to p. 11, wherein said modified so that encodes a serine residue instead of a cysteine residue.

13. Chimeric or modified TIE-2 ligand encoded by the nucleic acid molecule according to any one of the preceding paragraphs.

14. Chimeric TIE-ligand on p. 13 having the sequence shown in Fig.25 or 27.

15. Chimeric TIE-ligand on p. 13 having the sequence shown in Fig.27, modified in such a way as to have a different amino acid instead of a cysteine residue encoded by nucleotides 784-786.

16. The vector for expression of a chimeric or but associated with expressing regulatory sequence, capable of controlling the expression of the sequence in the cell host.

17. Vector under item 16, characterized in that it is a plasmid.

18. Method to get the vector-host to obtain chimeric or modified ligand according to any one of paragraphs.13, 14 or 15, comprising transforming the host cell with the vector according to p. 16 or 17.

19. The method according to p. 18, characterized in that a host cell is a bacteria cell, yeast, insect or mammalian.

20. A method of obtaining a chimeric or modified TIE-2 ligand according to any one of paragraphs.13, 14 or 15, which includes the cultivation of cells transformed by the vector according to PP.16 or 17, under conditions that allow to produce the ligand, and the allocation thus obtained ligand.

21. Conjugate chimeric or modified TIE-2 ligand comprising a ligand according to any one of paragraphs.13, 14 or 15, conjugated with a cytotoxic agent.

22. Conjugate under item 21, wherein the cytotoxic agent is a cytotoxic radioactive isotope or a toxin.

23. Pharmaceutical composition for the induction of hematopoiesis or neovascularization, containing chimeric or modified ligand according to any one of paragraphs.13, 14 or 15 and a pharmaceutically acceptable wear

 

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