RU2233330C2 - Nucleic acid molecule encoding ligand tie-2, ligand tie-2 and method for its preparing, vector, strain of cells cos, antibody, conjugate of ligand tie-2, ligand body, pharmaceutical composition, method for prophylaxis or attenuation of neovascularization and method for identification of tie-2 receptor antagonist - Google Patents

Abstract

FIELD: genetic engineering, molecular biology, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to genes encoding tyrosine kinases and can be used for diagnosis. Human ligand TIE-2 represents TIE-2 receptor antagonist. Ligand TIE-2 is prepared using vector pBluescript KS that comprises nucleic acid encoding ligand TIE-2. Conjugate is prepared by coupling ligand TIE-2 with cytotoxic agent. Ligand body represents ligand TIE-2 fused with immunoglobulin contact site. Pharmaceutical composition contains effective amount of ligand TIE-2 or ligand body and it is used in method for prophylaxis or attenuation of neovascularization. Invention provides diagnosis and treatment of diseases associated with endothelial cells comprising TIE-2 receptors. Invention provides the development of diagnostic systems for identification of TIE-2 receptor antagonist.

EFFECT: improved prophylaxis and treatment method, valuable medicinal properties of ligand.

16 cl, 14 dwg, 12 ex

Description

This international application claims the priority of pending US applications No. 418595, filed April 6, 1995, No. 373579, filed January 17, 1995, No. 353503, filed December 9, 1994, No. 348492, filed December 2, 1994, No. 330261, filed October 27, 1994 No. 319932, filed October 7, 1994, the contents of each of which are incorporated herein by reference. In the description there are links to various publications. The content of these publications is fully incorporated in the application under reference.

Introduction

In general terms, the present invention relates to the field of genetic engineering and more specifically to genes of tyrosine kinase receptors and their related ligands, to their incorporation into recombinant DNA vectors, and to the production of encoded proteins in recipient strains of microorganisms and recipient eukaryotic cells. More specifically, the present invention relates to new ligands, known as TIE-2 ligands that bind the TIE-2 receptor, as well as to methods for producing these TIE-2 ligands. The present invention also provides nucleic acid sequences encoding TIE-2 ligands and methods for producing nucleic acids encoding these TIE-2 ligands and their gene products. TIE-2 ligands, as well as the nucleic acids encoding them, can be useful in the diagnosis and treatment of certain diseases affecting endothelial cells and corresponding TIE receptors, such as neoplastic diseases, including tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis, and inflammatory diseases. More generally, biologically active TIE-2 ligands can be used to promote the growth, survival and / or differentiation of cells expressing the TIE-2 receptor. The biologically active TIE-2 ligand can be used to in vitro maintain a culture of cells expressing the TIE-2 receptor. Cells and tissues expressing the TIE-2 receptor include, for example, endothelial cells of the heart and blood vessels, lens epithelium and cardiac epicardium. In another embodiment, such a ligand can be used to support cells that are designed to express the TIE-2 receptor. Further, TIE-2 ligands and their corresponding receptors can be used in assay systems to identify TIE-2 receptor agonists or antagonists.

BACKGROUND OF THE INVENTION

The behavior of cells responsible for the development, preservation and restoration of differentiated cells and tissues is regulated to a large extent due to intercellular signals transmitted by growth factors and similar ligands and their receptors. These receptors are located on the surface of their respective cells, and they bind peptides or polypeptides, known as growth factors, as well as other hormone-like ligands. The result of this interaction is rapid biochemical changes in the corresponding cells, as well as a quick and long-lasting rearrangement of the expression of cell genes. Some receptors associated with various cell surfaces can bind specific growth factors.

Tyrosine phosphorylation on proteins due to tyrosine kinases is one of the key schemes by which signals are transmitted through the plasma membrane. Some currently known tyrosine kinase protein genes encode transmembrane receptors for polypeptide growth factors and hormones such as epidermal growth factor (EGF), insulin, insulin-like growth factor (IGF-1), platelet derived growth factors (PDGF-A u-B ) and fibroblast growth factors (FGFs) (Heldin et al., Cell Regulation, 1: 555-566 (1990); Ullrich, et al., Cell, 61: 243-54 (1990)).

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

Tyrosine kinase receptors are divided into evolutionary subfamilies based on the characteristic structures of their ectodomains (Ullrich., Et al .. Cell, 61: 243-54 (1990)). Such subfamilies include an EGF receptor-like kinase (subclass 1) and an insulin-like kinase kinase (subclass 11), each of which contains repeated homologous sequences with a high cysteine content in their extracellular domains. A separate site with a high cysteine content was also found in the extracellular domains of eph-like kinases. Hirai, et al., Science 238: 1717-1720 (1987); Lindberg, et al., Mol. Cell. Biol., 10: 6316-24 (1990); Lhotak, et al., Mol. Cell. Biol. 11: 2496-2502 (1991). PDGF receptors as well as c-fms and c-kit tyrosine kinase receptors can be grouped into subclass 111, while FGF receptors form subclass 1V. Typical for members of both of these subclasses are extracellular folding fragments stabilized by intrachain disulfide bonds. Such so-called immunoglobulin (Ig) -like folds are found in proteins of the immunoglobulin superfamily, which includes a wide range of other cell surface receptors having either cell-bound or soluble ligands. Williams, et al., Ann. Rev. Immunol., 6: 381-405 (1988).

Tyrosine kinase receptors differ in their specificity and affinity. Typically tyrosine kinase receptors are glycoproteins that consist of (1) an extracellular domain capable of binding specific growth factor (s); (2) a transmembrane domain, which is usually the alpha-helical part of the protein; (3) the juxtamembrane domain, where the receptor can be regulated, for example, by protein phosphorylation; (4) a tyrosine kinase domain, which is an enzymatic component of the receptor; and (5) a carboxyterminal tail, which in many receptors is involved in the recognition and binding of tyrosine kinase substrates.

It has been reported that processes such as alternative exon splicing and the alternative choice of a gene promoter or polyadenylation sites can lead to the formation of several different polypeptides from the same gene. Such polypeptides may or may not contain the various domains listed above. As a result, some extracellular domains can be expressed as separate secreted proteins and some forms of receptors may not contain a tyrosine kinase domain, but contain only the extracellular domain integrated into the plasma membrane due to the transmembrane domain plus a short carboxyterminal tail.

A gene encoding endothelial cell transmembrane tyrosine kinase, initially identified by RT-PCR as an unknown homologous tyrosine kinase cDNA fragment from human leukemia cells, is described by Partanen et al., Proc. Natl Acad. Sci. USA 87: 8913-8917 (1990). This gene and its encoded protein are called “tie,” which is short for “tyrosine kinase with domains homologous to Ig and EGF.” Partanen et al., Mol. Cell Biol. 12: 1698-1707 (1992).

It has been reported that tie mRNA is present in all tissues of the human fetus and mouse embryo. After studying the tie, the mediator was localized in the heart cells and in the vascular endothelial cells; the tie mRNAs were localized in the endothelium of the blood sousoses and the endocardium of mouse embryos on the 9.5-18.5 day of their existence. Enhanced tie expression has been shown during neovascularization associated with the development of ovarian follicles and granulation of tissues in skin wounds. Korhonen et al., Blood 80: 2548-2555 (1992). Thus, it is believed that tie plays a role in angiogenesis, which is important for the development of methods for treating solid tumors and some other diseases dependent on angiogenesis, such as diabetic retinopathy, psoriasis, atherosclerosis, and arthritis.

Two structurally related rat TIE receptor proteins have been reported to be encoded by different genes with related expression profiles. One gene, called tie-1, is the rat homologue of human tie. Maisonpierre, et al., OncogeneS: 1631-1637 (1993). Another gene, tie-2, may be the rat homologue of the mouse tek gene, which, like tie, was reported to be expressed in mice exclusively in endothelial cells and their putative predecessors. Dumout et al., Oncogene 8: 1293-1301 (1993).

Both genes were found to be widely expressed in endothelial cells of embryonic and postnatal tissues. Significant levels of tie-2 transcripts are also present in other populations of embryonic cells, including the lens epithelium, cardiac epicardium, and mesenchyme sites. Maisonpierre et al., Oncogene 8: 1631-1637 (1993).

The predominant expression of the TIE receptor in the vascular endothelium suggests that TIE plays a role in the development and maintenance of the vascular system. This includes roles in the determination, differentiation, proliferation of endothelial cells, in cell migration and copying into vascular elements. TIE-2 deficient mouse embryos have been reported to show that TIE-2 is important for angiogenesis, especially for the formation of a vascular network in endothelial cells. Sato, T.N. et al., Nature 376: 70-74 (1994). In a mature vascular system, TIEs can function as a factor in the survival, conservation of endothelial cells and in reactions to the influence of pathogens.

Summary of invention

The present invention provides a composition comprising a TIE-2 ligand substantially free of other proteins. The present invention also provides an isolated nucleic acid molecule encoding a TIE-2 ligand. An isolated nucleic acid may be DNA, cDNA or RNA. The present invention also provides a vector comprising an isolated nucleic acid molecule encoding a TIE-2 ligand. A host-vector system is also provided for producing a polypeptide having TIE-2 ligand activity in suitable host cells. Cells of suitable hosts may be bacterial, yeast, insect or mammalian cells. The present invention also provides a method for producing a polypeptide having the biological activity of a TIE-2 ligand, which comprises growing the host-vector system cells under conditions that produce the polypeptide and isolate the polypeptide thus obtained.

The present invention, which describes an isolated nucleic acid molecule encoding a TIE-2 ligand, further provides for the development of a ligand a fragment, or a derivative thereof, or another molecule that is a receptor agonist or antagonist, as a therapeutic agent for treating patients suffering from disorders, including cells, tissues or organs that express the TIE receptor. The present invention also provides an antibody that specifically binds such a therapeutic molecule. This antibody may be monoclonal or polyclonal. The present invention also provides a method of using such a monoclonal or polyclonal antibody to determine the number of therapeutic molecules in a sample taken from a patient in order to monitor the progress of treatment.

The present invention also provides an antibody that specifically binds a TIE-2 ligand. This antibody may be monoclonal or polyclonal. Thus, the present invention also provides therapeutic compositions comprising an antibody that specifically binds a TIE-2 ligand in a pharmaceutically acceptable carrier. The present invention also provides a method of blocking blood vessel growth in mammals by administering an effective amount of a therapeutic composition comprising an antibody that specifically binds a TIE-2 ligand in a pharmaceutically acceptable carrier.

The present invention further provides a therapeutic composition comprising a TIE-2 ligand in a pharmaceutically acceptable carrier. The present invention also provides a method for promoting neovascularization in a patient by administering to him an effective amount of a therapeutic composition containing a TIE-2 ligand in a pharmaceutically acceptable carrier. In one embodiment, the method can be used to promote wound healing. In another embodiment, the method can be used to treat ischemia.

In another embodiment, the present invention provides an embodiment in which a TIE-2 ligand can be conjugated to a cytotoxic agent and based on this a therapeutic composition can be prepared. The present invention also provides a receptor body that specifically binds a TIE-2 ligand. The present invention provides a therapeutic composition comprising a receptor body that specifically binds a TIE-2 ligand in a pharmaceutically acceptable carrier. A method is also provided for blocking the growth of blood vessels in mammals by administering an effective amount of a therapeutic composition comprising a receptor body that specifically binds a TIE-2 ligand in a pharmaceutically acceptable carrier.

The present invention also provides a TIE-2 receptor antagonist, as well as a method for inhibiting the biological activity of a TIE-2 ligand in mammals, comprising administering to the mammal an effective amount of a TIE-2 antagonist. According to the invention, the antagonist may be an antibody or other molecule capable of specifically binding either a TIE-2 ligand or a TIE-2 receptor. For example, the TlE-2 receptor body may be an antagonist.

A brief description of the drawings.

1A and 1B: TIE-2 receptor body (TIE-2 PB) inhibits the development of blood vessels in the chorioallantoic membrane of chicken (CAM). A separate piece of absorbent gel foam (Gelfoam) moistened with 6 μg PBS is injected immediately under the CAM of one-day chicken embryos. After three days of incubation, the four-day embryos and the CAMs surrounding them are removed and examined. Figa: embryos treated with ENK-1 RB (rENK-1 ecto / h IgGI Fc) were viable and had normally developed blood vessels in the surrounding CAM. Figv: all embryos treated with TIE-2 RB (r TIE-2 ecto / h IgGI Fc) died, decreasing in size and were almost completely devoid of surrounding blood vessels.

Figure 2: Vector pJFE14.

Figure 3: Restriction map λ gt10.

Figure 4: Nucleic acid and isolated amino acid sequences (single letter code) of the human TIE-2 ligand from clone λgt10 encoding htie-2 ligand 1.

Figure 5: Nucleic acid and isolated amino acid sequences (single letter code) of the human TIE-2 ligand from the T98G clone.

6: Nucleic acid and isolated amino acid sequences (single letter code) of human TIE-2 ligand from pBluescript KS clone encoding human TIE-2 ligand 2.

Fig. 7: Western blot results demonstrating activation of the TIE-2 receptor due to TIE-2 ligand 1 (L1 band), but not due to TIE-2 ligand 2 (L2 band), control (Mosk.) Activation is also absent.

Fig. 8: Western blot results demonstrating that pretreatment of HAEC cells with an excess of TIE-2 ligand 2 (lane 2) interferes with the subsequent ability of diluted TIE-2 ligand 1 to activate TIE-2 receptor (TIE-2-R) compared to what happens during the pretreatment of HAEC MOCK cells with medium (lane 1).

Figure 9: Bar graph representation of the binding to the immobilized surface of rat TIE2 IgG TIE2 ligand in C2C12 ras, Rat2 ras, SHEP and T98G concentrated (10x) conditioned medium. The specific binding of rat TIE2 (rTIE2) is demonstrated by a significant decrease in binding activity in the presence of 25 μg / ml soluble TIE2 RB compared with a smaller decrease in the presence of soluble trkB RB.

Figure 10: Binding of recombinant human TIE-2 ligand 1 (hTL1) and human TIE-2 ligand 2 (hTL2) in cos cell surface supernatants with immobilized surface of human TIE-2 RB. Specific binding of human TIE-2 is determined by incubating samples at 25 μg / ml of either soluble human TIE2 RB or trkB RB; a significant decrease in binding activity is observed not only for samples incubated with human TIE2 RB.

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

DETAILED DESCRIPTION OF THE INVENTION

As will be described in more detail below, the applicants isolated a new ligand that binds the TIE-2 receptor by expression cloning. The present invention includes the TIE-2 ligand, as well as its amino acid sequence and also functionally equivalent molecules, in which amino acid residues are replaced by residues within the sequence, resulting in a silent change. For example, one or more of the amino acid residues in the sequence can be replaced by another amino acid (s) of the same polarity, which acts as a functional equivalent, and get a silent replacement. The amino acid substituents in the sequence can be selected from other members of the class to which this amino acid belongs. For example, the class of non-polar (hydrophobic) amino acids includes alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present invention are proteins or fragments or derivatives thereof that exhibit the same or similar biological activities, and derivatives that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, binding to an antibody molecule or other cellular ligand, etc.

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

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

It will be apparent to one skilled in the art that the present invention encompasses DNA and RNA sequences that hybridize to an isolated sequence encoding a TIE-2 ligand under conditions of moderate stringency as defined, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ed . vol. 1, pp 101-104, Cold Spring Harbor Laboratory Press (1989). Thus, the nucleic acid molecule contemplated by the present invention includes a molecule having a sequence isolated from the TIE-2 ligand amino acid sequence obtained as indicated above, as well as a molecule having a nucleic acid sequence that hybridizes to such a nucleic acid sequence, and also a nucleic acid sequence that is degenerate with respect to the above sequences as a result of the genetic code, but which encodes a ligand that vyazyvaet TIE-2 receptor.

Any of the methods known to those skilled in the art for embedding DNA fragments into a vector can be used to construct an expression vector encoding a TIE-2 ligand by using appropriate transcriptional / translational control signals and protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (genetic recombination). The expression of a nucleic acid sequence encoding a TIE-2 ligand or its peptide fragment can be controlled by a second nucleic acid sequence such that the protein or peptide is expressed in a host transformed with a recombinant DNA molecule. For example, the expression of the TIE-2 ligand described herein can be controlled by any promoter / enhancer element known to those skilled in the art. Promoters that can be used to control ligand expression include, but are not limited to, a long terminal repeat, as described by Squinto et al. / Cell 65: 1-20 (1991) /; SV40 early promoter region (Bernoist and Chambon, Nature 290: 304-310), CMV promoter, M-MuLV 5 'terminal repeat, promoter contained in a 3' long Routh sarcoma virus terminal repeat (Yamamoto, et al .. Cell 22: 787-797 (1980)), herpes virus thymidine kinase gene promoter (Wagner et al., Proc. Nail Acad. Sci. USA 78: 144-1445 (1981)), adenovirus promoter, metallothionein gene regulatory sequences (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)) or the tac promoter (DeBoer, et al., Proc. Natl Acad. Sci. USA 80: 21-25 (1983)), see also "Useful proteins from recombinant bacteria" in Scientific American, 242: 74-94 (1980); promoter elements from yeast or other fungi, such as the Gal 4 promoter, ADH (alcohol dehydrogenase) promoter, RGK (phosphoglycerin kinate) promoter, alkaline phosphatase promoter and the following sections of transcriptional control of animals that demonstrate tissue specificity and were used in transgenic animals: control section of the elastase gene 1, which is active in pancreatic acinar cells (Swift et al., Cell. 38: 639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); MacDonald , Hepatology 7: 425-515 (1987); control plot of the insulin gene, which active in pancreatic beta cells (Hanahan, Nature 315: 115-122 (1985)), a control region of an immunoglobulin gene that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al ., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), a control section of the mouse breast tumor virus that is active in ovary, breast, lymphoid and mast cells cells (Leder et al., 1986, CEll 45: 485-495), a control portion of the albumin gene that is active in the liver (Pinkert et al., 1987, Genes and Devel 1: 268-276), a control portion of the alpha-fetoprotein gene that is active in the liver (Krum lauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); a control region of the alpha 1-antitrypsin gene that is active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), a control region of the beta-globin gene that is active in myeloid cells (Mogram et al., 1985 , Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); a control portion of the gene for myelin basic protein, which is active in oligodendrocyte brain cells (Readhead et al., 1987, Cell 48: 703-712); a control portion of the myosin light chain-2 gene that is active in skeletal muscle (Shani, 1985, Nature 314: 283-286), and a control portion of the gonadotropin releasing hormone gene that is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378). The invention further encompasses the production of antisense compounds that are capable of specifically hybridizing to an RNA sequence encoding a TIE-2 ligand to modulate its expression (Ecker, US Patent No. 5,166,195, issued November 24, 1992).

Thus, in accordance with the present invention, expression vectors capable of replication in bacterial or eukaryotic hosts containing a nucleic acid encoding a TIE-2 ligand as disclosed herein are used to transfect the host and thereby directly express such nucleic acid to obtain TGE-2 ligand, which can then be isolated in biologically active form. In the sense used here, the biologically active form includes a form capable of binding to the TIE-2 receptor and induce a biological response such as differentiated functioning or influencing the phenotype of a cell expressing this receptor. Such biologically active forms should, for example, include phosphorylation of the tyrosine kinase domain of the TIE-2 receptor.

Expression vectors containing the gene insert can be identified by four general approaches: (a) DNA-DNA hybridization, (b) the presence or absence of “marker” gene functions, (c) the expression of the inserted sequences, and (d) determination using PCR. In the first approach, the presence of a foreign gene insert in the expression vector can be detected by DNA-DNA hybridization using probes that include sequences that are homologous to the gene encoding the inserted TIE-2 ligand. In the second approach, the recombinant vector / host system can be identified and selected based on the presence or absence of certain "marker" gene functions (for example, thymidine kinase activity, antibiotic resistance, transformation phenotype, formation of occlusion bodies in baculoviruses, etc. ) caused by the incorporation of foreign genes into the vector. For example, if a TIE-2 ligand-encoding nucleic acid is inserted into the marker gene sequence of a vector, recombinants containing this insert can be identified by the absence of marker gene functions. In a third approach, recombinant expression vectors can be identified by analyzing a foreign gene product expressed by a recombinant. Such an analysis can be based, for example, on the physical or functional properties of the gene TIE-2 product, for example, by binding the ligand to the TIE-2 receptor or part thereof, which may be associated, for example, with a detectable antibody, or part thereof, or by binding to antibodies generated against the TIE-2 protein of the ligand or part thereof.

Cells of the present invention can briefly or preferably constitutively and continuously express TIE-2 ligands, as described herein. In a fourth approach, DNA nucleotide primers corresponding to tie-2 specific DNA sequences can be obtained. These primers can then be used in PCR tie-2 gene fragments (PCR Protocols: A Guide To Methods and Applications, Edited by Michael A. Innis et al .. Academic Press (1990)).

The recombinant ligand can be isolated by any method that provides the subsequent formation of a stable biologically active protein. For example (but not from the point of view of limitations), a ligand can be isolated from cells either as soluble proteins or as inclusion bodies, from which ligands can be extracted quantitatively using 8M Gaunidinium chloride and dialysis. For further purification of the ligand, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration can be used.

In further embodiments of the invention, the recombinant TIE-2 ligand coding gene can be used to inactivate or "knock out" endogenous genes through homologous recombination and thereby create TIE-2 deficient ligand, tissue or animal cells. For example, (and not for the purpose of limitation), the recombinant TIE-2 ligand coding gene can be designed to contain an inserted mutation, for example, a neogen that should inactivate the native TIE-2 ligand coding gene. Such a construct, under the control of a suitable promoter, can be inserted into cells such as embryonic stem cells, by methods such as transfection, transduction or injection. Cells containing this design can then be selected for resistance to G418. Cells lacking the intact gene encoding the TIE-2 ligand can then be identified, for example, by Southern blotting, PCR determination, Northern blotting, or expression analysis. Cells lacking the intact gene encoding the TIE-2 ligand can then be fused to the embryo cells at an early stage to create transgenic animals deficient in such a ligand. Such animals can be used to determine specific in vivo processes that usually depend on this ligand.

The present invention also provides antibodies to TIE-2 ligands that can be used to detect ligands, for example, for diagnostic purposes. To obtain monoclonal antibodies directed to the TIE-2 ligand, you can use any method that can provide antibody molecules during continuous cultivation of cell lines. For example, the scope of the invention encompasses a hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256: 495-497), as well as a triomeric technique, a technique using human B-cell hybridomas (Kozbor et al., 1983, Immunology Today 4: 72), and an EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96) and the like.

Monoclonal antibodies can be human monoclonal antibodies or chimeric human-mouse (or other types) monoclonal antibodies. Human monoclonal antibodies can be obtained by any of a variety of techniques known to those skilled in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80: 7308-7312; Kozbor et al., 1983, Immunology Today 4: 72- 79; Olsson et al., 1982, Meth. Enzymol. 92: 3-16). Chimeric antibody molecules can be made to contain a murine antigen-binding domain with constant human regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851; Takeda et al., 1985, Nature 314: 452).

Various procedures known to those skilled in the art can be used to produce polyclonal antibodies to the epitopes of the TIE-2 ligands described herein. To obtain antibodies, various host animals can be immunized by injecting a TIE-2 ligand or fragment or derivative thereof, such as (but not limited to) rabbits, mice, and rats.

To enhance the immunological reaction, various adjuvants may be used depending on the host species, which include (but not limited to) Freund's adjuvants (complete and incomplete), mineral gels, such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, cochlear lymphocytes hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium paryum.

Molecular antibody clones of selected TIE-2 ligand epitopes can be obtained by known methods. A recombinant technique (e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Sprind Harbor Laboratory, Cold Sprind Harbor, New York) can be used to construct nucleic acid sequences that encode monoclonal antibody molecules or their antigen binding sites.

The present invention provides antibody molecules as well as fragments of such antibody molecules. Antibody fragments that contain an idiotype of a molecule can be created by known methods. For example, such fragments include, but are not limited to the F (ab ') ₂ fragment, which can be obtained by digesting the pepsin of the antibody molecule; Fab '- fragments that can be obtained by restoring the disulfide bridges of F (ab') ₂ fragments; and Fab fragments that can be obtained by treating antibody molecules with papain and a reducing agent. Antibody molecules can be isolated by known methods, for example, by immunoabsorption or immunoaffinity chromatography, chromatographic techniques such as HPLC (high performance liquid chromatography), or combinations thereof.

The present invention also includes an immunoassay to determine the amount of TIE-2 ligand in biological samples due to:

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

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

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

a) contacting the biological sample with at least one ligand of the invention so that the ligand is complexed with the TIE-2 receptor, and

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

The present invention also provides the use of a TIE-2 ligand to support the survival and / or growth and / or differentiation of cells expressing the TIE-2 receptor. Thus, the ligand can be used as a supplement to maintain, for example, endothelial cell culture. Further, a related ligand for the TIE-2 receptor discovered by the applicants provides the use of assay systems suitable for identifying TIE-2 receptor agonists or antagonists. Such assay systems may be useful in identifying molecules capable of promoting inhibition of angiogenesis. For example, in one embodiment, TIE-2 receptor antagonists can be identified as test molecules that are capable of interfering with the interaction of the TIE-2 receptor with the biologically active TIE-2 ligand. Such antagonists are identified by their ability to 1) block the binding of biologically active TIE-2 ligand to the receptor when measured, for example, using BlAcore biosensor technology (BlAcore; Pharmacia Biosensor, Piscataway, NY), or 2) block the ability of biologically active TIE-2 ligand cause a biological reaction. Such biological reactions include, but are not limited to, phosphorylation of the TIE-2 receptor or the upstream components of the TIE-2 signal transduction pattern, or the survival, growth, or differentiation of TIE-2 containing cells.

In one embodiment, cells constructed to express the TIE-2 receptor could be growth dependent upon the addition of the TIE-2 ligand. Such cells provide suitable assay systems for identifying additional TIE-2 receptor agonists or antagonists capable of affecting the activity of TIE-2 ligand on such cells. In another embodiment, autocrine cells designed so that they are capable of co-expression of both TIE-2 ligand and receptor may represent suitable systems for the analysis of potential agonists or antagonists.

Therefore, the present invention can be used to introduce TIE-2 receptors into cells that usually do not express this receptor, thereby allowing these cells to exhibit strong and easily distinguishable reactions to the ligand that binds this receptor. The type of reaction chosen depends on the cells used, and not on the specific receptor introduced into these cells. Appropriate cell lines can be chosen so as to obtain a reaction of value for analysis, as well as to detect molecules that can act on tyrosine kinase receptors. These molecules can be any type of molecule, including but not limited to peptides and non-peptide molecules that will act in a receptor-specific manner in the systems described.

One of the most suitable systems for use is a system that includes the introduction of the TIE-2 receptor into the fibroblast cell line (for example, in NIH3T3 cells) in such a way that a receptor that is not usually involved in proliferative reactions may, after introduction into fibroblasts, not less, be analyzed by various well-known techniques on the quantitative characteristics of the effects of fibroblast growth factors (e.g., incorporation of thymidine or other types of proliferation assays; 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 analyzes differ the additional advantage is that any drugs can be analyzed either for a cell line that has an introduced receptor, or as a parent cell line in which this receptor is absent; only specific effects on a cell line with a receptor should be considered as controlled by the introduced receptor. Such cells can be further constructed to express the TIE-2 ligand, thereby creating an autocrine system suitable for the analysis of molecules that act as antagonists / agonists of such an interaction.

Thus, the present invention provides host cells comprising a nucleic acid encoding a TIE-2 ligand and a nucleic acid encoding a TIE-2 receptor.

The TIE-2 receptor / TIE-2 ligand interaction also provides a useful system for identifying small TIE-2 receptor agonist or antagonist molecules. For example, fragments, mutants or derivatives of the TIE-2 ligand can be identified that bind the TIE-2 receptor but do not induce biological activity. In another embodiment, characterization of the TIE-2 ligand allows the determination of the active moiety. Further, the identification of the ligand allows you to determine the crystal structure (using x-rays) of the receptor / ligand complex, thereby allowing you to determine the binding site on the receptor. Knowledge of the binding site allows for the rational construction of new agonists and antagonists.

The specific binding of the test molecule to the TIE-2 receptor can be determined in various ways. For example, the actual binding of a test molecule to tie-2 expressing cells can be determined or measured by determining or measuring (i) test molecules bound to the surface of intact cells, (ii) test molecules crosslinked with TIE-2 protein in cell lysates or (iii) in vitro test molecules linked to TIE-2. The specific interaction between the test molecule and TIE-2 can be evaluated using reagents that demonstrate the unique properties of this interaction.

As a specific non-limiting example, the methods of the present invention can be used as follows. Consider the case in which it is necessary to determine the content of TIE-2 ligand in the sample. Various dilutions of the sample (test molecules) in parallel with the negative control (NC) containing no TIE-2 ligand activity and the positive control (PC) containing a known amount of TIE-2 ligand can be treated with cells that express tie-2 in the presence of detectable labeled TIE-2 ligand (in this example, a radioiodinated ligand). The amount of TIE-2 ligand in the test sample can be estimated by determining the amount of ¹²⁵ I-labeled TIE-2 ligand that binds to the controls and in each dilution, and then compare the values obtained for the samples with the calibration curve. The more TIE-2 ligand is contained in the sample, the less ¹²⁵ I-ligand will be associated with TIE-2.

The amount of ¹²⁵ I-ligand associated with TIE-2 can be determined by measuring the amount of radioactivity per cell, or by binding of the TIE-2 ligand to cell surface proteins using DSS according to Meakin and Shooter, 1991, Neuron 6: 153-163 and determining the amount of labeled protein in cell extracts using, for example, polyacrylamide gel electrophoresis, which helps to identify labeled protein with sizes corresponding to the TIE-2 ligand / TIE-2 receptor. Specific interactions of the test molecule / TIE-2 can be further tested by adding various dilutions of the unlabeled control ligand to the assay, which does not bind the TIE-2 receptor and therefore does not significantly affect the competition between the labeled TIE-2 ligand and the test molecule for TIE-2 binding . In another embodiment, molecules that are known to be capable of disrupting TIE-2 ligand / TIE-2 binding, such as, but not limited to, an anti-TIE-2 antibody or TIE-2 receptor body, as described herein can be expected to compete between the ¹²⁵ I-TIE-2 ligand and the test molecule for binding of the TIE-2 receptor.

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

Alternatively, the specific binding of the test molecule to TIE-2 can be measured by evaluating the secondary biological effects of binding of the TIE-2 ligand / TIE-2 receptor, including but not limited to cell growth, and / or differentiation or immediate early gene expression, or phosphorylation of TIE-2. For example, the ability of a test molecule to induce differentiation can be tested in cells that lack tie-2 and in comparative cells that express tie-2; differentiation in tie-2 expressing cells, but not in comparative cells that do not contain tie-2, will serve as an indicator of the interaction of a specific test molecule with TIE-2. Similar assays can be performed by detecting the induction of immediately early genes (e.g., fos and jun) in tie2-minus and tie-2-plus cells or by detecting TIE-2 phosphorylation using a standard phosphorylation assay known to those skilled in the art. Such assays may be useful in identifying agonists or antagonists that competitively bind to TIE-2.

Similarly, the present invention provides a method for identifying molecules that have the biological activity of a TIE-2 ligand, comprising (i) exposing cells that express tie-2 to test molecules, and (ii) detecting specific binding of the test molecule to the TIE-2 receptor, where specific binding to TIE-2 positively correlates with TIE-2 similar activity. Specific binding can be determined either by directly analyzing the binding, or by the secondary biological effects of binding, as discussed previously. Such a method can be especially useful when identifying new members of the TIE family of ligands or in the pharmaceutical industry when screening a large number of peptides and non-peptide molecules (eg, peptidomimetics) by TIE of the corresponding biological activity. In a preferred specific non-limiting embodiment of the invention, it is possible to prepare a large plate of culture cells that contain in alternating rows of PC12 (or fibroblasts) cells that are either tie-2-minus or constructed to be tie-2-plus. You can then add test molecules so that each column of the plate or part of it contains a test molecule. Then each assess for the presence or absence of growth and / or differentiation. Thus, a very large number of test molecules can be screened for their activity.

In further embodiments, the present invention provides methods for determining or measuring TIE-like activity, or for identifying molecules as having such activity, comprising; (i) in vitro exposing the test molecule to the TIE-2 receptor protein under conditions that allow binding, and (ii) detecting the binding of the test molecule to the TIE-2 protein when binding of the test molecule to TIE-2 correlates with TIE-like activity. In accordance with such methods, TIE-2 may or may not be substantially purified, may be bound to a solid support (for example, in an affinity column or as in an ELISA assay), or may be incorporated into an artificial membrane. The binding of the test molecule to TIE-2 can be evaluated by any method known to those skilled in the art. In preferred embodiments, the binding of the test molecule can be detected or measured by evaluating its ability to compete with the detectably labeled known TIE-2 ligands for TIE-2 receptor binding.

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

The present invention further provides both a method for identifying antibodies or other molecules capable of neutralizing a ligand or blocking its binding to a receptor, as well as molecules identified by this method. By way of non-limiting example, this method can be carried out in an assay that is conceptually similar to an ELISA assay. For example, the TIE receptor body can be associated with a solid carrier, for example, with a plastic multicell plate. As a control, you can then introduce into the cells a known amount of TIE ligand that has been Mus-labeled, and then any TIE-labeled ligand that binds the receptor body can be identified using a reporter antibody directed against Myc-tag. This assay system can then be used to screen test samples for molecules that are capable of: (i) binding to the labeled ligand, or (ii) binding to the receptor body and thereby block binding to the receptor body via the labeled ligand. For example, a test sample containing molecules of presumably of interest, together with a known amount of labeled ligand, can be introduced into the cell and the amount of labeled ligand that binds to the receptor body can be determined. By comparing the amount of bound labeled ligand in the test sample with the amount in the control, samples containing molecules that are capable of blocking the binding of the ligand to the receptor can be determined. Molecules of interest defined in this way can be isolated using methods well known to those skilled in the art.

Once a ligand binding blocker has been determined, those skilled in the art should be aware of performing a second analysis to determine whether the blocker binds to the receptor or whether it binds to the ligand, as well as performing an analysis to determine whether the blocker molecule can neutralize the biological activity of the ligand. For example, using a BIAcore binding assay of biosensor technology (or equivalent) in which either the TIE receptor body or TIE ligand is covalently coupled to a solid support (e.g., carloxymethyldextran or a gold surface), one can determine if the blocker molecule binds specifically with a ligand or with a receptor body. To determine whether a blocker molecule can neutralize the biological activity of a ligand, a specialist can carry out the analysis by phosphorylation (Example 5) or, alternatively, by a functional bioassay such as survival analysis from primary cultures, for example endothelial cells. In another embodiment, the blocker molecules that bind to the receptor body may be agonists, and it should be clear to one skilled in the art how to determine this by performing an appropriate analysis to identify additional TIE-2 receptor agonists.

Since the TIE-2 receptor has been identified in association with endothelial cells and, as shown here, blocking the ligand appears to prevent vascularization, applicants have demonstrated that the TIE-2 ligand may be useful for inducing vascularization in diseases or disease states, in of which this vascularization is indicated. Such diseases or disorders include wound healing, ischemia, and diabetes. On the other hand, TIE-2 receptor antagonists, such as receptor bodies, as disclosed in Examples 2 and 3, and TIE-2 ligand 2, as disclosed in Example 9, may be useful in preventing or attenuating vascularization, thereby preventing or attenuating for example, tumor growth.

Since cells containing the TIE-2 receptor are likely to be adjusted to enhance vascularization, TIE-2 ligand bodies may also be useful in preventing or attenuating, for example, tumor growth. TIE-2 ligands may be useful for delivering toxins to receptor-containing cells. In another embodiment, other molecules such as growth factors, cytokines, or nutrients can be delivered to cells containing the TIE-2 receptor via TIE-2 ligands. TIE-2 ligands can also be used as a diagnostic reagent for the TIE-2 receptor, to determine the receptor in vivo or in vitro. Where the TIE-2 receptor is associated with a disease state, TIE-2 ligand bodies can be used as diagnostic reagents for determining a disease, for example, when staining tissues or when acquiring an entire body image.

The present invention also provides pharmaceutical compositions comprising TIE-2 ligands or ligand bodies described herein, their peptide fragments or derivatives in a pharmaceutically acceptable carrier. TIE-2 ligand proteins, peptide fragments or their derivatives can be administered systemically or locally. You can use any suitable types of administration known to those skilled in the art, including (but not limited to) intravenous, intracranial, intraarterial, intranasal, oral, subcutaneous, intraperitoneal, either by local injection or as surgical implants. Sustained release compositions may also be prepared.

The present invention also provides isolated and purified nucleic acid molecules containing nucleic acid sequences encoding a human TIE-2 ligand, wherein the nucleic acid sequence is selected from the group consisting of:

(a) nucleic acid sequences containing a region encoding a TIE-2 human ligand, as shown in FIGS. 4, 5 or 6,

(b) nucleic acid sequences that hybridize under moderate stringency to the nucleic acid sequence of (a), and which encode a TIE-2 ligand that binds the TIE-2 receptor, and

(c) nucleic acid sequences that are degenerate due to the genetic code with respect to nucleic acid sequences (a) or (b), and which encode a TIE-2 ligand that binds the TIE-2 receptor.

In another embodiment, the invention provides an isolated nucleic acid molecule that encodes a TIE-2 ligand, where the nucleic acid sequence is:

(a) a nucleic acid sequence comprising a region encoding a human TIE-2 ligand, as shown in FIGS. 4, 5 or 6,

(b) a nucleic acid sequence that hybridizes under moderate stringency to a nucleic acid sequence (a) and which encodes a TIE-2 ligand that binds the TIE-2 receptor, or

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

The present invention also provides isolated and purified TIE-2 ligands encoded by an isolated nucleic acid molecule of the present invention. The present invention also provides a vector that comprises isolating a nucleic acid molecule comprising a nucleic acid sequence encoding a human TIE-2 ligand. In one embodiment, a vector is designated as pBluescript KS encoding human TIE-2 ligand 2.

The present invention provides an expression vector comprising a DNA molecule encoding a human TIE-2 ligand, wherein the DNA molecule is operably linked to an expression control sequence. The present invention also provides a host-vector system for producing a polypeptide having the biological activity of a human TIE-2 ligand, which comprises an expression vector of the present invention in cells of a suitable host. In one embodiment, suitable host cells may be bacterial cells, yeast cells, insect cells, or mammalian cells. The present invention also provides a method for producing a polypeptide having the activity of a biologically active TIE-2 ligand, which comprises growing the host-vector system cells of the present invention under conditions that produce the polypeptide and isolating the polypeptide thus obtained.

The present invention describes an isolated nucleic acid molecule encoding a TIE-2 ligand, and proposes the creation of a ligand, its fragment or derivative or other molecule that is a receptor agonist or antagonist, as a therapeutic agent for treating patients suffering from diseases including cells, tissues or organs that express the TIE receptor. As a non-limiting example, the present invention provides a ligand body that specifically binds to a TIE-2 receptor or TIE-2. Alternatively, the present invention provides a ligand body that includes a TIE-2 ligand linked to a constant portion of an immunoglobulin. In one embodiment, the ligand body comprises TIE-2 ligand 1 or TIE-2 ligand 2 and the Fc portion of human IgGl. The body of the ligand can be used in a method for treating human or animal organisms or for diagnostic purposes.

The present invention also provides an antibody that specifically binds such therapeutic molecules. Such an antibody may be monoclonal or polyclonal. The present invention also provides a method of using such monoclonal or polyclonal antibodies to determine the number of therapeutic molecules in a sample taken from a patient in order to monitor the course of treatment.

The present invention further provides a therapeutic composition comprising a human TIE-2 ligand or a ligand body, and a cytotoxic agent conjugated thereto. In one embodiment, the cytotoxic agent may be a radioisotope or toxin.

The present invention also provides an antibody that specifically binds a human TIE-2 ligand. This antibody may be monoclonal or polyclonal.

The present invention also provides a method for isolating TIE-2 human ligand, comprising:

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

b) incubating the substrate a) with a cell lysate so that this substrate forms a complex with any TIE-2 ligand in the cell lysate,

c) washing the solid matrix, and

d) elution of the TIE-2 human ligand from the connected substrate. As a substrate, any substance that specifically binds human TIE-2 ligand can be used. In one embodiment, the substrate is selected from the group consisting of an anti-TIE-2 ligand antibody, a TIE-2 receptor, and a TIE-2 receptor body. The present invention also provides a receptor body that specifically binds a TIE-2 ligand, as well as a therapeutic composition comprising a receptor body in a pharmaceutically acceptable carrier and a method for blocking blood vessel growth in humans, comprising administering an effective amount of this therapeutic composition.

The present invention also provides a therapeutic composition comprising a human TIE-2 ligand or a ligand body in a pharmaceutically acceptable carrier, as well as a method for promoting neovascularization in a patient, comprising administering to the patient an effective amount of the therapeutic composition.

In addition, the present invention provides a method for identifying cells that express a TIE-2 receptor, which comprises contacting a cell with a detectably labeled TIE-2 ligand or a ligand body under conditions that allow the detection of the labeled ligand to bind to the TIE-2 receptor and determine whether a detectably labeled ligand with a TIE-2 receptor, thereby identifying cells as cells expressing a TIE-2 receptor. The present invention also provides a therapeutic composition comprising a TIE-2 ligand or a ligand body and a cytotoxic agent conjugated thereto. The cytotoxic agent may be a radioisotope or toxin.

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

The present invention also provides a method for determining TIE-2 ligand in tissue sections, which comprises contacting these tissue sections with labeled nucleic acid molecules encoding a TIE-2 ligand under hybridization conditions, determining the presence of mRNA hybridized with a labeled molecule, thereby determining thereby, the expression of TIE-2 ligand in tissue sections.

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

Adult BAE cells are registered with the European Cell Culture Depository under ECACC registration number 92010601 (PNAS 75: 2621 (1978)). Northern (RNA) analysis revealed moderate levels of tie-2 transcripts in ABAE (adult bovine arterial endothelium = Adult Bovine Arterial Endothelial) cell line, consistent with hybrid results, which demonstrates the almost exclusive localization of tie-2 RNA on vascular endothelial cells. Therefore, ABAE cell lysates were examined for the presence of TIE-2 protein, as well as for the extent to which this TIE-2 protein is tyrosine-phosphorylated under normal growth conditions. ABAE cell lysates are collected and immunoprecipitated, followed by Western blotting of immunoprecipitated proteins due to TIE-2 specific and phosphotyrosine-specific antiserum. The omission or inclusion of TIE-2 peptides as specifically blocking molecules during TIE-2 immunoprecipitation allows us to unambiguously identify TIE-2 as a moderately detectable protein of approximately 150 kDa, whose stable phosphotyrosine levels are reduced to almost undetectable levels due to previous serum starvation of cells.

The cultivation of ABAE cells and the collection of cell lysates is as follows. ABAE cells with a low number of passages are seeded as a monolayer with a density of 2 × 10 ⁶ cells / 150 mm plastic Petri dishes (Falcon) and cultured in Dyulbeko modified Needle medium (DMEM) containing 10% calf serum (10% BCS), 2 mm L-glutamine (9) and 1% of each of penicillin and streptomycin (PS) in an atmosphere of 5% CO ₂ . Before collecting the cell lysate, the cells are subjected to serum starvation for 24 hours in DMEM (Q) PS, followed by aspiration of the medium and washing the plates with ice-cold phosphate buffered saline (PB S) supplemented with sodium orthovanadate, sodium fluoride and sodium benzamidine. Cells are lysed in a small volume of this wash buffer, which was supplemented with 1% NP40 detergent and protease inhibitors, PMSF and aprotinin. Insoluble fragments are removed from cell lysates by centrifugation at 14,000 g for 10 minutes at 4 ° C and the supernatants are immunoprecipitated with an antiserum specific for the TIE-2 receptor with or without blocking peptides added to approximately 20 μg / ml lysate. The immunoprecipitated proteins are separated by PAGE (7.5% Laemmli gel), and then electrically transferred to the PVDF membrane and incubated with either different TIE-2 or phosphotyrosine-specific antiserum. TIE-2 protein is visualized by incubating membranes with an HPP-linked secondary antiserum, followed by treatment with an ECL reagent (Amersham).

EXAMPLE 2. Cloning and expression of TIE-2 receptor bodies for affinity-based studies of the interactions of TIE-2 ligand

An expression construct is created that can secrete a protein consisting of the entire extracellular portion of the rat TIE-2 receptor fused to a constant region of human gamma-2 immunoglobulin (IgGl Fc). This fusion protein is called TIE-2 “receptor body” (RB) and it can usually be expected to exist as a dimer in solution due to the formation of disulfide bonds between individual IgGl Fc tails. The Fc portion of TIE-2 RB is prepared as follows. The DNA fragment encoding the Fc portion of human IgGl, which extends from the hinge region to the carboxy terminus of the protein, was amplified from human placenta cDNA by PCR with oligonucleotides corresponding to the human IgGl sequence, the resulting DNA fragment is cloned into a plasmid vector. The corresponding DNA restriction fragments from the plasmid encoding the full-length TIE-2 receptor and from the human IgGl Fc plasmid are ligated on each side of the short PCR-derived fragment that was designed to frame TIE-2 and human IgGl Fc protein-encoding sequence. Thus, the resulting TIE-2 ectodomain-FC fusion protein exactly replaces IgGl Fc in place of the region covering the TIE-2 transmembrane and cytoplasmic domains. An alternative method for producing RB is disclosed in Goodwin et al., Cell 73: 447-456 (1993).

Milligram amounts of TIE-2 RB are obtained by cloning a TIE-2 RB DNA fragment into a pVL 1393 baculovirus vector and subsequent infection with an insect cell line Spodoptera frugiperda SF-21AE. In another embodiment, you can use the cell line SF-9 (ATCC registration N CRL-1711) or cell line BTI-TN-5bl-4. The DNA encoding TIE-2 RB is cloned as an Eco RI-Notl fragment into the baculovirus transfer plasmid pVL 1393. Plasmid DNA purified by centrifugation with a density gradient of cesium chloride is recombined with viral DNA by mixing 3 μg of plasmid DNA with 0.5 μg of Baculo- Gold DNA (Pharminigen), followed by introduction into liposomes using 30 μg lipofectin (Lipofectin, GIBCO-BRL). Mixtures of DNA liposomes are added to F-21AE cells (2 × 10 ⁶ cells / 60 mm dish) in TMN-FH medium modified Grace insect cell medium (GIBCO-BRL) for 5 hours at 27 ° C for 5 days in TMN-FH medium supplemented with 5% fetal calf serum. Tissue culture media were harvested for plaque purification of recombinant viruses, as described previously (O'Reilly, DR, LK Miller, and VA Luckow, Baculovirus Expression Vectors - A Laboratory Manual. 1992, New York: WHFreeman) except that agarose the surface layer contains 125 μg / ml X-ga1 (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, GIBCO-BRL). After 5 days of incubation at 27 ° C, non-recombinant plaques are evaluated by a positive chromogenic reaction on the X-ga1 substrate and their positions are noted. Recombinant plaques were then visualized by adding a second surface layer containing 100 μg / ml MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma). Putative recombinant viral plaques are selected by aspiration of the plaques and purified through multiple plaque isolation cycles to ensure homogeneity. The stock of viruses is created due to serial passage with a low multiplicity of passage of plaque-purified virus. A low passage stock of one viral clone (vTIE-2 receptor body) is obtained.

SF-21AE cells were cultured in serum-free medium (SF-900 II, Gibco BRL) containing antibiotic / antimycotic IX solution (Gibco BRL) and 25 mg / L gentamicin (Gibco BRL). As a surface-active agent, Pluronic F-68 is added to a final concentration of 1 g / L. Cultures (4 L) were grown in a bioreactor (Artisan Cell Station System) for at least three days before infection. Cells are grown at 27 ° C, aerated to 50% dissolved oxygen, at a gas flow rate of 80 ml / min (aeration with atomization in a ring). Mixing is carried out using a turbine at a speed of 100 rpm Cells are harvested in the middle logarithmic growth phase (approximately 2 × 10 ⁶ cells per ml), concentrated by centrifugation and infected with 5 plaque-forming units of vTIE-2 receptor body per cell. The cells and inoculum are adjusted to 400 ml with fresh medium and the virus is absorbed for 2 hours at 27 ° C in a spinner. The culture is then resuspended to a final volume in 8 L of fresh serum-free medium and the cells are incubated in a bioreactor using the previously described conditions.

The culture medium of SF21AE cells infected with the TIE-2 receptor body is harvested by centrifugation (500 g, 10 minutes) 72 hours after infection. The supernatants were adjusted to pH 8 with NaOH. EDTA was added to a final concentration of 10 mM and the supernatant pH was adjusted back to 8. The supernatant was filtered (0.45 μm, Millipore) and placed on a Protein A column (Protein A Sepharose 4, Fast Flow or HiTrapp Protein A, both from Pharmacia ) The columns are washed with PBS containing 0.5 M NaCl until the absorption at 280 nM drops to the baseline. The column was washed with PBS and eluted with 0.5 M acetic acid. Column fractions were immediately neutralized, eluting into ampoules containing 1 M Tris pH 9. Peak fractions containing TIE-2 PB were collected and dialyzed against PBS.

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

Given the lack of a known ligand for the TIE-2 receptor, it would be reasonable to assume that this may make it possible to clarify the functions of TIE-2 by introducing an “excess” of soluble TIE-2 receptor bodies (TIE-2 SB) into the developing system. The potential ability of TIE-2 SB to bind and, thereby, neutralize the available TIE-2 ligands, can lead to the observed disruption of the normal development of blood vessels and to the characterization of the ligand. To investigate whether TIE-2 RB can be used to impair vascular development in early chick embryos, small pieces of biologically absorbing foam are wetted with TIE-2 RB and immediately placed under the chorioallanto membrane in a position directly to the side of the primitive embryo.

An early chicken embryo develops on top of the yolk from a small disk of cells that is coated with a chorioallanoid membrane (CAM). Endothelial cells, which must line the vasculature in the embryo, are formed both from the outside and within the embryo cells. Extraembryonic obtained endothelial cells, which provide the main source of endothelial cells in the embryo, come from sections of the mesenchyme, which is located on the sides around the embryo itself, directly below the CAM. As these mesenchymal cells mature, they become sources of the common offspring of both endothelial and hematopoietic cell lines called hemangioblasts. In turn, hemangioblasts serve as a source of mixed populations of angioblasts (offspring of endothelial cells) and hemocytoblasts (polypotential hematopoietic precursor). The formation of the vestiges of the circulatory system begins when the offspring of endothelial cells is separated with the formation of a bubble one cell thick that surrounds the primitive blood cell. The proliferation and migration of these cellular components leads to the formation of a network of blood-filled microvessels under the CAM, which ultimately encompasses the embryo and combines with limited intraembryonic-derived vascular elements.

Recently Fertilized Chicken Eggs Obtained from Spafas, Inc. (Boston, MA) are incubated at 99.5 ° F (37.5 ° C), 55% RH. After approximately 24 hours of development, the eggshell is washed with 70% ethanol and a 1.5 cm hole is made in the blunt end of each egg using dental bur. The shell membrane is removed to create air space directly above the embryo. Small rectangular pieces of sterile Gelfoam (Upjohn) are cut with a scalpel and moistened with equal concentrations of either TIE-2 or EHC-1 receptor bodies. The ENK-1 receptor body is prepared, as indicated earlier in Example 2, using the ENK-1 extracellular domain instead of the TIE-2 extracellular domain (Maisonpierre et al., Oncogene 8: 3277-3288 (1993). Each piece of gel foam absorbs approximately 6 micrograms of protein in 30 μl. The watchmaker's sterile tweezers are used to create a small hole in the CAM at a position a few mm on the side of the primitive embryo. Most of the piece of RV-wetted Gelfoam is placed under the CAM and the eggshell is covered with pieces of adhesive tape. Other eggs of the same developmental stages handle parallel but RB is an unrelated neurally expressed receptor tyrosine kinase, EHC-1 (Maisonpierre et al., Oncogene 8: 3277-3288 (1993). Development is allowed to proceed for 4 days, and then the embryo is visually examined. The embryos are removed by carefully breaking the shell into heated cups RB, and the embryo is carefully cut out from the surrounding CAM. Of the 12 eggs treated with each RB, 6 TIE-2 RB and 5 ENK-1, the RB treated embryos developed after the observation stage at the beginning of the experiment. A striking difference is observed between these developed embryos, as can be seen in FIGS. 1A and 1B. Those that were treated with EHC-1 KB seem to have developed relatively normally. Four of the five EHC-1 embryos were viable, as judged by heartbeats. Moreover, the extraembryonic vasculature, which was visually evident due to the presence of red blood cells, penetrated and extended several centimeters sideways under the CAM. In contrast, those treated with TIE-2 RB were very limited, having only 2-5 mm in diameter compared to 10 mm in diameter for ENK-1 RB embryos. All embryos treated with TIE-2 RB died and their CAMs were devoid of blood vessels. The ability of TIE-2 RB to block the development of blood vessels in chickens demonstrates that the TIE-2 ligand is necessary for the development of the vascular system.

EXAMPLE 4. Identification of the activity of specific binding of TIE-2 in a conditioned medium from a ras-oncogen-transformed mouse myoblast C2C12 cell line

Screening 10 times of concentrated cell conditioned medium (10 × CCM) from various cell lines for the presence of soluble TIE-2 specific binding activity (BIAcore; Pharmacia Biosensor, Piscataway, NY) reveals the binding activity in serum-free medium from oncogene-ras- transformed C2C12 cells (C2C12-ras), RAT 2-ras (which is the ras transformed fibroblast cell line), human glioblastoma E98G, and a neuroblastoma cell line known as SHEP-1.

C2C12-ras 10x CCM, originally obtained from a stably-transfected C2C12 line of myoblasts that were tumorigenically transformed by transfection of T-24 with H-ras mutants using standard methods using calcium phosphate. Neomycin-resistant SV40-based expression plasmid was physically linked to the ras expression plasmid in order to enable selection of transfected clones. The obtained G418 resistant ras-C2C12 cells are routinely maintained as a monolayer on plastic plates in DMEM / glutamine / penicillin-streptomycin medium supplemented with 10% fetal calf serum (FCS). Serum-free C2C12-ras 10x CCMs are obtained by plating cells at 60% confluency on a serum-free specific medium for 12 hours. (Zhan and Goldfarb, Mol. Cell. Biol. 6: 3541-3544 (1986)); Zhan, et al. Oncogene 1: 369-376 (1987)). The medium is drained and replaced with fresh DMEM / Q / P-S for 24 hours. This medium is harvested and the cells are fed again with fresh DMEM / Q / P-S, which is also harvested after another 24 hours. These CCMs are supplemented with PMSF protease inhibitors (1 mM) and aprotinin (10 μg / ml) and are concentrated 10 times on sterile size exclusion membranes (Amicon). TIE-2 binding activity can be neutralized by incubating the medium with an excess of TIE-2 RB, but not by incubating with ENK-1 RB before BIAcore analysis.

The binding activity of 10x CCM is determined using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ) which allows real-time monitoring of biomolecular interactions due to surface plasmon resonance. Purified TIE-2 RBs are covalently coupled via primary amines to the CM5 carboxymethyldextran layer of a research grade sensor chip (Pharmacia Biosensor, Piscataway, NJ). The surface of the sensor chip is activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide (EDS), followed by immobilization of TIE-2 RB (25 μg / ml, pH 4.5) and deactivation unreacted sites due to 1.0 M ethanolamine (pH 8.5). The negative control surface of the EHC-1 receptor body is prepared in a similar manner.

The wash buffer in this system is HBS (10 mM Hepes 3.4 mM EDTA, 150 mM NaCl, 0.005% P20 surfactant, pH 7.4). 10x CCM samples were centrifuged for 15 minutes at 4 ° C, and then clarified using a sterile, low protein binding filter 0.45 μM Millipore, Bedford, MA). Dextran (2 mg / ml) and P20 surface active agent (0.005%) are added to each CCM sample. Aliquots of 40 μl are injected through the immobilized surface (either TIE-2 or ENK-1) at a flow rate of 5 μl / min, and receptor binding is recorded for 8 minutes. Binding activity (resonance unit, RU) is defined as the difference between the baseline value determined 30 seconds before injection of the sample and the value obtained 30 seconds after injection. Surface regeneration is carried out due to one 12-μl pulse 3M MgCl ₂ .

The instrument noise level is 20 RU, therefore, any binding activity with a signal above 20 RU can be interpreted as a real interaction with the receptor. For C2C12-ras conditioned medium, binding activities are in the range of 60-90 RU for the TIE-2 RB immobilized surface. For the same samples analyzed on an ENK-1 RB immobilized surface, the measured activities were less than 35 RU. Specific binding to the TIE-2 receptor body is assessed by incubating samples with an excess of either soluble TIE-2 or ENK-1 RB before binding activity analysis. The addition of soluble ENK-1 RB does not affect TIE-2 binding activity for any sample, whereas in the presence of soluble TIE-2, surface binding is 2-30 times less than the value measured in the absence of TIE-2. Repeated analysis using more than 50-fold concentrated C2Cl2-ras CCM leads to a four-fold increase compared to the background value for the specific binding signal TIE-2.

EXAMPLE 5. C2Cl2-ras CCM contains an activity that induces tyrosine phosphorylation of the TIE-2 receptor

C2Cl2-ras 10x CCM was tested for its ability to induce tyrosine phosphorylation of TIE-2 in ABAE cells. ABAE cells after serum starvation are briefly incubated with C2C12-ras CCM, lysed and immunoprecipitated, and then Western blotting is performed as previously described. Stimulation of starving for serum ABAE cells not containing serum C2C12-ras 10x SSMD is as follows. Fasting medium ABAE cells, as described previously, are removed and replaced with either a specific medium or 10x CCM that has been preheated to 37 ° C. After 10 minutes, the medium was removed, and the cells were washed twice on ice with excess chilled PBS supplemented with orthovanadate (NaF) benzamidine. Cell lysis and TIE-2 specific immunoprecipitation are carried out as previously described.

ABAE cells incubated for 10 minutes with the indicated medium induce TIE-2 tyrosine phosphorylation, while incubation with C2C12-ras CCM stimulates at least a hundred-fold increase in TIE-2 phosphorylation. This activity almost completely disappears upon preliminary incubation of C2C12-ras 10x CCM for 90 minutes at room temperature with 13 μg of TIE-2 BB fixed on protein 6-sepharose beads. When incubated with protein alone, 6-Sepharose, the medium does not lose this phosphorylation activity.

EXAMPLE 6

Expression Cloning of TIE-2 Ligand

COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% of each penicillin and streptomycin (P / S) and 2 mM glutamine in an atmosphere of 5% CO ₂ . The cell line of murine C2C12 ras myoblasts was cultured in the minimum necessary Eagle medium (EMEM) with 10% FBS, (P / S) and 2 mM glutamine. Clone cDNA clones of mouse TIE-2 ligands were obtained by screening the C2C12 ras cDNA library in the pJFE14 vector expressed in COS cells. This vector, as shown in FIG. 2, is a modified version of the pSRα vector (Takebe, et al. 1988, Mol. Cell. Biol. 8: 466-472). This library is created using two BSTX1 restriction sites in the pJFE14 vector.

COS-7 cells are briefly transfected with either a pJFE14 library or a control vector due to a DEAE-dextran transfection scheme. In short, COS-7 cells are seeded at a density of 1.0 × 10 ⁶ cells per 100 mm plate 24 hours before transfection. For transfection, cells were cultured in serum-free DMEM containing 400 μg / ml DEAE-dextran, 1 μM chloroquine and 2 mM glutamine and 1 μg of the corresponding DNA, for 3-4 hours at 37 ° C in an atmosphere of 5% CO ₂ . The transfection medium is aspirated and replaced with buffered phosphate saline with 10% DMSO for 2-3 minutes. After this DMSO “shock”, COS-7 cells are placed in DMEM with 10% FBS, 1% of each penicillin and streptomycin and 2 mM glutamine for 48 hours.

Since the TIE-2 ligand is secreted, it is necessary to make the cells permeable to determine the binding of the receptor body probe to the ligand. Two days after transfection, the cells are washed with PBS and then incubated with PBS containing 1.8% formaldehyde for 15-30 minutes at room temperature. The cells are then washed with XXX PBS and incubated for 15 minutes with PBS containing 0.1% Triton X-100 and 10% calf serum in order to make the cells permeable and block nonspecific binding sites. Screening is carried out by direct localization of staining using the TIE-2 receptor body, which consists of the extracellular domain of TIE-2, fused to a constant IgGl site. This receptor body is prepared as described previously in Example 2. 100 mm plates with transfected, fixed and made permeable COS cells are probed by incubating them for 30 minutes with TIE-2-RB. The cells are then washed twice with RBS and incubated for an additional 30 minutes with RBS (10% calf serum) anti-human IgG alkaline phosphatase conjugate. After three RBS washes, cells were incubated in an alkaline phosphatase substrate for 30-60 minutes. Then the cups are examined under the microscope for the presence of stained cells. For each of the stained cells, a small portion of the cells, including this stained cell, is excised from the dish using the tip of a plastic pipette, then plasmid DNA is isolated and used for electrostroporation of the bacterial cells. Separate bacterial colonies obtained by electroporation were selected and plasmid DNA obtained from these colonies was used to transfect COS-7 cells that were probed for TIE-2 ligand expression as evidence of binding to TIE-2 receptor bodies. This allows the identification of individual colonies encoding the TIE-2 ligand. Confirmation of the expression of the TIE-2 ligand is obtained by phosphorylation of the TIE-2 receptor using the method described previously in example 5. The plasmid clone encoding the TIE-2 ligand was deposited at ATCC on October 7, 1994 and designated as "pJFE14 encoding the TIE-2 ligand" under ATCC registration number 75910.

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

The human fetal lung cDNA library in lambda-gt 10 (FIG. 3) was obtained from Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were plated at a density of 1.25 × 10 ^6/20 × 20 cm plates and the filters with the prints removed in accordance with standard procedures (Sambrook, et al, Molecular Cloning :. A Laboratory Manual, 2nd Ed, page 8.46, Cold Spring Harbor. Laboratory, Cold Spring Harbor, New York).

Isolation of tie-2 clones to human ligands is as follows. 2.2 kb Xhol fragment from the deposited tie-2 ligand clone (ATCC No. 75910 - Example 6) was labeled by statistical priming to a specific activity of about 5 × 10 ⁸ cpm / ng. Hybridization is carried out at 65 ° C in a hybridization solution containing 0.5 mg / ml salmon sperm DNA. The filters are washed at 65 ° C in 2x SSC, 0.1% SDS and exposed to Kodak XAR-5 film overnight at -70 ° C. Positive phages are purified. High titer phage lysates from pure phages are used to isolate DNA on a Qiagen column using standard techniques (Qiagen, Inc., Chatsworth, CA, 1995 catalog, p. 36). Phage DNA is digested with EcoRl to isolate the cloned cDNA fragment for subsequent subcloning. A lambda phage vector containing human tie-2 ligand DNA was deposited in ATCC on October 26, 1994 under the name λgt10 encoding htie-2 ligand 1 (ATCC registration number 75928). Phage DNA can be directly subjected to DNA sequencing by a method using dideoxy chain termination (Sanger, et al., 1977, Proc. Natl. Acad. Sci. USA. 74: 5463-5467).

Subcloning TIE-2 human ligand into a mammalian expression vector

Clone λtgt10 encoding htie-2 ligand 1 contains an EcoRl site located at 490 base pairs in the forward direction from the start of the coding sequence for human tie-2 ligand. The coding region can be cut using unique restriction sites in the reverse and forward direction from the initiator and stop codon, respectively. For example, a Spel site located 70 bp 5 ′ from the initiator codon, and a Bpu1102 (also known as B1p1) site located 265 bp in the direction 3 ′ from the stop codon, can be used to cut out the complete coding region. It can then be subcloned into a pJFE14 cloning vector using Xbal (Spel compatible protrusion) and Pstl sites (Pstl and Bpu1102 sites both form blunt ends).

Human TIE-2 Ligand Sequencing

The coding region from λlgr10 clone encoding tie-2 ligand 1 is sequenced using an AB1 373A DNA sequencer and Taq Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The nucleotide and isolated amino acid sequence of the human TIE-2 ligand from clone λgt10 encoding tie-2 ligand 1, are presented in figure 4.

In addition, full-length human ligand TIE-2 cDNA clones are obtained by screening a T989 human glioblastoma cDNA library in a pJFEl4 vector. Clones encoding human tie-2 ligand were identified by DNA hybridization using a 2.2 kb Xhol fragment from a deposited tie-2 ligand clone (ATCC No. 75910) as a probe (Example 6). The coding region is sequenced using an ABl 373A DNA sequencer and the Taq Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). This sequence is almost identical to the sequence of clone λgt10 encoding tie-2 ligand 1. As shown in FIG. 4, clone λgt10 encoding tie-2 ligand 1 contains an additional glycine residue, which is encoded by nucleotides 1114-1116. The coding sequence of the T98G clone does not contain this glycine residue, but, on the other hand, is identical to the coding sequence of the gt10 clone encoding tie-2 ligand 1. Figure 5 shows the nucleotide and deduced amino acid sequences and the human TIE-2 ligand from the T98G clone.

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

A human fetal lung cDNA library in lambda gtl0 (FIG. 3) was obtained from Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were plated at a density of 1.25 × 10 ^6/20 × 20 cm plate and replica on the filters prepared in accordance with standard procedure (Sambrook, et al, Molecular Cloning :. A Laboratory Manual, 2nd Ed, page 8.46, Cold Spring Harbor. Laboratory, Cold Spring Harbor, New York). Duplicate filters are screened under mild conditions (2x SSC, 55 ° C) with probes obtained for tie-2 L-1 human sequences. One of the duplicate filters is probed with a 5 ′ probe encoding amino acids 25-265 of human tie-2 L-1, as shown in FIG. 4. The second duplicate filter is probed with a 3 'probe, which encodes amino acids 282-498 tie-2 L-1 of the human sequence (figure 4). Both probes hybridize at 55 ° C in a hybridization solution containing 0.5 mg / ml salmon sperm DNA. The filters are washed in 2x SSC at 55 ° C and exposed overnight to x-ray film. In addition, duplicate filters hybridize under normal stringency conditions (2x SSC, 65 ° C) with a full length mouse tie-2L coding probe (F3-15, Xhol insert). Three positive clones are selected that satisfy the following criteria: i) hybridization is not observed with the full length (mouse) probe under conditions of normal stringency, and ii) hybridization is observed under mild conditions with both 5'- and 3'-probes. EcoR1 digestion of phage DNA obtained from these clones indicates two independent clones with insert sizes of about 2.2 kb and about 1.8 kb. The 2.2 kb EcoRl insert is subcloned into the EcoR1 sites of both pBluescript KS (Stratagene) and a mammalian expression vector suitable for use in CO cells. Two orientations are determined for the mammalian expression vector. The 2.2 kb insert in pBluescript KS was deposited in ATCC on December 9, 1994 and designated as pBluescript KS encoding human TIE-2 ligand 2. The start site of the TIE-2 ligand 2 coding sequence contains approximately 355 base pairs in the forward direction from the pBluescript EcoR1 site.

COS-7 cells are transiently transfected with either an expression vector or a cloning vector according to the DEAE-dextran transfection scheme. In short, COS-7 cells are seeded at a density of 1.0 × 10 ⁶ cells per 100 mm plate 24 hours before transfection. For transfection, cells were cultured in serum-free DMEM containing 400 μg / ml DEAE-dextran, 1 μM chloroquine and 2 mM glutamine, and 1 μg of the corresponding DNA for 3-4 hours at 37 ° C in an atmosphere of 5% CO ₂ . The transfection medium is aspirated and replaced with buffered phosphate saline with 10% DMSO for 2-3 minutes. After this DMSO “shock”, COS-7 cells were placed in DMEM with 10% FBS, 1% of each penicillin and streptomycin and 2 mM glutamine for 48 hours.

Since the TIE-2 ligand is secreted, it is necessary to make the cells permeable in order to determine the binding of the receptor body probe to the ligand. Transfected COS-7 cells are seeded with a density of 1.0 × 10 ⁶ cells per 100 mm plate. These cells are wetted with PBS and then incubated with PBS containing 1.8% formaldehyde for 15-30 minutes at room temperature. The cells are then washed with PBS and incubated for 15 minutes in PBS containing 0.1% Triton X-100 and 10% calf serum to impart permeability to the cells and block nonspecific binding sites. Screening is performed by directly localizing staining using the TIE-2 receptor body, which consists of the extracellular domain of TIE-2 fused to a constant IgGl site. This receptor body was prepared as described in Example 2. Transfected COS cells were probed by incubating them for 30 minutes with TIE-2 RB. The cells are then washed twice with PBS, fixed with methanol, and then incubated for an additional 30 minutes with PBS / 10% calf serum anti-IgG human alkaline phosphatase conjugate. After three washes with PBS, cells are incubated in an alkaline phosphatase substrate for 30-60 minutes. The plates are then examined under a microscope to determine the presence of stained cells. Cells expressing one orientation of the clone, but not another orientation, are seen to bind the TIE-2 receptor body.

It will be apparent to one skilled in the art that the methods described can be used to further identify other related members of the TIE family of ligands.

Sequencing of the second TIE-2 human ligand

The coding region from pBluescript KS clone encoding human TIE-2 ligand 2 is sequenced using an AB1 373A DNA sequencer and Taq Dyedeoxy Terminator Cycle Sequencing Kit (Applid Biosystems, Inc., Foster City, CA). The nucleotide and deduced amino acid sequences of the human TIE-2 ligand from clone pBluesript KS encoding human TIE-2 ligand 2 are shown in Fig.6.

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

A conditioned medium of COS cells expressing either TIE-2 ligand 2 (TI2) or TIE-2 ligand 1 (TL1) is compared in terms of their ability to activate TIE-2 receptors naturally present in the human endothelial cell line.

Lipofectamine reagent (GIBCO-BRL, Inc.) and recommended regimens are used to transfect COS-7 cells with either one pJFE14 expression vector, pJFE14 vector containing human TIE-2 ligand 1 cDNA or with pMT21 expression vector (Kaufman, RJ, 1985, Proc . Natl. Acad. Sci. USA 82: 6892693) containing human TIE-2 ligand 2 cDNA. COS medium containing secreted ligands was collected three days later and concentrated 20 times by diafiltration (DIAFLO ultrafiltration membranes, Amicon, Inc.). The amount of active TIE-2 ligand 1 and TIE-2 ligand 2 present in these media is determined and expressed as the number (in resonance units, RU) of TIE-2 specific binding activity receptor as determined by the BlAcore binding assay.

Northern blotting (RNA) analysis revealed significant levels of TIE-2 transcripts in NAES (human aortic endothelial cells) of human primary endothelial cells (Clone-ties, Inc.), so these cells were used to determine whether the TIE-2 receptor becomes tyrosine-phosphorylated upon exposure to COS medium containing TIE-2 ligands. NAEC cells are maintained in a complete growth medium for growing endothelial cells (Clone-ties, Inc.), which contains 5% fetal calf serum, soluble bovine brain extract, 10 ng / ml human EGF, 1 mg / ml hydrocortisone, 50 μg / ml gentamicin and 50 ng / ml amphotericin-B. The determination of which of TL1 and TL2 can activate the TIE-2 receptor in HAEC cells is carried out as follows. NAEC semi-confluent cells are forced to fast on serum for two hours in Dulbecco high-glucose MEM supplemented with L-glutamine and penicillin-streptomycin at 37 ° C, followed by replacement of fasting medium with ligand-conditioned COS medium for 7 minutes at 37 ° C in an incubator with 5% CO ₂ . The cells are then lysed and the TIE-2 receptor protein is isolated by immunoprecipitation of TIE-2 lysates with a peptide antiserum followed by Western blotting with antiphosphothyrosine antiserum exactly as in Example 1. The results are shown in Fig.7. Phosphothyrosine levels at the TIE-2 receptor (TIE-2-B) are induced by treating HEAC cells with TIE-2 ligand 1 (L1 band) rather than TIE-2 ligand 2 (L2 band) of conditioned COS medium. MOCK represents an air-conditioned environment of COS transfected with JFE14 blank vector.

Evidence that both TL1 and TL2 specifically bind the TIE-2 receptor is demonstrated by using BlAcore to analyze TIE-2 receptor specific activities in transfected COS medium and by immunostaining TL1 and TL2 expressing COS TIE-2 receptor cells bodies.

Since TL2 does not activate the TIE-2 receptor, applicants decided to determine whether TIE-2 can act as an antagonist of TL1 activity. An HAEC phosphorylation assay is performed, in which cells are first incubated with an “excess” of TL2 followed by the addition of diluted TL1. It turned out to be reasonable that, prior to occupying the TIE-2 receptor, due to high levels of TL2, it can prevent subsequent stimulation of the receptor, followed by exposure to the TL1 present in limited concentrations.

The semi-confluent HAEC cells are starved for serum as previously indicated and then incubated for 3 minutes at 37 ° C. with 1-2 ml of 20x COS / JFE14-TL2 conditioned medium. The control plates only process 20x COS medium (MOCK). The plates are removed from the incubator, and then various dilutions of COS / JFE14-TL1 medium are added, followed by further incubation of the plates for 5-7 minutes at 37 ° C. Cells are then washed, lysed, and TIE-2 specific tyrosine phosphorylation in lysates is examined by immunoprecipitation of receptors and Western blotting, as previously indicated. TL1 dilutions are carried out using 20x COS / JFE14-TL1 medium diluted to 2x, 0.5x, 0.1x or 0.02x by adding only 20x COS / JFE14 medium. Analysis of the original 20x TL1 and 20x TL2 COS media using BlAcore biosensor technology shows that they contain similar amounts of TIE-2 specific binding activities, i.e. 445 RU and 511 RU for TL1 and TL2, respectively. The results of antiphosphothyrosine Western blotting shown in Fig. 8 show that, compared to pretreatment of HAEC MOCK cells with medium (lane 1), pretreatment of HAEC cells with excess TIE-2 ligand 2 (lane 2) counteracts the corresponding ability of diluted TIE-2 ligand 1 activate TIE-2 receptor (TIE-2-R).

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

EXAMPLE 10. Identification of TIE-2 specific binding activity in a coded medium and supernatant of COS cells

The binding activity of 10x CCM from C2C12 ras, Rat2 ras, SHEP and T986 cell lines or supernatant of C0S cells after transfection with either TIE-2 human ligand 1 (hTL1) or TIE-2 human ligand 2 (hTL2) is determined using biosensor technology ( BlAcore; Pharmacia Biosensor, Piscatway, NJ), which detects real-time biomolecular interactions through surface plasma resonance (SPR). Purified TIE-2 RBs in humans or rats are covalently coupled via primary amines to a layer of a research grade purity sensor carboxymethyldextran CM5 (Pharmacia Biosensor, Piscataway, NJ).

The surface of the sensor chip is activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide (EDS), followed by immobilization of TIE-2 RB (25 μg / ml, pH 4.5) and deactivation unreacted portions of 1.0 M ethanolamine (pH 8.5).

Typically, 9000-10000 RU of each of the receptor bodies is connected to a sensor chip.

HBS (10 mM Hepes, 150 mM NaCl, 0.005% P20 surface active agent, pH 7.4) is used as a flow buffer in the system. Samples were centrifuged for 15 minutes at 4 ° C and then cleaned using a sterile 0.45 μm low protein coupler filter (Millipore; Bedford, MA). Dextran (2 mg / ml) and surfactant P20 (0.005%) are added to each sample. Aliquots of 40 ml are injected through an immobilized surface (either human TIE2 or rat) at a flow rate of 5 μl / min and receptor binding is recorded for 8 minutes. Binding activity (resonance units, RU) is defined as the difference between the baseline, determined 30 seconds before injection of the sample, and the measured result obtained 30 seconds after injection. The surface regeneration is carried out due to one 15 μl of 3M MgCl ₂ input.

CCM samples (C2C12 ras, Rat2 ras, SHEP, T98G) are tested on the immobilized surface of rat TIE2 RB, while recombinant hTL1 and hTL2 are tested on the immobilized surface of TIE2 RB human. In each case, specific binding to the TIE-2 receptor body was evaluated by incubating samples of 25 μg / ml of either soluble TIE-2 (rat or human) RB or trkB RB before evaluating binding activity. As shown in FIGS. 9 and 10, the addition of soluble trkB RB causes a slight decrease in TIE-2 binding activity, while the addition of soluble TIE-2 RB significantly reduces binding activity compared to results in the absence of TIE-2 RB.

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

Applicants wanted to determine whether soluble TIE-2 RB can serve as a competing inhibitor when blocking TIE-2 receptor activation by TIE-2 ligand 1 (TL1). To accomplish this, the TLl-containing COS medium is preincubated with either TIE-2 or TgkB-RB, and then their ability to activate TIE-2 receptors naturally present in the human endothelial cell line is compared.

Conditioned COS medium is obtained from COS cells transfected with either pJPE14 single expression vector (MOCK) or pJFE14 vector containing human TIE-2 ligand 1 cDNA (TL1) and collected as shown in Example 9, except that the medium is filtered sterile conditions, but do not concentrate. The amount of TL1 is determined and expressed as the amount (in resonance units, RU) of TIE-2 receptor-specific binding activity determined in the BlAcore binding assay.

Northern blotting (RNA) reveals significant levels of TIE-2 transcripts in HUVES (human umbilical vein endothelial cells), human primary endothelial cells (Cloneties, Inc). Therefore, these cells are used to determine whether the TIE-2 receptor can be tyrosine phosphorylated when exposed in the presence of TIE-2 or ThqB-RB in a COS medium containing TL1. HUVEC cells are maintained at 37 ° C, 5% CO ₂ in a complete medium for growing endothelial cells (Clonetics, Inc.), which contains 5% fetal calf serum, soluble bovine brain extract with 10 μg / ml heparin, 10 ng / ml EGF human, 1 μg / ml hydrocortisone, 50 μg / ml gentamicin and 50 ng / ml amphotericin-B. Determining whether TL1 can activate the TIE-2 receptor in HUV EC cells is carried out as follows. Confluent plates of HUV EC cells are starved for two to four hours in Dulbecco MEM low glucose at 37 ° C, 5% CO ₂ followed by ten-minute incubation in starvation medium containing 0.1 mM sodium orthovanadate, a potential inhibitor phosphothyrosine phosphatase. Meanwhile, conditioned COS medium, pre-incubated for 30 minutes at room temperature with either TIE-2 or TrkB-RB, was added up to 50 μg / ml. Fasting medium is removed from the HUVEC plates and incubated with RB-containing COS medium for 7 minutes at 37 ° C. The HUV EC cells are then lysed and the TIE-2 receptor protein is isolated by immunoprecipitation of the TIE-2 peptide antiserum followed by Western blotting with an antiphosphothyrosine antibody, as described in Example 1. The results are shown in FIG. 11. TIE-2 receptor phosphotyrosine levels are induced by treating HUVEC cells with TIE-2 ligand 1 (TL1) relative to those observed for control medium (MOCK), and this induction is specifically blocked by pre-incubation with TIE2-RB (TIE2-Fc ), but not due to incubation with TrkB-RB (TrkB-Fc). These data show that soluble TIE-2 RB can serve as a selective inhibitor to block the activation of the TIE-2 receptor due to TIE-2 ligand 1.

EXAMPLE 12. Construction of TIE-2 ligand bodies

The expression construct is designed to provide a secreted protein consisting of the complete coding sequence of human TIE-2 ligand 1 (TL1) or TIE-2 ligand 2 (TL2) fused to the constant region of human gamma-1 immunoglobulin (IgGl Fc). These fusion proteins are called TIE-2 "ligand bodies" (TL1- or TL2-). The Fc portion of TL1-Fc and TL2-Fc are prepared as follows. The DNA fragment encoding the Fc portion of human IgGl that extends from the hinge to the carboxy terminus of the protein is amplified from human placenta cDNA by PCR with oligonucleotides corresponding to the published human IgGl sequence, the resulting DNA fragment is cloned into a plasmid vector. The corresponding DNA restriction fragments from the plasmid encoding full length TL1 or TL2 and from the Fc of the human IgGl plasmid are ligated on each side of the short PCR fragment that was designed to be fused in frame of TL1 or TL2 with the human FG IgGl protein coding sequence .

Milligram amounts of TL2-Fc are obtained by cloning the DNA of the TL2-Fc fragment into the pVL 1393 baculovirus vector and then culturing the infected insect cell line Spodoptera Frugiperda SF-21AE. In another embodiment, you can use the cell line SF-9 (ATCC registration number CRL-1711) or cell line BPl-TN-5bl-4. DNA encoding TL2-FC is cloned as an EcoRl-Notl fragment into the baculovirus transfer plasmid pVL 1393. Plasmid DNA is recombined into viral DNA by mixing 3 mg of plasmid DNA with 0.5 mg of Baculo-Gold DNA (Pharminigen), followed by liposomes using 30 mg lipofectin (GIBCO-BRL). DNA liposome mixtures are added to SF-21AE cells (2 x 106 cells) in a 60 mm dish (in TMN-FH medium). Fraces Modified Insect Cell Medium (GIBCO-BRL): for 5 hours at 27 ° C followed by incubation at 27 ° C for 5 days in TMN-FH medium supplemented with 5% fetal calf serum. The tissue culture medium is harvested for purification of plaques of recombinant viruses, which is carried out using the methods described previously (O'Reilly, DR, LK Miller, and VA Luckow, Baculovirus Expression Vectors - A Laboratory Manual. 1952, New York: WH Freeman) except that the agarose surface layer contains 125 mg / ml X-gal (5-bromo-4-chloro-3-indolyl-bD-galactopyranoside, GIBCO-BRL). After 5 days of incubation at 27 ° C, non-recombinant plaques were counted for a positive chromogenic reaction with the X-gal substrate and their positions were noted. Recombinant plaques are then visualized by adding an additional second surface layer containing 100 mg / ml MTT (3- / 4,5-dimethylthiazol-2-yl / -2,5-diphenyltetrazolium bromide, Sigma).

Plaques of putative recombinant viruses are aspirated and purified in multiple plaque isolation cycles to ensure homogeneity. The stock of viruses is created as a result of a series of passages with a low multiplicity of plaque-purified virus. Get a stock with a small number of passages of one viral clone (vTL2-Fc clone No. 7).

F-21AE cells are cultured in serum-free medium (SF-900 II, Gibco BRL) containing 1x antibiotic / antimycotin solution (Gibco BRL) and 25 mg / L gentamicin (Gibco BRL). As a surface-active agent, Pluronic F-68 is added to a final concentration of 1 g / L. Cultures (4 L) were grown in a bioreactor (Artisan Cell Station System) for at least three days before infection. Cells are grown at 27 ° C, carbonated with up to 50% dissolved oxygen at a gas flow rate of 80 ml / min (aeration along the spray ring). Mixing is carried out with a turbine at a speed of 100 rpm. Cells are harvested in the mid-log phase of growth (approximately 2 × 10 ⁶ cells per ml), concentrated by centrifugation and infected with 5 plaque-forming units of vTL2-Fc per cell. The cells and inoculum were adjusted to 400 ml by adding fresh medium, and the virus was adsorbed for 3 hours at 27 ° C in a spinner. The culture is then resuspended in a final volume of 8 L of fresh serum-free medium, and the cells are incubated in the bioreactor using the previously described conditions.

The culture medium from vTL2-Fc infected with SF21AE cells is collected by centrifugation (500 vol., 10 minutes) at 72 hours after infection. The cell supernatant was adjusted to pH 8 with NaOH. EDTA was added to a final concentration of 10 mM, and the pH of the supernatant was adjusted to 8. The supernatant was filtered (0.45 mm, Millipore) and introduced into a protein A column (Protein A, Sepharose 4, flow rate or HiTrap Protein A, both from Pharmacia). The column is washed with PBS containing 0.5 M NaCl until the absorbance at 280 nm drops to the baseline. The column was washed with PBS and eluted with 0.5 M acetic acid. Fractions from the column are immediately neutralized, eluting into ampoules containing 1 M Tris, pH 9. Peak fractions containing TL2-FC are collected and dialyzed against PBS.

Deposits

The following were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 in accordance with the Budapest Agreement. The plasmid clone encoding the TIE-2 ligand was deposited with ATCC on October 7, 1994 and designated as "pJFE14 encoding the TIE-2 ligand" under ATCC registration No. 75910. Recombinant baculovirus Autographa californica encoding the TIE-2 receptor body was deposited with ATCC October 7, 1994 and designated as "vTIE-2 receptor body" under ATCC registration number VR2484. A lambda phage vector containing human tie-2 ligand DNA was deposited with ATCC on October 26, 1994 and designated λgt10 encoding tie-2 ligand 1 under ATCC registration No. 75928. The plasmid clone encoding the second TIE-2 ligand was deposited with ATCC on December 9, 1994 and designated as "pBluescript KS encoding the human TIE-2 ligand 2" under ATCC registration number 75963.

The present invention is not limited by the scope of the specific embodiments disclosed herein. Naturally, various modifications of the invention, in addition to those disclosed herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications should be included in the scope of the attached claims.

Claims (16)

1. The selected nucleic acid molecule encoding a TIE-2 ligand, comprising a nucleotide sequence encoding the amino acid sequence shown in Fig.6. 2. Isolated TIE-2 ligand, which is a TIE-2 ligand having the amino acid sequence shown in Fig.6, essentially free of other proteins. 3. Vector pBluescript KS encoding TIE-2 ligand 2 person and deposited in ATCC with registration No. 75963. 4. The COS cell line transformed with the vector according to claim 3, used to produce the TIE-2 ligand according to claim 2. 5. A method of producing a TIE-2 ligand, comprising growing host cells transformed with a vector comprising the nucleic acid molecule of claim 1, under conditions providing for the production of a ligand, and isolating the ligand thus obtained. 6. The antibody specifically binds to the TIE-2 ligand according to claim 2, obtained using the ligand according to claim 2. 7. The antibody according to claim 6, which is a monoclonal antibody. 8. The TIE-2 ligand conjugate comprising the TIE-2 ligand according to claim 2 and a cytotoxic agent conjugated thereto. 9. The conjugate of claim 8, wherein the cytotoxic agent is a radioisotope or toxin. 10. The ligand body specifically binding to the TIE-2 receptor, which includes the TIE-2 ligand according to claim 2, fused to the constant region of immunoglobulin. 11. The ligand body of claim 10, wherein the constant portion of the immunoglobulin is the Fc part of human IgG1. 12. A pharmaceutical composition for preventing or attenuating neovascularization in a mammal, comprising an effective amount of a TIE-2 ligand according to claim 2 or a ligand body according to claim 10 and a pharmaceutically acceptable carrier. 13. A method for preventing or attenuating neovascularization in a human or animal subject, comprising administering to the subject a pharmaceutical composition according to claim 12. 14. The method of claim 13 for attenuating or preventing tumor growth. 15. The method according to p. 13 or 14, characterized in that the subject is a person. 16. A method for identifying a TIE-2 receptor antagonist, comprising contacting cells expressing a TIE-2 receptor with (a) a test compound and (b) a TIE-2 ligand according to claim 2, under conditions providing for binding of the ligand to the receptor and determining whether the test compound is able to prevent ligand binding to the receptor.

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