KR100392937B1 - Tie-2 ligands, methods of making and uses thereof - Google Patents

Abstract

The present invention provides isolated nucleic acid molecules encoding human TIE-2 ligands. In addition, the present invention provides a receptor body that specifically binds a human TIE-2 ligand. The invention also provides antibodies that specifically bind human TIE-2 ligands. The invention also provides antagonists of human TIE-2 ligands. The present invention also provides a method of blocking vascular growth, as well as a therapeutic composition, a method for promoting neovascularization, a method for promoting growth or differentiation of cells expressing a TIE-2 receptor, a growth of cells expressing a TIE-2 receptor. Or methods of blocking differentiation, and methods of delaying or preventing tumor growth in humans.

Description

TIE-2 ligand, its preparation method and use {TIE-2 LIGANDS, METHODS OF MAKING AND USES THEREOF}

The cellular actions that contribute to the development, maintenance and repair of differentiated cells and tissues are largely regulated by growth factors and pseudoligands and intercellular signals carried through their receptors. The receptors are located on the cell surface of the response cells and they bind peptides or polypeptides known as growth factors and also other hormone-like ligands. The result of this interaction is not only rapid biochemical changes in the responding cells but also rapid and long-term reregulation of cellular gene expression. Several receptors associated with various cell surfaces bind specific growth factors.

Phosphorylation of tyrosine in proteins by tyrosine kinases is one of the key ways in which signals are transmitted across cell membranes. Some currently known protein tyrosine kinase genes include polypeptide growth factor and epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF-A and -B), and fibroblast growth. Encode transmembrane receptors for hormones such as 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 function by binding to the extracellular portion of their homologous receptors, which results in the activation of the native tyrosine kinases present in the cytoplasmic portion of the receptor. Endothelial growth factor receptors are of particular interest due to possible attendance of growth factors in some important physiological and pathological processes such as angiogenesis, angiogenesis, thromboembolic diseases and inflammatory diseases (Folkman, et al., Science, 235: 442-447 (1987). In addition, the receptors of several hematopoietic growth factors are tyrosine kinases and these are the colony stimulator 1 receptors reported in Sherr, et al., Cell, 41: 665-676 (1985), c-fms and Huang, et al., Cell. 63: 225-33 (1990), c-kit, the primitive hematopoietic growth factor receptor reported.

Receptor tyrosine kinases are divided into evolutionary subfamily based on the characteristic structure of their outer domains (Ullrich, et al., Cell, 61: 243-54 (1990)). Such subfamily include EGF receptor-like kinases (subclass I) and insulin receptor-like kinases (subclass II), each of which contains homologous cysteine rich sequences repeated in their extracellular domain. Single cysteine rich regions are also found in the extracellular domain 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)). In addition to c-fms and c-kit receptor tyrosine kinases, PDGF receptors are classified as subclass III, while FGF receptors form subclass IV. Representative of both of these subclasses are extracellular folding units stabilized by intrachain disulfide bonds. These so called immunoglobulin (Ig) -like folds are found in proteins of the immunoglobulin superfamily containing a wide variety of other cell surface receptors with cell binding or soluble ligands (William, et. al., Ann. Rev. Immunol., 6: 381-405 (1988).

Receptor tyrosine kinases differ in their specificity and affinity. Generally receptor tyrosine kinases are glycoproteins, which include (1) an extracellular domain capable of binding specific growth factors; (2) a transmembrane domain, usually the alpha helix portion of a protein; (3) a membrane proximity domain in which the receptor can be regulated, for example by protein phosphorylation; (4) a tyrosine kinase domain that is an enzymatic component of the receptor; And (5) a carboxy terminal tail that is involved in the recognition and binding of a substrate for tyrosine kinase in many receptors.

It has been reported that methods such as alternating exon splicing and alternating selection of gene promoters or polyadenylation sites can produce several distinct polypeptides from the same gene. These polypeptides may or may not have the various domains listed above. As a result, some extracellular domains may be expressed as separate secreted proteins and some types of receptors lack tyrosine kinase domains and have only extracellular domains and short carboxyl termini inserted through the transmembrane domain into the cytoplasmic membrane.

Genes encoding endothelial transmembrane tyrosine kinases originally identified by RT-PCR as unknown tyrosine kinase homologous cDNA fragments from human leukemia cells are described in Partanen, et al., Proc. Natl. Acad. Sci. USA, 87: 8913-8917 (1990). This gene and its encoded protein is called "tie", which stands for "tyrosine kinase with Ig and EGF homolog domains" (Partanen, et al., Mol. Cell. Biol. 12: 1698-1707 (1992) )).

Tie mRNA has been reported to be present in all human fetal and mouse embryonic tissues. At examination, tie messages were localized to the heart and vascular endothelial cells. tie mRNA was localized in the endothelium and endocardium of blood vessels of 9.5-18.5 day mouse embryos. Enhanced tie expression has been shown during neovascularization associated with developing granulation tissue in follicles and skin wounds (Korhonen, et al., Blood 80: 2548-2555 (1992)). Thus, the tie has been proposed to play a role in angiogenesis, which is important for elucidating the treatment of angiogenic dependent diseases such as solid tumors and several others, diabetic retinopathy, psoriasis, thromboembolism and arthritis.

It has been reported that two structurally related rat TIE receptor proteins are encoded with related expression profiles by separate genes. One gene called tie- 1 is the rat homologue of human tie (Maisonpierre, et al., Oncogene 8: 1631-1637 (1993)). Another gene tie- 2 is a rat homologue of the murine tek gene, which, like the tie , has been reported to be expressed in mice only in endothelial cells and their putative ancestors (Dumont, et al., Oncogene 8: 1293-). 1301 (1993)).

Both genes were found to be highly expressed in endothelial cells of embryonic and postnatal tissues. There were also significant levels of tie- 2 transcripts in other germ cell populations including lens epithelium, cardiac epicardium and mesenchymal region (Maisonpierre, et al., Oncogene 8: 1631-1637 (1993)).

Predominant expression of TIE receptors in the vascular endothelium suggests that TIE plays a role in the development and maintenance of the vascular system. This may include a role in endothelial cell determination, proliferation, differentiation and cell migration and patterning into vascular elements. Analysis of mouse embryos lacking TIE-2 has been shown to indicate that TIE-2 is important for angiogenesis, particularly for vascular network formation in endothelial cells (Sato, TN, et al., Nature 376: 70 -74 (1994)). In mature vascular systems, TIE can act on endothelial cell survival, maintenance, and response to pathogen effects.

The present invention relates generally to the field of genetic engineering, and more specifically to genes for receptor tyrosine kinases and their homologous ligands, insertion into their recombinant DNA vectors, proteins encoded in microbial recipient strains and eukaryotic cells. It relates to the manufacture of. More specifically, the present invention relates to a novel ligand called TIE-2 ligand, which also binds a TIE-2 receptor, and also a method of making and using a TIE-2 ligand. The present invention also provides a nucleic acid sequence encoding a TIE-2 ligand and a method of producing a nucleic acid encoding a TIE-2 ligand and their gene products. TIE-2 ligands, and also the nucleic acids encoding them, may be used for endothelial cells such as neoplastic disease, wound healing, thromboembolic disorders, atherosclerosis and inflammatory diseases involving tumor angiogenesis and certain diseases involving related TIE receptors. It is useful for diagnosis and treatment. More generally, biologically active TIE-2 ligands are used to promote the growth, survival and / or differentiation of cells expressing TIE-2 receptors. Biologically active TIE-2 ligands can also be used for in vitro maintenance of TIE-2 receptor expressing cells in culture. Cells and tissues expressing TIE-2 receptors include, for example, heart and vascular endothelial cells, lens epithelial cells and cardiac epicardium. Alternatively, such ligands may be used to support cells that are engineered to express TIE-2 receptors. TIE-2 ligands and their homologous receptors may also be used in assay systems to identify agonists or antagonists of TIE-2 receptors.

1A and 1B: TIE-2 receptor body (TIE-2 RB) inhibits the development of blood vessels in chicken embryo chorionic villus (CAM). A piece of resorbable gelfoam moistened with 6 μg of RB was immediately inserted under the CAM of the 1 day old chick embryo. After 3 more days of incubation, the 4 day old embryos and circumferential CAMs were removed and examined. 1A: Embryos treated with EHK-1 RB (rEHK-1 ecto / h IgG1 Fc) were able to survive and had blood vessels normally developing in the circumferential CAM. 1B: All embryos treated with TIE-2 RB (rTIE-2 ecto / h IgG1 Fc) were dead, reduced in size and almost completely lacking circumferential blood vessels.

2 is a vector pJFE 14

3 is a limit map of λgt10

4 shows the nucleic acid and deduced amino acid (letter code) sequences of human TIE-2 ligand from clone λgt10 encoding htie-2 ligand1.

5 shows nucleic acid and deduced amino acid (single-letter code) sequences of human TIE-2 ligand from T98G clone.

Figure 6 shows the nucleic acid and inferred amino acid (letter code) sequences of human TIE-2 ligand from clone pBluescript KS encoding human TIE 2 ligand 2.

7 is a western blot showing that the TIE-2 receptor is activated by TIE-2 ligand 1 (Lane L1) and not by TIE-2 ligand 2 (Lane L2) or control (Mock).

FIG. 8 shows that pretreatment of HAEC cells with excess TIE-2 ligand 2 (Lane2) is a dilute TIE-2 ligand1 that activates the TIE-2 receptor (TIE2-R) compared to pretreatment of HAEC cells with MOCK medium (Lane1). Western blot showing antagonistic ability of

9 is a histogram diagram of binding to rat TIE-2 IgG immobilized surface by TIE-2 ligand in C2C12 ras, Rat2 ras, SHEP, and T98G concentrated (10-fold) conditioned media. Rat TIE2 (rTIE2) specific binding is evidenced by a significant decrease in binding activity in the presence of 25 μg / ml soluble rat TIE2 RB compared to a small decrease in the presence of soluble trkB RB.

10 Binding of recombinant human TIE-2 ligand 1 (hTL1) and human TIE-2 ligand 2 (hTL2) in cos cell supernatant to human TIE-2 RB immobilized surface. Human TIE-2-specific binding was determined by incubating the sample with 25 μg / ml of soluble human TIE 2 RB or trkB RB; A significant decrease in binding activity is only observed for samples incubated with human TIE-2 RB.

11 shows that the TIE-2 receptor body (labeled TIE-2 RB or TIE2-Fc as here) blocks activation of the TIE-2 receptor by TIE-2 ligand1 (TL1) in HUVEC cells, while Western blot indicating that the receptor body (TRKB-Fc) does not block this activation.

(Detailed Description of the Invention)

As will be described in more detail below, Applicants have isolated novel ligands that bind the TIE-2 receptor by expression cloning. The present invention consists not only of the amino acid sequence but also of the TIE-2 ligand and functionally equivalent molecules in which the amino acid residues are substituted with residues which bring about a silent change in the sequence. For example, one or more amino acid residues in the sequence can be substituted with another amino acid of similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substituents for amino acids in the sequence may be selected from other members of the class to which the amino acid belongs. For example, classes of nonpolar (hydrophobic) amino acids include 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 arginine, lysine and histidine. Negatively charged (acidic) 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 activity, antibody molecules or other cellular ligands, and the like, for example, during translation by glycosylation, proteolytic cleavage, ligation, or Derivatives which are otherwise modified later.

The invention also relates to, for example, transfection, transduction of nucleotide sequences encoding proteins described herein as TIE-2 ligand 1 and nucleic acids encoding TIE-2 ligand 1 described herein in suitable expression vectors. Cells that are genetically engineered by infection, electroporation, or microinjection to produce proteins.

The invention also relates to, for example, transfection, transduction of nucleotide sequences encoding proteins described herein as TIE-2 ligand 2 and nucleic acids encoding TIE-2 ligand 2 described herein in suitable expression vectors. Cells that are genetically engineered by infection, electroporation, or microinjection to produce proteins.

Those skilled in the art will appreciate that the present invention is described, for example, in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. It will be appreciated that it includes DNA and RNA sequences that hybridize to TIE-2 ligand coding sequences inferred under conditions of stringency, as defined by 101-104, Cold Spring Harbor Laboratory Press (1989). Thus, a nucleic acid molecule intended in the present invention has a sequence inferred from the amino sequence of a TIE-2 ligand prepared as described herein, and also has a molecule having a sequence of nucleic acid hybridizing to such nucleic acid sequence, and also a genetic code. Results in degeneracy of the sequences but includes a nucleic acid sequence encoding a ligand that binds a TIE-2 receptor.

Any method known to those skilled in the art for inserting DNA fragments into a vector can be used to prepare expression vectors encoding TIE-2 ligands using appropriate transcriptional / translational control signals and protein coding sequences. These methods include ex vivo recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination). Expression of a nucleic acid sequence encoding a TIE-2 ligand or peptide fragment thereof can be adjusted by the 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 in the art. Promoters that can be used to modulate the expression of a ligand include, but are not limited to, the following: long terminal repeats 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 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22 : 787-797 (1980)), herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78 : 144-1445 (1981)), adenovirus promoter, regulation of metallothionein gene Sequence (Brinster at al., Nature 296 : 39-42 (1982)); β-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad, Sci. USA 75 : 3727-3731 (1978)) or tac promoter (DeBoer, et al., Proc. Natl, Acad. Sci. Prokaryotic expression vectors (see also "Protein Useful from Recombinant Bacteria", in USA 80 : 21-25 (1983)), in Scientific American, 242 : 74-94 (1980)); The following animal transcriptional regulatory regions exhibiting tissue specificity used in the Gal 4 promoter, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter and transgenic animals: Active elastase I gene regulatory region (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)); Insulin gene regulatory region active in pancreatic beta cells (Hanahan, Nature 315: 115-122 (1985)), immunoglobulin gene regulatory region 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), mouse breast cancer virus active in testes, breast, lymphoid and mammary cells Regulatory region (Leder et al., 1986, Cell 45 : 485-495), albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Devel. 1 : 268-276), alpha active in the liver Fetoprotein gene regulatory region (Krumlauf et al., 1985, Mol. Cell. Biol. 5 : 1639-1648; Hammer et al., 1987, Science 235 : 53-58); Alpha 1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1 : 161-171), beta-globin gene regulatory region active in bone marrow cells (Mogram et al., 1985, Nature 315 : 338-340; Kollias et al., 1986, Cell 46 : 89-94); Myelin basic protein gene regulatory regions active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48 : 703-712); Myosin light chain-2 gene regulatory region active in skeletal muscle (Shani, 1985, Nature 314 : 283-286), and gonadotropin releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234) . Promoter elements from yeast or other fungi, such as 1372-1378). The present invention encompasses the preparation of antisense compounds that can hybridize specifically to sequences of RNA encoding TIE-2 ligands to regulate their expression (Ecker, US Patent No. 5,166,195, issued November 24, 1992). .

As such, according to the present invention, an expression vector capable of replicating in a bacterial or eukaryotic host consisting of a nucleic acid encoding a TIE-2 ligand described herein is used for transfection into a host and thus such nucleic acid is directly expressed to express the TIE-2 ligand. Which is then recovered in biologically active form. As used herein, biologically active forms include forms that can bind to a TIE-2 receptor and can cause a biological response, such as a differentiated function, or affect the phenotype of a cell expressing the receptor. Such biologically active forms induce phosphorylation of, for example, the tyrosine kinase domain of the TIE-2 receptor.

Expression vectors containing gene inserts have four general approaches: (a) DNA-DNA hybridization, (b) the presence or absence of "marker" gene function, (c) expression of the inserted gene, and (d) PCR detection. Can be identified by In the first approach, the presence of the foreign gene inserted into the expression vector can be detected by DNA-DNA hybridization using a probe consisting of a sequence homologous to the inserted TIE-2 ligand coding gene. In the second approach, the recombinant vector / host system functions by some "marker" gene function (eg thymidine kinase activity, antibiotic resistance, transgenic phenotype, and occlusal formation in baculovirus) due to the insertion of a foreign gene into the vector. Can be identified and selected based on their presence or absence. For example, if a nucleic acid encoding a TIE ligand is inserted into the marker gene sequence of the vector, the recombinant containing the insert can be identified by the absence of marker gene function. In a third approach, recombinant expression vectors can be identified by analyzing foreign gene products expressed by the recombinant. Such an assay may, for example, bind a ligand to a TIE-2 receptor or part thereof, which may be tagged with a detectable antibody or part thereof, or an antibody produced against a TIE-2 ligand protein or By binding to portions of it, one can base the physical or functional properties of the TIE-2 ligand gene product. The cells of the present invention express TIE-2 ligands temporarily or preferably, essentially and permanently, as described herein. In a fourth approach, DNA nucleotide primers corresponding to tie- 2 specific DNA sequences can be prepared. These primers can then be used for PCR tie- 2 gene fragments (PCR Protocols: A Guide To Methods and Applications, Michael A. Innis et al., Academic Press (1990)).

Recombinant ligands can be purified by any technique that allows for the subsequent formation of stable, biologically active proteins. For example, and not by way of limitation, ligands can be recovered from cells as soluble proteins or as inclusion bodies from which they can be quantitatively extracted by 8M guanidinium hydrochloride and dialysis. To further purify the ligand, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration can be used.

In a further embodiment of the invention, recombinant TIE-2 ligand coding genes are used to inactivate or "knock out" endogenous genes by homologous recombination, thereby TIE-2 ligand deficient cells, tissues, or Make an animal By way of example and not by way of limitation, recombinant TIE-2 ligand coding genes may be engineered to contain insertion mutations, eg, neo genes which will inactivate the innate TIE-2 ligand coding gene. Under the control of an appropriate promoter, such constructs can be introduced into cells such as embryonic liver cells by techniques such as transfection, transduction or injection. Cells containing the construct may then be selected for G418 resistance. Cells that lack the native TIE-2 ligand coding gene can then be identified, for example, by Southern blot, PCR detection, Northern blot or analysis of expression. Cells that lack the native TIE-2 ligand coding gene can then be fused to early germ cells to generate transgenic animals that lack these ligands. Such animals are used to define specific in vivo processes that usually depend on ligands.

The invention also provides antibodies to the TIE-2 ligands described herein useful for the detection of ligands, for example in diagnostic use. For the preparation of monoclonal antibodies directed against TIE-2 ligands, any technique that provides for the production of antibody molecules by continuous cell lines in culture is used. For example, the hybridoma technology originally developed by Kohler and Milstein (1975, Nature 256 : 495-497), and also trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983). , Immunology Today 4 : 72), and EBV-Hybridoma Technology (Cole et al., 1985, "Monoclonal Antibodies and Cancer Therapies", Producing Human Monoclonal Antibodies, Alan R. Liss. Inc. pp. 77 -96) is within the scope of the present invention.

The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies are made with any of a number of techniques known in the art (eg, 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 containing mouse antigen binding domains with human constant regions are also prepared (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851, Takeda et al., 1985, Nature 314: 452). ).

Several procedures of the art can be used for the production of polyclonal antibodies directed against epitopes of the TIE-2 ligand described herein. For the production of antibodies, various host animals, including but not limited to rabbits, mice and rats, can be immunized by injection with a TIE-2 ligand or fragment or derivative thereof. Depending on the host species, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface activators such as lysocithin, pluronic polyols, polyanions, peptides, oil emulsions, keyholepet hemocyanin, di Various adjuvants, including but not limited to nitrophenols and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum , can be used to increase the immunological response.

Molecular clones of antibodies to selected TIE-2 ligand epitopes can be prepared by known techniques. Recombinant DNA methods (eg, Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring) to prepare nucleic acid sequences encoding monoclonal antibody molecules, or antigen binding regions thereof Harbor, New York) may be used.

The present invention provides antibody molecules and also fragments of such antibody molecules. Antibody fragments containing genotypes of molecules can be generated by known techniques. For instance, papain these fragments F that can be produced by pepsin digestion of the antibody molecule (ab ') ₂ fragments, F (ab') Fab 'fragments which saenggilsu by reducing the disulfide bridges of the _second fragment, and an antibody molecule and Fab fragments that can occur by treatment with a reducing agent include, but are not limited to. Antibody molecules are purified by known techniques such as immunoabsorption or immunoaffinity chromatography, chromatography methods such as HPLC (high performance liquid chromatography), or combinations thereof.

The present invention also

a) contacting a biological sample with at least one antibody that specifically binds a TIE-2 ligand such that the antibody complexes with any TIE-2 ligand present in the sample,

b) by measuring the amount of the complex thereby measuring the amount of TIE-2 ligand in the biological sample

Immunoassays to determine the amount of TIE-2 ligand in a biological sample.

The present invention also

a) contacting at least one ligand of the present invention with a biological sample such that the ligand forms a complex with the TIE-2 receptor,

b) by measuring the amount of the complex thereby measuring the amount of TIE-2 receptor in the biological sample

Assays for measuring the amount of TIE-2 receptor in a biological sample.

The invention also provides for the use of TIE-2 ligands to support the survival and / or growth and / or differentiation of TIE-2 receptor expressing cells. Thus, ligands are used, for example, as supplements to support endothelial cells in culture.

In addition, the applicant's discovery of homologous ligands for the TIE-2 receptor allows the use of useful assay systems for the identification of agonists or antagonists of the TIE-2 receptor. Such an assay system will be useful for identifying molecules that can promote or inhibit angiogenesis. For example, in one embodiment, the antagonist of the TIE-2 receptor may be identified as a test molecule that may interfere with the interaction of the TIE-2 receptor with the biologically active TIE-2 ligand. These antagonists

1) the ability to block binding of the biologically active TIE-2 ligand to the receptor, as measured using, for example, BIAcore (Pharmacia Biosensor, Piscataway, NJ); or

2) Biologically active The ability of the TIE-2 ligand to block its ability to elicit a biological response is identified. Such biological responses include, but are not limited to, phosphorylation of components downstream of the TIE-2 receptor or TIE-2 signaling pathway, or survival, growth, or differentiation of cells with the TIE-2 receptor.

In one embodiment, cells engineered to express a TIE-2 receptor have growth dependent on the addition of a TIE-2 ligand. Such cells provide a useful assay system for identifying additional agonists of TIE-2 receptors, or antagonists that may interfere with the activity of TIE-2 ligands in such cells. Alternatively, autocrine cells engineered to co-express both TIE-2 ligand and receptor may provide a useful system for analyzing potential agonists or antagonists.

Therefore, the present invention provides for the introduction of TIE-2 receptors into cells that normally do not express these receptors, thus allowing these cells to exhibit profound and easily distinguished responses to ligands that bind to these receptors. The type of inferred response depends on the cell used and not on the specific receptor introduced into the cell. In addition to finding molecules that can act on tyrosine kinase receptors, the appropriate cell lines can be selected to obtain the responses of the largest use for analysis. Molecules can be any type of molecule, including but not limited to peptide and nonpeptide molecules, which will act in a system to be described in a receptor specific method.

One of the more useful systems to study involves the introduction of TIE-2 receptors into fibroblast cell lines (e.g., NIH3T3 cells) and thus these receptors that normally do not mediate proliferative responses, nevertheless are introduced into fibroblasts. This can then be analyzed by a variety of well established methods for quantifying the effects of fibroblast growth factors (eg, thymidine incorporation or other types of proliferation assays: van Zoelen, 1990, "For Detection of Polypeptide Growth Factors). Use of Biological Assays "Progress Factor Research, Vol. 2, pp. 131-152; Zhan and M. Goldfarb, 1986, Mol. Cell. Biol., Vol. 6, pp. 3541-3544. These assays have the added advantage that any agent can be analyzed in both the cell line with the receptor as well as the parent cell line lacking the receptor. Only specific effects in cell lines with receptors will be judged as being mediated through the introduced receptors. Such cells may be further engineered to express a TIE-2 ligand and thus make a useful autosecretion system for analysis of molecules that act as antagonists / agonists of this interaction. Accordingly, the present invention provides a host cell consisting of a nucleic acid encoding a TIE-2 ligand and a nucleic acid encoding a TIE-2 receptor.

TIE-2 receptor / TIE-2 ligand interactions also provide a useful system for identifying small molecule agonists or antagonists of TIE-2 receptors. For example, fragments, mutations or derivatives of TIE-2 ligands that bind TIE-2 receptors but do not induce biological activity can be identified. Alternatively, the characterization of the TIE-2 ligand allows for the determination of the active moiety of the molecule. In addition, the identification of the ligand allows the determination of X-ray crystal structure in the receptor / ligand complex, and thus the identification of the binding site on the receptor. Knowledge of binding sites will provide useful insights into the rational design of novel agonists and antagonists.

Specific binding of test molecules to TIE-2 receptors can be measured in many ways. For example, the actual binding of a test molecule to a cell expressing tie- 2 may include (i) a test molecule bound to the surface of the cell intact; (ii) test molecules crosslinked to TIE-2 protein in cell lysate; Or (iii) by detecting or measuring a test molecule bound to TIE-2 ex vivo. Specific interactions between test molecules and TIE-2 are assessed by using reagents demonstrating the unique nature of the interactions.

As a specific unlimited embodiment, the present invention method is used as follows. Consider the case where the TIE-2 ligand in the sample is measured. TIE-2 ligand detectably labeled several diluent samples (test molecules) in combination with a negative control (NC) containing no TIE-2 ligand activity and a positive control (PC) containing a known amount of TIE-2 ligand. (In this example, radioiodinated ligand) is exposed to cells expressing tie- 2. The test sample compared to the ¹²⁵ I- labeled with ¹²⁵ I- determining the amount of the TIE-2 ligand, and then cover the sample values standard curves combined in a volume of each dilution of the TIE-2 ligand binding to TIE-2 ligand in the amount of the control group It is calculated by. The more TIE-2 ligands in the sample, the less ¹²⁵ I-ligands that bind to TIE-2.

The amount of bound ¹²⁵ I-ligand can be determined by measuring the amount of radioactivity per cell or by crosslinking the TIE-2 ligand to the cell surface protein using DSS as described in Meakin and Shooter, 1991 , Neuron 6: 153-163. For example, it is determined by detecting the amount of labeled protein in the cell extract using SDS polyacrylamide gels representing labeled proteins having a size corresponding to the TIE-2 ligand / TIE-2 receptor. Specific test molecule / TIE-2 interactions do not bind the TIE-2 receptor to the assay and thus should not have a substantial effect on contention between the labeled TIE-2 ligand and the test molecule for TIE-2 binding. It may be tested by adding several dilutions of the ligand. Alternatively, a molecule known to be capable of disrupting TIE-2 ligand / TIE-2 binding, such as but not limited to an anti-TIE-2 antibody, or the TIE-2 receptor body described herein, may be ¹²⁵ I-TIE. It is expected to interfere with competition between the -2 ligand and the test molecule for TIE-2 receptor binding.

Detectable labeled TIE-2 ligands or preferably for radioactive substances, fluorescent substances, substances with enzymatic activity, substances which serve as substrates for enzymes (preferably enzymes and substrates associated with colorimetrically detectable reactions) Includes, but is not limited to, a TIE-2 ligand that is covalently or non-covalently linked to a substance recognizable by an antibody molecule that is a detectably labeled antibody molecule.

Alternatively, specific binding of a test molecule to TIE-2 may include, but is not limited to, cell growth and / or differentiation or direct initial gene expression or phosphorylation of TIE-2. It is measured by evaluating the secondary biological effects of binding. For example, in the comparison it may be tested in a cell for testing molecules that express the ability to tie -2 cells in the absence of tie -2 to induce differentiation, cell comparative to the lack tie -2 andoena tie -2- Differentiation that occurs in expressing cells will be an indication of specific test molecule / TIE-2 interactions. tie -2- minus and tie -2- positive cells directly from the early gene (e. g., fos and jun) induction by detecting, or phosphorylation of TIE-2 using standard phosphorylation analysis known in the art Similar analysis can be performed by detecting. Such an assay may be useful for identifying agonists or antagonists that do not competitively bind to TIE-2.

Likewise, the present invention possesses the biological activity of a TIE-2 ligand consisting of (i) exposing cells expressing tie- 2 to a test molecule and (ii) detecting specific binding of the test molecule to a TIE-2 receptor. Methods of identifying molecules are provided wherein specific binding to TIE-2 correlates positively with TIE-2 like activity. Specific binding can be detected by assaying for direct binding or by the secondary biological effect of binding as discussed above. This method is particularly useful for identifying new members of the TIE ligand family or for screening large arrays of peptide and nonpeptide molecules (eg peptide analogs) for the TIE related biological activity in the pharmaceutical industry. In a preferred specific unlimited number of embodiments of the invention, or the negative or tie -2- (refer to or fibroblasts,) -2- tie plus the PC12 cell operation such that the prepared culture well of a large grit containing alternately open do. Various test molecules may then be added so that each column of the grit, or part of it, contains different test molecules. Each well may then be recorded for the presence or absence of growth and / or differentiation. Very many test molecules can be screened for this activity in this way.

In a further embodiment, the present invention provides a method of detecting or measuring TIE-like activity or identifying a molecule having such activity, wherein the method comprises (i) ex vivo TIE-2 under conditions that allow binding to occur. Exposing the test molecule to the receptor protein and (ii) detecting the binding of the test molecule to the TIE-2 protein, wherein the binding of the test molecule to TIE-2 is correlated to TIE-like activity. According to this method, TIE-2 may or may not be substantially purified, attached to a solid support (eg, as an affinity column or as an ELISA assay), or incorporated into an artificial membrane. The binding of the test molecule to TIE-2 can be estimated by any method known in the art. In a preferred embodiment, the binding of a test molecule can be detected or measured by calculating its ability to compete with known TIE-2 ligands that are detectably labeled for TIE-2 receptor binding.

The present invention also detects the ability of test molecules to function as antagonists of TIE-like activity, which consists in detecting the ability of test molecules to inhibit the effect of TIE ligands binding to TIE-2 in cells expressing tie -2. Provide a way to. Such antagonists may or may not interfere with TIE-2 ligand / TIE-2 receptor binding. The effect of the TIE-2 ligand binding to the TIE-2 receptor preferably is biological or biochemical, including but not limited to cell survival or proliferation, cell transformation, direct early gene induction, or TIE-2 phosphorylation Effect.

The present invention also provides methods for identifying antibodies or other molecules that can neutralize ligands or block binding to receptors, and also provide molecules identified by the methods. By way of unlimited examples, this is conceptually carried out via an assay similar to the ELISA assay. For example, the TIE receptor body may be coupled to a solid support such as a plastic multiwell plate. As a control, any tagged TIE ligand that introduces a Myc-tagged known amount of TIE ligand into the well and binds the receptor body may be identified by a reporter antibody against Myc-tag. The assay system can then be used to screen test samples for molecules that can i) bind to the tagged ligand or ii) bind to the receptor body and block binding to the receptor body by the tagged ligand. For example, a test sample containing the putative molecule of interest along with a known amount of tagged ligand is introduced into the wells and the amount of tagged ligand bound to the receptor body is measured. By comparing the amount of tagged ligand bound in the test sample with that in the control, a sample containing molecules capable of blocking the ligand binding to the receptor can be identified. The molecules of interest thus identified may be isolated using methods well known to those skilled in the art.

Once a blocker of ligand binding has been found, one of skill in the art will conduct secondary assays to determine whether the blocker is binding to the receptor or to the ligand, and also to determine whether the blocker molecule can neutralize the biological activity of the ligand. You will know what to do. For example, one skilled in the art would use a binding assay using the BIAcore biosensor technology (or equivalent) in which the TIE receptor body or TIE ligand is covalently attached to a solid support (e.g., carboxymethyldextran on a gold surface). It may be determined whether the molecule specifically binds to the ligand or the receptor body. To determine if the blocker molecule can neutralize the biological activity of the ligand, one skilled in the art can perform a phosphorylation assay (see Example 5) or alternatively, for example, by using a primary culture of endothelial cells, The same functional bioassay can be performed. Alternatively, the blocker molecule that binds to the receptor body may be an agonist and one skilled in the art will know how to determine this by performing appropriate assays to identify additional agonists of TIE-2 receptors.

Since TIE-2 receptors can be identified in association with endothelial cells, and as demonstrated herein, blocking of ligands has been shown to prevent angiogenesis, the present applicant has found that TIE-2 ligands may be used in diseases or disorders that require angiogenesis. It has proven useful for inducing angiogenesis. Such diseases or disorders would include wound healing, ischemia and diabetes. On the other hand, antagonists of TIE-2 receptors, such as the receptor body described herein in Examples 2 and 3, and TIE-2 ligand 2, such as described in Example 9, prevent or delay angiogenesis, and thus, for example, tumor growth. It will be useful to prevent or delay.

Since cells with TIE-2 receptors appear to be upregulated in angiogenesis, the TIE-2 ligand body may also be useful for preventing or delaying tumor growth, for example. The TIE-2 ligand body may also be useful for the delivery of toxins to cells with receptors. Alternatively, other molecules, such as growth factors, cytokines or nutrients, are delivered through the TIE-2 ligand body to cells carrying the TIE-2 receptor. The TIE-2 ligand body can also be used as a diagnostic reagent for TIE-2 receptors to detect receptors in vivo or in vitro. If the TIE-2 receptor is associated with a disease state, the TIE-2 ligand body may be used as a diagnostic reagent for detecting a disease, for example by tissue staining or systemic imaging.

The present invention also provides a pharmaceutical composition consisting of the TIE-2 ligand in a pharmacologically acceptable excipient, or the ligand body described herein, peptide fragments thereof, or derivatives thereof. TIE-2 ligand proteins, peptide fragments, or derivatives are administered systemically or locally. Any suitable mode of administration known in the art is used and includes, but is not limited to, those by intravenous, intravesical, intraarterial, intranasal, oral, subcutaneous, intraperitoneal or topical or surgical implantation. Sustained release formulations are also provided.

The invention also provides isolated and purified nucleic acid molecules consisting of nucleic acid sequences encoding human TIE-2 ligands, wherein the nucleic acid sequences are selected from the group consisting of:

(a) a nucleic acid sequence consisting of a coding region of a human TIE-2 ligand as shown in FIG. 4, 5 or 6,

(b) a nucleic acid sequence encoding a TIE-2 ligand that binds the nucleic acid sequence of (a) to a TIE-2 receptor that hybridizes, usually under tight conditions, and

(c) a nucleic acid sequence encoding a TIE-2 ligand that binds a TIE-2 receptor, which is degenerate as a result of the genetic coding for the nucleic acid sequence of (a) or (b).

In another embodiment, the present invention provides an isolated nucleic acid molecule encoding a TIE-2 ligand, wherein the nucleic acid sequence is

(a) a nucleic acid sequence consisting of a coding region of a human TIE-2 ligand as shown in FIG. 4, 5 or 6,

(b) a nucleic acid sequence encoding a TIE-2 ligand that binds the nucleic acid sequence of (a) to a TIE-2 receptor that hybridizes, usually under tight conditions, or

(c) Without degeneracy of the genetic code, it will hybridize to the nucleic acid sequence of (a) or (b) and is a nucleic acid sequence encoding a TIE-2 ligand that binds a TIE-2 receptor.

The present invention also provides isolated and purified human TIE-2 ligands encoded by the isolated nucleic acid molecules of the present invention. The present invention also provides vectors consisting of isolated nucleic acid molecules consisting of nucleic acid sequences encoding human TIE-2 ligands. In one embodiment, the vector is referred to as pBluescript KS encoding human TIE-2 ligand2.

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

The invention of an isolated nucleic acid molecule encoding a TIE-2 ligand described herein relates to a ligand, fragment or derivative thereof, receptor receptor as a therapeutic agent for the treatment of a patient suffering from a disorder involving a cell, tissue or organ expressing a TIE receptor. Provides the development of another molecule that is a nysted or antagonist. By way of unlimited example, the present invention provides a ligand body that specifically binds a TIE-2 receptor or TIE-2. In another embodiment the invention provides a ligand body consisting of a TIE-2 ligand fused to an immunoglobulin constant region. In one embodiment, the ligand body consists of TIE-2 ligand 1 or TIE-2 ligand 2 and the Fc portion of human IgG1. Ligand bodies are used for the treatment of human or animal bodies or for diagnostic methods.

The present invention also provides antibodies that specifically bind such therapeutic drug molecules. The antibody is monoclonal or polyclonal. The present invention also provides a method of using such monoclonal or polyclonal antibodies to determine the amount of therapeutic drug molecule in a sample taken from a patient for the purpose of monitoring the course of treatment.

The present invention also provides a therapeutic composition consisting of a human TIE-2 ligand or ligand body and a cytotoxic agent incorporated therein. In one embodiment, the cytotoxic agent may be a radioisotope or toxin.

The present invention also provides antibodies that specifically bind human TIE-2 ligands. The antibody may be monoclonal or polyclonal.

The present invention also,

a) coupling at least one TIE-2 binding substrate to the solid matrix;

b) incubating the substrate of a) with a cell lysate to form a complex with the human TIE-2 ligand in the cell solution,

c) washing the solid matrix,

d) a method of purifying a human TIE-2 ligand consisting of eluting a human TIE-2 ligand from the bound substrate.

The substrate may be any substance that specifically binds a human TIE-2 ligand. 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 blocks the growth of blood vessels in a person consisting of administering an effective amount of a composition comprising a receptor body specifically binding a human TIE-2 ligand, a receptor body in a pharmaceutically acceptable excipient, and a therapeutic composition. Provide a way to.

The present invention also provides a therapeutic composition comprising a human TIE-2 ligand or ligand body in a pharmaceutically acceptable excipient, and also 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 of contacting a cell with a detectably labeled TIE-2 ligand or ligand body under conditions that allow binding of the detectably labeled ligand to a TIE-2 receptor, and Provided are methods for identifying cells expressing a TIE-2 receptor comprising binding to a TIE-2 receptor thereby identifying the cell as expressing a TIE-2 receptor. The present invention also provides a therapeutic composition consisting of a TIE-2 ligand or ligand body and a cytotoxic agent incorporated therein. The cytotoxic agent may be a radioisotope or toxin.

The present invention also obtains mRNA from a cell and, under hybridization conditions, contacts the mRNA thus obtained with a labeled nucleic acid molecule encoding a TIE-2 ligand and determines the presence of mRNA hybridized to the labeled molecule and thereby intracellular TIE-2. A method of detecting expression of a TIE-2 ligand by a cell comprising detecting expression of a ligand is provided.

The present invention is also directed to contacting a tissue section with a labeled nucleic acid molecule encoding a TIE-2 ligand under hybridization conditions and determining the presence of hybridized mRNA to the labeled molecule thereby detecting the expression of the TIE-2 ligand in the tissue section. It provides a method for detecting the expression of the TIE-2 ligand in the tissue section.

(Summary of invention)

The present invention provides a composition consisting of a TIE-2 ligand substantially free of other proteins. The present invention also provides isolated nucleic acid molecules encoding TIE-2 ligands. The isolated nucleic acid is DNA, cDNA or RNA. The present invention also provides a vector consisting of isolated nucleic acid molecules encoding a TIE-2 ligand. The present invention also provides a host-vector system for the production of suitable host cells of a polypeptide having the biological activity of the TIE-2 ligand. Suitable host cells are bacteria, yeasts, insects or mammals. The present invention also provides a method for producing a polypeptide having a biological activity of a TIE-2 ligand, which consists of growing cells of a host-vector system under conditions that allow the production of the polypeptide and recovering the polypeptide so produced.

The invention of an isolated nucleic acid molecule encoding a TIE-2 ligand described herein is a therapeutic agent for the treatment of a patient suffering from a disorder involving a cell, tissue or organ expressing a TIE receptor, the ligand, fragment or derivative or ligand of the ligand being known. Provides the development of another molecule that is a nysted or antagonist. The present invention also provides antibodies that specifically bind such therapeutic drug molecules. The antibody is monoclonal or polyclonal. The present invention also provides methods of using such monoclonal or polyclonal antibodies for measuring the amount of therapeutic molecule in a sample taken from a patient for the purpose of monitoring the progress of the treatment.

The present invention also provides an antibody that specifically binds to a TIE-2 ligand. The antibody is monoclonal or polyclonal. Accordingly, the present invention provides a therapeutic composition consisting of an antibody that specifically binds a TIE-2 ligand in a pharmaceutically acceptable excipient. The present invention also provides a method of blocking vascular growth in a mammal by administering an effective amount of a therapeutic composition consisting of an antibody that specifically binds a TIE-2 ligand in a pharmaceutically acceptable excipient.

The present invention also provides a therapeutic composition consisting of TIE-2 ligands in pharmaceutically acceptable excipients. The present invention also provides a method of promoting neovascularization in a patient by administering an effective amount of a therapeutic composition consisting of a TIE-2 ligand in a pharmaceutically acceptable excipient. In one embodiment, the present invention is used to promote wound healing. In another embodiment, the present methods are used to treat ischemia.

Alternatively, the present invention provides a conjugate wherein the TIE-2 ligand is incorporated into a cytotoxic drug and a therapeutic composition prepared therefrom. The present invention also provides a receptor body that specifically binds to a TIE-2 ligand. The present invention also provides a therapeutic composition consisting of a receptor body that specifically binds a TIE-2 ligand in a pharmaceutically acceptable excipient. The present invention also provides a method of blocking vascular growth in a mammal by administering an effective amount of a therapeutic composition consisting of a receptor body that specifically binds a TIE-2 ligand in a pharmaceutically acceptable excipient.

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

Example 1

Identification of ABAE Cell Lines as Reporter Cells for TIE-2 Receptors

Adult BAE cells are registered as ECACC # 92010601 in the European Cell Culture Repository (see PNAS 75: 2621 (1978)). Intermediate levels of tie-2 transcription in ABAE (Adult Bovine Arterial Endothelial) cell lines in line with in situ hybridization results demonstrating nearly exclusive localization of tie-2 RNA to vascular endothelial cells. Sieve appeared. We therefore investigated ABAE cell lysates for the presence of TIE-2 protein as well as for the degree to which tyrosine phosphorylates this TIE-2 protein under normal versus serum deficient growth conditions. ABAE cell lysates were harvested and immunoprecipitated and then immunoprecipitated proteins were Western blot analyzed with TIE-2 specific and phosphotyrosine specific antisera. Deletion or inclusion of the TIE-2 peptide as a specific blocking molecule during TIE-2 immunoprecipitation results in a moderately detectable protein of ˜150 kD in which steady-state phosphotyrosine levels are reduced to levels almost undetectable by prior serum deprivation of cells. As a result, TIE-2 was clearly identified.

Culture of ABAE cells and harvest of cell lysates were performed as follows. Low-passage-number ABAE cells were obtained as monolayers at a density of 2 × 10 ⁶ cells / 150 mm plastic Petri plate (Falcon). Dulbecco's modified Eagle's medium plated and containing 10% calf serum (10% BCS), 2 mM L-glutamine (Q) and 1% of penicillin and streptomycin (PS) in an atmosphere of 5% CO ₂ Incubated in DMEM). Ice harvested phosphate buffered cells were deficient in serum for 24 hours in DMEM / Q / PS before harvesting cell lysates, then the medium was aspirated and the plates supplemented with sodium orthovanadate, sodium fluoride and sodium benzamidine Washed with brine (PBS). Cells were lysed in a small amount of this wash buffer supplemented with 1% NP40 detergent and protease inhibitor PMSF and aprotinin. Insoluble debris was removed from the cell lysate by centrifugation at 14,000 × G for 10 minutes at 4 ° C. and the supernatant was specific for the TIE-2 receptor with or without blocking peptide added at ˜20 μg / ml per 1 lysate. Immunoprecipitated with antiserum. Immunoprecipitated proteins were digested with PAGE (7.5% Laemmli gel) and then electrophoresed into PVDF membranes and incubated with various TIE-2 specific or phosphotyrosine specific antisera. The membrane was incubated with HRP-linked secondary antiserum and then treated with ECL reagent (Amersham) to visualize TIE-2 protein.

Example 2

Cloning and Expression of TIE-2 Receptor Body for Affinity-based Studies of TIE-2 Ligand Interactions

An expression construct was constructed that could yield a secreted protein consisting of the entire extracellular portion of the rat TIE-2 receptor fused to a human immunoglobulin gamma-1 constant region (IgG1 Fc). This fusion protein is called TIE-2 “receptor body” (RB) and can be expected to exist as a dimer in solution, usually based on the disulfide bond formation between each IgG1 Fc tail. The Fc portion of TIE-2 RB was prepared as follows. DNA fragments encoding the Fc portion of human IgG1 extending from the hinge portion of the protein to the carboxy terminus were amplified from human placental cDNA by PCR with oligonucleotides corresponding to the published sequence of human IgG1. The resulting DNA fragment was cloned in a plasmid vector. Appropriate DNA restriction fragments from plasmids encoding full-length TIE-2 receptors and human IgG1 Fc plasmids were linked to either side of a short PCR-derived fragment designed to fuse the TIE-2 and human IgG1 Fc protein coding sequences in frame. . Thus, the resulting TIE-2 ectodomain-Fc fusion protein was precisely substituted with IgG1 Fc instead of the region extending the TIE-2 transmembrane and cytoplasmic domain. Alternative methods of making RBs are described in Goodwin, et. al. Cell 73: 447-456 (1993).

Milligrams of TIE-2 RB were obtained by cloning the TIE-2 RB DNA fragment into the pVL1393 baculovirus vector and subsequently infecting the Spodoptera frugiperda SF-21AE insect cell line. Alternatively, cell line SF-9 (ATCC Accession No. CRL-1711) or cell line BTI-TN-5b1-4 may be used. DNA encoding TIE-2 RB was cloned into the baculovirus transfer plasmid pVL1393 as an EcoRI-NotI fragment. Plasmid DNA purified by cesium chloride density gradient centrifugation was obtained by mixing 3 μg of plasmid DNA with 0.5 μg of Baculo-Gold DNA (Pharminigen) and then introducing it into liposomes using 30 μg of lipofectin (GIBCO-BRL). Recombinant to DNA. The DNA-liposomal mixture was added to SF-21AE cells (2 × 10 ⁶ cells / 60 mm plate) in TMN-FH medium (GIBCO-BRL modified Grace) for 5 hours at 27 ° C. and then 5%. Cultured for 5 days at 27 ℃ in TMN-FH medium supplemented with fetal calf serum. Tissue culture medium was harvested for plaque purification of the recombinant virus, which had agarose top layer of 125 μg / mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyrano). Seed; performed using previously described methods (O'Reilly, DR, LK Miller, and VALuckow, Baculovirus Expression Vectors-A Laboratory Manual. 1992, New York: WHFreeman), except that it contained GIBCO-BRL It was. After 5 days of incubation at 27 ° C., non-recombinant plaques were assessed by positive chromosome response to X-gal substrate and marked for their location. The recombinant plaque was then visualized by adding a second phase layer containing 100 μg / mL MTT (3- [4,5-dimethylthiazol-2-yl] 2,5, diphenyltetrazolium bromide; Sigma). Putative recombinant virus plaques were picked out by plug aspiration and purified by several rounds of plaque isolation to confirm homology. Virus stocks were generated with a series of low-multiplicity passages of plaque purified virus. Low pass stocks of one virus clone (vTIE-2 receptor body) were prepared.

SF-21AE cells were cultured in serum-free medium (SF-900II, Gibco BRL) containing 1 × antibiotic / antibacterial solution (Gibco BRL) and 25 mg / L gentamycin (Gibco BRL). Pluronic F-68 was added as surfactant at a final concentration of 1 g / L. Cultures (4 L) were grown in an Artisan Cell Station System for at least 3 days prior to infection. Cells were grown at 27 ° C. (vented in a spray ring) with gas evolution with 50% dissolved oxygen at a gas flow rate of 80 mL / min. Stirring was performed at a speed of 100 rpm by a marine impeller. Cells were harvested in the mid-log growth phase (˜2 × 10 ⁶ cells per mL), concentrated by centrifugation and infected with the vTIE-2 receptor body of 5 plaque forming units per cell. Cells and inoculation material were taken to 400 mL fresh medium and the virus was adsorbed in a spinner flask at 27 ° C. for 2 hours. The culture was then resuspended to final volume at 8 L with fresh serum-free medium and cells were cultured in a bioreactor using the conditions previously described.

Culture medium from vTIE-2 receptor body-infected SF21AE cells was collected by centrifugation (500 × g, 10 minutes) 72 hours after infection. The cell supernatant was brought to pH 8 with NaOH. EDTA was added to a final concentration of 10 mM and the supernatant pH was readjusted to 8. The supernatant was filtered (0.45 μm, Millipore) and loaded onto a Protein A column (Protein A Sepharose 4 Fast Flow or Hitrap Protein A, both manufactured by Pharmacia). The column was washed with 0.5 M NaCl containing PBS until the absorbance of 280 nm decreased to baseline. The column was washed with PBS and eluted with 0.5M acetic acid. The column fractions were immediately neutralized by eluting with a tube containing 1M Tris pH 9. Peak fractions containing TIE-2 RB were pooled and dialyzed against PBS.

Example 3

Evidence that TIE-2 plays an important role in the development of the vascular system

Absence of known ligands for TIE-2 receptors may be possible to penetrate the function of TIE-2 by introducing a "excess" soluble TIE-2 receptor body (TIE-2 RB) into the developmental system. It was because. The potential ability of TIE-2 RB to bind and thus neutralize available TIE-2 ligands could lead to observable cleavage of normal vascular development and characterization of the ligands. To investigate whether TIE-2 RB can be used to disrupt vascular development in early chicken embryos, a small piece of biologically absorbable foam is impregnated with TIE-2 RB and just below the chorionic membrane in a position next to the embryonic embryo. Inserted in

Early chicken embryos develop on top of egg yolk from small discs of cells covered by the chorionic ureter (CAM). Endothelial cells that will support the vascular system in the embryo arise from extracellular and intraembryonic cell sources. Extracellular endothelial cells, which provide a major source of endothelial cells in the embryo, originate in the growth of mesenchyme located around the side just below the CAM, suitable for the embryo. These mesenchymal cells produce a common progenitor of both endothelial and hematopoietic lineage, termed hemangioblasts, as they mature. Hemangioblasts then produce a mixed population of vasculature (endothelial progenitors) and hematopoietic cells (multipotential hematopoietic progenitors). Progeny of the circulatory system is initiated when endothelial progeny are separated to form one-cell-thick vesicles that surround the neoplastic blood cells. The proliferation and movement of these cellular components eventually creates a vast network of blood-filled microvascular vessels under the CAM that eventually invade the vessel and bind to defined, intra-embedded vascular elements.

Spafas, Inc. Freshly fertilized chicken eggs from (Boston, Mass.) Were incubated at 99.5 ° F. 55% RH. After about 24 hours of development, the egg shells were cleaned with 70% ethanol and a dentist's drill was used to make a 1.5 cm hole in the blunt top of each egg. The shell was removed to reveal the space directly above the vessel. Small rectangular pieces of sterile gel foam (Upjohn) were cut with a surgical scalpel and impregnated into the TIE-2- or EHK-1 receptor body at the same concentration. The EHK-1 receptor body was prepared as described in Example 2 using the EHK-1 extracellular domain instead of the TIE-2 extracellular domain (Maisonpierre et al., Oncogene 8: 3277-3288 (1993)). Each gelfoam piece absorbed about 6 μg of protein in 30 μl. Slightly tearing the CAM at a position next to a few millimeters of the embryonic embryo using sterile clock tweezers. Most of the RB-impregnated gelfoam pieces were inserted under the CAM and the eggshell was sealed with adhesive tape pieces. Eggs undergoing other similar steps were treated side by side with the RB of the neuron-expressed receptor tyrosine kinase EHK-1 (Maisonpierre et al., Oncogene 8: 3277-3288 (1993)). After four days of development, the embryos were examined by visual inspection. Pears were removed by carefully breaking the shells in a dish of warmed PBS and cutting the pears carefully with the surrounding CAM. Of the 12 eggs treated with each RB, 6 TIE-2 RB and 5 EHK-1 RB treated embryos developed beyond the stages observed at the beginning of the experiment. Dramatic differences were seen between these developed embryos as shown in FIGS. 1A and 1B. Those treated with EHK-1 RB seemed to develop relatively normally. Of the five EHK-1 vessels, four were viable, judging by the presence of a pulsating heart. Furthermore, the presence of erythrocytes was rich in the extravasated vascular system and extended several centimeters below the CAM. In contrast, those treated with TIE-2 RB significantly inhibited growth in the range of 2-5 mm in diameter compared to 10 mm or more in diameter for EHK-1 RB times. All vessels treated with TIE-2 RB died and all of their CAMs had no vessels. The ability of TIE-2 RB to block vascular development in chickens demonstrates that TIE-2 ligand is required for the development of the vascular system.

Example 4

Ras Oncoregulation from oncogene-transformed C2C12 mouse myoblast cell line Identification of TIE-2-specific Binding Activity in Medium

Ten-fold enriched cell-conditioned media (10 × CCM) from several cell lines were screened for soluble TIE-2-specific binding activity (BIAcore; Pharmacia Biosensor, Piscataway, NJ), oncogene-ras-trait Binding activity was shown in serum-free medium from converted C2C12 cells (C2C12-ras), RAT 2-ras (this is the ras transformed fibroblast cell line), human neuroblastoma T98G and human neuroblastoma cell lines known as SHEP-1. .

C2C12-ras 10 × CCMs were derived from stably transfected lines of C2C12 source cells that were transformed to be oncogene by transfection with T-24 mutant of H-ras by standard calcium phosphate-based methods. The SV40 basal neomycin-resistant expression plasmid was physically linked with the ras expression plasmid to enable the selection of transfected clones. The resulting G418-resistant ras-C2C12 cells were maintained in monolayers on plastic dishes in DMEM / glutamine / penicillin-streptomycin supplemented with 10% fetal calf serum (FCS) customarily. Serum-free C2C12-ras 10 × CCMs were prepared by plating cells at a density of 60% in serum-free medium for 12 hours (Zhan and Goldfarb, Mol. Cell. Biol. 6: 3541-3544 (1986); Zhan, et al. Oncogene 1: 369-376 (1987)). This medium was discarded and replaced with fresh DMEM / Q / P-S for 24 hours. This medium was harvested and freshly replenished with fresh DMEM / Q / P-S, which was also harvested after 24 hours. These CCMs were supplemented with protease inhibitors PMSF (1 mM) and aprotinin (10 μg / ml) and concentrated 10-fold on sterile size exclusion membranes (Amicon). TIE-2 binding activity can be neutralized by incubating the medium with excess TIE-2 RB prior to BIAcore assay. However, incubation with EHK-1 RB cannot neutralize.

The binding activity of 10 × CCM was measured using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ), which monitors biomolecular interactions in real time via surface plasmon resonance. Purified TIE-2 RB was covalently bound to the carboxymethyl dextran layer of the CM5 Research Grade Sensor Chip (Pharmacia Biosensor; Piscataway, NJ) via primary amine. The sensor chip surface was activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide (EDC) followed by TIE-2 RB (25 μg). / mL, pH 4.5) was fixed and the unreacted site was inactivated with 1.0 M ethanolamine (pH 8.5). The negative control surface of the EHK-1 receptor body was also prepared in a similar manner.

The flowable buffer used in this system was HBS (10 mM Hepes, 3.4 mM edta, 150 mM NaCl, 0.005% P20 surfactant, pH 7.4). 10 × CCM samples were centrifuged at 4 ° C. for 15 minutes and further clarified using a sterile low protein-bound 0.45 μm filter (Millipore; Bedford, Mass.). Dextran (2 mg / ml) and P20 surfactant (0.005%) were added to each CCM sample. 40 μL aliquots were injected across the fixed surface (TIE-2 or EHK-1) at a flow rate of 5 μL / min and receptor binding was monitored for 8 minutes. Binding activity (resonance unit, RU) was measured as the difference between the baseline value measured 30 seconds before the sample was injected and the measurement taken 30 seconds after the injection. Surface regeneration consisted of one 12 μL pulse of 3M MgCl ₂ .

The noise level of the instrument is 20 RU. Therefore, the binding activity with a signal of 20RU or more can be interpreted as the actual interaction with the receptor. For C2C12-ras conditioned media the binding activity was in the range of 60-90 RU for TIE-2 RB immobilized surface. For the same sample analyzed for EHK-1 RB immobilized surface the activity measured was less than 35 RU. Specific binding to the TIE-2 receptor body was assessed by incubating the sample with excess soluble TIE-2 or EHK-1 RB prior to analyzing binding activity. The addition of soluble EHK-1 RB did not affect the TIE-2 binding activity of any of the samples, in the presence of soluble TIE-2 the binding to the surface was 2/3 less than measured in the absence of TIE-2. . Repeated analysis using> 50 × concentrated C2C12-ras CCM resulted in a four-fold increase in TIE-2 specific binding signal beyond background.

Example 5

C2C12-ras CCM has activity to induce tyrosine phosphorylation of TIE-2 receptor have.

C2C12-ras 10 × CCM was tested for its ability to induce tyrosine phosphorylation of TIE-2 in ABAE cells. Serum-starved ABAE cells were briefly incubated with C2C12-ras CCM and lysed as described above for immunoprecipitation and Western analysis. Stimulation of serum deficient ABAE cells by serum free C2C12-ras 10 × CCM was performed as follows. The medium of deficient ABAE cells as described above was removed and replaced with definite medium or 10 × CCM pre-warmed to 37 ° C. After 10 minutes the medium was removed and cells were washed twice on ice with excess cold PBS supplemented with orthovanadate / NaF / benzamidine. Cytolysis and TIE-2 specific immunoprecipitation were performed as follows.

ABAE cells incubated for 10 minutes with defined medium showed no induction of TIE-2 tyrosine phosphorylation, whereas incubation with C2C12-ras CCM stimulated at least a 100-fold increase in TIE-2 phosphorylation. This activity was reduced almost entirely by incubating C2C12-ras 10 × CCM for 90 minutes at room temperature with 13 ug of TIE-2 RB bound to protein G-Sepharose beads.

Example 6

Expression Cloning of TIE-2 Ligands

COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin (P / S), and 2 mM glutamine under an atmosphere of 5% CO ₂ . . Mouse myoblast C2C12-ras cell lines were cultured in Eagle's minimal essential medium (EMEM) containing 10% FBS, (P / S) and 2 mM glutamine. Full-length mouse TIE-2 ligand cDNA clones were obtained by screening C2C12 ras cDNA libraries in pJFE14 vectors expressed in COS cells. This vector as shown in FIG. 2 is a modification of the vector pSR _α (Takebe, et al. 1988, Mol. Cell. Biol. 8: 466-472). The library was made using two BSTX1 restriction sites in the pJFE14 vector.

COS-7 cells were transiently transfected into the pJFE14 library or regulatory vector by the DEAE-dextran transfection protocol. Briefly, COS-7 cells were plated at a density of 1.0 × 10 ⁶ cells per 100 mm plate 24 hours prior to transfection. Cells were harvested for 3-4 hours at 37 ° C. in an atmosphere of 5% CO ₂ in serum free DEME containing 400 μg / ml of DMEM-dextran, 1 μM chloroquine and 2 mM glutamine and 1 μg of appropriate DNA for transfection. Incubated. Transfection medium was aspirated and replaced with 10% DMSO containing phosphate buffered saline for 2-3 minutes. Following this DMSO "shock", COS-7 cells were plated in DMEM containing 10% FBS, 1% of penicillin and streptomycin and 2 mM glutamine for 48 hours.

Since the TIE-2 ligand is secreted, it has been necessary to make the cells permeable to detect binding of the receptor body probe to the ligand. Two days after transfection, cells were washed with PBS and incubated with PBS containing 1.8% formaldehyde for 15-30 minutes at room temperature. Cells were then washed with PBS and incubated with PBS containing 0.1% Triton X-100 and 1% calf serum for 15 minutes to make cells permeable and block nonspecific binding sites. Screening was performed by direct localization of staining using a TIE-2 receptor body consisting of the extracellular domain of TIE-2 fused to an IgGl constant region. This receptor body was prepared as described in Example 2. 100 mm dishes of the cells were probed by incubating transformed, fixed and permeable COS cells with TIE-2-RB for 30 minutes. The cells were then washed twice with PBS and incubated with PBS / 10% calf serum / anti-human IgG-alkaline phosphatase conjugate for another 30 minutes. After washing three times with PBS, cells were incubated for 30-60 minutes in alkaline phosphatase substrate. The dish was then examined under the microscope for the presence of stained cells. For each stained cell, a small portion of the cell site containing the stained cells was scraped from the dish using the tip of a plastic pipette and then plasmid DNA was taken to electroporate the bacterial cells. Single bacterial colonies generated by electroporation were isolated and transfected with COS-7 cells using plasmid DNA prepared from these colonies, which were demonstrated to bind to the TIE-2 receptor body, as demonstrated by TIE- Probe for 2 ligand expression. This made it possible to identify monoclonals encoding TIE-2 ligands. Confirmation of TIE-2 ligand expression was made by phosphorylating the TIE-2 receptor using the method described in Example 5. The plasmid clone encoding the TIE-2 ligand was deposited with the ATCC on October 7, 1994 and was named "pJFE14 encoding the TIE-2 ligand" under ATCC Accession No. 75910.

Example 7

Isolation and Sequencing of Full-length cDNA Clones Encoding Human TIE-2 Ligands

Human fetal lung cDNA libraries in lambda gt-10 (see FIG. 3) were obtained from Clontech Laboratories, Inc. (Palo Alto, Calif.). Plaques play a density of 1.25 × 10 ^6/20 × 20㎝ plate plated and replicate filter is a standard method (Sambrook, et al, Molecular Cloning :.. A Laboratory Manual, 2nd Ed, page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Isolation of human tie-2 ligand was performed as follows. 2.2 kb XhoI fragments from deposited tie-2 ligand clones (ATCC NO. 75910—see Example 6 above) were labeled with specific activity of about 5 × 10 ⁸ cpm / ng by random priming. Hybridization was performed at 65 ° C. in a hybridization solution containing 0.5 mg / ml salmon sperm DNA. The filter was washed in 2 × SSC, 0.1% SDS at 65 ° C. and exposed to Kodak XAR-5 film overnight at −70 ° C. Positive phage were plaque purified. High titer phage lysates of pure phage were used to separate DNA through Qiagen columns using standard techniques (Qiagen, Inc., Chatsworth, CA, 1995 catalog, page 36). Phage DNA was digested with EcoRI to release cloned cDNA fragments for subsequent subcloning. A lambda phage vector containing human tie-2 ligand DNA was deposited with the ATCC on October 26, 1994 under the name λgt10 encoding htie-2 ligand 1 (ATCC Accession No. 75928). Phage DNA can be DNA sequencing directly by dideoxy chain termination (Sanger, et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467).

Subcloning of Human TIE-2 Ligands into Mammalian Expression Vectors

Clone λgt 10 encoding htie-2 ligand 1 contains EcoRI located 490 base pairs downstream from the start of the coding sequence for the human tie-2 ligand. The coding region can be excised using specific restriction enzymes upstream and downstream of the start codon and the stop codon, respectively. For example, the SpeI site located at 70 bp 5 'for the start codon and the Bpu1102i (also known as BlpI) site at 265 bp 3' for the stop codon can be used to ablate the complete coding region. This can then be subcloned into the pJFE14 cloning vector using XbaI (suitable for SpI overhang) and PstI sites (both PstI sites and Bpu1102i sites have blunt ends).

Sequencing of Human TIE-2 Ligands

Coding regions from clone λgt10 encoding htie-2 ligand 1 were sequenced using ABI 373A DNA sequencer and Taq dideoxy terminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, Calif.). The nucleotide and deduced amino acid sequences of the human TIE-2 ligand from clone λgt10 encoding htie-2 ligand 1 are shown in FIG. 4.

In addition, full-length human TIE-2 ligand cDNA clones were obtained by screening the human glioma cell T98G cDNA library in the pJFE14 vector. Clones encoding human tie-2 ligands were identified by DNA hybridization using 2.2 kb XhoI fragments from deposited tie-2 ligand clones (ATCC NO. 75910) as probes (see Example 6 above). Coding regions were sequenced using ABI 373A DNA Sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, Calif.). This sequence was nearly identical to that of clone λgt10 encoding htie-2 ligand 1. As shown in FIG. 4, the clone λgt10 encoding htie-2 ligand 1 contains additional glycine residues encoded by nucleotides 1114-1116. The coding sequence of the T98G clone does not contain this glycine residue but the others are identical to the coding sequence of clone λgt10 encoding htie-2 ligand 1. Figure 5 depicts the nucleotide and inferred amino acid sequences of human TIE-2 ligands from the T98G clone.

Example 8

Isolation and Sequencing of Second Full Length cDNA Clone A Encoding Human TIE-2 Ligand

The lambda gt-10 human fetal lung cDNA library (see FIG. 3) was obtained from Clontech Laboratories, Inc. Obtained from (Palo Alto, Calif.). Plaques 1.25 × 10 ^6/20 × density plated and replicate filter to the plate 20㎝ standard methods (Sambrook et al, Molecular Cloning: .. A Laboratory Manual, 2nd Ed, page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Duplicate filters were screened for low shrinkage (2 × SSC, 55 ° C.) with probes made for human tie2L-1 sequences. One of the duplicate filters was probed with a 5 'probe encoding amino acids 25-265 of human tie 2L-1 as described in FIG. The second duplicate filter was probed with a 3 'probe encoding amino acids 282-498 of the human tie 2L-1 sequence (see Figure 4). Both probes hybridized at 55 ° C. in a hybridization solution containing 0.5 mg / ml salmon sperm DNA. The filter was washed with 2 × SSC at 55 ° C. and exposed to X-ray film overnight. In addition, the duplicate filter was hybridized with moderate constriction (2 × SSC, 65 ° C.) to the full length coding probe of mouse tie 2L (F3-15, XhoI insert). Three positive clones were selected that met the following conditions: i. There was no hybridization to the full length (mouse) probe at normal contractility. ii. Hybridization was seen at low shrinkage for both 5 'and 3' probes. EcoRI digestion of phage DNA from these clones suggested two independent clones with insert sizes of about 2.2 kb and about 1.8 kb. The 2.2 kb EcoRI insert was subcloned into EcoRI sites of both mammalian expression vectors and pBluescript KS (Stratagene) suitable for COS cells. Two orientations have been identified for mammalian expression vectors. The 2.2 kb insert in pBluescript KS was deposited with ATCC on Dec. 9, 1994 and was called pBluescript KS encoding human TIE-2 ligand 2. The initiation site of the TIE-2 ligand 2 coding sequence is about 355 base pairs downstream of the pBluescript EcoRI site.

COS-7 cells were transiently transfected with expression or control vectors by DEAE-dextran transfection protocol. Briefly, COS-7 cells were plated at a density of 1.0 × 10 ⁶ cells per 100 mm plate 24 hours prior to transfection. Incubate cells in serum-free DMEM containing 400 μg / ml of DEAE-dextran, 1 μM chloroquine and 2 mM glutamine and 1 μg of appropriate DNA for 3-4 hours at 37 ° C. in a 5% CO ₂ atmosphere for transfection. It was. Transfection medium was aspirated and replaced with phosphate buffered saline containing 10% DMSO for 2-3 minutes. Following this DMSO "vi", COS-7 cells were placed in DMEM containing 10% FBS, 1% each of penicillin and streptomycin and 2 mM glutamine for 48 hours.

Since the TIE-2 ligand is secreted, it has been necessary to make the cells permeable to detect binding of the receptor body probe to the ligand. Transfected COS-7 cells were plated at a density of 1.0 × 10 ⁶ cells per 100 mm plate. The cells were washed with PBS and then incubated with PBS containing 1.8% formaldehyde for 15-30 minutes at room temperature. Cells were then washed with PBS and incubated with PBS containing 0.1% Triton X-100 and 10% calf serum for 15 minutes to make cells permeable and block nonspecific binding sites. Screening was performed by direct localization of staining using a TIE-2 receptor body consisting of the extracellular domain of TIE-2 fused to an IgGl constant region. This receptor body was prepared as described in Example 2. Transformed COS cells were probed by incubating them with TIE-2-RB for 30 minutes. Cells were then washed twice with PBS, fixed with methanol and incubated for 30 minutes with PBS / 10% calf serum / anti-human IgG-alkaline phosphatase conjugate. After washing three times with PBS, cells were incubated for 30-60 minutes in alkaline phosphatase substrate. The dish was then examined under the microscope for the presence of stained cells. It was found that cells expressing one orientation of the clone but no other orientation bind the TIE-2 receptor body.

Those skilled in the art will readily appreciate that the described method can be used to further identify other relevant members of the TIE ligand.

Sequencing Second Human TIE-2 Ligands

Coding regions from clone pBluescript KS encoding human TIE-2 ligand 2 were sequenced using an ABI 373A DNA sequencer and Taq dideoxy terminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA). Nucleotide and inferred amino acid sequences of human TIE-2 ligand from clone pBluescript KS encoding human TIE-2 ligand 2 are shown in FIG. 6.

Example 9 TIE-2 Ligand 2 is a receptor antagonist.

Conditioned media from COS cells expressing TIE-2 ligand 2 (TL2) or TIE-2 ligand 1 (TL1) were compared for their ability to activate TIE-2 receptors that are naturally present in human endothelial cell lines. PMT21 expression containing pJFE14 expression vector alone, pJFE14 vector containing human TIE-2 ligand 1 cDNA or human TIE-2 ligand 2 cDNA, using lipofectamine reagent (GIBCO-BRL, Inc.) and the recommended protocol. COS-7 cells were transfected with the vector (Kaufman, RJ, 1985, Proc. Natl. Acad. Sci. USA 82: 689-693). After 3 days COS medium containing the secreted ligand was collected and concentrated 20 times with permeation (DIAFLO ultrafiltration membrane, Amicon, Inc.). The amounts of active TIE-2 ligand 1 and TIE-2 ligand 2 present in these media were measured and expressed as the amount of TIE-2 receptor specific binding activity (in resonance unit R.U.) as determined by BIAcore binding assay.

Northern (RNA) analysis showed significant levels of TIE-2 transcripts in Human Aortic Endothelial Cell (HAEC) human primary endothelial cells (Clonetics, Inc.). Thus, these cells were used to investigate whether tyrosine phosphorylation was when the TIE-2 receptor was exposed to COS medium containing the TIE-2 ligand. HAEC cells were complete containing 5% fetal bovine serum, soluble cerebellar extract, 10 n / ml human EGF, 1 mg / ml hydrocortisone, 50 mg / ml gentamicin and 50 ng / ml amphotericin-B. Endothelial cell growth medium (Clonetics, Inc.) was maintained. Evaluation of whether TL1 and TL2 can activate the TIE-2 receptor in HAEC cells was carried out as follows. Semi-dense HAEC cells were deficient in serum for 2 hours in MEM of high glucose Dulbecco added L-glutamine and penicillin-streptomycin at 37 ° C. and then depleted medium was removed at 37 ° C. in a 5% CO ₂ incubator at 7 ° C. Substituted with ligand containing conditioned COS medium for minutes. Cells were then lysed and the lysates immunoprecipitated with TIE-2 peptide antiserum to recover the TIE-2 receptor protein, followed by western blotting with antiphosphotyrosine antiserum exactly as described in Example 1. The results are shown in FIG. Phosphotyrosine levels on the TIE-2 receptor (TIE2-R) were induced by treating HEAC cells with TIE-2 ligand 1 (lane L1) but not with TIE-2 ligand 2 (lane L2) conditioned COS medium. . MOCK is a conditioning medium from COS transfected with a JFE14 free vector.

Both TL1 and TL2 are specific for the TIE-2 receptor by measuring TIE-2 receptor specific binding activity in a transformed COS medium using BIAcore and immunostaining TL1 and TL2-expressing COS cells with the TIE-2 receptor body. Prove evidence of binding.

Since TL2 did not activate the TIE-2 receptor, we set out to determine whether TL2 could serve as an antagonist of TL1 activity. HAEC phosphorylation assays were performed where cells were first incubated with "excess" TL2 and then diluted TL1. The reason for this is that pre-occupancy of the TIE-2 receptor due to high levels of TL2 may interfere with subsequent stimulation of the receptor after exposure to TL1 present in limited concentrations.

Semi-dense HAEC cells were serum deficient as described above and then incubated for 3 minutes at 37 ° C. with 1-2 ml of 20 × COS / JFE14-TL2 conditioned medium. Control plates were treated with 20 × COS / JFE14 medium alone (MOCK). The plates were removed from the incubator and then several dilutions of COS / JFE14-TL1 medium were added and the plates were then further incubated at 37 ° C. for 5-7 minutes. Cells were then washed and lysed and TIE-2 specific tyrosine phosphorylation in the lysates was examined by receptor immunoprecipitation and western blotting as described above. TL1 dilutions were made using 20 × COS / JFE14-TL1 medium diluted to 2 ×, 0.5 ×, 0.1 × or 0.02 × with 20 × COS / JFE14 alone. Analysis of 20 × TL1 and 20 × TL2 COS media using BIAcore biosensor technology revealed that they had similar amounts of TIE-2 specific binding activity, 445 R. U. and 511 R. U., respectively for TL1 and TL2. The results of the antiphosphotyrosine western blot shown in FIG. 8 show that pretreatment of HAEC cells with excess TIE-2 ligand 2 (lane 2) compared to pretreatment of HAEC cells with MOCK medium (lane 1). It suggests that dilute TIE-2 ligand 1 antagonizes the subsequent ability to activate the TIE-2 receptor (TIE2-R).

These data show that, unlike TL1, TL2 cannot stimulate TIE-2 receptor kinase activity in HAEC cells. Furthermore, pre-culture of endothelial cells with high concentrations of TL2 followed by addition of TL1 blocked the ability of TL1 to stimulate TIE-2 receptors, indicating that TL2 is a TIE-2 receptor antagonist.

Example 10

Identification of TIE-2 Specific Binding Activity in Conditioned Medium and COS Cell Supernatant

The binding activity of 10 × CCMs from cell lines C2C12 ras, Rat2 ras, SHEP and T98G or COS cell supernatants after transfection with human TIE-2 ligand 1 (hTL1) or human TIE-2 ligand 2 (hTL2) was determined by biomolecule interaction. Action was measured using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ), which monitors in real time via surface plasmon resonance (SPR). Purified rat or human TIE-2 RB was covalently bonded via primary amine to the carboxymethyl dextran layer of CM5 Research Grade Sensorchip (Pharmacia Biosensor, Piscataway, NJ). Sensor chip surface was activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide (EDC), followed by TIE-2 RB (25 μg). / mL, pH 4.5) was fixed and the unreacted site was inactivated with 1.0 M ethanolamine (pH 8.5). Typically, each receptor body of 9000-10000RU is coupled to a sensor chip.

The flowable buffer used in this system was HBS (10 mM Hepes, 150 mM NaCl, 0.005% P20 surfactant, pH 7.4). Samples were centrifuged at 4 ° C. for 15 minutes and further clarified using a sterile low protein bound 0.45 μm filter (Millipore; Bedford, Mass.). Dextran (2 mg / ml) and P20 surfactant (0.005%) were added to each sample. 40 μL aliquots were injected at a flow rate of 5 μL / min across the fixed surface (rat or human TIE2) and receptor binding was monitored for 8 minutes. Binding activity (resonance unit, RU) was determined as the difference between the baseline value measured 30 seconds before the sample injection and the measurement taken 30 seconds after the injection. Surface regeneration consisted of one 15 μL pulse of 3M MgCl ₂ .

CCM samples (C2C12ras, Rat 2 ras, SHEP, T98G) were tested on rat TIE2 RB fixed surfaces, while recombinant hTL1 and hTL2 were tested on human TIE 2 RB fixed surfaces. In each case specific binding to the TIE-2 receptor body was assessed by incubating the sample with 25 μg / ml of soluble TIE-2 (rat or human) RB or trkB RB prior to analyzing binding activity. As shown in FIGS. 9 and 10, addition of soluble trkB RB slightly reduced TIE-2 binding activity, whereas addition of soluble TIE-2 RB significantly increased binding activity compared to that measured in the absence of TIE-2 RB. Decrease.

Example 11

TIE-2 RB specifically blocks activation of the TIE-2 receptor by TIE-2 ligand 1.

Applicants sought to determine if soluble TIE-2 RB could serve as a competitive inhibitor to block activation of TIE-2 receptor by TIE-2 ligand 1 (TL1). To do this, TL1 containing COS medium was pre-cultured with TIE2- or TrkB-RB and then compared for their ability to activate TIE-2 receptors that are naturally present in human endothelial cell lines.

Conditioned COS medium was generated from COS-7 cells transfected with pJFE14 expression vector alone (MOCK) or pJFE14 vector containing human TIE-2 ligand 1 cDNA (TL1), and the medium was sterile filtered but not concentrated. Except as described in Example 9 above. The amount of TL1 was measured and expressed as the amount of TIE-2 receptor specific binding activity (in resonance unit R.U.) measured by BIAcore binding assay.

Northern (RNA) analysis showed significant levels of TIE-2 transcripts in human Umbilical Vein Endothelial Cell (HUVEC) human primary endothelial cells (Clonetics, Inc.). Thus, these cells were used to investigate whether TIE-2 receptors could be tyrosine phosphorylated when exposed to TL1-containing COS medium in the presence of TIE2- or TrkB-RB. HUVEC cells were soluble cerebellum with 5% fetal bovine serum, 10 ug / ml heparin, 10 ng / ml human EGF, 1 ug / ml hydrocortisone, 50 ug / ml gentamicin and 50 ng / ml amphotericin-B Complete endothelial growth medium containing extracts (Clonetics, Inc.) was maintained at 37 ° C. in 5% CO ₂ . Evaluation of whether TL1 can activate TIE-2 receptor in HUVEC cells was carried out as follows. The dense dish of HUVEC cells was deficient in serum for 2-4 hours at 37 ° C., 5% CO ₂ in MEM of low-glucose Dulbecco, followed by 0.1 mM sodium orthovanadate, a potent inhibitor of phosphotyrosine phosphatase. Incubation was performed for 10 minutes in deficient medium. Conditioned COS medium was pre-incubated for 30 minutes at room temperature by adding TIE2- or TrkB-RB at 50 ug / ml. The depleted medium was then removed from the HUVEC dish and incubated with RB containing COS medium at 37 ° C. for 7 minutes. HUVEC cells were then lysed and the TIE-2 receptor protein was recovered by immunoprecipitation against TIE-2 peptide antiserum as described in Example 1 and then western blotting with antiphosphotyrosine antibody. The results are shown in FIG. Phosphotyrosine levels on TIE-2 receptors were induced by treatment of HUVEC cells with TIE-2 ligand 1 (TL1) as compared to control medium (MOCK), and this induction was previously performed with TIE2-RB (TIE2-Fc). It is specifically blocked by culture, but not by culture with TrkB-RB (TrkB-Fc). These data show that soluble TIE-2 RB can serve as a selective inhibitor that blocks the activation of TIE-2 receptor by TIE-2 ligand 1.

Example 12 Construction of TIE-2 Ligand Body

Expression constructs capable of producing secreted proteins consisting of the entire coding sequence of human TIE-2 ligand 1 (TL1) or TIE-2 ligand 2 (TL2) fused to human immunoglobulin gamma-1 constant region (IgG1 Fc) Made. These fusion proteins are termed TIE-2 "ligand bodies" (TL1-Fc or TL2-Fc). The Fc portion of TL1-Fc and TL2-Fc was prepared as follows. DNA fragments encoding the Fc portion of human IgG1 extending from the hinge portion of the protein to the carboxy terminus were amplified from human placental cDNA by PCR with oligonucleotides corresponding to the published sequence of human IgG1. The resulting DNA fragment was cloned in plasmid vector. Appropriate DNA restriction fragments from plasmids encoding full length TL1 or TL2 and human IgG1 Fc plasmids were linked to either side of a short PCR-derived fragment designed to fuse TL1 or TL2 into the human IgG1 Fc protein coding sequence in frame.

Milligrams of TL2-Fc were obtained by cloning the TL2-Fc DNA fragment into the pVL1393 baculovirus vector and subsequently infecting the Spodoptera frugiperda SF-21AE insect cell line. Alternatively, cell line SF-9 (ATCC Accession No. CRL-1711) or cell line BTI-TN-5b1-4 may be used. DNA encoding TL2-Fc was cloned into the baculovirus delivery plasmid pVL1393 as an EcoRI-NotI fragment. Plasmid DNA was recombined into viral DNA by mixing 3 mg of plasmid DNA with 0.5 mg of Baculo-Gold DNA (Pharminigen) and then introducing it into liposomes using 30 mg of lipofectin (GIBCO-BRL). DNA-liposomal mixture was added to SF-21AE cells (2 × 10 ⁶ cells / 60 mm plates) in TMN-FH medium (modified Grace's Insect Cell Medium (GIBCO-BRL)) for 5 hours at 27 ° C. and then 5%. Cultured for 5 days at 27 ℃ in TMN-FH medium supplemented with fetal calf serum. Tissue culture medium was harvested for plaque purification of the recombinant virus, which was purified from 125 mg / mL X-gal (5-bromo-4-chloro-3-indolyl-bD-galactopyranoside in the agarose layer). Performed using previously described methods (O'Reilly, DR, LK Miller, and VA Luckow, Baculovirus Expression Vectors-A Laboratory Manual. 1992, New York: WHFreeman), except that they contain GIBCO-BRL). It was. After 5 days of incubation at 27 ° C., non-recombinant plaques were assessed by positive chromosome response to X-gal substrate and marked for their location. Recombinant plaques were then visualized by addition of a second phase containing 100 mg / mL MTT (3- [4,5-dimethylthiazol-2-yl] 2,5, diphenyltetrazolium bromide; Sigma). Putative recombinant virus plaques were picked out by plug aspiration and purified by multiple plaque isolation to confirm homology. Virus stocks were generated in a series of low multiplicity passages of plaque purified virus. Low pass stocks of one viral clone (vTL2-Fc clone # 7) were prepared.

SF-21AE cells were cultured in serum-free medium (SF-900II, Gibco BRL) containing 1 × antibiotic / antibacterial solution (Gibco BRL) and 25 mg / L gentamycin (Gibco BRL). Pluronic F-68 was added as surfactant at a final concentration of 1 g / L. Cultures (4 L) were grown in Artisan Cell Station System for at least 3 days prior to infection. Cells were grown at 27 ° C. (vented in a spray ring) with gas evolution with 50% dissolved oxygen at a gas flow rate of 80 mL / min. The marine impeller was stirred at a speed of 100 rpm. Cells were harvested in mid-log growth phase (˜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 brought to 400 mL with fresh medium and the virus was adsorbed in a spinner flask at 27 ° C. for 2 hours. The cultures were then resuspended to a final volume of 8 L with fresh serum-free medium and cells were incubated in a bioreactor using the conditions previously described.

Culture medium from vTL2-Fc-infected SF21AE cells was collected by centrifugation (500 × g, 10 minutes) 72 hours after infection. The cell supernatant was brought to pH 8 with NaOH. EDTA was added to a final concentration of 10 mM and the supernatant pH was readjusted to 8. The supernatant was filtered (0.45 mm, Millipore) and loaded on a Protein A column (Protein A Sepharose 4 Fast Flow or Hyblap Protein A, both manufactured by Pharmacia). The column was washed with 0.5 M NaCl containing PBS until the absorbance at 280 nm decreased to baseline. The column was washed with PBS and eluted with 0.5M acetic acid. Column fractions were immediately neutralized by eluting with a tube containing 1M Tris pH 9. Peak fractions containing TL2-Fc were pooled and dialyzed against PBS.

submission

The following was deposited in the American Type Culture Collection of 20852 Rockville Parkron Drive 12301, Maryland, under the Budapest Treaty. The plasmid clone encoding the TIE-2 ligand was deposited with the ATCC on October 7, 1994 and was designated as "pJFE14 encoding the TIE-2 ligand" under ATCC Accession No. 75910. The recombinant Autographa californica baculovirus encoding the TIE-2 receptor body was deposited with the ATCC on October 7, 1994 and was named "vTIE-2 receptor body" under ATCC accession number VR2484. A lambda phage vector containing human tie-2 ligand DNA was deposited with the ATCC on October 26, 1994 and named as λgt10 encoding htie-2 ligand 1 under ATCC accession number 75928. The plasmid clone encoding the second TIE-2 ligand was deposited with ATCC on Dec. 9, 1994 and was named ATCC Accession No. 75963 as "pBluescript KS encoding human TIE2 ligand 2."

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention other than those described herein will become apparent from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims (38)

An isolated nucleic acid molecule encoding a TIE-2 ligand, wherein the nucleic acid sequence is (a) a nucleic acid sequence consisting of a coding region of a human TIE-2 ligand as shown in FIG. 4 or 5, (b) a nucleic acid sequence encoding a TIE-2 ligand that hybridizes to the nucleic acid sequence of (a) under conditions of tightness and which binds a TIE-2 receptor, or (c) a nucleic acid molecule that is hybridized to the nucleic acid sequence of (a) or (b) if there is no degeneracy of the gene code and that encodes a TIE-2 ligand that binds a TIE-2 receptor. The nucleic acid molecule of claim 1, wherein the encoded TIE-2 ligand is a TIE-2 agonist. An isolated nucleic acid molecule encoding a TIE-2 ligand, wherein the nucleic acid sequence is (a) a nucleic acid sequence consisting of a coding region of a human TIE-2 ligand as shown in FIG. 6, (b) a nucleic acid sequence encoding a TIE-2 ligand that hybridizes to the nucleic acid sequence of (a) under conditions of tightness and which binds a TIE-2 receptor, or (c) a nucleic acid molecule that is hybridized to the nucleic acid sequence of (a) or (b) if there is no degeneracy of the gene code and that encodes a TIE-2 ligand that binds a TIE-2 receptor. 4. The nucleic acid molecule of claim 3 wherein the encoded TIE-2 ligand is a TIE-2 antagonist. A nucleic acid molecule encoding a ligand that is degenerate to the sequence of claims 1 or 3 and binds a TIE-2 receptor. Nucleic acid molecules that hybridize to sequences encoding TIE-2 ligands as shown in FIG. 4, 5, or 6 under conditions of tightness. A vector comprising the nucleic acid molecule of any one of claims 1 to 6. 8. The vector of claim 7, wherein the nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. The vector according to claim 7, which is a plasmid. The vector according to claim 8, which is a plasmid. The plasmid according to claim 9, referred to as pJFE14 (ATCC Accession No. 75910) encoding TIE-2 ligand or pBluescript KS (ATCC Accession No. 75963) encoding human TIE-2 Ligand2. The vector (ATCC Accession No. 75928) according to claim 7, which is referred to as λgt10 encoding hTIE-2 ligand1. An isolated TIE-2 ligand having an amino acid sequence set forth in FIG. 4, 5 or 6 or a fragment or derivative thereof that binds to a TIE-2 receptor, substantially free of other proteins. An isolated TIE-2 ligand, encoded by the nucleic acid according to claim 1, substantially free of other proteins. An isolated TIE-2 ligand, encoded by the nucleic acid according to claim 3, substantially free of other proteins. A host-vector system for the production of a ligand according to any one of claims 13 to 15 consisting of a vector according to any one of claims 7 to 12 in a host cell. 17. The host-vector system according to claim 16, wherein the host cell is a bacteria, yeast, insect or mammalian cell. A host-vector system comprising the host-vector system of claim 16 and a nucleic acid encoding a TIE-2 receptor. A method for preparing a ligand as defined in any one of claims 13 to 15, comprising growing the cells of the host-vector system according to claim 16 and recovering the ligand thus produced under conditions that allow the production of the ligand. . The antibody which specifically binds the ligand of any one of Claims 13-15. The antibody of claim 20, which is a monoclonal antibody. A ligand body consisting of a TIE-2 ligand as defined in any of claims 13 to 15 fused to an immunoglobulin constant region. The ligand body of claim 22, wherein the TIE-2 ligand has the amino acid sequence set forth in FIG. 4, 5, or 6 and wherein the immunoglobulin constant portion is the Fc portion of human IgG1. A conjugate comprising a ligand according to any one of claims 13 to 15 and a cytotoxic agent incorporated therein. The conjugate of claim 24, wherein the cytotoxic agent is a radioisotope or toxin. A pharmaceutical composition comprising the TIE-2 ligand according to any one of claims 13 to 15 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the antibody according to claim 20 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the ligand body according to claim 22 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the conjugate of claim 24 and a pharmaceutically acceptable carrier. A method for maintaining TIE-2 receptor expressing cells in a culture medium comprising administering an effective amount of the ligand of claim 14 to TIE-2 receptor expressing cells. 31. The method of claim 30, wherein the TIE-2 receptor expressing cells are endothelial cells. Cells expressing a TIE-2 receptor, a) test compound and b) a TIE-2 consisting of contacting the ligand of claim 14 or 15 with conditions that allow binding of the ligand to the receptor and determining whether the test compound can interfere with binding of the ligand to the receptor. How to Identify Receptor Antagonists. A receptor body that specifically binds a TIE-2 ligand according to any one of claims 13-15. An isolated nucleic acid molecule encoding the receptor body according to claim 33. A vector consisting of the nucleic acid molecule according to claim 34. 36. The vector of claim 35 which is a plasmid. A plasmid according to claim 36 (ATCC deposited VR2484), referred to as the vTIE-2 receptor body. A pharmaceutical composition comprising the receptor body according to claim 33 and a pharmaceutically acceptable carrier.

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