WO1995013387A1 - Tie-2, a novel receptor tyrosine kinase - Google Patents

Abstract

The present invention relates to a novel receptor tyrosine kinase, herein referred to as tie-2, to nucleotide sequences and expression vectors encoding tie-2, and to methods of inhibiting tie-2 activity. The invention also relates to other members of the tie-2 receptor tyrosine kinase family. Genetically engineered host cells that express tie-2 may be used to evaluate and screen drugs involved in tie-2 activation and regulation. The invention relates to the use of such drugs as agonists or antagonists of tie-2 activity.

Description

TIE-2 . A NOVEL RECEPTOR TYROSINE KINASE

1. INTRODUCTION The present invention relates to a novel receptor tyrosine kinase, herein referred to as tie-2, to nucleotide sequences and expression vectors encoding tie-2, and to methods of inhibiting tie-2 activity. The invention also relates to other members of the tie-2 receptor tyrosine kinase family. Genetically engineered host cells that express tie-2 may be used to evaluate and screen drugs involved in tie-2 activation and regulation. The invention relates to the use of such drugs as agonists or antagonists of tie-2 activity.

2. BACKGROUND Receptor tyrosine kinases comprise a large and evolutionarily conserved family of proteins comprised of an extracellular ligand-binding domain, a transmembrane domain and an intraceUular tyrosine kinase domain. Receptor tyrosine kinases are involved in a variety of critical cellular processes such as cellular differentiation and proliferation. The binding of ligand to receptor tyrosine kinases induces the formation of receptor dimers followed by activation of receptor tyrosine kinase activity. This in turn results in phosphorylation of a number of intraceUular substrates leading to a cascade of events eventually resulting in cellular responses such as changes in gene expression, cell morphology and cell proliferation.

The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, play an important role in a variety of physiological processes such as embryonic development, wound healing and organ regeneration. In particular, endothelial cell proliferation plays an important role during the development of the vascular system. In in vitro systems of angiogenesis, molecules that regulate tyrosine kinase activity have been found to modulate the invasion and tube formation of endothelial cells indicating that tyrosine phosphorylation plays a critical role in the regulation of endothelial cell proliferation and morphogenesis.

The cloning and characterization of two novel tyrosine kinases, expressed in endothelial cells, has recently been reported in the literature. Partanean et al. (1992, Mol. Cell. Biol. 12:1698-1707) identified a receptor tyrosine kinase, referred to as tie , which has 76% amino acid sequence homology with tie-2 in the cytoplasmic domain. The sequence homology between the two proteins diverges and is less pronounced in the extracellular and transmembrane domain (33% and 37% respectively) . In addition, Dumont et al. (1992, Oncogene 7:1471-1480) reported the cloning and characterization of a partial cDNA clone encoding only the cytoplasmic domain of a novel receptor tyrosine kinase. The cloning of a full length representative of this particular clone, referred to as tek, has recently been reported and the deduced amino acid sequence of the tek tyrosine kinase receptor indicates broad homology with tie-2 (Ziegler et al. , 1993, Oncongene 8:663-670).

The identification of novel tyrosine kinase receptors meditating physiological processes such as vasculogenesis and angiogenesis will lead to a more complete understanding of the molecular mechanisms controlling blood vessel formation. In addition, the identification of novel receptors involved in these processes will provide targets for development of therapeutic applications designed to treat disorders associated with aberrant blood vessel formation.

^ _ 3. SUMMARY OF THE INVENTION

The present invention relates to a novel receptor tyrosine kinase receptor, herein referred to as tie-2, to nucleotide sequences and expression vectors encoding tie-2, and to methods of inhibiting tie-2 activity. The invention is based, in part, on the isolation of a cDNA clone, from a brain capillary cDNA library, encoding the tie-2 receptor tyrosine kinase.

The invention also relates to novel members of the tie-2 family of receptor tyrosine kinases. More specifically, the invention relates to members of the tie-2 family of receptor tyrosine kinases that are defined, herein, as those receptors demonstrating 80% homology at the amino acid level in substantial stretches of DNA sequence with tie-2. In addition, members of the tie-2 family of receptor tyrosine kinases are defined as those receptors containing an intraceUular tyrosine kinase domain and, in the extracellular region of the protein, EGF-like repeats flanked by a single amino terminal immunoglobulin (Ig) domain and a carboxyl-terminal triplet of fibronectin (Fn) type III domains.

Northern blot analysis and in situ hybridization indicates that tie-2 is expressed in endothelial cell precursors (angioblasts) and in endothelial cells of the sprouting blood vessels throughout development and in all organs and tissues so far examined. Pharmaceutical reagents designed to modulate tie-2 activity may be useful for treating diseases and/or processes associated with angiogenesis and vasculogenesis.

The invention relates to expression systems designed to produce tie-2 receptor and/or cell lines expressing tie-2 receptor. For example, engineered cell lines expressing tie-2 on their surface may be advantageously used to identify tie-2 ligands and to screen and identify agonist and antagonist of the tie- 2 receptor. Additionally, expression of soluble recombinant tie-2 may be used to generate antibodies against specific epitopes of the tie-2 protein and/or to screen peptide libraries for molecules that bind tie-2.

4. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1A, IB and IC. Nucleotide Sequence of murine tie-2. The AUG START codon and AGA STOP codon are underlined.

Fig. 2A. Deduced amino acid sequence and structure of tie-2. Amino acid sequence of tie-2 in single letter code. The potential signal sequence cleavage site is indicated by an arrowhead. Black dots mark the two cysteine residues, possible involved in sulfhydryl bonding of the immunoglobulin domain. The three EGF-like repeats are boxed. The three fibronectin type III domains are underlined. The transmembrane region is given in bold face letters. The tyrosine kinase domain is indicated by shaded boxes. The RGD triplet is marked by an asterisk.

Fig. 2B. Schematic diagram of the structure of tie-2. Ig. immunoglobulin domain; EGF, EGF-like repeats; FN, fibronectin type III domains; Kinase, tyrosine kinase domain; KI, kinase insertion.

Fig. 3. Amino acid sequence comparison of tek , TEK and tie with tie-2. Amino acid residues identical to tie-2 are represented by (-) . Gaps are indicated by (.).

Fig. 4. Northern analysis of tie-2 expression in brain capillaries and total brain tissue from postnatal day 4 (P4) mice. A single transcript of approximately 4.7 kb, highly enriched in the capillary fraction was detected.

Fig. 5. tie-2 expression during brain development. Sagittal sections of adult brain (A,B) , postnatal day 4 brain (C,D,G,H) and embryonic day 12.5

1- brain (E,F) were hybridized with a tie-2 probe. Bright-field (A,C,E) and corresponding dark-field micrographs (B,D,F) Arrowheads indicate expression in capillaries and arrows indicate hybridization signals over meningeal blood vessels. Higher magnification of a capillary sprout at the dorsal surface of a postnatal day 4 telencephalon (G) . Higher magnification of a capillary in a deeper layer of the brain (H) . No other than the vascular elements of the brain were found to synthesize tie-2 mRNA. LM, leptomeninges; LV, lateral ventricle; TE, telencephalon; (A,B,C,D,E,F) bar represents 110 μm; (G,H) bar represents 10 μm;

Fig. 6. Colocalization of tie-2 expression with immunostaining for PECAM (CD31) . Adjacent section of E12.5 embryos were hybridize with a tie-2 probe (A) or stained with antibody recognizing PECAM (CD31) (B) . Arrowheads indicate expression of tie-2 in the endothelial cell layer of a medium-sized blood vessel from the head region. Bar represents 10 μm.

Fig. 7. Tie-2 expression in organs and tissues of E 12.5 embryos and colocalization with immunostaining for PECAM (CD31) . Adjacent sections of E 12.5 embryos were stained with an antibody recognizing PECAM (CD31) (A,C,E,G) or hybridized with a tie-2 probe (B,D,F,H). The expression pattern of tie-2 was found to be identical to the staining pattern fort PECAM (CD31) . TE, telencephalon; LV, lateral ventricle; CP, choroid plexus of the lateral ventricle; DA, dorsal aorta; SC, spinal cord; EN, endocardium; MY, myocardium; BR, bronchus; SO, somite; Bar represents 110 μm.

Fig. 8. Tie-2 expression at embryonic day 8.5. Transverse section of an E 8.5 embryo and adjacent yolk sac. Hybridization signals were detectable in the endocardium, dorsal aorta, cardinal vein and the mesodermal (inner layer) of the yolk sac (A,B) .

- _ Arrowheads indicate hybridization signal over the marginal cells of an advanced stage blood island (C,D) . Note the absence of hybridization signals in the neuroectoderm. CV, cardinal vein (head vein); BI, blood island; DA, dorsal aorta; EN, endocardial tissue; TE, telencephalon; YS, yolk sac. Bar, represents 50 μm.

5. DETAILED DESCRIPTION

The present invention relates to a novel receptor tyrosine kinases referred to herein as tie-2. The invention is based, in part, on the isolation of a cDNA clone encoding the tie-2 receptor tyrosine kinase from a brain capillary cDNA library. The invention also relates to novel members of the tie-2 receptor tyrosine kinase family as defined herein.

Results from Northern Blot analysis and in situ hybridization indicates that tie-2 is expressed specifically in the endothelial cell lineage. Tie-2 transcripts could be detected in endothelial cell precursors (angioblasts) and in endothelial cells of sprouting blood vessels throughout development and in all organs and tissues so far examined.

The invention relates to expression of the tie-2 receptor and/or cell lines that express the tie-2 receptor which may be used to screen for antibodies, peptides, or other ligands that act as agonists or antagonists of the tie-2 receptor. For example, anti-tie-2 antibodies may be used to inhibit tie-2 function. Alternatively, screening of peptide libraries with recombinantly expressed soluble tie-2 protein or cell lines expressing tie-2 protein may be useful for identification of therapeutic molecules that function by regulating the biological activity of tie-2.

For clarity of discussion, the invention is described in the subsections below by way of example

_rf_ for the murine tie-2 . However, the principles may be analogously applied to clone and express the tie-2 receptor of other species including humans.

5.1. THE TIE-2 CODING SEQUENCE The nucleotide coding sequence and deduced amino acid sequence of the tie-2 gene is depicted in

Figures 1A, IB, IC (SEQ. ID NO: ) and Figure 2A

(SEQ. ID NO: ) , respectively. In accordance with the invention, any nucleotide sequence which encodes the amino acid sequence of the tie-2 gene product can be used to generate recombinant molecules which direct the expression of tie-2. In additional embodiments of the invention, nucleotide sequences which selectively hybridize to the tie-2 nucleotide sequence shown in

FIG. 1 (SEQ ID NO: ) may also be used to express gene products with tie-2 activity. Hereinafter all such variants of the tie-2 nucleotide sequence will be referred to as the tie-2 DNA sequence.

In a specific embodiment described herein, the tie-2 gene was isolated by performing a polymerase chain reaction (PCR) in combination with two degenerate oligonucleotide primer pools that were designed from conserved protein regions of tyrosine kinases. As a template cDNA synthesized by reverse transcription of purified mRNA from capillaries of pooled P4-P8 mice brains was used. Gel-purified reaction products of the expected size were radiolabelled and used directly to screen a cDNA library constructed from the remainder of the capillary mRNA. Sequence analysis of the inserts from positive phages revealed that 13 of the cDNAs were derived from one mRNA species. The longest of these 13 clones, a fragment of 4640 bp, was sequenced completely. It contained a long open reading frame encoding a protein of 1123 amino acid residues.

- ? - Within the extracellular part, the FASTA program detected homologies to proteins including TAN-1 (Ellisen et al., 1991 Cell 66:649-661), Xotch (Coffman et al., 1990), La inin (Sasaki et al., 1988 J. Biol. Chem. 263:16536-16544). Delta (Vassin et al. , 1987 EMBO J. 6:3431-3440) and Perlecan (Noonan et al., 1991 J. Biol. Chem. 266:22939-22947) (around 30% sequence identity) . The structural basis for these homologies are three EGF-like repeats in the center of the extracellular portion of tie-2 (Fig. 2A) . EGF-like repeats consist of 30 to 40 amino acid residues often found in the extracellular parts of membrane-bound proteins or secreted proteins (Davies, 1990 New Biol 5:410-419). A common feature of these domains are six conserved cysteine residues, which are known to be involved in disulfide bonds. The three EGF domains in tie-2 contain two additional cysteine residues at the carboxy1-terminal end of each repeat.

The central EGF-like repeats are flanked by two different structural motifs, a single amino-terminal immunoglobulin (Ig) domain and a carboxy1-terminal triplet of fibronectin (FN) type III domains. Although the Ig domain does not exhibit clear sequence similarities to other known proteins (except for tie and TEK) , the Ig domain has the conserved features of a C2-set domain including typical residues surrounding the first cysteine residue and the canonical GxYxC found at the second cysteine residue (Williams and Barclay, 1988 Ann. Rev. Immunol. 6:381-405).

The three fibronectin type III (FN III) domains are most closely related to FN III repeats present in the protein-tyrosine phosphatases Delta (Krueger et al., 1990 EMBO J. 9:3241-3252), LAR (Streuli et al. , 1988 J. Exp. Med. 168:1553-1562) and DLAR (Streuli et al., 1989 Proc. Natl. Acad. Sci USA 86:8698-8702) and to FN III repeats of the axonal glycoprotein TAG-1 (Furley et al., 1990 Cell 61:157-170) (around 20%

- <S^> - sequence identity) . These domains have been described as units of approximately 90 amino acids containing hydrophobic residues at characteristic positions. In addition, a total of nine potential N-glycosylation sites are located in the extracellular part of tie-2.

The invention also relates to tie-2 genes iso¬ lated from other species, including humans. Members of the tie-2 family are defined herein as those receptors containing an intraceUular tyrosine kinase domain, and, in the extracellular domain, EGF-like repeats flanked by two different structural motifs, a single amino-terminal immunoglobulin (Ig) domain and a carboxyl-terminal triplet of fibronectin (FN) type III domains. Such receptors may demonstrate about 80% homology at the nucleotide level, and even 90% homology at the amino acid level in substantial stretches of DNA sequence.

To isolate the tie-2 gene from other species a bacteriophage cDNA library may be screened, under conditions of reduced stringency, using a radioactively labeled fragment of the murine tie-2 clone. Alternatively the murine tie-2 sequence can be used to design degenerate or fully degenerate oligonucleotide probes which can be used as PCR probes or to screen bacteriophage cDNA libraries. A poly- merase chain reaction (PCR) based strategy may be used to clone, for example, the human tie-2. Two pools of degenerate oUgonucleotides, corresponding to conserved motifs within the murine tie-2 may be designed to serve as primers in a PCR reaction. Conserved motifs may include the tyrosine kinase domain, the EGF-like repeats, the immunoglobulin (Ig) domain or the fibronectin (Fn) type III domains. The template for the reaction is cDNA obtained by reverse transcription of mRNA prepared from cell lines or tissue known to express human tie-2 such as blood capillaries or endothelial cells. The PCR product may

-_ be subcloned and sequenced to insure that the amplified sequences represent the tie-2 sequences.

The PCR fragment may be used to isolate a full length tie-2 cDNA clone by radioactively labeling the amplified fragment and screening a bacteriophage cDNA library. Alternatively, the labeled fragment may be used to screen a genomic library. For a review of cloning strategies which may be used, see e.g. ,

Maniatis, 1989, Molecular Cloning, A Laboratory

Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology,

(Green Publishing Associates and Wiley Interscience,

N.Y.)

Northern Blot analysis and in situ hybridization indicated that tie-2 was expressed in blood vessels during brain angiogenesis. Figure 4 demonstrates the presence of a 4.7 Mb mRNA highly enriched in the capillary fraction. A single RNA species could also be detected in organs like the brain, kidney and heart.

In situ hybridization experiments indicated that tie-2 mRNA was exclusively synthesized in the vasculature of adult brain, P4 brain, E12.5 brain E8.5 neuroectoderm (FIG. 5A-H) . In P4 brain, tie-2 expression was detected in capillaries that are about to invade the neural tissue as well as in vessels that have already reached deep layers of neuroectoderm. In addition to capillaries, eningeal blood vessels and the choroid plexus were also found to synthesize tie-2 mRNA at comparable levels (FIGS. 5, 7B) . At E12.5 the overall expression pattern was virtually identical to that observed at P4, although the density of labelled structures in E12.5 brain was reduced (Fig. 5C,0) . This observation correlates with the less extensive vascularization of the embryonic brain (Bar, 1980 Adv. Anat. Embryol Cell Biol. 59:1-62). No hybridization signals were observed in the neuroectoderm of E8.5

_/o — embryos, because at that stage this tissue is still avascular (Fig. 8) . In the adult brain, tie-2 expression was detectable, although it seemed to be reduced when compared to postnatal or embryonic stages. Hybridization signals persisted over larger vessels especially those of the meninges (Fig. 5A,B) . The synthesis of tie-2 mRNA in smaller vessels and capillaries was barely detectable.

To identify the cell type, expressing tie-2 mRNA, in situ hybridization and immunohistochemistry was performed. The pattern of tie-2 expression was compared with the staining of cells with a monoclonal antibody against the endothelial cell-specific adhesion molecule PECAM (CD31) . Figure 6 demonstrates the overlap between cells stained by the PECAM antibody and those cells labeling with a tie-2 probe. Figure 7 presents a survey on the coexpression of tie- 2 and PECAM in several organs and tissues and it is clear from the results that tie-2 is coexpressed with PECAM in several organs and tissues.

The possible role of tie-2 in early stages of vascular development was investigated by in situ hybridization studies with E8.5 sections. The probe detected tie-2 mRNA in the mesodermal component of the yolk sac (FIG. 8) . This finding is of particular interest, because it is in the mesodermal component of the yolk sac that the first signs of blood vessel development are evident. Clusters of mesenchymal cells form the so-called blood islands. At the margin of these aggregates cells, the so-called angioblasts, adopt an endothelial-like phenotype, whereas in the center cells differentiate into embryonic hemoblasts. Fig. 8 demonstrates that the peripheral angioblasts synthesize high levels of tie-2 mRNA.

Within the embryo proper, expression was seen in the anlagen of the vascular system, e.g. in the developing endocardium, the dorsal aortae and the cardinal veins (Fig. 8A,B) . It is likely that these signals stem from intraembryonic endothelial cell precursors, which are present in these structures at that stage, tie-2 expression could also be detected in the allantois and in blood vessels of the maternal decidua.

5.2. EXPRESSION OF TIE-2 RECEPTOR

AND GENERATION OF CELL LINES THAT EXPRESS TIE-2

In accordance with the invention, tie-2 nucleotide sequences which encode tie-2, peptide fragments of tie-2, tie-2 fusion proteins or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression of tie-2 protein or a functionally equivalent thereof, in appropriate host cells. Alternatively, nucleotide sequences which hybridize to portions of the tie-2 sequence may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of the tie-2 protein. Such DNA sequences include those which are capable of hybridizing to the murine tie-2 sequence under selective conditions.

Altered DNA sequences which may be used in accor¬ dance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. These alterations would in all likelihood be in regions of tie-2 that do not constitute functionally conserved regions. In contrast, alterations, such as deletions, additions or substitutions of nucleotide residues in functionally conserved tie-2 regions would be expected to result in a nonfunctional tie-2 receptor. The gene product itself may contain deletions, additions or substitutions of amino acid residues within the tie-2 sequence, which result in a silent change thus produ¬ cing a functionally equivalent tie-2. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipatic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine. The DNA sequences of the invention may be engineered in order to alter the tie- 2 coding sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g. site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc. For example, in certain expression systems such as yeast, host cells may over glycosylate the gene product. When using such expression systems it may be preferable to alter the tie-2 coding sequence to eliminate any N-linked glycosylation site.

In another embodiment of the invention, the tie-2 or a modified tie-2 sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries it may be useful to encode a chimeric tie-2 protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the tie-2 sequence and the heterologous protein sequence, so that the tie-2 may be cleaved away from the heterologous moiety.

In an alternate embodiment of the invention, the coding sequence of tie-2 could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers, et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers,

1980, Tetrahedron Letters 21:719; and Chow and Kempe,

1981, Nuc. Acids Res. 9 (12) :2807-2817. Alternatively, the protein itself could be produced using chemical methods to synthesize the tie-2 amino acid sequence in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high perform¬ ance liquid chromatography. (e.g. , see Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60) . The composi¬ tion of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g. , the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49.

In order to express a biologically active tie-2, the nucleotide sequence coding for tie-2, or a func¬ tional equivalent, is inserted into an appropriate expression vector, i.e.. a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. The tie-2 gene products as well as host cells or cell lines transfected or transformed with recombinant tie-2 expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e. , monoclonal or polyclonal)

-- - that bind to the receptor, including those that competitively inhibit binding of tie-2 ligand and "neutralize" activity of tie-2 and the screening and selection of drugs that act via the tie-2 receptor; etc.

5.2.1. EXPRESSION SYSTEMS Methods which are well known to those skilled in the art can be used to construct expression vectors containing the tie-2 coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and jLn vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to express the tie-2 coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the tie-2 coding sequence; yeast transformed with recombinant yeast expression vectors containing the tie-2 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g.. baculovirus) containing the tie-2 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g.. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g.. Ti plasmid) containing the tie-2 coding sequence; or animal cell systems. The expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g.. heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g. , the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g.. metallothionein promoter) or from mammalian viruses (e.g.. the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of the tie-2 DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.

In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the tie-2 expressed. For example, when large quantities of tie-2 are to be produced for the generation of antibodies or to screen peptide libraries, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited to the E__. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2.:1791), in which the tie-2 coding sequence may be ligated into the vector in frame with the lacZ coding region so

- ^_ that a hybrid tie-2/lacZ protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) . In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.

In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II.

In cases where plant expression vectors are used, the expression of the tie-2 coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.

-^•? - 3:1671-1680; Broglie et al. , 1984, Science 224:838- 843); or heat shock promoters, e.g.. soybean hspl7.5-E or hspl7.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transfor¬ mation, microinjec ion, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421- 463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

An alternative expression system which could be used to express tie-2 is an insect system. In one such system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The tie-2 coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) . Successful insertion of the tie-2 coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e. , virus lacking the proteinaceous coat coded for by the polyhedrin gene) . These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g.. see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Patent No. 4,215,051).

In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the tie-2 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g. , the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by jLn vitro or _Ln vivo recombination. Inser¬ tion in a non-essential region of the viral genome (e.g.. region El or E3) will result in a recombinant virus that is viable and capable of expressing tie-2 in infected hosts. (E.g. - See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659). Alterna¬ tively, the vaccinia 7.5K promoter may be used. (See, e.g.. Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

Specific initiation signals may also be required for efficient translation of inserted tie-2 coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire tie-2 gene, including its own initiation codon and adjacent sequences, is inserted into the appro¬ priate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the tie-2 coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the tie-2 coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544) .

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g.. glycosylation) and processing (e.g. , cleavage)

- 'Jc\~

" of protein products may be important for the function of the protein. The presence of nine consensus N- glycosylation sites in the tie-2 extracellular domain indicate that proper modification may be important for tie-2 function. Different host cells have charac¬ teristic and specific mechanisms for the post-transla- tional processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.

For long-term, high-yield production of recombi¬ nant proteins, stable expression is preferred. For example, cell lines which stably express the tie-2 may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the tie-2 DNA controlled by appropriate expression control elements (e.g.. promoter, enhancer, sequences, transcription termina¬ tors, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the tie-2 on the cell surface. Such engineered cell lines are

_ 2.o - particularly useful in screening for drugs that affect tie-2.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be employed in tk", hgprt" or aprt" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al. , 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981) , Proc. Natl. Acad. Sci. USA 78:2072); neo , which confers resistance to the aminoglycoside G-418 (Colberre- Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L. , 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS THAT EXPRESS THE TJ.E-2

The host cells which contain the coding sequence and which express the biologically active gene product

- 2. 1 may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expres¬ sion of tie-2 mRNA transcripts in the host cell; and

(d) detection of the gene product as measured by immunoassay or by its biological activity.

In the first approach, the presence of the tie-2 coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the tie-2 coding sequence, respectively, or portions or derivatives thereof.

In the second approach, the recombinant expres¬ sion vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g. , thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the tie-2 coding sequence is inserted within a marker gene sequence of the vector, recombinants containing the tie-2 coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the tie-2 sequence under the control of the same or different promoter used to control the expression of the tie-2 coding sequence. Expression of the marker in response to induction or selection indicates expression of the tie-2 coding sequence.

In the third approach, transcriptional activity for the tie-2 coding region can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to the tie-2 coding sequence or particular portions thereof. Alternatively, total nucleic acids

Z2. - of the host cell_.may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of the tie-2 protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.

5.3. USES OF THE TIE-2 RECEPTOR AND ENGINEERED CELL LINES

The uses of the tie-2 receptor and engineered cell lines, described in the subsections below, may be employed equally well for the tie-2 family of receptor tyrosine kinases.

In an embodiment of the invention, engineered cell lines which express the entire tie-2 coding region or its ligand binding domain may be utilized to screen and identify the natural ligand and/or ligand antagonists as well as agonists. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways.

5.3.1 IDENTIFICATION OF TIE-2 LIGAND USING ENGINEERED CELL LINES

Engineered cell lines that express the tie-2 receptor, may be advantageously used to identify and characterize the natural tie-2 ligand(s) . For example, a genomic or cDNA library may be transfected into the engineered cell lines. Any cell that expresses and secretes the tie-2 ligand, due to the transfer of a DNA clone capable of encoding the tie-2 ligand, will stimulate the biological activity of the recombinantly expressed the tie-2 receptor. The resulting autocrine loop will lead to preferential proliferation of cells expressing tie-2 ligand and the

-.2.3 _ resulting cell colonies may be used to isolate the DNA encoding the tie-2 ligand.

The engineered cell lines may also be used to assay tissue or cell extracts for their ability to activate tie-2 receptor tyrosine kinase activity. Activation of tie-2 tyrosine kinase activity may be assayed using a variety of methods. For example, in cells overexpressing the tie-2 receptor, overal incorporation of labeled P0₄ into the cell may be measured after contacting cells with extracts. Alternatively, the tie-2 protein may be immunoprecipitated following stimulation with extracts, using anti-tie-2 antibodies, followed by Western blot analysis using anti-tyrosine phosphate antibodies to determine whether the immunoprecipitated tie-2 has been tyrosine phosphorylated.

Once an extract is identified which contains tie-2 ligand, various procedures and techniques known in the art which include but are not limited to chromatography (e.g., reverse phase liquid, gell permeation, liquid exchange, ion exchange, size exclusion, affinity chromatography) , centrifugation, electrophoretic procedures, differential solubility, or other standard techniques may be used to purify tie-2 ligand.

In a preferred embodiment, affinity chromatography using recombinantly expressed tie-2 covalently attached to a column matrix may be used to purify tie-2 ligand. Alternatively, a recombinantly expressed hybrid protein comprised of the tie-2 extracellular domain fused to the immunoglobulin protein A binding domain may be used to purify tie-2 ligand. The fusion protein may be used to prepare a column matrix over which cell extracts may be added, or, the fusion protein may be used to immunoprecipitate the tie-2 ligand from cell extracts.

- ².4- Once purified, the tie-2 ligand may be subjected to microsequencing, using techniques routinely used by those skilled in the art to determine the amino acid sequence of a protein. If the tie-2 ligand molecule is blocked at the amino terminus, the protein may be chemically cleaved or partially enzymatically digested to yield peptide fragments that may be purified and sequenced.

A mixture of degenerate oligonucleotide probes may be designed using the information derived from the protein sequencing of the purified tie-2 ligand. The oUgonucleotides may be labeled and used directly to screen a cDNA library for clones containing inserts with sequence homology to the oligonucleotide sequences. Alternatively, the oUgonucleotides may be used as primers in a polymerase chain reaction. The amplified DNA fragment may be labeled and used to screen a library for isolation of full length clones.

5.3.2. SCREENING OF PEPTIDE LIBRARY WITH TIE-2 PROTEIN OR ENGINEERED CELL LINES

Random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to the ligand binding site of a given receptor or other functional domains of a receptor such as kinase domains (Lam, K.S. et al., 1991, Nature 354: 82-84). The screening of peptide libraries may have therapeutic value in the discovery of pharmaceutical agents that act to inhibit the biological activity of receptors through their interactions with the given receptor.

Identification of molecules that are able to bind to the tie-2 may be accomplished by screening a peptide library with recombinant soluble tie-2 protein. Methods for expression and purification of

- 2. -3 _ tie-2 are described in Section 5.2.1 and may be used to express recombinant full length tie-2 or fragments of tie-2 depending on the functional domains of interest. For example, the kinase and extracellular ligand binding domains of tie-2 may be separately expressed and used to screen peptide libraries.

To identify and isolate the peptide/solid phase support that interacts and forms a complex with tie-2, it is necessary to label or "tag" the tie-2 molecule. The tie-2 protein may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase or to other reagents such as fluorescent labels which may include fluorescein isothyiocynate (FITC) , phycoerythrin (PE) or rhodamine. Conjugation of any given label, to tie-2, may be performed using techniques that are routine in the art. Alternatively, tie-2 expression vectors may be engineered to express a chimeric tie-2 protein containing an epitope for which a commercially available antibody exist. The epitope specific antibody may be tagged using methods well known in the art including labeling with enzymes, fluorescent dyes or colored or magnetic beads.

To screen a peptide library the "tagged" tie-2 conjugate is incubated with the random peptide library for 30 minutes to one hour at 22°C to allow complex formation between tie-2 and peptide species within the library. The library is then washed to remove any unbound tie-2 protein. If tie-2 has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing substrates for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3 ' ,4,4"-diamnobenzidine (DAB), respectively. After incubating for several minutes, the peptide/solid phase-tie-2 complex changes color, and can be easily identified and isolated physically

- a - under a dissecting microscope with a micromanipulator. If a fluorescent tagged tie-2 molecule has been used, complexes may be isolated by fluorescent activated sorting. If a chimeric tie-2 protein expressing a heterologous epitope has been used, detection of the peptide/tie-2 complex may be accomplished by using a labeled epitope specific antibody. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing.

In addition to using soluble tie-2 molecules, in another embodiment, it is possible to detect peptides that bind to cell surface receptors using intact cells. The use of intact cells is preferred for use with receptors that are multi-subunits or labile or with receptors that require the lipid domain of the cell membrane to be functional. Methods for generating cell lines expressing tie-2 are described in Sections 5.2.1. and 5.2.2. The cells used in this technique may be either live or fixed cells. The cells will be incubated with the random peptide library and will bind to certain peptides in the library to form a "rosette" between the target cells and the relevant solid phase support/peptide. The rosette can thereafter be isolated by differential centrifugation or removed physically under a dissecting microscope.

As an alternative to whole cell assays for membrane bound receptors or receptors that require the lipid domain of the cell membrane to be functional, the receptor molecules can be reconstituted into liposomes where label or "tag" can be attached.

5.3.3. ANTIBODY PRODUCTION AND SCREENING

Various procedures known in the art may be used for the production of antibodies to epitopes of the recombinantly produced tie-2 receptor. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by an Fab expression library. Neutralizing antibodies i.e.. those which compete for the ligand binding site of the receptor are especially preferred for diagnostics and therapeutics.

Monoclonal antibodies that bind tie-2 may be radioactively labeled allowing one to follow their location and distribution in the body after injection. Radioactivity tagged antibodies may be used as a non- invasive diagnostic tool for imaging de novo cells expressing tie-2.

Immunotoxins may also be designed which target cytotoxic agents to specific sites in the body. For example, high affinity tie-2 specific monoclonal antibodies may be covalently complexed to bacterial or plant toxins, such as diphtheria toxin, ab in or ricin. A general method of preparation of antibody/hybrid molecules may involve use of thiol- crosslinking reagents such as SPDP, which attack the primary amino groups on the antibody and by disulfide exchange, attach the toxin to the antibody. The hybrid antibodies may be used to specifically eliminate tie-2 expressing cells.

For the production of antibodies, various host animals may be immunized by injection with the tie-2 protein including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacteriu parvum.

- z<_? - Monoclonal antibodies to tie-2 may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the EBV-hybridoma tech¬ nique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce tie-2-specific single chain antibodies.

Antibody fragments which contain specific binding sites of tie-2 may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab*)₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alterna¬ tively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to tie-2.

- 2. ° 5.4. USES OF TIE-2 CODING SEQUENCE The tie-2 coding sequence may be used for diagnostic purposes for detection of tie-2 expression. Included in the scope of the invention are oligoribonucleotide sequences, that include antisense RNA and DNA molecules and ribozymes that function to inhibit translation of tie-2. In addition, mutated forms of tie-2, having a dominant negative effect, may be expressed in targeted cell populations to inhibit the activity of endogenously expressed tie-2.

5.4.1. USE OF TIE-2 CODING SEQUENCE

IN DIAGNOSTICS AND THERAPEUTICS

The tie-2 DNA may have a number of uses for the diagnosis of diseases resulting from aberrant expression of tie-2. For example, the tie-2 DNA sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of tie-2 expression; e.g.. Southern or Northern analysis, including in situ hybridization assays.

Also within the scope of the invention are oligo¬ ribonucleotide sequences, that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of tie-2 mRNA. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g.. between -10 and +10 regions of the tie-2 nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mecha¬ nism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complemen¬ tary target RNA, followed by a endonucleolytic cleav¬ age. Within the scope of the invention are engineered

- 3θ - hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of tie-2 RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligo¬ nucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complemen¬ tary oUgonucleotides, using ribonuclease protection assays.

Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a means of increasing intraceUular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phospho- diesterase linkages within the oligodeoxyribonucleo- tide backbone.

5.4.2. USE OF DOMINANT NEGATIVE

TIE-2 MUTANTS IN GENE THERAPY

Receptor dimerization induced by ligands, is thought to provide an allosteric regulatory signal that functions to couple ligand binding to stimulation of kinase activity. Defective receptors can function as dominant negative mutations by suppressing the activation and response of normal receptors by formation of unproductive heterodimers. Therefore, defective receptors can be engineered into recombinant viral vectors and, used in gene therapy in individuals that inappropriately express tie-2.

In an embodiment of the invention, mutant forms of the tie-2 molecule having a dominant negative effect may be identified by expression in selected cells. Deletion or missense mutants of tie-2 that retain the ability to form dimers with wild type tie-2 protein but cannot function in signal transduction may be used to inhibit the biological activity of the endogenous wild type tie-2. For example, the cytoplasmic kinase domain of tie-2 may be deleted resulting in a truncated tie-2 molecule that is still able to undergo dimerization with endogenous wild type receptors but unable to transduce a signal.

Recombinant viruses may be engineered to express dominant negative forms of tie-2 which may be used to inhibit the activity of the wild type endogenous tie-2. These viruses may be used therapeutically for treatment of diseases resulting from aberrant expression or activity of tie-2, such as cancers. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of recombinant tie-2 into the targeted cell population. Methods which are well known to those skilled in the art can be used to construct > those recombinant viral vectors containing tie-2 coding sequence. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning

A Laboratory Manual, Cold Spring Harbor Laboratory,

N.Y. and Ausubel et al., 1989, Current Protocols in

Molecular Biology, Greene Publishing Associates and

Wiley Interscience, N.Y. Alternatively, recombinant tie-2 molecules can be reconstituted into liposomes for delivery to target cells.

6. EXAMPLES: CLONING AND CHARACTER¬ IZATION OF THE TIE-2 RECEPTOR TYROSINE KINASE

6.1. MATERIALS AND METHODS

6.1.1. ANIMALS AND TISSUES Balb/c mice were mated overnight and the morning of vaginal plug detection was defined as 0.5 day of gestation. Organs were removed, frozen immediately in liquid nitrogen and stored at -80°C. Capillary 5 fragments from pooled P4-P8 mice brains were prepared according to Risau et al. (1990 J. Cell Biol. 110:1757-1766). For in situ hybridization and immunohistochemistry, whole embryos or organs were fixed for 12 hours in freshly prepared 4% 0 paraformaldehyde in PBS at 4°C, rinsed for 24-48 hours in 0.5 M sucrose at PBS at 4°C, embedded in TissueTek (Miles) and stored frozen at -80°C.

5 6.1.2- RNA EXTRACTION AND ANALYSIS Total cytoplasmic RNA was isolated according to the method of Chomczynksi and Sacchi (1987 Anal. Biochem. 162:156-159). Enrichment for poly(A) ⁺ containing fractions was achieved by oligo(dT) chromatography using push columns (Stratagene) . Aliquots of poly(A)⁺ RNA were electrophoresed in agarose gels containing 0.66 M formaldehyde and transferred to Zeta-Probe membrane (Bio Rad) in 2Ox SSPE. Hybridizations were performed overnight in 0.5 M sodium phosphate butter, 5% SDS, 1% BSA, pH 7.5 at 68°C with lxlO⁶ cts/minute/ml of probe, which had been labelled with ³²P-dCTP according to the protocol of a random-primed DNA-labeling kit (Bohringer Mannheim) . Membranes were washed under high-stringency conditions at 68°C in 0.1XSSPE. 0.5% SDS and autoradiographed at -80°C on Fuji films. Poly(A) ⁺ RNA from brain capillary fragments was isolated using a QuickPrep Micro mRNA purification kit from Pharmacia.

6.1.3. PCR AND cDNA CLONING

Capillary poly(A) ⁺ RNA was reverse transcribed and aliquots of the cDNA were used as templates in PCR reactions. The following oUgonucleotides were used as primers: 5 ^»-CAC/TCGIGAC/TC/TTIGCIGCIA/CG-3 ^• ,5•-

AC/TICCIAA/CIC/GA/TCCAIACA/CTC-3¹ (I stands for inosine) in addition to the following premiss described by Wilks et al. (1989, DPNAS Proc. Natl.

Acad. Sci. USA 86:1603-1607):

5 ^•-CGGATCCACAGNGACCT-3 • and C TT

3 '-CTGCAGACCAGGAAACCTTAAGG-5 ^• A C T

The amplification products were separated in acrylamide gels. Fragments of the expected size were purified, labeled and used as probes to screen a random hexanucleoride-primed cDNA library constructed

- 3-9 _ with a Time Saver cDNA Synthesis Kit (Pharmacia) . cDNA fragments were subcloned into Bluescript vectors (Stratagene) . Sequencing was done by using nested oligonucleotide primers in combination with a 373 DNA Sequencer (Applied Biosystems) and with the Convention Sequenase System (USB) .

6.1.4. IN SITU HYBRIDIZATION Preparation of tissue sections and in situ hybridizations with single-stranded DNA probes or single-stranded RNA probes was performed as described by Schnϋrch and Risau (1991, Development 111:1143- 1154) . The probe for all hybridization experiments was derived from a 1179 bp DNA fragment encoding a portion of the putative tie-2 protein from amino acid 419 to amino acid 812.

6.1.5. IMMUNOHISTOCHEMISTRY Embedded embryos and whole organs were sectioned on a Leitz cryostat 8 μm sections were mounted on organosilane-treated slides, dried overnight under vacuum and stored desiccated at -80°C. Sections were brought to room temperature, rehydrated in PBS for 5 minutes and incubated in 0.1% H₂0₂ in methanol for 15 minutes. After washing three times in PBS for 5 minutes each, nonspecific antibody binding was blocked by application of 20% normal goat serum in PBS for 20 minutes. Sections were washed, incubated with a rat monoclonal antibody against mouse PECAM for one hour, washed again and then incubated with a viotinylated gout anti-rat IgG (Dianova) for one hours. Color development was performed with a Vectastain ABC Kit (Vector Laboratories) according to the vendor's protocol. Sections were slightly counterstained with toluene blue, dehydrated and mounted.

_ 3 *5 6. 2 . RESULTS

6.2.1. CDNA CLONING AND STRUCTURE OF THE TIE-2 PROTEIN

The approach for the isolation of receptor tyrosine kinases expressed in endothelial cells of sprouting blood vessels involved the following steps. First, mRNA was purified from capillaries of pooled

P4-P8 mice brains. A portion of the mRNA was reverse transcribed and used in PCR reactions with degenerate primers deduced from conserved protein regions of tyrosine kinases. Gel-purified reaction products of the expected size were radiolabelled and used directly as hybridization probes to screen a cDNA library constructed from the remainder of the capillary mRNA. Partial sequencing of the inserts from positive phages revealed that 13 of the cDNAs were derived from one mRNA species. The longest of these 13 cDNAs, a fragment of 4640 bp, was sequenced completely (FIG. 1A, IB, IC) . It contained a long open reading frame encoding a protein of 1123 amino acid residues (FIG. 2A) . This deduced polypeptide has all features of a receptor tyrosine kinase: an amino terminal signal sequence followed by a long extracellular domain, a single hydrophobic transmembrane region and a cytoplasmic portion that contains a tyrosine kinase domain (Fig. 2B) . A survey for homologous proteins revealed that the predicted protein is most closely related to the two recently identified tyrosine kinases tie and tek (Partanen et al., 1992 Mol. Cell. Biol. 12:1698-1707; Dumont et al., 1992 Oncogene 7:1471-1480). With the exception of two residues, the protein sequence published for tek is identical to the intraceUular part of our polypeptide, from position 823 to the carboxyl-terminal residue 1123. Both an extracellular domain and a transmembrane region are missing in the tek polypeptide (Fig. 3) . It is therefore possible

- - - that tek represents a partial sequence or that the mRNA encoding tek is the result of an alternative splice event, which results in the production of a cytoplasmic tyrosine kinase. Comparison of our protein with tie reveals a high degree of similarity especially in the cytoplasmic part (76% sequence identify) . In the extracellular domain and the transmembrane region, the similarity is less pronounced (33% and 37% respectively; Fig. 3) . Ziegler et al. (1993 Oncogene 8:663-670) have also reported the cloning of a cDNA whose translation product is a receptor tyrosine kinase with an overall similarity of more than 90% when compared to tie-2 (Fig. 3) . It is therefore likely that this protein, which they called TEK, is the human homolog of tie-2. The intraceUular part of tie-2 is also related to the product of the human ret protooncogene and to FGF receptors (Takahashi et al., 1989 Oncogene 4:805-806; Partanen et al., 1991 EMBO J. 10:1347-1354; Stark et al., 1991 Development 113:641-651). The similarity to both is about 43%. The intraceUular part can be divided into three characteristic regions: the juxtamembrane sequence, the catalytic domain and the cytoplasmic tail. The kinase domain, which is split by an 14 amino acid insertion contains the GxGxxG consensus sequence that is part of the ATP binding site. Typical tyrosine kinase motifs like HRDLAARN and DFGL are present.

Within the extracellular part, the FASTA program detected homologies to proteins including TAN-1 (Ellisen et al., 1991 Cell 66:649-661), Xotch (Coffman et al., 1990 Science 249:1438-1440), Laminin (Sasaki et al., 1988 J. Biol. Chem. 263:16536-16544). Delta (Vassin et al., 1987 EMBO J. 6:3431-3440) and Perlecan (Noonan et al., 1991 J. Biol. Chem. 266:22939-22947) (around 30% sequence identity) . The structural basis for these homologies are three EGF-like repeats in the

— 3

' center of the extracellular portion of tie-2 (Fig. 2A and 2B) . EGF-like repeats consist of 30 to 40 amino acid residues often found in the extracellular parts of membrane-bound proteins or secreted proteins

(Davies, 1990 New Biol. 5:410-419) . A common feature of these domains are six conserved cysteine residues, which are known to be involved in disulfide bonds.

The three EGF domains in tie-2 contain two additional cysteine residues at the carboxyl-terminal end of each repeat.

The central EGF-like repeats are flanked by two different structural motifs, a single amino-terminal immunoglobulin (Ig) domain and a carboxyl-terminal triplet of fibronectin (FN) type III domains. Although the Ig domain does not exhibit clear sequence similarities to other known proteins (except for tie and TEK) , the Ig domain has the conserved features of a C2-set domain including typical residues surrounding the first cysteine reside and the canonical GxYxC found at the second cysteine residue (Williams and Barclay, 1988 Ann. Rev. Immunol. 6:381-405).

The three fibronectin type III (FN III) domains are most closely related to FN III repeats present in the protein-tyrosine phosphatases Delta (Krueger et al., 1990 EMBO J. 9:3241-3252), LAR (Streuli et al., 1988 J. Exp. Med. 168:1553-1562) and DLAR (Streuli et al., 1989) and to FN III repeats of the axonal glycoprotein TAG-1 (Furley et al., 1990 Cell 61:157- 170) (around 20% sequence identity) . These domains have been described as units of approximately 90 amino acids containing hydrophobic residues at characteristic positions. In addition, a total of nine potential N-glycosylation sites are located in the extracellular part of tie-2.

_ 3 & - 6.2.2. TIE-2 EXPRESSION IN BRAIN CAPILLARIES

The amount of tie-2 in mRNA from P4-P8 brain capillaries was compared with total P4 brain by Northern analysis. To avoid artifactual results, the part of the cDNA that encodes the least conserved regions of the protein, namely the three FN III domains, the transmembrane region and the juxtamembrane portion was used. Fig. 4 demonstrates the presence of a 4.7 kd mRNA highly enriched in the capillary fraction. RNA expression was also detected in organs like brain, kidney and heart.

6.2.3. TIE-2 EXPRESSION DURING BRAIN DEVELOPMENT

Investigation of the spatial and temporal expression profile of tie-2 during brain development was performed by in situ hybridization. When analyzed, it became evident that tie-2 mRNA is exclusively synthesized in the vasculature of (Fig. 5A-H) adult brain, P4 brain, E12.5 brain and E8.5 neuroectoderm. No other brain components were labelled. In P4 brain, tie-2 expression was detected in capillaries that are about to" invade the neural tissue as well as in vessels that have already reached deeper layers of the neuroectoderm. High magnifications in Fig. 5G,H clearly show the high concentration of silver grains over the vascular perikarya. In addition to capillaries, meningeal blood vessels and the choroid plexi were also found to synthesize tie-2 mRNA at comparable levels (Figs 5, 7B) . At 312.5 the overall expression pattern was virtually identical to that observed at P4, although the density of labelled structures in E12.5 brain was reduced (Fig. 5C,D) . This observation correlates with the less extensive vascularization of the embryonic brain (Bar, 1980 Adv. Anat. Embryol. Cell Biol 59:1-

— 3θ\- 62) . No hybridization signals were observed in the neuroectoderm of E8.5 embryos, because at that stage this tissue is still avascular (Fig. 8) . In the adult brain, tie-2 expression was detectable, although it seemed to be reduced when compared to postnatal or embryonic stages. Hybridization signals persisted over larger vessels especially those of the meninges (Fig. 5A,B). The synthesis of tie-2 mRNA in smaller vessels and capillaries was barely detectable.

6.2.4. ENDOTHELIAL CELL-SPECIFIC EXPRESSION OF TIE-2

To identify the cellular source of tie-2 mRNA, in situ hybridization and immunohistochemistry on adjacent sections were performed. The pattern of tie-2 hybridization signals was compared with the immunohistochemical staining of a monoclonal antibody against the endothelial cell-specific adhesion molecule PECAM (CD31) (Newman et al., 1990, Science 247:1219-1222). Fig. 6 shows a representative example of one such experiment. The antibody stains the continuous layer of endothelial cells surrounding the lumen of a medium-sized blood vessel in the head region of an E12.5 embryo. On the adjacent section, the tie-2-specific probe labels the vessel in an identical way. These results, together with the northern hybridization signal detected in RNA from a capillary fraction highly enriched for endothelial cells, provide strong evidence for endotheliu - specific expression of tie-2 and PECAM in several organs and tissues. It is clear that tie-2 mRNA is present in endothelial cells all over the body. Strong hybridization signals were associated with the heart endocardium as well with the myocardial blood vessels (Fig. 7E,F) . The same holds true for the endothelium of the dorsal aorta (Fig. 7C,D) , the intersomitic vasculature, the vessels surrounding the

_ -vo — lung bronchia and the capillaries perforating the spinal cord (Fig. 7H,D). In summary, tie-2 gene expression seems to be a general feature of endothelial cells.

6.2.5. TIE-2 EXPRESSION DURING

EARLY STAGES OF DEVELOPMENT

The possible role of tie-2 in early stages of vascular development was investigated by in situ hybridization studies with E8.5 sections. The probe detected tie-2 mRNA in the mesodermal component of the yolk sac (Fig. 8) . This finding is of particular interest, because it is in the mesodermal component of the yolk sac that the first signs of blood vessel development are evident. Clusters of mesenchymal cells form the so-called blood islands. At the margin of these aggregates cells, the so-called angioblasts, adopt an endothelial-like phenotype, whereas in the center cells differentiate into embryonic hemoblasts. Fig. 8 demonstrates that the peripheral angioblasts synthesize high levels of tie-2 mRNA.

Within the embryo proper, expression was seen in the anlagen of the vascular system, e.g. in the developing endocardium, the dorsal aortae and the cardinal veins (Fig. 8A,B) . It is likely that these signals stem from intraembryonic endothelial cell precursors, which are present in these structures at that stage, tie-2 expression could also be detected in the allantois and in blood vessels of the maternal decidua.

- / — SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Risau, Werner

Schnurch, Harald

(ii) TITLE OF INVENTION: TIE-2 A NOVEL TYROSINE RECEPTOR KINASE

(iii) NUMBER OF SEQUENCES: 5

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pennie & Edmonds

(B) STREET: 1155 Avenue of the Americas

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: USA

(F) ZIP: 10036-2711

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/152,552

(B) FILING DATE': 12-NOV-1993

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Coruzzi, Laura A.

(B) REGISTRATION NUMBER: 30,742

(C) REFERENCE/DOCKET NUMBER: 7683-059

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 790-9090

(B) TELEFAX: (212) 869-9741

(C) TELEX: 66141 PENNIE

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4640 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 341..3712

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AGCAGGAGCC GGAGCAGGAG CAGAAGATAA GCCTTGGATG AAGGGCAAGA TGGATAGGGC 60

TCGCTCTGCC CCAAGCCCTG CTGATACCAA GTGCCTTTAA GATACAGCCT TTCCCATCCT 120

AATCTGCAAA GGAAACAGGA AAAAGGAACT TAACCCTCCC TGTGCTCAGA CAGAAATGAG 180

- 4 * ACTGTTACCG CCTGCTTCTG TGGTGTTTCT CCTTGCCGCC AACTTGTAAA CAAGAGCGAG 240

TGGACCATGC GAGCGGGAAG TCGCAAAGTT GTGAGTTGTT GAAAGCTTCC CAGGGACTCA 300

TGCTCATCTG TGGACGCTGG ATGGGGAGAT CTGGGGAAGT ATGGACTCTT TAGCCGGCTT 360

AGTTCTCTGT GGAGTCAGCT TGCTCCTTTA TGGAGTAGTA GAAGGTGCCA TGGACCTGAT 420

CTTGATCAAT TCCCTACCTC TTGTGTCTGA TGCCGAAACA TCCCTCACCT GCATTGCCTC 480

TGGGTGGCAC CCCCATGAGC CCATCACCAT AGGAAGGGAC TTTGAAGCCT TAATGAACCA 540

GCACCAAGAT CCACTGGAGG TTACTCAAGA TGTGACCAGA GAATGGGCGA AAAAAGTTGT 600

TTGGAAGAGA GAAAAGGCCA GTAAGATTAA TGGTGCTTAT TTCTGTGAAG GTCGAGTTCG 660

AGGACAGGCT ATAAGGATAC GGACCATGAA GATGCGTCAA CAAGCGTCCT TCCTACCTGC 720

TACTTTAACT ATGACCGTGG ACAGGGGAGA TAATGTGAAC ATATCTTTCA AAAAGGTGTT 780

AATTAAAGAA GAAGATGCAG TGATTTACAA AAATGGCTCC TTCATCCACT CAGTGCCCCG 840

GCATGAAGTA CCTGATATTT TAGAAGTTCA CTTGCCGCAT GCTCAGCCCC AGGATGCTGG 900

TGTGTACTCG GCCAGGTACA TAGGAGGAAA CCTGTTCACC TCAGCCTTCA CCAGGCTGAT 960

TGTTCGGAGA TGTGAAGCTC AGAAGTGGGG GCCCGACTGT AGCCGTCCTT GTACTACTTG 1020

CAAGAACAAT GGAGTCTGCC ATGAAGATAC CGGGGAATGC ATTTGCCCTC CTGGGTTTAT 1080

GGGGAGAACA TGTGAGAAAG CTTGTGAGCC GCACACATTT GGCAGGACCT GTAAAGAAAG 1140

GTGTAGTGGA CCAGAAGGAT GCAAGTCTTA TGTGTTCTGT CTCCCAGACC CTTACGGGTG 1200

TTCCTGTGCC ACAGGCTGGA GGGGGTTGCA GTGCAATGAA GCATGCCCAT CTGGTTACTA 1260

CGGACCAGAC TGTAAGCTCA GGTGCCACTG TACCAATGAA GAGATATGTG ATCGGTTCCA 1320

AGGATGCCTC TGCTCTCAAG GATGGCAAGG GCTGCAGTGT GAGAAAGAAG GCAGGCCAAG 1380

GATGACTCCA CAGATAGAGG ATTTGCCAGA TCACATTGAA GTAAACAGTG GAAAATTTAA 1440

CCCCATCTGC AAAGCCTCTG GGTGGCCACT ACCTACTAGT GAAGAAATGA CCCTAGTGAA 1500

GCCAGATGGG ACAGTGCTCC AACCAAATGA CTTCAACTAT ACAGATCGTT TCTCAGTGGC 1560

CATATTCACT GTCAACCGAG TCTTACCTCC TGACTCAGGA GTCTGGGTCT GCAGTGTGAA 1620

CACAGTGGCT GGGATGGTGG AAAAGCCTTT CAACATTTCC GTCAAAGTCC TTCCAGAGCC 1680

CCTGCACGCC CCAAATGTGA TTGACACTGG ACATAACTTT GCTATCATCA ATATCAGCTC 1740

TGAGCCTTAC TTTGGGGATG GACCCATCAA ATCCAAGAAG CTTTTCTATA AACCTGTCAA 1800

TCAGGCCTGG AAATACATTG AAGTGACGAA TGAGATTTTC ACTCTCAACT ACTTGGAGCC 1860

GCGGACTGAC TACGAGCTGT GTGTCCAGCT GGCCCGTCCT GGAGAGGGTG GAGAAGGGCA 1920

TCCTGGGCCT GTGAGACGAT TTACAACAGC GTCTATCGGA CTCCCTCCTC CAAGAGGTCT 1980

CAGTCTCCTG CCAAAAAGCC AGACAGCTCT AAATTTGACT TGGCAACCGA TATTTACAAA 2040

CTCAGAAGAT GAATTTTATG TGGAAGTCGA GAGGCGATCC CTGCAAACAA CAAGTGATCA 2100

GCAGAACATC AAAGTGCCTG GGAACCTGAC CTCGGTGCTA CTGAGCAACT TAGTCCCCAG 2160

GGAGCAGTAC ACAGTCCGAG CTAGAGTCAA CACCAAGGCG CAGGGGGAGT GGAGTGAAGA 2220

- ^ -3 _ ACTCAGGGCC TGGACCCTTA GTGACATTCT CCCTCCTCAA CCAGAAAACA TCAAGATCTC 2280

CAACATCACT GACTCCACAG CTATGGTTTC TTGGACAATA GTGGATGGCT ATTCGATTTC 2340

TTCCATCATC ATCCGGTATA AGGTTCAGGG CAAAAATGAA GACCAGCACA TTGATGTGAA 2400

GATCAAGAAT GCTACCGTTA CTCAGTACCA GCTCAAGGGC CTAGAGCCAG AGACTACATA 2460

CCATGTGGAT ATTTTTGCTG AGAACAACAT AGGATCAAGC AACCCAGCCT TTTCTCATGA 2520

ACTGAGGACG CTTCCACATT CCCCAGCCTC TGCAGACCTC GGAGGGGGAA AGATGCTACT 2580

CATAGCCATC CTTGGGTCGG CTGGAATGAC TTGCATCACC GTGCTGTTGG CGTTTCTGAT 2640

TATGTTGCAA CTGAAGAGAG CAAATGTCCA AAGGAGAATG GCTCAGGCAT TCCAGAACGT 2700

GAGAGAAGAA CCAGCTGTGC AGTTTAACTC AGGAACTCTG GCCCTTAACA GGAAGGCCAA 2760

AAACAATCCG GATCCCACAA TTTATCCTGT GCTTGACTGG AATGACATCA AGTTTCAAGA 2820

CGTGATCGGA GAGGGCAACT TTGGCCAGGT TCTGAAGGCA CGCATCAAGA AGGATGGGTT 2880

ACGGATGGAT GCCGCCATCA AGAGGATGAA AGAGTATGCC TCCAAAGATG ATCACAGGGA 2940

CTTCGCAGGA GAACTGGAGG TTCTTTGTAA ACTTGGACAC CATCCAAACA TCATCAATCT 3000

CTTGGGAGCA TGTGAACACC GAGGCTATTT GTACCTAGCT ATTGAGTATG CCCCGCATGG 3060

AAACCTCCTG GACTTCCTGC GTAAGAGCAG AGTGCTAGAG ACAGACCCTG CTTTTGCCAT 3120

CGCCAACAGT ACAGCTTCCA CACTGTCCTC CCAACAGCTT CTTCATTTTG CTGCAGATGT 3180

GGCCCGGGGG ATGGACTACT TGAGCCAGAA ACAGTTTATC CACAGGGACC TGGCTGCCAG 3240

AAACATTTTA GTTGGTGAAA ACTACATAGC CAAAATAGCA GATTTTGGAT TGTCACGAGG 3300

TCAAGAAGTG TATGTGAAAA AGACAATGGG AAGGCTCCCA GTGCGTTGGA TGGCAATCGA 3360

ATCACTGAAC TATAGTGTCT ATACAACCAA CAGTGATGTC TGGTCCTATG GTGTATTGCT 3420

CTGGGAGATT GTTAGCTTAG GAGGCACCCC CTACTGCGGC ATGACGTGCG CGGAGCTCTA 3480

TGAGAAGCTA CCCCAGGGCT ACAGGCTGGA GAAGCCCCTG AACTGTGATG ATGAGGTGTA 3540

TGATCTAATG AGACAGTGCT GGAGGGAGAA GCCTTATGAG AGACCATCAT TTGCCCAGAT 3600

ATTGGTGTCC TTAAACAGGA TGCTGGAAGA ACGGAAGACA TACGTGAACA CCACACTGTA 3660

TGAGAAGTTT ACCTATGCAG GAATTGACTG CTCTGCGGAA GAAGCAGCCT AGAGCAGAAC 3720

TCTTCATGTA CAACGGCCAT TTCTCCTCAC TGGCGCGAGA GCGCCTTGAC ACCTGTACCA 3780

AGCAAGCCAC CCACTGCCAA GAGATGTGAT ATATAAGTGT ATATATTGTG CTGTGTTTGG 3840

GACCCTCCTC ATACAGCTCG TGCGGATCTG CAGTGTGTTC TGACTCTAAT GTGACTGTAT 3900

ATACTGCTCG GAGTAAGAAT GTGCTAAGAT CAGAATGCCT GTTCGTGGTT TCATATAATA 3960

TATTTTTCTA AAAGCATAGA TTGCACAGGA AGGTATGAGT ACAAATACTG TAATGCATAA 4020

CTTGTTACTG TCCTAGATGT GTTTGATATT TTTCCTTTAC AACTGAATGC TATAAAAGTG 4080

TTTTGCTGTG TACACATAAG ATACTGTTCG TTAAAATAAG CATTCCCTTG ACAGCACAGG 4140

AAGAAAAGCG AGGGAAATGT ATGGATTATA TTAAATGTGG GTTACTACAC AAGAGGCCGA 4200

ACATTCCAAG TAGCAGAAGA GAGGGTCTCT CAACTCTGCT CCTCACCTGC AGAAGCCAGT 4260 TTGTTTGGCC ATGTGACAAT TGTCCTGTGT TTTTATAGCA CCCAAATCAT TCTAAAATAT 4320

GAACATCTAA AAACTTTGCT AGGAGACTAA GAACCTTTGG AGAGATAGAT ATAAGTACGG 4380

TCAAAAAACA AAACTGCGGG ACTTACATTT ATTTTCTATA GTAATCTGTT GTACATTTTA 4440

AGAAGTAAAA CTAGGAATTT AGGAGTGATG TGTGACATTT CTGACATGGA GTTACCATCC 4500

CCACATGTAT CACATACTGT CATATTCCCA CATGTATCAC ACATGTATTG TAAAATTTTG 4560

TAGTTTTGAT CACTTGTGAA TTTACTGTTG ATGTGGTAGC CACCTGCTGC AATGGTTCCT 4620

CTTGTAGGTG AATAAATGTC 4640 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1123 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Asp Ser Leu Ala Gly Leu Val Leu Cys Gly Val Ser Leu Leu Leu 1 5 10 15

Tyr Gly Val Val Glu Gly Ala Met Asp Leu lie Leu lie Asn Ser Leu 20 25 30

Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys lie Ala Ser Gly 35 40 45

Trp His Pro His Glu Pro lie Thr lie Gly Arg Asp Phe Glu Ala Leu 50 55 60

Met Asn Gin His Gin Asp Pro Leu Glu Val Thr Gin Asp Val Thr Arg 65 70 75 80

Glu Trp Ala Lys Lys Val Val Trp Lys Arg Glu Lys Ala Ser Lys lie 85 90 95

Asn Gly Ala Tyr Phe Cys Glu Gly Arg Val Arg Gly Gin Ala lie Arg 100 105 110 lie Arg Thr Met Lys Met Arg Gin Gin Ala Ser Phe Leu Pro Ala Thr 115 120 125

Leu Thr Met Thr Val Asp Arg Gly Asp Asn Val Asn lie Ser Phe Lys 130 135 140

Lys Val Leu lie Lys Glu Glu Asp Ala Val lie Tyr Lys Asn Gly Ser 145 150 155 160

Phe lie His Ser Val Pro Arg His Glu Val Pro Asp lie Leu Glu Val 165 170 . 175

His Leu Pro His Ala Gin Pro Gin Asp Ala Gly Val Tyr Ser Ala Arg 180 185 190

Tyr lie Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr Arg Leu lie Val

_ ^5 _ 195 200 205

Arg Arg Cys Glu Ala Gin Lys Trp Gly Pro Asp Cys Ser Arg Pro Cys 210 215 220

Thr Thr Cys Lys Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu Cys 225 230 235 240

He Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys Glu 245 250 255

Pro His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Pro Glu 260 265 270

Gly Cys Lys Ser Tyr Val Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser 275 280 285

Cys Ala Thr Gly Trp Arg Gly Leu Gin Cys Asn Glu Ala Cys Pro Ser 290 295 300

Gly Tyr Tyr Gly Pro Asp Cys Lys Leu Arg Cys His Cys Thr Asn Glu 305 310 315 320

Glu He Cys Asp Arg Phe Gin Gly Cys Leu Cys Ser Gin Gly Trp Gin 325 330 335

Gly Leu Gin Cys Glu Lys Glu Gly Arg Pro Arg Met Thr Pro Gin He 340 345 350

Glu Asp Leu Pro Asp His He Glu Val Asn Ser Gly Lys Phe Asn Pro 355 360 365

He Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Ser Glu Glu Met Thr 370 375 380

Leu Val Lys Pro Asp Gly Thr Val Leu Gin Pro Asn Asp Phe Asn Tyr 385 390 395 400

Thr Asp Arg Phe Ser Val Ala He Phe Thr Val Asn Arg Val Leu Pro 405 410 415

Pro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr Val Ala Gly Met 420 425 430

Val Glu Lys Pro Phe Asn He Ser Val Lys Val Leu Pro Glu Pro Leu 435 440 445

His Ala Pro Asn Val He Asp Thr Gly His Asn Phe Ala He He Asn 450 455 460

He Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pro He Lys Ser Lys Lys 465 470 475 480

Leu Phe Tyr Lys Pro Val Asn Gin Ala Trp Lys Tyr He Glu Val Thr 485 490 495

Asn Glu He Phe Thr Leu Asn Tyr Leu Glu Pro Arg Thr Asp Tyr Glu 500 505 510

Leu Cys Val Gin Leu Ala Arg Pro Gly Glu Gly Gly Glu Gly His Pro 515 520 525

Gly Pro Val Arg Arg Phe Thr Thr Ala Ser He Gly Leu Pro Pro Pro 530 535 540

Arg Gly Leu Ser Leu Leu Pro Lys Ser Gin Thr Ala Leu Asn Leu Thr 545 550 555 560

- 4 ^- Trp Gin Pro He Phe Thr Asn Ser Glu Asp Glu Phe Tyr Val Glu Val 565 570 575

Glu Arg Arg Ser Leu Gin Thr Thr Ser Asp Gin Gin Asn He Lys Val 580 585 590

Pro Gly Asn Leu Thr Ser Val Leu Leu^* Ser Asn Leu Val Pro Arg Glu 595 600 605

Gin Tyr Thr Val Arg Ala Arg Val Asn Thr Lys Ala Gin Gly Glu Trp 610 615 620

Ser Glu Glu Leu Arg Ala Trp Thr Leu Ser Asp He Leu Pro Pro Gin 625 630 635 640

Pro Glu Asn He Lys He Ser Asn He Thr Asp Ser Thr Ala Met Val 645 650 655

Ser Trp Thr He Val Asp Gly Tyr Ser He Ser Ser He He He Arg 660 665 670

Tyr Lys Val Gin Gly Lys Asn Glu Asp Gin His He Asp Val Lys He 675 680 685

Lys Asn Ala Thr Val Thr Gin Tyr Gin Leu Lys Gly Leu Glu Pro Glu 690 695 700

Thr Thr Tyr His Val Asp He Phe Ala Glu Asn Asn He Gly Ser Ser 705 710 715 720

Asn Pro Ala Phe Ser His Glu Leu Arg Thr Leu Pro His Ser Pro Ala 725 730 735

Ser Ala Asp Leu Gly Gly Gly Lys Met Leu Leu He Ala He Leu Gly 740 745 750

Ser Ala Gly Met Thr Cys He Thr Val Leu Leu Ala Phe Leu He Met 755 760 765

Leu Gin Leu Lys Arg Ala Asn Val Gin Arg Arg Met Ala Gin Ala Phe 770 775 780

Gin Asn Val Arg Glu Glu Pro Ala Val Gin Phe Asn Ser Gly Thr Leu 785 790 795 800

Ala Leu Asn Arg Lys Ala Lys Asn Asn Pro Asp Pro Thr He Tyr Pro 805 810 815

Val Leu Asp Trp Asn Asp He Lys Phe Gin Asp Val He Gly Glu Gly 820 ' 825 830

Asn Phe Gly Gin Val Leu Lys Ala Arg He Lys Lys Asp Gly Leu Arg 835 840 845

Met Asp Ala Ala He Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp Asp 850 855 860

His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly His 865 870 875 880

His Pro Asn He He Asn Leu Leu Gly Ala Cys Glu His Arg Gly Tyr 885 890 895

Leu Tyr Leu Ala He Glu Tyr Ala Pro His Gly Asn Leu Leu Asp Phe 900 905 910

Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala He Ala 915 920 925

Asn Ser Thr Ala Ser Thr Leu Ser Ser Gin Gin Leu Leu His Phe Ala 930 935 940

Ala Asp Val Ala Arg Gly Met Asp Tyr Leu Ser Gin Lys Gin Phe He 945 950 955 960

His Arg Asp Leu Ala Ala Arg Asn He Leu Val Gly Glu Asn Tyr He 965 970 975

Ala Lys He Ala Asp Phe Gly Leu Ser Arg Gly Gin Glu Val Tyr Val 980 985 990

Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala He Glu Ser 995 1000 1005

Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly 1010 1015 1020

Val Leu Leu Trp Glu He Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly 1025 1030 1035 1040

Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gin Gly Tyr Arg Leu 1045 1050 1055

Glu Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr Asp Leu Met Arg Gin 1060 1065 1070

Cys Trp Arg Glu Lys Pro Tyr Glu Arg Pro Ser Phe Ala Gin He Leu 1075 1080 1085

Val Ser Leu Asn Arg Met Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr 1090 1095 1100

Thr Leu Tyr Glu Lys Phe Thr Tyr Ala Gly He Asp Cys Ser Ala Glu 1105 1110 1115 1120

Glu Ala Ala

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 301 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

He Lys Phe Gin Asp Val He Gly Glu Gly Asn Phe Gly Gin Val Leu 1 5 10 15

Lys Ala Arg He Lys Lys Asp Gly Leu Arg Met Asp Ala Ala He Lys 20 25 30

Arg Met Lys Glu Tyr Ala Ser Lys Asp Asp His Arg Asp Phe Ala Gly 35 40 45

Glu Leu Glu Val Leu Cys Lys Leu Gly His His Pro Asn He He Asn

__ 5>--_ 50 55 60

Leu Leu Gly Ala Cys Glu His Arg Gly Tyr Leu Tyr Leu Ala He Glu 65 70 75 80

Tyr Ala Pro Met Gly Asn Leu Leu Asp Phe Leu Arg Lys Ser Arg Val 85 90 95

Leu Glu Thr Asp Pro Ala Phe Ala He Ala Asn Ser Thr Ala Ser Thr 100 105 110

Leu Ser Ser Gin Gin Leu Leu His Phe Ala Ala Asp Val Ala Arg Gly 115 120 125

Met Asp Tyr Leu Ser Gin Lys Gin Phe He His Arg Asp Leu Ala Ala 130 135 140

Arg Asn He Leu Val Gly Glu Asn Tyr He Ala Lys He Ala Asp Phe 145 150 155 160

Gly Leu Ser Arg Gly Gin Glu Val Tyr Val Lys Lys He Met Gly Arg 165 170 175

Leu Pro Val Arg Trp Met Ala He Glu Ser Leu Asn Tyr Ser Val Tyr 180 185 190

Thr Thr Asn Ser Asp Val Trp Ser Tyr Gly Val Leu Leu Trp Glu He 195 200 205

Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly Met Thr Cys Ala Glu Leu 210 215 220

Tyr Glu Lys Leu Pro Gin Gly Tyr Arg Leu Glu Lys Pro Leu Asn Cys 225 230 235 240

Asp Asp Glu Val Tyr Asp Leu Met Arg Gin Cys Trp Arg Glu Lys Pro 245 250 255

Tyr Glu Arg Pro Ser Phe Ala Gin He Leu Val Ser Leu Asn Arg Met 260 265 270

Leu Glu Glu Arg Lys Thr Tyr Val Asn Thr Thr Leu Tyr Glu Lys Phe 275 280 285

Thr Tyr Ala Gly He Asp Cys Ser Ala Glu Glu Ala Ala 290 295 300

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1124 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Asp Ser Leu Ala Ser Leu Val Leu Cys Gly Val Ser Leu Leu Leu 1 5 10 15

Ser Gly Thr Val Glu Gly Ala Met Asp Leu He Leu He Asn Ser Leu 20 25 30

Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys He Ala Ser Gly 35 40 45

Trp Arg Pro His Glu Pro He Thr He Gly Arg Asp Phe Glu Ala Leu 50 55 60

Met Asn Gin His Gin Asp Pro Leu Glu Val Thr Gin Asp Val Thr Arg 65 70 75 80

Glu Trp Ala Lys Lys Val Val Trp Lys Arg Glu Lys Ala Ser Lys He 85 90 95

Asn Gly Ala Tyr Phe Cys Glu Gly Arg Val Arg Gly Glu Ala He Arg 100 105 110

He Arg Thr Met Lys Met Arg Gin Gin Ala Ser Phe Leu Pro Ala Thr 115 120 125

Leu Thr Met Thr Val Asp Lys Gly Asp Asn Val Asn He Ser Phe Lys 130 135 140

Lys Val Leu He Lys Glu Glu Asp Ala Val He Tyr Lys Asn Gly Ser 145 150 155 160

Phe He His Ser Val Pro Arg His Glu Val Pro Asp He Leu Glu Val 165 170 175

His Leu Pro His Ala Gin Pro Gin Asp Ala Gly Val Tyr Ser Ala Arg 180 185 190

Tyr He Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr Arg Leu He Val 195 200 205

Arg Arg Cys Glu Ala Gin Lys Trp Gly Pro Glu Cys Asn His Leu Cys 210 215 220

Thr Ala Cys Met Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu Cys 225 230 235 240

He Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys Glu 245 250 255

Leu His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Gin Glu 260 265 270

Gly Cys Lys Ser Tyr Val Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser 275 280 285

Cys Ala Thr Gly Trp Lys Gly Leu Gin Cys Asn Glu Ala Cys His Pro 290 295 300

Gly Phe Tyr Gly Pro Asp Cys Lys Leu Arg Cys Ser Cys Asn Asn Gly 305 310 315 320

Glu Met Cys Asp Arg Phe Gin Gly Cys Leu Cys Ser Pro Gly Trp Gin 325 330 335

Gly Leu Gin Cys Glu Arg Glu Gly He Pro Arg Met Thr Pro Lys He 340 345 350

Val Asp Leu Pro Asp His He Glu Val Asn Ser Gly Lys Phe Asn Pro 355 360 365

He Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Asn Glu Glu Met Thr 370 375 380

so - Leu Val Lys Pro Asp Gly Thr Val Leu His Pro Lys Asp Phe Asn His 385 390 395 400

Thr Asp His Phe Ser Val Ala He Phe Thr He His Arg He Leu Pro 405 410 415

Pro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr Val Ala Gly Met 420 425 430

Val Glu Lys Pro Phe Asn He Ser Val Lys Val Leu Pro Lys Pro Leu 435 440 445

Asn Ala Pro Asn Val He Asp Thr Gly His Asn Phe Ala Val He Asn 450 455 460

He Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pro He Lys Ser Lys Lys 465 470 475 480

Leu Leu Tyr Lys Pro Val Asn His Tyr Glu Ala Trp Gin His He Gin 485 490 495

Val Thr Asn Glu He Val Thr Leu Asn Tyr Leu Glu Pro Arg Thr Glu 500 505 510

Tyr Glu Leu Cys Val Gin Leu Val Arg Arg Gly Glu Gly Gly Glu Gly 515 520 525

His Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Ser He Gly Leu Pro 530 535 540

Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys Ser Gin Thr Thr Leu Asn 545 550 555 560

Leu Thr Trp Gin Pro He Phe Pro Ser Ser Glu Asp Asp Phe Tyr Val 565 570 575

Glu Val Glu Arg Arg Ser Val Gin Lys Ser Asp Gin Gin Asn He Lys 580 585 590

Val Pro Gly Asn Leu Thr Ser Val Leu Leu Asn Asn Leu His Pro Arg 595 600 605

Glu Gin Tyr Val Val Arg Ala Arg Val Asn Thr Lys Ala Gin Gly Glu 610 615 620

Trp Ser Glu Asp Leu Thr Ala Trp Thr Leu Ser Asp He Leu Pro Pro 625 630 635 640

Gin Pro Glu Asn He Lys He Ser Asn He Thr His Ser Ser Ala Val 645 650 655

He Ser Trp Thr He Leu Asp Gly Tyr Ser He Ser Ser He Thr He 660 665 670

Arg Tyr Lys Val Gin Gly Lys Asn Glu Asp Gin His Val Asp Val Lys 675 680 685

He Lys Asn Ala Thr He He Gin Tyr Gin Leu Lys Gly Leu Glu Pro 690 695 700

Glu Thr Ala Tyr Gin Val Asp He Phe Ala Glu Asn Asn He Gly Ser 705 710 715 720

Ser Asn Pro Ala Phe Ser His Glu Leu Val Thr Leu Pro Glu Ser Gin 725 730 735

Ala Pro Ala Asp Leu Gly Gly Gly Lys Met Leu Leu He Ala He Leu

- S i 740 745 750

Gly Ser Ala Gly Met Thr Cys Leu Thr Val Leu Leu Ala Phe Leu He 755 760 765

He Leu Gin Leu Lys Arg Ala Asn Val Gin Arg Arg Met Ala Gin Ala 770 775 780

Phe Gin Asn Val Arg Glu Glu Pro Ala Val Gin Phe Asn Ser Gly Thr 785 790 795 800

Leu Ala Leu Asn Arg Lys Val Lys Asn Asn Pro Asp Pro Thr He Tyr 805 810 815

Pro Val Leu Asp Trp Asn Asp He Lys Phe Gin Asp Val He Gly Glu 820 825 830

Gly Asn Phe Gly Gin Val Leu Lys Ala Arg He Lys Lys Asp Gly Leu 835 840 845

Arg Met Asp Ala Ala He Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp 850 855 860

Asp His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly 865 870 875 880

His His Pro Asn He He Asn Leu Leu Gly Ala Cys Glu His Arg Gly 885 890 895

Tyr Leu Tyr Leu Ala He Glu Tyr Ala Pro His Gly Asn Leu Leu Asp 900 905 910

Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala He 915 920 925

Ala Asn Ser Thr Ala Ser Thr Leu Ser Ser Gin Gin Leu Leu His Phe 930 935 940

Ala Ala Asp Val Ala Arg Gly Met Asp Tyr Leu Ser Gin Lys Gin Phe 945 950 955 960

He His Arg Asp Leu Ala Ala Arg Asn He Leu Val Gly Glu Asn Tyr 965 970 975

Val Ala Lys He Ala Asp Phe Gly Leu Ser Arg Gly Gin Glu Val Tyr 980 985 990

Val Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala He Glu 995 1000 1005

Ser Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp Ser Tyr 1010 1015 1020

Gly Val Leu Leu Trp Glu He Val Ser Leu Gly Gly Thr Pro Tyr Cys 1025 1030 1035 1040

Gly Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gin Gly Tyr Arg 1045 1050 1055

Leu Glu Lys Pro Leu Asn Cys Asp Asp Glu Val Tyr Asp Leu Met Arg 1060 1065 1070

Gin Cys Trp Arg Glu Lys Pro Tyr Glu Arg Pro Ser Phe Ala Gin He 1075 1080 1085

Leu Val Ser Leu Asn Arg Met Leu Glu Glu Arg Lys Thr Tyr Val Asn 1090 1095 1100 Thr Thr Leu Tyr Glu Lys Phe Thr Tyr Ala Gly He Asp Cys Ser Ala 1105 1110 1115 1120

Glu Glu Ala Ala

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Val Trp Arg Val Pro Pro Phe Leu Leu Pro He Leu Phe Leu Ala 1 5 10 15

Ser His Val Gly Ala Ala Val Asp Leu Thr Leu Leu Ala Asn Leu Arg 20 25 30

Leu Thr Asp Pro Gin Arg Phe Phe Leu Thr Cys Val Ser Gly Glu Ala 35 40 45

Gly Ala Gly Arg Gly Ser Asp Ala Trp Gly Pro Pro Leu Leu Leu Glu 50 55 60

Lys Asp Asp Arg He Val Arg Thr Pro Pro Gly Pro Pro Leu Arg Leu 65 70 75 80

Ala Arg Asn Gly Ser His Gin Val Thr Leu Arg Gly Phe Ser Lys Pro 85 90 95

Ser Asp Leu Val Gly Val Phe Ser Cys Val Gly Gly Ala Gly Ala Arg 100 105 110

Arg Thr Arg Val He Tyr Val His Asn Ser Pro Gly Ala His Leu Leu 115 120 125

Pro Asp Lys Val Thr His Thr Val Asn Lys Gly Asp Thr Ala Val Leu 130 135 140

Ser Ala Arg Val His Lys Glu Lys Gin Thr Asp Val He Trp Lys Ser 145 150 155 160

Asn Gly Ser Tyr Phe Tyr Thr Leu Asp Trp His Glu Ala Gin Asp Gly 165 170 175

Arg Phe Leu Leu Gin Leu Pro Asn Val Gin Pro Pro Ser Ser Gly He 180 185 190

Tyr Ser Ala Thr Tyr Leu Glu Ala Ser Pro Leu Gly Ser Ala Phe Phe 195 200 205

Arg Leu He Val Arg Gly Cys Gly Ala Gly Arg Trp Gly Pro Gly Cys 210 215 220

Thr Lys Glu Cys Pro Gly Cys Leu His Gly Gly Val Cys His Asp His 225 230 235 240

- a -i — Asp Gly Glu Cys Val Cys Pro Pro Gly Phe Thr Gly Thr Arg Cys Glu 245 250 255

Gin Ala Cys Arg Glu Gly Arg Phe Gly Gin Ser Cys Gin Glu Gin Cys 260 265 270

Pro Gly He Ser Gly Cys Arg Gly Leu Thr Phe Cys Leu Pro Asp Pro 275 280 285

Tyr Gly Cys Ser Cys Gly Ser Gly Trp Arg Gly Ser Gin Cys Gin Glu 290 295 300

Ala Cys Ala Pro Gly His Phe Gly Ala Asp Cys Arg Leu Gin Cys Gin 305 310 315 320

Cys Gin Asn Gly Gly Thr Cys Asp Arg Phe Ser Gly Cys Val Cys Pro 325 330 335

Ser Gly Trp His Gly Val His Cys Glu Lys Ser Asp Arg He Pro Gin 340 345 350

He Leu Asn Met Ala Ser Glu Leu Glu Phe Asn Leu Glu Thr Met Pro 355 360 365

Arg He Asn Cys Ala Ala Ala Gly Asn Pro Phe Pro Val Arg Gly Ser 370 375 380

He Glu Leu Arg Lys Pro Asp Gly Thr Val Leu Leu Ser Thr Lys Ala 385 390 395 400

He Val Glu Pro Glu Lys Thr Thr Ala Glu Phe Glu Val Pro Arg Leu 405 410 415

Val Leu Ala Asp Ser Gly Phe Trp Glu Cys Arg Val Ser Thr Ser Gly 420 425 430

Gly Gin Asp Ser Arg Arg Phe Lys Val Asn Val Lys Val Pro Pro Val 435 440 445

Pro Leu Ala Ala Pro Arg Leu Leu Thr Lys Gin Ser Arg Gin Leu Val 450 455 460

Val Ser Pro Leu Val Ser Phe Ser Gly Asp Gly Pro He Ser Thr Val 465 470 475 480

Arg Leu His Tyr Arg Pro Gin Asp Ser Thr Met Asp Trp Ser Thr He 485 490 495

Val Val Asp Pro Ser Glu Asn Val Thr Leu Met Asn Leu Arg Pro Lys 500 505 510

Thr Gly Tyr Ser Val Arg Val Gin Leu Ser Arg Pro Gly Glu Gly Gly 515 520 525

Glu Gly Ala Trp Gly Pro Pro Thr Leu Met Thr Thr Asp Cys Pro Glu 530 535 540

Pro Leu Leu Gin Pro Trp Leu Glu Gly Trp His Val Glu Gly Thr Asp 545 550 555 560

Arg Leu Arg Val Ser Trp Ser Leu Pro Leu Val Pro Gly Pro Leu Val 565 570 . 575

Gly Asp Gly Phe Leu Leu Arg Leu Trp Asp Gly Thr Arg Gly Gin Glu 580 585 590

Arg Arg Glu Asn Val Ser Ser Pro Gin Ala Arg Thr Ala Leu Leu Thr

_ 5^ _ 595 600 605

Gly Leu Thr Pro Gly Thr His Tyr Gin Leu Asp Val Gin Leu Tyr His 610 615 620

Cys Thr Leu Leu Gly Pro Ala Ser Pro Pro Ala His Val Leu Leu Pro 625 630 635 640

Pro Ser Gly Pro Pro Ala Pro Arg His Leu His Ala Gin Ala Leu Ser 645 650 655

Asp Ser Glu He Gin Leu Thr Trp Lys His Pro Glu Ala Leu Pro Gly 660 665 670

Pro He Ser Lys Tyr Val Val Glu Val Gin Val Ala Gly Gly Ala Gly 675 680 685

Asp Pro Leu Trp He Asp Val Asp Arg Pro Glu Glu Thr Ser Thr He 690 695 700

He Arg Gly Leu Asn Ala Ser Thr Arg Tyr Leu Phe Arg Met Arg Ala 705 710 715 720

Ser He Gin Gly Leu Gly Asp Trp Ser Asn Thr Val Glu Glu Ser Thr 725 730 735

Leu Gly Asn Gly Leu Gin Ala Glu Gly Pro Val Gin Glu Ser Arg Ala 740 745 750

Ala Glu Glu Gly Leu Asp Gin Gin Leu He Leu Ala Val Val Gly Ser 755 760 765

Val Ser Ala Thr Cys Leu Thr He Leu Ala Ala Leu Leu Thr Leu Val 770 775 780

Cys He Arg Arg Ser Cys Leu His Arg Arg Arg Thr Phe Thr Tyr Gin 785 790 795 800

Ser Gly Ser Gly Glu Glu Thr He Leu Gin Phe Ser Ser Gly Thr Leu 805 810 815

Thr Leu Thr Arg Arg Pro Lys Leu Gin Pro Glu Pro Leu Ser Tyr Pro 820 825 830

Val Leu Glu Trp Glu Asp He Thr Phe Glu Asp Leu He Gly Glu Gly 835 840 845

Asn Phe Gly Gin Val He Arg Ala Met He Lys Lys Asp Gly Leu Lys 850 855 860

Met Asn Ala Ala He Lys Met Leu Lys Glu Tyr Ala Ser Glu Asn Asp 865 870 875 880

His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly His 885 890 895

His Pro Asn He He Asn Leu Leu Gly Ala Cys Lys Asn Arg Gly Tyr 900 905 910

Leu Tyr He Ala He Glu Tyr Ala Pro Tyr Gly Asn Leu Leu Asp Phe 915 920 925

Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Arg Glu 930 935 940

His Gly Thr Ala Ser Thr Leu Ser Ser Arg Gin Leu Leu Arg Phe Ala 945 950 955 960

-.-5 ε-_ Ser Asp Ala Ala Asn Gly Met Gin Tyr Leu Ser Glu Lys Gin Phe He 965 970 975

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gly Glu Asn Leu Ala 980 985 990

Ser Lys He Ala Asp Phe Gly Leu Ser Arg Gly Glu Glu Val Tyr Val 995 1000 1005

Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala He Glu Ser 1010 1015 1020

Leu Asn Tyr Ser Val Tyr Thr Thr Lys Ser Asp Val Trp Ser Phe Gly 1025 1030 1035 1040

Val Leu Leu Trp Glu He Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly 1045 1050 1055

Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gin Gly Tyr Arg Met 1060 1065 1070

Glu Gin Pro Arg Asn Cys Asp Asp Glu Val Tyr Glu Leu Met Arg Gin 1075 1080 1085

Cys Trp Arg Asp Arg Pro Tyr Glu Arg Pro Pro Phe Ala Gin He Ala 1090 1095 1100

Leu Gin Leu Gly Arg Met Leu Glu Ala Arg Lys Ala Tyr Val Asn Met 1105 1110 1115 1120

Ser Leu Phe Glu Asn Phe Thr Tyr Ala Gly He Asp Ala Thr Ala Glu 1125 1130 1135

Glu Ala

-_s l

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleotide sequence encoding a tie-2 protein.

2. A cDNA nucleotide sequence encoding a tie-2 protein.

3. A cDNA nucleotide sequence encoding a member of the MCK-10 family of proteins in which the nucleotide sequence encodes the amino acid sequence of FIG. 2 (SEQ. ID NO: ) , or which is capable of selectively hybridizing to the DNA sequence of FIG. 1 (SEQ. ID NO: ) .

4. A recombinant DNA vector containing a nucleotide sequence that encodes a tie-2 protein.

5. A recombinant DNA vector containing a nucleotide sequence that encodes a tie-2 fusion protein.

6. The recombinant DNA vector of Claim 4 in which the tie-2 nucleotide sequence is operatively associated with a regulatory sequence that controls the tie-2 gene expression in a host.

7. The recombinant DNA vector of Claim 5 in which the tie-2 fusion protein nucleotide sequence is operatively associated with a regulatory sequence that controls the tie-2 fusion protein gene expression in a host.

8. The DNA of Claim 2, 3, 4, 5, 6 or 7 in which the nucleotide sequence is capable of hybridizing under standard conditions, or which would be capable of hybridizing under standard conditions but for the

Ψ degeneracy of the genetic code to the DNA sequence of FIG. 1.

9. An engineered host cell that contains the recombinant DNA vector of Claims 4, 5, 6, or 7.

10. An engineered cell line that contains the recombinant DNA expression vector of Claim 6 and expresses tie-2.

11. An engineered cell line that contains the recombinant DNA expression vector of Claim 7 and expresses tie-2 fusion protein.

12. The engineered cell line of Claim 10 which expresses the tie-2 on the surface of the cell.

13. The engineered cell line of Claim 11 that expresses the tie-2 fusion protein on the surface of the cell.

14. A method for producing recombinant tie-2, comprising:

(a) culturing a host cell ^'transformed with the recombinant DNA expression vector of Claim 9 and which expresses the tie-2; and

(b) recovering the tie-2 gene product from the cell culture.

15. A method for producing recombinant tie-2 fusion protein, comprising:

(a) culturing a host cell transformed with the recombinant DNA expression vector of Claim 11 and which expresses the tie-2 fusion protein; and

(b) recovering the tie-2 fusion protein from the cell culture.

- 59 -

16. An isolated recombinant tie-2 receptor protein.

17. A fusion protein comprising tie-2 linked to a heterologous protein or peptide sequence.

18. An oligonucleotide which encodes an antisense sequence complementary to the tie-2 nucleotide sequence, and which inhibits translation of the tie-2 gene in a cell.

19. The oligonucleotide of Claim 18 which is complementary to a nucleotide sequence encoding the amino terminal region of the tie-2.

20. A monoclonal antibody which iirvmunospecifically binds to an epitope of the tie-2.

21. The monoclonal antibody of Claim 20 which competitively inhibits the binding of ligand to the tie-2.

22. The monoclonal antibody of Claim 20 which is linked to a cytotoxic agent.

23. The monoclonal antibody of Claim 20 which is linked to a radioisotope.

24. A method for screening and identifying antagonists of tie-2, comprising:

(a) contacting a cell line that expresses tie-2 with a test compound; and

(b) determining whether the test compound inhibits the binding of tie-2 ligand and the cellular effects of ligand binding on the cell line,

- 5Λ- fy in which antagonists are identified as those compounds that inhibit both the binding and cellular effects of tie-2 ligand binding on the cell line.

25. A method for screening and identifying agonists of tie-2 ligand, comprising:

(a) contacting a cell line that expresses the tie-2 with a test compound in the presence and in the absence of ligand;

(b) determining whether, in the presence of ligand, the test compound inhibits the binding of ligand to the cell line and;

(c) determining whether, in the absence of the ligand, the test compound mimics the cellular effects of ligand on the cell line in which agonists are identified as those test compounds that inhibit the binding but mimic the cellular effects of ligand on the cell line.

26. The method according to Claim 24 or 25 in which the cell line is a genetically engineered cell line.

27. The method according to Claim 24 or 25 in which the cell line endogenously expresses the tie-2.

28. A method for screening and identifying antagonists of tie-2 activity comprising:

(a) contacting tie-2 protein with a random peptide library such that tie-2 will recognize and bind to one or more peptide species within the library;

(b) isolating the tie-2/peptide combination;

(c) determining the sequence of the peptide isolated in step c; and (d) determining whether the test compound inhibits the biological activity of tie-2. in which agonists are identified as those peptides that inhibit the binding but mimic the cellular ef ects of tie-2.

29. A method for screening and identifying agonists of tie-2 comprising:

(a) contacting tie-2 protein with a random peptide library such that tie-2 will recognize and bind to one or more peptide species within the library;

(b) isolating the tie-2/peptide combination;

(c) determining the sequence of the peptide isolated in step c; and

(d) determining whether, in the absence of the tie-2 ligand, the peptide mimics the cellular effects of tie-2.

30. The method according to Claim 28 or 29 in which the tie-2 protein is genetically engineered.

31. A method of modulating the endogenous enzymatic activity of the tyrosine kinase tie-2 receptor in a mammal comprising administering to the mammal an effective amount of a ligand to the tie-2 receptor protein to modulate the enzymatic activity.

32. The method of Claim 31 in which the enzymatic activity of the receptor protein is decreased.

33. A recombinant vector containing a nucleotide sequence that encodes a truncated tie-2 which has

-m - dominant-negative activity which inhibits the biological activity tie-2.

34. The recombinant vector of claim 33 in which the vector is a retrovirus vector.

35. An engineered cell line that contains the recombinant DNA vector of Claim 34 and expresses truncated tie-2.

36. An engineered cell line that contains the recombinant vector of Claim 35 and produces infectious retrovirus particles expressing truncated tie-2.

37. An isolated recombinant truncated tie-2 receptor protein which has dominant-negative activity which inhibits the biological activity of tie-2.

38. A method of modulating the biological activity of tie-2 in a mammal comprising administrating to the mammal an effective amount of truncated tie-2 receptor protein which inhibits the biological activity of tie-2 activation.

. _ _