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WO1995013387A1 - Nouvelle tyrosine-kinase receptrice appelee tie-2 - Google Patents

Nouvelle tyrosine-kinase receptrice appelee tie-2

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Publication number
WO1995013387A1
WO1995013387A1 PCT/EP1994/003767 EP9403767W WO9513387A1 WO 1995013387 A1 WO1995013387 A1 WO 1995013387A1 EP 9403767 W EP9403767 W EP 9403767W WO 9513387 A1 WO9513387 A1 WO 9513387A1
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WO
WIPO (PCT)
Prior art keywords
tie
leu
gly
ala
glu
Prior art date
Application number
PCT/EP1994/003767
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English (en)
Inventor
Werner Risau
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication date
Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority to AU81430/94A priority Critical patent/AU8143094A/en
Publication of WO1995013387A1 publication Critical patent/WO1995013387A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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.
  • vasculogenesis and angiogenesis play an important role in a variety of physiological processes such as embryonic development, wound healing and organ regeneration.
  • endothelial cell proliferation plays an important role during the development of the vascular system.
  • 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.
  • 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.
  • novel receptors involved in these processes will provide targets for development of therapeutic applications designed to treat disorders associated with aberrant blood vessel formation.
  • 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.
  • tie-2 a novel receptor tyrosine kinase receptor
  • 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.
  • Ig immunoglobulin
  • Fn fibronectin
  • 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.
  • 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.
  • 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.
  • 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
  • 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 ⁇ m.
  • 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 ⁇ m.
  • 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) .
  • C,D advanced stage blood island
  • CV cardinal vein (head vein); BI, blood island; DA, dorsal aorta; EN, endocardial tissue; TE, telencephalon; YS, yolk sac. Bar, represents 50 ⁇ m.
  • 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.
  • 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.
  • anti-tie-2 antibodies may be used to inhibit tie-2 function.
  • 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.
  • TIE-2 CODING SEQUENCE The nucleotide coding sequence and deduced amino acid sequence of the tie-2 gene is depicted in
  • 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.
  • FIG. 1 may also be used to express gene products with tie-2 activity.
  • tie-2 DNA sequence all such variants of the tie-2 nucleotide sequence will be referred to as the tie-2 DNA sequence.
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • 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).
  • FN III fibronectin type III domains
  • 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%
  • 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.
  • a bacteriophage cDNA library may be screened, under conditions of reduced stringency, using a radioactively labeled fragment of the murine tie-2 clone.
  • 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.
  • conserveed 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
  • 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.
  • the labeled fragment may be used to screen a genomic library.
  • FIG. 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.
  • tie-2 mRNA was exclusively synthesized in the vasculature of adult brain, P4 brain, E12.5 brain E8.5 neuroectoderm (FIG. 5A-H) .
  • 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.
  • eningeal blood vessels and the choroid plexus were also found to synthesize tie-2 mRNA at comparable levels (FIGS. 5, 7B) .
  • 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
  • tie-2 mRNA 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.
  • tie-2 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • host cells may over glycosylate the gene product.
  • the tie-2 or a modified tie-2 sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence 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.
  • 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,
  • the protein itself could be produced using chemical methods to synthesize the tie-2 amino acid sequence in whole or in part.
  • 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.
  • the nucleotide sequence coding for tie-2 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.
  • 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)
  • 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...).
  • 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
  • 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.
  • inducible promoters such as pL of bacteriophage ⁇ , 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.
  • promoters derived from the genome of mammalian cells e.g.. metallothionein promoter
  • mammalian viruses e.g. the adenovirus late promoter
  • the vaccinia virus 7.5K promoter e.g. the vaccinia virus 7.5K promoter
  • 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.
  • 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
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) .
  • GST glutathione S-transferase
  • 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.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • 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.
  • the expression of the tie-2 coding sequence may be driven by any of a number of promoters.
  • 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. 3:1311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.
  • An alternative expression system which could be used to express tie-2 is an insect 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) .
  • 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.
  • 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) .
  • 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)
  • telomeres 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.
  • 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.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.
  • cell lines which stably express the tie-2 may be engineered.
  • 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.
  • appropriate expression control elements e.g. promoter, enhancer, sequences, transcription termina ⁇ tors, polyadenylation sites, etc.
  • 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
  • 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.
  • 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.
  • hygro which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147) genes.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • DFMO 2-(difluoromethyl)-DL-ornithine
  • 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
  • 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.
  • 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.).
  • certain "marker" gene functions e.g. , thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • certain "marker” gene functions e.g. , thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • 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
  • transcriptional activity for the tie-2 coding region can be assessed by hybridization assays.
  • RNA can be isolated and analyzed by Northern blot using a probe homologous to the tie-2 coding sequence or particular portions thereof.
  • total nucleic acids can be assessed by hybridization assays.
  • Z2. - of the host cell . may be extracted and assayed for hybridization to such probes.
  • 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.
  • tie-2 receptor and engineered cell lines may be employed equally well for the tie-2 family of receptor tyrosine kinases.
  • 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.
  • Engineered cell lines that express the tie-2 receptor may be advantageously used to identify and characterize the natural tie-2 ligand(s) .
  • 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
  • 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 P0 4 into the cell may be measured after contacting cells with extracts.
  • 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.
  • affinity chromatography using recombinantly expressed tie-2 covalently attached to a column matrix may be used to purify tie-2 ligand.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the receptor molecules can be reconstituted into liposomes where label or "tag" can be attached.
  • antibodies to epitopes of the recombinantly produced tie-2 receptor 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.
  • 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.
  • SPDP thiol- crosslinking reagents
  • the hybrid antibodies may be used to specifically eliminate tie-2 expressing cells.
  • 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.
  • BCG Bacilli Calmette-Guerin
  • Corynebacteriu parvum bacilli Calmette-Guerin
  • 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).
  • Antibody fragments which contain specific binding sites of tie-2 may be generated by known techniques.
  • 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.
  • 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.
  • 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.
  • the tie-2 DNA may have a number of uses for the diagnosis of diseases resulting from aberrant expression of tie-2.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • 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.
  • 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.
  • 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.
  • 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
  • tie-2 molecules can be reconstituted into liposomes for delivery to target cells.
  • 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.
  • 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 lxlO 6 cts/minute/ml of probe, which had been labelled with 32 P-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.
  • 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-3 1 (I stands for inosine) in addition to the following premiss described by Wilks et al. (1989, DPNAS Proc. Natl.
  • 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
  • IMMUNOHISTOCHEMISTRY Embedded embryos and whole organs were sectioned on a Leitz cryostat 8 ⁇ m 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% H 2 0 2 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.
  • 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
  • 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).
  • 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) .
  • 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 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.
  • 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
  • EGF-like repeats consist of 30 to 40 amino acid residues often found in the extracellular parts of membrane-bound proteins or secreted proteins
  • 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.
  • 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).
  • 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.
  • RNA expression was also detected in organs like brain, kidney and heart.
  • 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.
  • 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.
  • tie-2 hybridization signals were 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.
  • the tie-2-specific probe labels the vessel in an identical way.
  • tie-2 gene expression seems to be a general feature of endothelial cells.
  • tie-2 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.
  • 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.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • Lys Val Leu lie Lys Glu Glu Asp Ala Val lie Tyr Lys Asn Gly Ser 145 150 155 160
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO

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Abstract

Nouvelle tyrosine-kinase réceptrice appelée tie-2, séquence nucléotidique et vecteurs d'expression codant la tie-2, et procédés d'inhibition de l'activité de la tie-2. On a également prévu d'autres membres de la famille des tyrosine-kinases réceptrices tie-2. On peut utiliser des cellules hôtes manipulées génétiquement exprimant la tie-2, dans l'évaluation et le criblage des médicaments impliqués dans l'activation et la régulation de la tie-2. En outre, on a prévu l'utilisation de ces médicaments comme agonistes ou antagonistes de l'activité de la tie-2.
PCT/EP1994/003767 1993-11-12 1994-11-12 Nouvelle tyrosine-kinase receptrice appelee tie-2 WO1995013387A1 (fr)

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