Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• the present invention relates generally to the field of genetic engineering and more particularly to genes for receptor tyrosine kinases, their insertion into recombinant DNA vectors, and the production of the resulting proteins in recipient strains of micro-organisms and recipient eukaryotic cells. More specifically, the present invention is directed to tie, a novel receptor tyrosine kinase, to nucleotide sequences encoding tie, and to methods for the generation of DNAs encoding tie and their gene products.
• Tie DNAs and polypeptides of the invention may be useful in the diagnosis and treatment of certain diseases involving endothelial cells and associated tie receptors, such as neoplastic diseases involving tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis and inflammatory diseases.
• diseases involving endothelial cells and associated tie receptors such as neoplastic diseases involving tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis and inflammatory diseases.
• the cellular behavior responsible for the development, maintenance, and repair of differentiated cells and tissues is regulated, in large part, by intercellular signals conveyed via growth factors and similar ligands and their receptors.
• the receptors are located on the cell surface of responding cells and they bind peptides or polypeptides known as growth factors as well as other hormone-like ligands. The results of this interaction are rapid biochemical changes in the responding cells, as well as a rapid and a long-term readjustment of cellular gene expression.
• Several receptors associated with various cell surfaces may bind specific growth factors.
• Tyrosine phosphorylation is one of the key modes of signal transduction across the plasma membrane.
• Several currently known protein tyrosine kinase genes encode transmembrane receptors for polypeptide growth factors and hormones such as epidermal growth factor (EGF) , insulin, insulin-like growth factor-I (IGF-I) , platelet derived growth factors (PDGF-A and -B) , and fibroblast growth factors (FGFs) .
• EGF epidermal growth factor
• IGF-I insulin-like growth factor-I
• PDGF-A and -B platelet derived growth factors
• FGFs fibroblast growth factors
• Growth factor receptors of endothelial cells are of particular interest due to the possible involvement of growth factors, such as FGFs, in several important physiological and pathological processes, such as vasculogenesis, angiogenesis, atherosclerosis, and inflammatory diseases. Folkman, et al . Science, 235 : 442-447 (1987) .
• the receptors of several hematopoietic growth factors are tyrosine kinases; these include c-fms, which is the colony stimulating factor 1 receptor, Sherr, et al. r Cell , 41 : 665-676 (1985) , and c-kit, a primitive hematopoietic growth factor receptor reported in Huang, et al . , Cell , 63 : 225-33 (1990) .
• the receptor tyrosine kinases may be divided into evolutionary subfamilies.
• Such subfamilies include, EGF receptor-like kinase (subclass I) and insulin receptor-like (subclass II) kinase , each of which contains repeated homologous cysteine-rich sequences in their extracellular domains.
• a single cysteine-rich region is also found in the extracellular domains of the eph-like kinases.
• PDGF receptors as well as c-fms and c-kit receptor tyrosine kinases may be grouped into subclass III; while the FGF receptors form subclass IV. Typical for the members of both of these subclasses are extracellular folding units stabilized by intrachain disulfide bonds. These so-called immunoglobulin (Ig)-like folds are found in the proteins of the immunoglobulin superfamily which contains a wide variety of other cell surface receptors having either cell-bound or soluble ligands. Williams, et al .
• Receptor tyrosine kinases differ in their specificity and affinity.
• receptor tyrosine kinases are glycoproteins, which consist of (1) an extracellular domain capable of binding the specific growth factor(s) ; (2) a transmembrane domain which usually is an alpha-helical portion of the protein; (3) a juxtame brane domain where the receptor may be regulated by, e . g .
• tyrosine kinase domain which is the enzymatic component of the receptor
• carboxyterminal tail which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.
• the present invention provides a novel endothelial cell receptor tyrosine kinase which was originally identified as an unknown tyrosine kinase-homologous PCR-cDNA fragment from human leukemia cells by Partanen, et al . , Proc . Natl . Acad . Scl . USA, 87: 8913-8917 (1990).
• This gene and its encoded protein are called tie which is an abbreviation for the "tyrosine kinase containing immunoglobulin- and EGF-like repeats”.
• a DNA or RNA segment of defined structure encoding the tie receptor tyrosine kinase may be produced synthetically or isolated from natural sources and may be used in the production of desired recombinant DNA vectors or may be used to recover related genes from other sources. It is a further object of the present invention to provide a recombinant-DNA vector containing a heterologous segment encoding the tie receptor tyrosine kinase or a related protein which is capable of being inserted into a microorganism or eukaryotic cell for expression of the encoded protein.
• the present invention also provides eukaryotic cells capable of producing useful quantities of the tie receptor tyrosine kinase and proteins of similar function from multiple species.
• peptides which may be produced synthetically in a laboratory or by a microorganism which mimic the activity of the natural tie receptor tyrosine kinase protein and which may be used to produce the tie receptor tyrosine kinase or a portion thereof in eukaryotic cells in a reproducible and standardized manner are disclosed.
• Particularly preferred are peptides selected from the group consisting of: (a) a first sequence:
• DNA and RNA molecules comprising a nucleotide which encodes any of the peptides indicated above are also contemplated in the present invention.
• sequences comprising all or part of the following two DNA sequences, their complements, or corresponding RNA sequences are preferred:
• DNA and RNA molecules containing segments of the larger sequence are also provided for use in carrying out preferred aspects of the invention relating to the production of such peptides by the techniques of genetic engineering and the production of oligonucleotide probes. Since the DNA sequence encoding the tie protein has been fully identified, it is possible to produce an entire gene by, for example, polymerase chain reaction or by synthetic chemistry using commercially available equipment, after which the gene can be inserted into any of the many available DNA vectors using known techniques of recombinant DNA technology. Furthermore, automated equipment is also available which makes direct synthesis of any of the peptides disclosed herein readily available. Thus, the present invention may be carried out using reagents, plasmids, and microorganism which are readily available to the skilled artisan.
• Figure 1 Nucleic and deduced amino acid sequence of the tie cDNA.
• the 3845 bp nucleotide sequence compiled from two overlapping cDNA clones isolated from HEL library contains an open reading frame of 1138 amino acids (marked in the single-letter code) .
• the tie precursor begins from nucleotide number 37 and the mature tie protein from amino acid 22 (nucleotide number 100) .
• the hydrophobic signal sequence and the putative transmembrane domain are underlined (thick lines) as are the sites for potential N-linked glycosylation (thin lines) .
• the mature tie protein Cysteine residues found in the extracellular domain have been boxed, the tyrosine kinase domain is shown by horizontal arrows and the kinase insert with italics.
• the three cysteine-rich segments having homology to EGF-like domains are also boxed (EGFH I-III) . Their alignment is shown in Fig. 2.
• the first of the EGF repeats missing in clone 3a is indicated by vertical arrows.
• the sequence has been deposited to GenBank/EMBL (Accession no. X60957) .
• A is alanine ⁇ C is cysteine, D is aspartate, E is glutamate, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, ⁇ L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine.
• FIG. 1 A. Alignment of the EGF-like domains of tie. Comparison is made with human EGF sequence (amino acid residues 1-44) and homologous sequence ' s in the growth factor CRIPTO (67-108), laminin A chain (1092-1138), Drosophila melanogaster Notch (897-945) and Caenorhabditis elegans Lin-12 (204-246) developmental control proteins, human blood coagulation factor IXa (83-130) and mouse urokinase type plasminogen activator (18-65) . The asterisks point out conserved residues and the homologous cysteine residues are boxed.
• FIG. 3 Expression of tie cDNA in COS cells.
• COS cells were transfected with SV40-based expression vectors for tie (SV14-1, SV14-2) and FGFR-4 (C, Partanen, J. , T. P. Makela, E. Eerola, J. Korhonen, H. Hirvonen, L. Claesson-Welsh, and K. Alitalo, EMBO J . 10 : 1347-1354, 1991), labelled with 35 S-methionine, lysed and immunoprecipitated as described in materials and methods of example 3. Autoradiograms of the SDS-PAGE analysis of the precipitated proteins are shown.
• HI immune serum against s-gal-tie fusion protein
• HO preim une serum.
• the immune serum was blocked with the antigen where indicated (+) .
• B Effect of tunicamycin on the molecular weight of the tie protein. MI, immune serum.against a carboxyl terminal tie peptide; MO preimmune serum. Where indicated (+) , the transfected cell cultures were labelled in the presence of tunicamycin. Mobilities of the molecular weight markers are shown on the left.
• FIG. 4 Immunoblot analysis of cell lines expressing the tie protein.
• Cell lysates of NIH3T3 cells transfected (LTR14-2) or not transfected (NE01) with a tie expression vector as well as porcine aortic endothelial cells (PAE) were analyzed by immunoblotting with antiserum against a carboxyterminal tie peptide.
• the samples in the two right most lanes (aPY, IP) were immunoprecipitate with ahti-phosphotyrosine antibodies prior to immunoblotting.
• FIG. 5 Chromosomal mapping of the tie locus. Radiolabeled JTK14 DNA was hybridized to normal human male peripheral lymphocyte metaphase preparations; slides were washed, developed after exposure and chromosomes were G-banded to distinguish individual chromosomes. Grain localization is illustrated on the schematic chromosome 1 where each dot represents 3 grains. Some nonspecific background signal was detected on the other chromosomes; 12,6% (40/317) on other chromosomes of group A, 8,5% (27/317) on chromosomes of group B, 29.6% (94/317) on C-group chromosomes and 14.8% (47/317) on the other chromosome groups.
• RNA expression in leukemia cell lines Poly (A)+ RNA from the indicated cell lines was analyzed by Northern blotting and hybridization with the tie cDNA probe. Hybridization with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used as an internal control for the loading of even amounts of RNA to the analysis.
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• Figure 7 tie mRNA expression in endothelial cell lines. Northern blot analysis of tie mRNA expression in PAE and EA hy926 endothelial cell lines. A lane containing poly(A)+ RNA from Dami cells was included as a positive control.
• Figure 8 Location of tie mRNA in endothelium of the kidney vessels by in situ hybridization.
• the dark field image showing the hybridization signal is on the top (A) .
• a corresponding phase-contrast micrograph is shown below (B) .
• FIG. 9 Comparison of the structure of the tie protein with some other receptor tyrosine kinases containing immunoglobulin and fibronectin type III repeats.
• the open circles represent immunoglobulin loops, the open boxes fibronectin type III repeats and the filled ovals EGF homology domains.
• the shaded box represents the cysteine rich region of the eph-like kinases.
• the cytoplasmic tyrosine kinase domains are drawn as black boxes.
• Figure 10. Schematic structure of the human tie receptor tyrosine kinase and comparison of its deduced amino acid sequence with two mouse tie cDNA clones (1C1D and D10E5) .
• the tie receptor consists of two immunoglobulin-like loops (Ig) , three (or two) epidermal growth factor domains (EGF) followed by three fibronectin III like domains, a transmembrane region (TM) and two cytoplasmic tyrosine kinase domains (TK1 and TK2) .
• Amino acid homology between mouse and human tie amino acid sequences is 96% and 95% for the segments 1C1D and D10E5, respectively. Amino acid residue symbols are as in Fig. 1.
• FIG. 11 Expression of tie mRNA in human tissues.
• Total RNA isolated from 17-19 week fetal tissues was analyzed by Northern blotting (A) .
• Hybridization of polyadenylated RNA from human adult tissues is shown in B.
• the s-actin and GAPDH probes were used as internal controls for the amount of RNA loaded.
• Figure 12 In situ hybridization analysis of tie mRNA expression in 12 day p.c. mouse embryo.
• Expression of tie mRNA is restricted to the endothelium of blood vessels. Used abbreviations: br (brain) , mg ( eninges) , lg (lung) , mb (mandible) , ht (heart) , vn (ventricle) , at (atrium) , sc (spinal cord) , pv (prevertebra) , and cv (posterior cardinal vein) .
• Figure 13 Comparison of tie mRNA (A) and factor VIII (B) expression in a 8 day p.c. mouse placenta.
• Factor VIII is seen as the dark deposit surrounding the blood lacunae in (A) and the tie signal in a similar but separate section (B) is seen as white grains. As can be seen from the figure, both signals are localized to endothelial cells of blood lacunae which form the labyrinth.
• rDNA reco binant DNA
• RNA transcribed from a gene may or may not code for a protein.
• RNA that codes for a protein is termed messenger RNA (mRNA) and, in eukaryotes, is transcribed by RNA polymerase II.
• mRNA messenger RNA
• RNA polymerase II messenger RNA
• antisense RNA gene Such a gene construct is herein termed an “antisense RNA gene” and such a RNA transcript is termed an “antisense RNA.” Antisense RNAs are not normally translatable due to the presence of translational stop codons in the antisense RNA sequence.
• a "complementary DNA” or “cDNA” gene includes recombinant genes synthesized by reverse transcription of mRNA lacking intervening sequences (introns) .
• Cloning vehicle A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning.
• the cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance. The word “vector” is sometimes used for "cloning vehicle.”
• Expression vector A vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
• the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
• Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
• the present invention pertains both to expression of recombinant tie protein, and to the functional derivatives of this protein.
• a “functional derivative” of tie protein is a protein which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of non-recombinant tie protein.
• a functional derivative of tie protein may or may not contain post-translational modifications such as covalentiy linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function.
• the term “functional derivative” is intended to include the "fragments,” “variants,” “analogues,” or “chemical derivatives” of a molecule.
• a molecule is said to be a "chemical derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule.
• Such moieties may improve the molecule's solubility, absorption, biological half life, etc.
• the moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc.
• Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980) . Procedure for coupling such moieties to a molecule are well known in the art.
• a "fragment" of a molecule such as tie protein is meant to refer to any variant of the molecule, such as the peptide core, or a variant of the peptide core.
• Variant A “variant” of a molecule such as tie protein is meant to refer to a molecule substantially similar in structure and biological activity to either the entire molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.
• Analog An "analog" of tie protein or genetic sequences is meant to refer to a protein or genetic sequence substantially similar in function to the tie protein or genetic sequence herein.
• the present invention is directed to "tie", a novel receptor tyrosine kinase, tie-encoding nucleic acid molecules (e . g. , cDNAs, genomic DNAs, RNAs, anti-sense RNAs, etc.), production of tie peptides or tie protein from a tie gene and its product, recombinant tie expression vectors, tie analogs and derivatives, and diagnostic and/or therapeutic uses of tie and related proteins, tie -encoding nucleic acid molecules, tie ligands, tie antagonists and anti-tie antibodies.
• tie-encoding nucleic acid molecules e . g. , cDNAs, genomic DNAs, RNAs, anti-sense RNAs, etc.
• production of tie peptides or tie protein from a tie gene and its product
• recombinant tie expression vectors e.g., recombinant tie expression vectors, tie analogs and derivatives, and diagnostic and/or therapeutic uses of tie and related proteins
• Biologically-active tie may be produced by the cloning and expression of a tie-encoding nucleotide or its functional equivalent in a suitable host cell.
• Production of tie using recombinant DNA technology may be divided into a step-wise process for the purpose of description, which process includes: (1) isolating or generating the coding sequence (gene) for the desired tie; (2) constructing an expression vector capable of directing the synthesis of the desired tie;
• the nucleotide coding sequence of tie, or functional equivalents thereof, may be used to construct recombinant expression vectors which direct the expression of the desired tie product.
• the nucleotide coding sequence for tie is depicted in SEQ ID NO. 1.
• the nucleotide sequence depicted therein, or fragments or functional equivalents thereof, may be used to generate the recombinant molecules which direct the expression of the recombinant tie product in appropriate host cells.
• Tie-encoding nucleotide sequences may be obtained from a variety of cell sources which produce products with tie-like activities and/or which express tie-encoding mRNA. The Applicants have identified a number of suitable human cell sources for tie including endothelial cells, leukemia cells, and rhabdomyosarcoma and fibrosarcoma cells.
• the tie coding sequence may be obtained by cDNA cloning from RNA isolated and purified from such cell sources or by genomic cloning.
• the tie sequence may be amplified by polymerase chain reaction from cDNA or genomic DNA material using techniques well-known in the art.
• Either cDNA or genomic libraries of clones may be prepared using techniques well-known in the art and may be screened for particular tie DNAs with nucleotide probes which are substantially complementary to any portion of the tie gene.
• Full length clones i.e., those containing the entire coding region of the desired tie gene, may be selected for use in constructing expression vectors.
• tie-encoding DNAs may be synthesized, in whole or in part, by chemical synthesis using standard techniques.
• tie nucleotide sequences include deletions, additions, or substitutions of different nucleotides resulting in a sequence that encodes the same or a functionally equivalent gene product.
• the gene product may contain deletions, additions, or substitutions of amino acid residues within the sequence which result in "silent" changes thus producing a bioactive tie product.
• Such amino acid deletions, additions, or substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the a phipathic nature of the amino acids involved.
• negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids indude lysine and arginine; amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values indude the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
• the host cells which contain recombinant coding sequences and which express the biologically active, mature product may be identified by at least four general approaches: (a) DNA-DNA, DNA-RNA or RNA-antisense RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of tie mRNA transcripts in the host cell; and (d) detection of the mature gene product as measured by immunoassay and, ultimately, by its biological activities.
• the presence of tie coding sequences inserted into expression vectors may be detected by DNA-DNA hybridization using probes comprising nucleotide sequences that are homologous to the tie coding sequence.
• the recombinant expression vector/host system may 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.
• a marker gene may be placed in tandem with the tie sequence under the control of the same or different promoter used to control the expression of the tie coding sequence. Expression of the marker in response to induction or selection indicates expression of the tie coding sequence.
• transcriptional activity of the tie coding region may be assessed by hybridization assays.
• polyadenylated RNA may be isolated and analyzed by Northern blotting using a probe homologous to the tie coding sequence or particular portions thereof.
• the total nucleic acid of the host cell may be extracted and assayed for hybridization to such probes.
• the expression of tie may be assessed immunologically, for example, by Western blots, immunoassays such as radioimmunoprecipitation, enzyme-linked immunoassays and the like.
• the ultimate test of the success of the expression system involves the detection of the biologically-active tie gene product.
• a cell-free media obtained from the cultured transfectant host cell may be assayed for tie activity when the gene product is secreted.
• cell lysates may be assayed for such activity. In either case, assays which measure ligand binding to tie or other bioactivities of tie may be used.
• tie-related derivatives, analogs, and peptides of the invention may be produced by a variety of means known in the art. Procedures and manipulations at the genetic and protein levels are within the scope of the invention. Peptide synthesis, which is standard in the art, may be used to obtain tie peptides. At the protein level, numerous chemical modifications may be used to produce tie like derivatives, analogs, or peptides by techniques known in the art, including but not limited to, specific chemical cleavage by endopeptidases (e . g. cyanogen bromides, trypsin, chymotrypsin, V8 protease, and the like) or exopeptidases, acetylation, formulation, oxidation, etc.
• endopeptidases e g. cyanogen bromides, trypsin, chymotrypsin, V8 protease, and the like
• exopeptidases acetylation, formulation, oxidation, etc.
• polyclonal and monoclonal antibodies which recognize tie or related proteins.
• Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of tie.
• various host animals may be immunized by injection with tie, or a synthetic tie peptide, including but not limited to, rabbits, mice, and rats.
• adjuvants may be used to increase the immunological response, depending upon the host species, including but not limited, to Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum .
• BCG Bacillus Calmette-Guerin
• a monoclonal antibody directed against an epitope of tie 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, 256 : 495-497 (1975) , and the more recent human B-cell hybridoma technique of Kosbor et al . , Immunology Today, 4 : 12 (1983) and the EBV-hybridoma technique of Cole et al . , Monoclonal Antibodies and Cancer Therapy, 77-96 (Alan R. Liss, Inc. 1985) .
• Antibody fragments which contain the idiotype of the molecule may be generated by known techniques.
• such fragments include, but are not limited to, the F(ab')2 fragment which may be produced by pepsin digestion of the antibody molecule; the Fab' fragments which may be generated by reducing the disulfide bridges of the F(ab')2 fragment; and the two Fab fragments which may be generated by treating the antibody molecule with papain and a reducing agent.
• Antibodies to tie may find use in the qualitative and quantitative detection of mature tie and its precursor and subcomponent forms, in the affinity purification of tie polypeptides, and in the elucidation of tie biosynthesis, metabolism and function. Detection of tie tyrosine kinase activity may be used as an enzymatic means of generating and amplifying a tie-specific signal in such assays. Antibodies to tie may also be useful as diagnostic and therapeutic agents.
• compositions of the present invention may be applied to a wide variety of uses, including diagnostic and/or therapeutic uses of tie, tie analogs and derivatives, tie-encoding nucleic acid molecules, antisense nucleic acid molecules and anti-tie antibodies.
• Tie-encoding nucleic acid molecules or fragments thereof may be used as probes to detect and quantify mRNAs encoding tie.
• Assays which utilize nucleic acid probes to detect sequences comprising all or part of a known gene sequence are well-known in the art. Tie mRNA levels may indicate emerging and/or exiting neoplasias as well as the onset and/or progression of other human diseases. Therefore, assays which detect and quantify tie mRNA may be of considerable diagnostic value.
• Anti-sense tie RNA molecules may be useful therapeutically to inhibit the translation of tie-encoding mRNAs where the therapeutic objective involves the elimination of the presence of tie or to downregulate its levels.
• Tie anti-sense RNA for example, may be useful as a tie- antagonizing agent in the treatment of diseases for which tie is involved as a causative agent, for example due to its overexpression.
• tie anti-sense RNAs may be useful in elucidating tie functional mechanisms.
• Tie-encoding nucleic acid molecules may be used for the production of recombinant tie proteins and related molecules as separately discussed in this application.
• Anti-tie antibodies may be used to diagnose and quantify tie in various contexts. For example, antibodies against various domains of tie may be used as a basis for tie immunoassays or immunohistochemical assessment of tie. Tyrosine kinase activity of tie may be useful in these assays as an enzymatic amplification reaction for the generation of a tie signal. Anti-tie antibodies may also be useful in studying the amount of tie on cell surfaces.
• Antibodies may be produced which function as tie ligand agonists or antagonists whereby the regulation of tie activity becomes possible. Since tie apparently is located on endothelial surfaces facing the vascular lumen, introduction of tie extracellular domain, its fragments or analogs, ligands or anti-tie extracellular domain antibodies into the bloodstream may allow the manipulation of tie activity and function in vivo with consequences on endothelial cell behavior and disease onset/progression.
• endothelial cells in vivo The introduction and expression of genes in endothelial cells in vivo is envisioned and will allow further manipulation of tie activity via expression vectors producing tie or its various functional derivatives in endothelial cells.
• receptor tyrosine kinases in which the tyrosine kinase domain has been specifically inactivated by in vitro mutagenesis function as dominant inhibitors of receptor function.
• Cloning of the tie promoter and regulatory sequences may allow targeting of gene expression mainly to endothelial cells in vivo .
• EGF-like, immunoglobulin-like, fibronectin-like and tyrosine kinase domains tie belongs to four different gene superfamilies.
• a combination of motifs from all immunoglobulin, fibronectin and EGF-homology superfamilies in the extracellular domain is a unique feature among known receptor tyrosine kinases.
• the EGF-like domain is a commonly found structural motif in cell surface and extracellular proteins involved in protein-protein interactions. Davis, The New Biologist, 2 : 410-419, (1990). Many transmembrane receptors for either soluble or cell bound ligands contain EGF repeats. Furthermore, two of the six EGF repeats of thrombomodulin, an endothelial cell surface glycoprotein, have been reported to be responsible for thrombin binding, Stearns, et al . , J. Biol . Chem . , 264 : 3352-3356, (1989), and the EGF domain of the lymph node homing receptor has been implicated in the adhesion of lymphocytes to high endothelial venules.
• EGF repeats are found also in extracellular matrix proteins mediating cell adhesion, such as laminin and tenascin.
• EGF repeats are a common motif in secreted proteins involved in blood clotting, including coagulation factors VII, IX, X, proteins C and S as well as tissue- and urokinase-type plasminogen activators. Furie and Furie, Cell, 53 : 505-518, (1988). The EGF like domain of urokinase-type plasminogen activator has been reported to be responsible for its receptor binding. Appella, et al . , J. Biol . Chem . , 262 : 4437-4440, (1987).
• the EGF-like repeats of tie contain eight cysteine residues instead of the usual six. Although eight cysteines are also found in the EGF repeats of laminin, the tie repeats are clearly most related to each other. None of the repeats of tie contains the consensus sequence required for asparagine/aspartate s-hydroxylation and calcium binding.
• the finding of a tie cDNA clone which encoded a protein lacking the first of the EGF-like repeats further suggests that these domains are located in separate exons and that the repeat structure was presumably created by exon duplication in the course of the molecular evolution of the tie receptor tyrosine kinase.
• the observation of several tie mRNA forms in EAhy926 cells supports the notion that various forms of the tie receptor are produced, presumably due to differential splicing.
• the immunoglobulin and fibfonectin superfamilies also comprise glycoproteins implicated in extracellular protein-protein interactions with either soluble or cell bound molecules. Williams and Barclay, Ann . Rev. Immunol . , 6: 381-405, (1988) .
• Many receptor tyrosine kinases such as PDGF, CSF-1 receptors, c-kit proto-oncogene as well as the FGF receptors contain Ig-like loops. Ullrich and Schlessinger, Cell, 61 : 243-54, (1990). In many cases both immunoglobulin and fibronectin type III domains are found in the same protein. This type of multidomain structure has recently been reported to be present in some receptor tyrosine kinases.
• the regional localization of the tie gene at Ip33-p34 indicates that the tie locus is telomeric of the jun locus since the PB5-5 hybrid which is negative for tie is positive for jun .
• Haluska, et al . Proc . Natl . Acad . Sci . USA, 85 : 2215-2218, (1988).
• the chromosomal region Ip32-p34 is involved in deletions in neuroblastoma, malignant lymphoma, glioma and other malignancies. Trent, et al . , Cytogenet . Cell Genet . , 51 : 533-562, (1989).
• tie mRNA expression is characteristic of the bipotential hematopoietic cell lineage retaining erythroid and megakaryoblastic differentiation capacities as well as for the endothelial cell lineage.
• Several differentiation antigens shared between megakaryoblastic and endothelial cells have been shown to exist, one example being the platelet glycoprotein Ilia Blood, 72 : 1478-1486, (1988); Kieffer, et al.. Blood, 72 : 1209-1215, (1988); Berridge, et al . , Blood, 66: 76-85, (1985) .
• the observed expression pattern of tie mRNA is rather interesting as EGF motifs are a common theme of proteins controlling hemostasis as well as proteins mediating associations with the endothelium.
• EXAMPLE 1 Isolation and characterization of cDNA clones encoding tie An oligo-dT primed human HEL cell cDNA library in bacteriophage Igtll (A kind gift from Dr. Mortimer Poncz, Childrens Hospital of Philadelphia, PA; (Poncz, et al., Blood, 69 : 219-223, 1987)) and a random primed human endothelial cell cDNA library (Clontech Cat. #107Ob) were screened with the JTK14 cDNA fragment PCR-amplified from the reverse-transcribed polyadenylated RNA of K562 leukemia cells. Partanen, et al . , Proc . Natl . Acad .
• a 200 bp long tie cDNA fragment isolated by a PCR cloning method from K562 cell cDNA was used as a molecular probe to screen an oligo dT-primed human erythroleukemia cell cDNA library and a random-primed human endothelial cell cDNA library.
• Nucleotide sequence analysis of clones HE11-1 and 12a isolated from the HEL library revealed an open reading frame of 1138 amino acids (Fig. 1) .
• the translational initiator, methionine, marked in Figure 1 is surrounded by a typical consensus sequence (Kozak, Nucleic Acids Res .
• Figure 2A also shows the comparison of the tie cysteine-rich domains with the epidermal growth factor (EGF) and CRIPTO growth factor proteins and the EGF-like repeats of laminin A chain, the Notch and Linl2 developmental control proteins of Drosophila melanogaster and Caenorhabditis elegans , respectively, and blood coagulation factor IXa.
• EGF epidermal growth factor
• CRIPTO CRIPTO growth factor proteins
• laminin A chain the Notch and Linl2 developmental control proteins of Drosophila melanogaster and Caenorhabditis elegans
• IXa blood coagulation factor
• a cDNA clone was isolated from HEL cell cDNA library, which lacked the first of the EGF repeats (marked between the arrowheads in Fig. 1) without otherwise affecting the reading frame.
• the amino-terminal region of the tie extracellular domain shows weak, but significant, homology to the amino terminus of chicken N-CAM protein (Cunningham, et al., Science, 236: 799-806, 1987).
• N-CAM a pair of cysteine residues surrounded by consensus motifs characteristic for the proteins of the immunoglobulin superfamily (Williams and Barclay, Ann. Rev. Immunol .
• FN2 contains a pair of cysteine residues as well as some other features of an immunoglobulin domain (Fig. 2B) and thus represents an intermediate of a FNIII repeat and an immunoglobulin domain.
• NXS/T any amino acid
• Amino acids 761-787 form a hydrophobic region of the sequence, which is likely to function as the transmembrane domain of the receptor, followed by several basic residues on the putative cytoplasmic side of the polypeptide.
• the juxtamembrane domain is 50 residues long before the beginning of a tyrosine kinase sequence homology at amino acid 837. With the interruption of homology in the kinase insert sequence of 14 aa (indicated by italics in the Fig.
• a peptide corresponding to 15 amino acids from the carboxyl terminus of the predicted tie protein was synthesized and cross-linked by glutaraldehyde to keyhole limpet hemocyanin (KLH, Calbiochem) .
• the immunizations were performed as in Example 1. Briefly, 7.5 mg carrier protein was dissolved in 0.5 ml of 0.1 M phosphate, pH 8.0, mixed with 7.5 mg of peptide and 5 ml of 20 mM glutaraldehyde was added. After mixing the solution, it was left for 15 min. at room temperature, after which 2.5 ml of glutaraldehyde was again added and the 15 min. incubation was repeated.
• the full-length tie protein coding sequence (combined from two overlapping clones, HE11-1 and 12a) was inserted into the -EcoRI site of an SV poly-mammalian expression vector (Stacey and Schnieke, Nucleic Acids Res . , 18 : 1829, 1990; construct SV14-2) .
• the SV14-1 vector lacks the first seven amino acids from its signal sequence, but it is initiated from an ATG codon present in the SV-poly vector.
• the expression vectors (SV14-2, SV14-1) were introduced into COS-1 cells by the DEAE-dextran transfection method (McCutchan and Pagano, ⁇ J. Natl Cancer Inst.
• the structural predictions of the tie cDNA sequence were tested by cloning the full-length tie protein coding region into the -EcoRI site of the pSVpoly expression vector (constructs pSV14-2 and pSV14-l) , and these expression vectors were then transfected into COS cells.
• the proteins produced by these two constructs differ in their signal sequence as noted above, but the predicted mature protein products are identical.
• Fig. 3 shows analysis of the immunoprecipitated radioactive polypeptides by SDS-polyacrylamide gel electrophoresis. As can be seen from Fig. 3A, the HI immune serum precipitated some weakly labeled polypeptides from untransfected COS cells. These polypeptides were probably not related to tie because the COS cells do not express its mRNA.
• Cells transfected with the pSV14 expression vector show an additional specific polypeptide of 117 kD (marked tie in figure 3) .
• This tie polypeptide was not precipitated with the preimmune serum or the antiserum blocked with the immunogen.
• the 117 kD polypeptide was recognized also by the MI antiserum against a carboxyl terminal peptide (Fig. 3B) .
• Immunoprecipitation of tie polypeptides from transfected COS cells metabolically labeled in the presence of tunicamycin to prevent N-linked glycosylation of proteins gave a specific polypeptide of approximately 105 kD apparent molecular weight (marked tie* in Fig. 3B) .
• tie in NIH3T3 cells The full-length tie cDNA was subcloned under the control of Moloney murine leukemia virus long terminal repeat promoter. This expression vector was used to co-transfect NIH3T3 cells with the pSVneol marker plasmid and G418 resistant clones were analyzed for tie expression. Cells on one confluent plate were lysed in 2.5% SDS, 125mM Tris, pH 6.5 for immunoblot analysis. Cell lysates were electrophoresed on SDS-page and electroblotted on nitrocellulose membrane.
• the membrane was incubated with the anti-peptide antiserum against the tie carboxyterminus and bound antibodies were visualized using horseradish peroxidase conjugated swine anti-rabbit antiserum (Dako) and ECL reagents (Amersham) .
• Tyrosine phosphorylated proteins were immunoprecipitated as described (Frackelton, et al., 1991, In T. Hunterand B. M. Sefton (ed.), Protein phosphorylation part B, Meth. Enzymol. 201:79-91).
• the 117 kD tie protein was detected by immunoblotting with the antiserum raised against the peptide corresponding the tie carboxyterminus (Fig. 4) .
• endogenous tie protein of a similar molecular weight was detected in PAE (porcine aortic endothelial) cells.
• the tie protein was also detected in anti-phosphotyrosine immunoprecipitates of the tie -transfected cells.
• the slides were developed with Kodak Dektol developer and Kodafix solution, and chromosomes were first G-banded with Wright-Giemsa stain (Cannizarro and Emanuel, Cytogenet . Cell Genet . , 38 : 308-309) , and if necessary, rebanded by the trypsin-Giemsa (GTG) technique.
• GTG trypsin-Giemsa
• In situ hybridization of radiolabeled tie probe to normal human metaphase chromosomes localized tie sequences to chromosome 1.
• a total of 317 chromosomally-localized grains were scored on 145 metaphases. Thirty-four percent (109/317) of the grains were on chromosome 1 with 69% (75/109) of chromosome 1 grains localized to Ip33-p34.
• Grain localization on chromosome 1 is illustrated schematically in Fig. 5, where each dot represents 3 grains. This narrows the localization to lp33 - p34, with the highest concentration of grains close to the border between bands lp33 and p34.
• Chromosomal localization using a panel of somatic mouse-human hybrid cell lines also placed the tie locus to human chromosome 1.
• the leukemia cells were grown in RPMI containing 10% FCS and antibiotics. Dami cells were cultivated in Iscoves modified DMEM with 10% horse serum.
• a permanent hybrid cell line (EA.hy926) obtained by fusing first-passage human umbilical vein endothelial cells with the A549 lung carcinoma cells (Edgell, et al., Proc . Natl . Acad . Sci . USA, 50 : 3734-3737) was cultured in DMEM-HAT medium containing 10% FCS and antibiotics.
• the PAE cells (a kind gift from Dr. Lena Claesson-Welsh, Ludwig Institute for Cancer Research, Uppsala, Sweden) were grown in Ham's F12 medium containing 10% FCS Poly(A)+ RNA was extracted from the cell lines as described in Sambrook, et al . , Molecular cloning - a laboratory manual, Cold Spring Harbor Laboratory Press, 1989. Five grams of the Poly(A)+ RNA samples were electrophoresed in agarose gels containing formaldehyde and blotted using standard conditions (Sambrook, et al. , supra) . The insert of the HE11-1 cD ⁇ A clone was labelled by the random priming method and hybridized to the blots.
• Hybridization was carried out in 50% formamide, 5 x Denhardt's solution (lOOx Denhardt's solution comprises 2% each of Ficoll, polyvinylpyrrolidone and bovine serum albumin), 5 x SSPE (3M ⁇ aCl, 200mM ⁇ aH2P04 . H20, 20 mM EDTA, pH 7.0), 0.1% SDS (sodium dodecyl sulphate), and 0.1 mg/ l of sonicated salmon sperm DNA at 42°C for 18-24 h.
• the filters were washed at 65°C in IxSSC (150 mM NaCl, 15mM sodium citrate, pH 7.0), 0,1% SDS and exposed to Kodak XAR-5 film.
• Figure 6 shows the results of analysis of tie mRNA expression in ten leukemia cell lines. Only the HEL erythroleukemia cells, KG-1 myeloid leukemia cells and Dami megakaryoblastic leukemia cells expressed a 4.4 kb tie mRNA, as detected with the 3.8 kb tie cDNA probe. The Jurkat and MOLT-4 T-cell leukemias, as well as HL-60 promyelocytic leukemia, U937 and RC-2A onocytic leukemias, JOK-1 hairy cell leukemia and ML-2 myeloid leukemia cells were negative for the tie mRNA.
• the tie mRNA was also induced after TPA treatment of the K562 cells, when the cells undergo megakaryoblastoid differentiation.
• porcine aortic endothelial cells PAE
• porcine aortic endothelial cells PAE
• EA.hy926 porcine aortic endothelial cell line
• the EA.hy926 cell line was created by the fusion of human umbilical vein endothelial cells with A549 lung carcinoma cell line.
• the A549 cells were negative for tie mRNA expression.
• the EA.hy926 cells expressed tie mRNA species of 3.9, 4.2 and 4.7 kb.
• the results of Northern blot analyses of the tie mRNA expression in cell lines are summarized in table 1.
• the hybridizations were carried out at 42°C for 24 h using 35 S-deoxy(thio)ATP-labeled probes, followed by washing, autoradiography at +4°C for 5-25 days, and staining of the sections with hematoxylin.
• the probe was labelled with [a- 35 P]dCTP by the random priming method.
• the nitrocellulose replicas of each phage-infected plate were hybridized in 50 % deionized formamide, 5 x Denhardt's solution, 5xSSPE, 0.1 % SDS and 100 mg/ml ssDNA. Seven positive clones were purified out of which four were subcloned into pGEM 3Zf(+) (Promega) and sequenced. DNA sequencing was performed by the dideoxy chain termination method of Sanger, .et al . , Proc . Natl . Acad . Sci .
• RNA (5 g) and total RNA (20 g) were electrophoresed in 0.8 % agarose gels containing formaldehyde and blotted into Hybond-N (Amersha ) filters using standard conditions. After transfer, the filters were exposed to ultraviolet radiation for 4 minutes, hybridized and washed in stringent conditions (Sambrook, et al . , Molecular cloning - a laboratory manual, Cold Spring Harbor Laboratory Press, 1989) . In situ hybridization of sections was performed according to Wilkinson, et al .
• RNA and polyadenylated RNA were isolated from various human fetal and adult tissues as well as mouse tissues and subjected to Northern blotting and hybridization with the tie cDNA probes.
• Figure 11A shows that all fetal human tissues tested contain a 4.4 kb tie mRNA.
• the tie signal is most prominent in the highly vascularized lung, placenta and heart, but a weaker signal can also be recognized in other tissues as well, particularly in long exposure of the autoradiogram (Fig. 11B)
• the expression of tie begins very early; from 9 to 10 days of gestation tie is expressed weakly, then the number of tie transcripts increases (maximum at 14 days gestation) .
• FIG. 12A shows the brightfield image of a representative section probed with antisense RNA.
• Figure 12A illustrates that the autoradiographic grains decorate the linings of major blood vessels. These signals were, however, better visualized in the darkfield microscopy of the same section (Fig. 12B) .
• the cells responsible for tie expression were endothelial cells as shown by Factor VIII immunostaining, which is specific to endothelial cells.
• the sense probe did not give signals above background, as can be seen from Fig. 12C.
• the tie hybridization signal in a 8-day p.c. mouse placenta (Fig. 13A) was very similar and practically superi posable with the pattern of factor VIII staining of adjacent sections (Fig. 13B) .
• Lys Asp Asp Arg lie Val Arg Thr Pro Pro Gly Pro Pro Leu Arg Leu 65 70 75 80
• Leu Tyr lie Ala lie Glu Tyr Ala Pro Tyr Gly Asn Leu Leu Asp Phe 915 920 925
• Val Leu Leu Trp Glu lie Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly 1045 1050 1055
• Lys Asp Asp Arg lie Val Arg Thr Pro Pro Gly Pro Pro Leu Arg Leu 65 70 75 80
• GTGTCTGCCA CCTGCCTCAC CATCCTGGCC GCCCTTTTAA CCCTGGTGTG CATCCGCAGA 2400 AGCTGCCTGC ATCGGAGACG CACCTTCACC TACCAGTCAG GCTCGGGCGA GGAGACCATC 2460
• CTGAGCTACC CAGTGCTAGA GTGGGAGGAC ATCACCTTTG AGGACCTCAT CGGGGAGGGG 2580
• CTTAAGCTGC CTCAAGGAAT TTTTTTAACT TAAGGGAGAA AAAAAGGGAT CTGGGGATGG 3600
• AAATTGGGGC ATCACCCCAA CATCATCAAC CTCCTGGGGG CCTGTAAGAA CCGAGGTTAC 2640

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