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WO1994010197A1 - Nouvelle proteine tyrosine-kinase - Google Patents

Nouvelle proteine tyrosine-kinase Download PDF

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Publication number
WO1994010197A1
WO1994010197A1 PCT/AU1993/000560 AU9300560W WO9410197A1 WO 1994010197 A1 WO1994010197 A1 WO 1994010197A1 AU 9300560 W AU9300560 W AU 9300560W WO 9410197 A1 WO9410197 A1 WO 9410197A1
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Prior art keywords
extracellular domain
domain
jil
protein
repeats
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PCT/AU1993/000560
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English (en)
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Andrew Frederick Wilks
Andrew Stewart Runting
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Ludwig Institute For Cancer Research
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Application filed by Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to AU53663/94A priority Critical patent/AU678413B2/en
Priority to JP6510469A priority patent/JPH08504760A/ja
Priority to EP93923978A priority patent/EP0674658A4/fr
Publication of WO1994010197A1 publication Critical patent/WO1994010197A1/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
    • 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
    • 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
    • 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 proteins having receptor-type tyrosine kinase-like properties which represent a novel sub-family of proteins related to protein tyrosine kinases.
  • the present invention relates to the full length proteins and to subunits, mutants, derivatives and /or analogues thereof and to nucleotide sequences encoding same.
  • the present invention also extends to ligands for the above proteins and to pharmaceutical compositions comprising the proteins and/or mutants, derivatives and/or analogues thereof and/or ligands thereto.
  • an intracellular tyrosine kinase domain a single hydrophobic transmembrane domain and an extracellular domain, usually composed of a particular constellation of structural domains, of which the Immunoglobulin-like (Ig) domain (9), the fibronectin type III (FNIII) repeat (10) and a number of distinct cysteine rich domains (11, 12, 13) are the most widely known.
  • Ig Immunoglobulin-like domain
  • FNIII fibronectin type III
  • cysteine rich domains 11, 12, 13
  • one aspect of the present invention provides an isolated protein having receptor-type protein tyrosine kinase (RTK)-like properties including an extracellular domain, wherein said extracellular domain comprises at least three fibronectin (FN) type III repeats, an immunoglobulin (Ig)-like domain and an epidermal growth factor (EGF)-like domain, with the proviso that said RTK is not tie
  • RTK receptor-type protein tyrosine kinase
  • the extracellular domain comprises at least three FN type III repeats N- terminal to a transmembrane domain and at least two Ig-like domains which flank cysteine-rich EGF-like repeats.
  • the extracellular domain comprises at least three cysteine-rich EGF-like repeats.
  • Another aspect of the present invention contemplates an isolated protein: (i) having RTK-like properties including an extracellular domain, wherein said extracellular domain comprises at least three FN type III repeats, an Ig-like domain and an EGF-like domain; (ii) which is encoded by a nucleotide sequence capable of hybridising under low stringent conditions to all or a part of the nucleotide sequence set forth in Figure 1; and (iii) which is not tie
  • the polypeptide comprises at least three cysteine-rich EGF-like repeats.
  • Yet another aspect of the present invention relates to an isolated nucleic acid molecule:
  • III repeats an Ig-like domain and an EGF-like domain; (ii) which hybridises under low stringent conditions to the nucleotide sequence set forth in Figure 1; and (iii) which is not tie
  • Figure 1 is a representation of the nucleotide and predicted amino acid sequence of the JEL cDNA.
  • the pJIL clone was then used to screen a mouse lung cDNA library (Stratagene Catalogue # 936307, Stratagene, CA) using conventional techniques (28). Overlapping clones obtained from screening the library three times were sequenced as above in both directions, either by the creation of nested deletions using the Erase-A-Base system (Promega, WI), or by the production of specific oligonucleotide primers.
  • the putative translation initiation codon (ATG) at nucleotide position 16 has been designated amino acid number 1 in the translated sequence.
  • Figure 2 is a photographic representation showing northern Analysis of JIL.
  • mRNA from mouse tissues was isolated using standard methods (29) and poly A+ selected using Oligo-dT sepharose. 1 ⁇ g of mRNA was subjected to electrophoresis in a 1% agarose gel containing formaldehyde and transferred to nitrocellulose (Hybond-C, Amersham International, U.K.) in 20x SSC (lx SSC is 0.15M Sodium Chloride / 0.015M Sodium Citrate, pH 7.0).
  • An antisense riboprobe was transcribed from a clone of JIL which encoded the extracellular portion of the protein using a Message Maker kit (Bresatec, Sydney, Australia) and ⁇ - ⁇ P-UPT (Amersham International, U.K.).
  • Hybridisation was carried out at 42 °C in 50% formamide containing 4x Denhardt's solution (lx Denhardt's solution is 0.02% each of bovine serum albumin / Ficoll / polyvinylpyrrolidone), 5x SSC, 100 ⁇ g/ml denatured herring sperm DNA and 4 mM EDTA After hybridisation for 16 hours and washing for three hours at 65 ⁇ C in 0.1% SDS, O.lx SSC, the membrane was treated with 1 ⁇ g/ml RNaseA in 2x SSC at room temperature for five minutes, and then washed for one hour in O.lx SSC, 0.1% SDS at 50 ⁇ C.
  • 4x Denhardt's solution is 0.02% each of bovine serum albumin / Ficoll / polyvinylpyrrolidone
  • 5x SSC 100 ⁇ g/ml denatured herring sperm DNA and 4 mM EDTA
  • the membrane was treated with 1 ⁇ g/m
  • the treated membrane was then exposed to a Phosphorimager Screen (Molecular Dynamics, Sunnyvale, CA) for 48 hours and the signal detected using a Phosphorimager Analyser (Molecular Dynamics, Sunnyvale, CA) and v3.2 Phosphorimager software. Arrows indicate the position of the 28S and 18S subunits.
  • Figure 3 is a representation showing alignment of the kinase domains of mouse JIL (amino acids 837-1116) and human tie (18) (amino acids 822 - 1090). Residues conserved between the two species are shown by asterisks.
  • Figure 4 is a representation comparing the EGF homology domains of mouse JIL (amino acids 214-346) and human tie (18) (amino acids 210-342). Residues conserved between the EGF homology domains in the two proteins are shown by asterisks. conserveed cysteine residues are shown in bold type. Gaps represented by a period were introduced to align the cysteine residues.
  • Figure 5 is a diagrammatic representation of the arrangement of structural features in JIL.
  • the present invention is directed to proteins including polypeptides which have RTK-like properties including an extracellular domain wherein said extracellular domain comprises at least three fibronectin (FN) type III repeats, an immunoglobulin (Ig)-like domain and an epidermal growth factor (EGF)-like domain, provided said RTK is not tie.
  • proteins include recombinant or synthetic molecules and further include sub-units, fragments, mutants, derivatives, analogues and homologues thereof.
  • the proteins of the present invention differ from these known RTKs by the arrangement of the structural domains in the extracellular domain by having at least three FN type III repeats, an Ig domain and an EGF-like domain.
  • the present invention provides an extracellular domain of an RTK or an RTK having said extracellular domain comprising at least three and not more than five FN type III repeats N-terminal to a transmembrane domain and at least two and not more than four Ig-like domains which flank cysteine rich EGF-like repeats.
  • the molecule comprises at least three and not more than six cysteine rich EGF-like repeats.
  • JIL The RTK of the present invention is designated herein "JIL".
  • the present invention extends to the sub-family of RTKs represented by JIL and JIL-like molecules but does not extend to the RTK tie Accordingly, reference herein to “JIL” extends to this sub-family but excluding tie
  • JIL also encompasses sub-units, fragments, mutants, derivatives, homologues and analogues of JIL as discussed below.
  • the JIL molecule is a biologically pure and isolated preparation meaning that it has undergone some purification away from other proteins and/or non-proteinacious material.
  • the purity of the preparation may be represented as at least 40% JIL, preferably at least 60% JIL, more preferably at least 75% JIL, even more preferably at least 85% JIL and still more preferably at least 95% JIL relative to non-JIL material as determined by weight, activity, amino acid homology, antibody reactivity or other convenient means.
  • the JIL of the present invention may be naturally occurring or may be synthetic meaning that it is prepared by recombinant DNA or chemical synthetic techniques.
  • the present invention encompasses JIL molecules having the naturally occurring amino acid sequence as well as molecules having single or multiple amino acid substitutions, deletions and/or additions.
  • Amino acid insertional derivatives of the JIL of the present invention include amino and /or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table 1:
  • JIL is derivatised by amino acid substitution
  • the amino acids are generally replaced by other amino acids having like properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.
  • Amino acid substitutions are typically of single residues.
  • Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues.
  • deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
  • the amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield synthesis) and the like, or by recombinant DNA manipulations.
  • substitution mutations at predetermined sites in DNA having known or partially known sequence include, for example, Ml 3 mutagenesis.
  • the manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently elsewhere described (for example, see ref 28).
  • recombinant or synthetic mutants and derivatives of the JIL of the present invention include single or multiple substitutions, deletions and/or additions of any molecule naturally or artificially associated with the molecule such as carbohydrates, lipids and/or proteins or polypeptides.
  • different glycosylation patterns or elimination of glycosylation can result from expressing the JIL in different host cells.
  • reference herein to "mammalian JIL” or “mammalian RTK” includes RTK produced by recombinant means in bacteria or in animals cells or produced by chemical synthetic means. Accordingly, a mammalian JIL or RTK is a RTK of mammalian origin but not necessarily produced in mammalian cells.
  • mutants include truncated mutants, i.e. a JIL molecule absent N- terminal and/or C-terminal portions conveniently made using cDNA molecules truncated at the 5' and/or 3' ends, respectively.
  • the present invention extends to subunits or fragments of mammalian JIL carrying one or more of the extracellular domain, transmembrane domain and/or cytoplasmic domain (also referred to as kinase catalytic domain or intracellular domain).
  • a subunit or fragment containing the extracellular domain is particularly useful for screening for ligands of JIL or antagonists to JIL-ligand binding or may be useful as an antagonist itself.
  • the present invention extends to a subunit or fragment of mammalian JIL containing the extracellular domain or portion or derivative thereof.
  • subunit or fragment is meant a non-full length JIL molecule.
  • the subunit or fragment is the extracellular domain portion of JIL.
  • the present invention extends to an isolated extracellular domain or part or derivative thereof, said domain characterised in that it is isolatable from mammalian JIL and comprises at least three FN type III repeats, an Ig-like domain and an EGF- like domain.
  • the FN type III repeats are N-terminal to a transmembrane domain and at least two Ig-like domains flank cysteine-rich EGF-like repeats. Most preferably, there are at least three cysteine-rich EGF-like repeats.
  • a hybrid JIL molecule includes a molecule with at least part of one domain from a JIL from a first species of mammal fused or otherwise associated with at least part of another domain from a JIL from a second different species of mammal.
  • the extracellular domain or part thereof of human JIL may be fused or associated with other domains of mouse JIL.
  • the JIL hybrid or fusion molecules may be with regions of growth factor receptors such as Epidermal Growth Factor Receptor (EGFR).
  • EGFR Epidermal Growth Factor Receptor
  • the present invention also extends to functional chemical equivalents or analogues of JIL herein described.
  • Analogues of the JIL protein contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatising the molecule and the use of crosslinkers and other methods which impose conformational constraints on the molecule.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6 trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed by reduction with NaBH
  • the guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via Oacylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • peptides could be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention also extends to fragments, derivatives, homologues, analogues and immunological, functional and/or structural relatives of the JIL contemplated herein. Accordingly, reference herein to JIL, JIL molecules or JIL-like molecules is to be taken as covering the full length molecule or any sub-units, fragments, derivatives, homologues, analogues, and/or relatives thereof including proteins having JIL-like properties.
  • the JIL of the present invention has an amino acid sequence substantially as set forth in Figure 1, or having amino acid similarity to all or a region thereof such as in the order of at least 50- 70%, preferably at least 80% and most preferably at least 90% provided that the JIL or JIL-like molecule does not extend to tie
  • the JIL molecule is approximately 1120-1125 amino acids, in length with a molecular weight of the unglycosylated form of from about 120,000 to about 130,000 daltons as determined by SDS-PAGE.
  • the glycosylated form of the molecule has a molecular weight in the range 135,000-145,000 daltons as determined by SDS-PAGE.
  • the glycosylated form of the molecule includes both natural glycosylation patterns and altered glycosylation patterns.
  • the JIL of the present invention may be of normal cell origin or may be from a genetically modified cell such as, for example, tumour cells.
  • Types of cells carrying the JIL molecule include but are not limited to cells from one or more of the following sources: colon, kidney, brain, placenta, ovary, lung, thymus and spleen.
  • the JIL protein may conveniently be defined by reference to the ability for an encoding nucleotide sequence to hybridise to the sequence in Figure 1. According to this aspect of the present invention, there is provided an isolated protein:
  • RTK-like properties including an extracellular domain, wherein said extracellular domain comprises at least three FN type III repeats, an Ig-like domain and an EGF-like domain;
  • conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25-0.5% w/v SDS at ⁇ 45 ⁇ C for 2-3 hours or high stringent conditions as disclosed by Sambrook et al (28).
  • the JIL and/or nucleic acid molecules encoding same of the present invention have important utility in modulating growth and differentiation of cells. Accordingly, the present invention extends to the ligand for JIL and to any agonists and antagonists of the JIL. Since the JIL or a genetically modified form thereof may be an oncogenic protein, antagonists to the JIL are of particular relevance. Such antagonists include antibodies (monoclonal or polyclonal), the enzyme itself in soluble form including its extracellular domain, specific peptides or proteins and/or carbohydrates amongst others (e.g. the fragments, derivatives, analogues, homologues and relatives of JIL as contemplated above).
  • a method of inhibiting, reducing or otherwise interfering with interaction between a protein having JIL-like properties and a ligand thereof in a mammal comprising the administration of a ligand binding interfering effective amount of an antagonist to said ligand interaction for a time and under conditions sufficient to inhibit, reduce or otherwise interfere with said interaction.
  • the present invention extends to agonists to JIL which facilitate ligand-JIL interaction and to pharmaceutical compositions comprising same.
  • the active molecules of the pharmaceutical composition are contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the animal and the active molecule. For example, from about 0.05 ⁇ g to about 20 mg of JIL ligand or JIL-ligand antagonist may be administered per kilogram of body weight per day to distrupt JIL- ligand interaction. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Sterile injectable solutions are prepared by incorporating the active molecules in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-diying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in the pharmaceutical compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared,so that an oral dosage unit form contains between about 0.05 ug and 20 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as pepper
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutical compositions is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions is contemplated. Supplementary active molecules can also be incorporated into the compositions.
  • the present invention also extends to nucleic acid molecules in the form of oligonucleotide probes or primers useful for detecting genomic sequences encoding a mammalian JIL molecule and in particular human JIL. More particularly, the oligonucleotide probes will be specific to particular regions of the genomic sequence such as those sequences encoding the extracellular domain, transmembrane domain or intracellular domain (including kinase catalytic domain) of JEL. Even more particularly, the oligonucleotide probes will be useful in screening a genomic sequence for abnormalities in relation to the JIL coding sequence which result in an abnormal or mutant JIL which might in turn result in or facilitate JIL related tumours or sarcomas.
  • Another aspect of the present invention contemplates an assay for identifying or otherwise diagnosing abnormalities in JIL or for identifying or otherwise screening for a normal JIL molecule in a human.
  • a source of genetic material is isolated from a human subject to be tested and subjected to Southern blot analysis, Northern blot analysis, Western blot analysis, radioimmunoassay (RIA) and other immunological techniques or variations or combinations of such analyses.
  • a method for detecting an abnormal genomic coding sequence for a protein having JIL-like properties in a human subject comprising contacting a genetic sample from said human subject with one or more oligonucleotide primers specific for a part of the naturally occurring genomic sequence for said protein or for an abnormal coding sequence for said protein for a time and under conditions sufficient for said oligonucleotides to hybridise to said genomic sequence and then screening for said hybridisation.
  • a human subject is screened for a normal or abnormal JIL gene by isolating a genetic sample including genomic DNA from said human subject, subjecting said genetic sample to restriction endonuclease digestion to produce digested or partially digested DNA, subjecting said digested DNA to electrophoresis to separate the digested DNA based on length of fragments in the DNA digestion and screening the separated DNA digest to Southern blot analysis to screen for the presence or absence of particular regions of the JIL gene.
  • an oligonucleotide probe can be generated capable of screening for a nucleotide sequence corresponding to a "normal" extracellular region of JIL.
  • the restriction pattern of this region may alter or contain deleted or duplicated sequences. Such an assay will screen for these modifications.
  • abnormal JIL is defined inter alia at the genetic level as an alteration in the nucleotide sequence encoding normal JIL such as to result in a JIL molecule with an altered amino acid sequence such as an insertion, deletion and/or substitution.
  • the altered JIL may also have a different glycosylation pattern relative to the naturally occurring (i.e. normal) JIL molecule. Such a change in glycosylation patterns can result from a change in a single amino acid residue.
  • An "abnormal JIL” can be defined inter alia at the functional level as a molecule having altered ligand binding characteristics. Frequently, this can result in a tumour or sarcoma or a predisposition thereto.
  • the human subject may be an adult, adolescent, child, infant or a foetus.
  • the assay may be particularly useful in screening members of a family with a pre ⁇ disposition to cancer based on a defective or modified JIL molecule.
  • the ligand may be isolated in any number of ways including screening a cDNA library with a labelled receptor.
  • Yet another embodiment of the present invention extends to the use of JIL to phosphorylate tyrosine residues on a protein substrate. This will be useful for in vitro labelling.
  • a PCR screen of colonic cDNA led to the isolation of 2 individual isolates of a protype PTK-related clone, 6S-5. This clone was subsequently renamed pJIL. This clone was used to screen a mouse lung cDNA library (see legend to Figure 1). Overlapping clones encoding a significant portion of the cDNA sequence were isolated, and further screening of the library with probes closest to the 5' end of the accumulated known sequence were used to rescreen the same library. Overlapping clones encoding the entire cDNA sequence of 4364 base pairs were thereby obtained. These clones were derived from a randomly primed adult mouse lung cDNA library and are consistent with the existence of a single transcript in this tissue. The complete nucleotide sequence of murine JIL covered by these clones is shown in Figure 1.
  • This kinase domain exhibits a high degree of sequence identity (82%) to a human clone (JTK 14; ref 17) named fie (18).
  • the "insert” domain does not appear to have any consensus sequences for the binding of other signal transduction molecules.
  • the extracellular component of JIL contains five potential N-linked glycosylation sites, (-NXT/S-) and several structural features in common with tie, even though the identity with tie in this area is low (31%).
  • Ig domains the first located between amino acids 44 and 102, and the second between amino acids 370 and 424, characterised by the presence of cysteine residues surrounded by a consensus sequence from the C2 set (similar to N-cam)(9) are present in the extracellular structure.
  • EGF like motifs are located in the N-terminal portion of the extracellular domain (amino acids 210-342). This type of motif is a feature of the structures of several other classes of protein such as the growth factors EGF (20) and CRIPTO (21), the cell surface proteins notch (22) and lin 12 (23). The function of these motifs in the context of the JIL protein remains to be established.
  • the most C-terminal portion of the extracellular domain bears three domains with homology to the FNIII repeat structure.
  • This element is characteristic of a number of different classes of transmembrane proteins, including other growth factor receptors (24) and the tumour suppressor gene DCC (25).
  • JIL JIL-like protein
  • Northern analysis see Figure 2. JIL exhibits its highest level of expression in lung, with lower levels of transcript found in heart and kidney. A single transcript of approximately 4.5 kB, was detected in each tissue expressing JIL, consistent with the size of the cDNA isolated. It is concluded, therefore, that the sequence of JIL presented in Figure 1 represents the major transcript in adult issues and that JIL appears to be expressed predominantly in tissues with a high content of endothelial cells.
  • the structure of the protein encoded by JIL cDNAs bears a resemblance to that of the tie protein (18).
  • the PTK catalytic domains of these two proteins exhibits 82% amino acid sequence identity (see Figure 3).
  • the extracellular domains are significantly less well conserved (31% amino acid sequence identity) and it is most likely that these two genes are distinct from one another.
  • the constellation of structural domains found in the extracellular domains of these two receptors is very similar and distinct from that found in any other class of growth factor receptor.
  • both tie and JIL have three fibronectin type III repeats N-terminal to the transmembrane domain.
  • both of these receptors have two immunoglobulin-like (Ig.) domains which flank three cysteine rich epidermal growth factor like (EGF-like) repeats (Figure 4).
  • Figure 5 shows the structure of this class of RTK's. The evolution of this class of molecules has thus apparently occurred by the admixture of at least four distinct structural domains found elsewhere, namely, the PTK domain (21), fibronectin type III repeats (10), Ig domain (9) and EGF like repeat (20).

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Abstract

La présente invention se rapporte à des protéines présentant des propriétés analogues à celles de la tyrosine-kinase de type récepteur, qui représentent une nouvelle sous-famille de protéines apparentées aux protéines tyrosines-kinases. La présente invention se rapporte également à des protéines complètes et à des sous-unités, des mutants, des dérivés et/ou analogues de ces protéines, ainsi qu'à des séquences nucléotidiques qui les codent. La présente invention se rapporte en outre à des ligands destinés à ces protéines, et à des compositions pharmaceutiques comprenant les protéines et/ou les mutants, leurs dérivés et/ou leurs analogues, et/ou des ligands qui leur sont destinés.
PCT/AU1993/000560 1992-10-30 1993-10-29 Nouvelle proteine tyrosine-kinase WO1994010197A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU53663/94A AU678413B2 (en) 1992-10-30 1993-10-29 A novel protein tyrosine kinase
JP6510469A JPH08504760A (ja) 1992-10-30 1993-10-29 新規なタンパク質チロシンキナーゼ
EP93923978A EP0674658A4 (fr) 1992-10-30 1993-10-29 Nouvelle proteine tyrosine-kinase.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPL5581 1992-10-30
AUPL558192 1992-10-30

Publications (1)

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WO1994010197A1 true WO1994010197A1 (fr) 1994-05-11

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PCT/AU1993/000560 WO1994010197A1 (fr) 1992-10-30 1993-10-29 Nouvelle proteine tyrosine-kinase

Country Status (3)

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EP (1) EP0674658A4 (fr)
JP (1) JPH08504760A (fr)
WO (1) WO1994010197A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585269A (en) * 1994-06-02 1996-12-17 The University Of North Carolina At Chapel Hill Isolated DNA encoding c-mer protooncogene
WO1997007209A3 (fr) * 1995-08-21 1997-05-15 Imperial College Recepteur
US6521424B2 (en) 1999-06-07 2003-02-18 Immunex Corporation Recombinant expression of Tek antagonists
US7485297B2 (en) 2003-08-12 2009-02-03 Dyax Corp. Method of inhibition of vascular development using an antibody

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3353293A (en) * 1992-01-09 1993-08-03 Helsinki University Holding, Ltd. Tie, a novel endothelial cell receptor tyrosine kinase

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
US5466596A (en) * 1992-08-25 1995-11-14 Mount Sinai Hospital Corporation Tissue specific transcriptional regulatory element

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Publication number Priority date Publication date Assignee Title
AU3353293A (en) * 1992-01-09 1993-08-03 Helsinki University Holding, Ltd. Tie, a novel endothelial cell receptor tyrosine kinase

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Biochemical and Biophysical Research Communications, Vol. 189, No. 3, 1992 (December 30), pp. 1747-1753, "A Novel Tyrosine Kinase, Hyk, Expressed in Murine Embryonic Stem Cells" (see entire documents). *
Oncogene, Vol. 7, 1992, pp. 1471-1480, DUMONT, J.D. et al., "Tek, a Novel Tyrosine Kinase Gene Located on Mouse Chromosome 4, is Expressed in Endothelial Cells and their Presumptive Precursors" (whole document, see in particular fig. 1(a) and pages 1476-1478). *
Oncogene, Vol. 8, 1993, pp. 1293-1301, DUMONT, D.J. et al., "The Endothelial-Specific Receptor Tyrosine Kinase, Tek, is a Member of a New Subfamily of Receptors" (see entire document). *
Oncogene, Vol. 8, 1993, pp. 663-670, ZIEGLER, S.F. et al., "Molecular Cloning and Characterization of a Novel Receptor Protein Tyrosine Kinase from Human Placenta" (see entire document). *
See also references of EP0674658A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585269A (en) * 1994-06-02 1996-12-17 The University Of North Carolina At Chapel Hill Isolated DNA encoding c-mer protooncogene
WO1997007209A3 (fr) * 1995-08-21 1997-05-15 Imperial College Recepteur
US6521424B2 (en) 1999-06-07 2003-02-18 Immunex Corporation Recombinant expression of Tek antagonists
US7067475B2 (en) 1999-06-07 2006-06-27 Immunex Corporation Tek antagonists
US7485297B2 (en) 2003-08-12 2009-02-03 Dyax Corp. Method of inhibition of vascular development using an antibody

Also Published As

Publication number Publication date
JPH08504760A (ja) 1996-05-21
EP0674658A1 (fr) 1995-10-04
EP0674658A4 (fr) 1997-12-03

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