WO1994010197A1

WO1994010197A1 - A novel protein tyrosine kinase

Info

Publication number: WO1994010197A1
Application number: PCT/AU1993/000560
Authority: WO
Inventors: Andrew Frederick Wilks; Andrew Stewart Runting
Original assignee: Ludwig Institute For Cancer Research
Priority date: 1992-10-30
Filing date: 1993-10-29
Publication date: 1994-05-11
Also published as: JPH08504760A; EP0674658A1; EP0674658A4

Abstract

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.

Description

A NOVEL PROTEIN TYROSINE KINASE

FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION The role of growth factors and their receptors in the processes of cellular growth and development is well documented in organisms as widely divergent as the fruit fly (1,2), nematode worm (3), mouse (4,5) and human (6). Of particular importance in this respect, are members of the growth factor receptor protein tyrosine kinase (RTK) family (7,8). To date, there are in excess of fifty members of this family, each of which share a number of characteristic features, namely: 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. The arrangement of the various sub-domains found in the extracellular portion of this class of molecules is characteristic of each sub-family of RTK. One example of an RTK has the designation "tie" (18).

Due to the role of members of the RTK family in various cellular processes and their potential use in the development of therapeutics and diagnostics, it is important to further identify new members of this family. In accordance with the present invention, a new sub-family of RTK's has been discovered.

SUMMARY OF THE INVENTION

Accordingly, 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

Preferably, 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.

Preferably, 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

Still another aspect of the present invention is directed to an isolated polypeptide having extracellular domain-like properties of an RTK wherein said isolated polypeptide comprises three FN type III repeats, an Ig-like domain and an EGF-like domain. Preferably, the polypeptide comprises at least three FN type III repeats and at least two Ig-like domains which flank cysteine-rich EGF-like repeats.

Preferably, 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:

(i) encoding a protein 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 hybridises under low stringent conditions to the nucleotide sequence set forth in Figure 1; and (iii) which is not tie

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a representation of the nucleotide and predicted amino acid sequence of the JEL cDNA. Poly A+ mRNA from neonatal mouse colon prepared using 'Fast Track" mRNA isolation kit (Amersham International, U.K.) underwent reverse transcription using a cDNA synthesis kit (Amersham International, U.K.). This cDNA was amplified using PCR and degenerate oligonucleotides as described elsewhere (14). Clones were sequenced using the dideoxy chain termination method (27) using Sequenase V2.0 (United States Biochemical, OH) and [[cr³⁵Si]thio]dATP (Amersham International, U.K.). 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. Cysteine residues in the extracellular region are shown in shaded boxes and arrows indicate the borders of the tyrosine kinase domain (TK), interrupted by the Kinase Insert (KI). The transmembrane domain (TM) is underlined. N-linked glycosylation sites (-N-X-S/T-) have lines marked over the residues, and rectangles outline the EGF homology areas (EGFH 1-3). The FNIII repeats are designated FNIIIa FNIIIb and FNIIIc. The ATP-binding site (-G-X-G- X-X-G-) is indicated by inverted triangles.

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 μg 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, Adelaide, Australia) and α-^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 μg/ml denatured herring sperm DNA and 4 mM EDTA After hybridisation for 16 hours and washing for three hours at 65 ^βC in 0.1% SDS, O.lx SSC, the membrane was treated with 1 μg/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 ^βC. 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. Conserved 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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. Such proteins include recombinant or synthetic molecules and further include sub-units, fragments, mutants, derivatives, analogues and homologues thereof. The proteins of the present invention have "RTK-like properties" since by comparison with known RTKs, they possess an intracellular tyrosine kinase domain, a single hydrophobic transmembrane domain and an extracellular domain, usually composed of structural domains such as an Ig domain, an FN type III repeat and a cysteine-rich domain.

However, 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.

More particularly, 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. Preferably, the molecule comprises at least three and not more than six cysteine rich EGF-like repeats.

The RTK of the present invention is designated herein "JIL". The present invention, however, 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 The designation "JIL" also encompasses sub-units, fragments, mutants, derivatives, homologues and analogues of JIL as discussed below.

Although the present invention is exemplified by a murine JIL, the subject invention extends to JIL homologues from other mammalian species with like characteristics as described herein in relation to murine JIL. All such JEL molecules homologous to murine JIL are encompassed by the present invention. Preferred mammalian sources of JIL and JIL-like molecules include humans, livestock animals (e.g. sheep, goats, bovine animals, horses and pigs), companion animals (e.g. cats and dogs), laboratory test animals (e.g. rabbits and murine animals) and wild or free range animals.

Advantageously, 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. In any event, 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:

TABLE 1 Suitable residues for amino acid substitutions

here 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. Preferably, 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. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and 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).

Other examples of 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. For example, different glycosylation patterns or elimination of glycosylation can result from expressing the JIL in different host cells. It should be noted that 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.

Particularly useful 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. Furthermore, 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. Accordingly, the present invention extends to a subunit or fragment of mammalian JIL containing the extracellular domain or portion or derivative thereof. By "subunit" or "fragment" is meant a non-full length JIL molecule. Preferably, the subunit or fragment is the extracellular domain portion of JIL. More particularly, 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. Preferably, 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.

Other useful mutants include hybrid molecules and fusion molecules. 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. For example, the extracellular domain or part thereof of human JIL may be fused or associated with other domains of mouse JIL. Alternatively, the JIL hybrid or fusion molecules may be with regions of growth factor receptors such as Epidermal Growth Factor Receptor (EGFR).

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. Examples of 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. Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH2)_n spacer groups with n = 1 to n = 6, glutaraldehyde, N-hydroxysuccinimide esters and hetero- bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety or carbodiimide. In addition, peptides could be conformationally constrained by, for example, incorporation of C_β and N_β-methylamino acids, introduction of double bonds between C_β 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. In its most preferred form, 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 present invention is also directed to nucleic acid molecules encoding JIL including its fragments, derivatives, analogues, homologues and/or relatives. The nucleic acid molecules may be RNA or DNA (e.g. cDNA), single or double stranded, hnear or a covalently closed circle. The nucleotide sequence may correspond to the naturally occurring sequence or may contain single or multiple nucleotide substitutions, deletions and/or additions. The nucleic acid molecules may also be part of a vector such -as an expression and/ or cloning vector and may contain extraneous nucleic acid material encoding a signal peptide, fusion peptide, purification peptide and/or marker peptide. The preferred nucleotide sequence is set forth in Figure 1 and includes molecules having at least 50-70%, preferably at least 80% and most preferably at least 90% similarity to all or a region thereof provided that such molecules to not include tie

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:

(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) 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 In a related embodiment, there is provided an isolated nucleotic acid molecule: (i) encoding a protein 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 hybridises under low stringent conditions to the nucleotide sequence set forth in Figure 1; and (iii) which is not tie.

The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al (28) at pp 387- 389 which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1- 0.5% w/v SDS at 37-45 ^βC for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative 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 ^βC 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). These types of antagonists are useful in developing ariti-tvunour agents where the growth or maintenance of the tumour itself is supported by the JIL of the present invention. Accordingly, the addition of an effective amount of an antagonist to the tumour-associated JIL will inhibit, reduce or otherwise interfere with JIL activity and thus prevent, reduce and/or inhibit tumour growth. The present invention, therefore, also extends to pharmaceutical compositions comprising one or more antagonists to JIL. According to this aspect of the present invention, there is contemplated a method of inhibiting, reducing or otherwise interfering with interaction between a protein having JIL-like properties and a ligand thereof in a mammal said method 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.

In other circumstances, however, it may be useful to promote ligand-JIL binding and /or interaction. Accordingly, the present invention extends to agonists to JIL which facilitate ligand-JIL interaction and to pharmaceutical compositions comprising same.

Accordingly, the present invention contemplates a pharmaceutical composition comprising as active ingredient, JIL or fragments, parts or derivatives thereof, JIL fusion or hybrid molecules, JIL ligands, JIL-ligand antagonists and/or JIL-ligand agonists, depending on the condition to be treated. For example, a JIL-ligand antagonist or a JIL ligand may be useful as an anti-cancer agent such as for treatment of carcinomas. For convenience, and as a short hand notation for the following description of pharmaceutical compositions, all of the above molecules and referred to herein after as "active molecules". The use of the term "active molecule(s)" therefore should be read as one or more of the above molecules depending on the condition to be treated.

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 μg 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 active molecules may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active molecules may be required to be coated in a material to protect said molecules from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, a low lipophilicity of JIL or its ligands may allow these to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. Accordingly, in order to administer the pharmaceutical composition by other than parenteral administration, the active molecules may be coated by, or administered with, a material to prevent its inactivation.

The active molecules may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

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. In all cases 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. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example.

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. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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.

When the active molecules are suitably protected as described above, the pharmaceutical composition may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the 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. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active molecule, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

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.

Still yet another aspect of the present invention is directed to the use of mammalian JIL or antagonists or agonists thereof in the manufacture of a medicament for the treatment against cancer or tumour growth.

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. In accordance with this aspect of the invention, 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.

In one embodiment, there is provided a method for detecting an abnormal genomic coding sequence for a protein having JIL-like properties in a human subject said method 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.

In a more particular embodiment, 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. For example, an oligonucleotide probe can be generated capable of screening for a nucleotide sequence corresponding to a "normal" extracellular region of JIL. In an abnormal JIL, the restriction pattern of this region may alter or contain deleted or duplicated sequences. Such an assay will screen for these modifications.

An "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.

Another embodiment relates to a ligand to JIL. 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.

The present invention is further described by reference to the following Examples. EXAMPLE 1 Isolation and Sequence Analysis of Murine JIL

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.

The structural features of the protein encoded by the JIL cDNA are as follows. At the 5' end of the clone, a start codon is found at position 16 embedded in a good Kozak consensus sequence (19), namely -GGAAGTATGG-. This initiates an open reading frame of 1122 amino acids terminated at position 3381. A hydrophobic transmembrane domain bisects the protein at residues 746 to 771. The intracellular portion of the protein contains all the features of a protein tyrosine kinase with a 14 residue kinase insert (amino acids. 921-933). 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%).

Two 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.

Three 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).

EXAMPLE 2 Expression of JIL mRNA

Expression of JIL mRNA has been examined by 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.

EXAMPLE 3 Analysis of JIL

SDS-PAGE analysis of unglycosylated murine JIL showed it to have a molecular weight of approximately 126,000 daltons. Molecular weight of the glycosylated form is about 140,000 daltons. Amino acid analysis showed the molecule to be 1122 amino acids in length. EXAMPLE 4 JIL defines a new class of RTK

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. Thus, both tie and JIL have three fibronectin type III repeats N-terminal to the transmembrane domain. Similarly, 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).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. REFERENCES:

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5. Stephenson, D ., Mercola, M., Anderson, E., Wang, C, Stiles, CD., Bowen- Pope, D.F., and Chapman, V.M., (1991) Proc. Nad. Acad. Sci. (USA) 8& 6- 10.

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9. Williams, AF., and Barclay, AN., (1988) Ann. Rev. Immunol. 6. 381-405.

10. Peterson, T.E., Thorgerson, H.C., Skorstengaard, K., Vibe-Peterson, K., Sahl, P., Sottrup-Jensen, L., and Magnusson, S., (1983) Proc. Nail. Acad. Sci. (USA) 80. 137-141. 11. Ullrich, A, Coussens, L., Hayflick, J.S., Dull, T.J., Gray, A, Tarn, A.W., Lee, J., Yarden, Y., Libermann, TA., Schlessinger, J., Downward, J., Mayes, EX. V., Whitde, N., Waterfield, M.D., and Seeburg, P.H. (1984) Nature (London) 309. 418-425.

12. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y.W., Godfine, I.D., Roth, RA, and Rutter, WJ., (1985) Cell 40. 747-758.

13. Lamballe, F., Klein, R., and Barbacid, M., (1991) Cell 66. 967-979.

14. Wilks, AF., (1989) Proc. Nail. Acad. Sci. (USA) 86: 1603-1607.

15. Wilks, AF., (1991) Methods in Enzymology, 200: 533-545.

16. Hovens, CM., Stacker, SA, Andres, A-C, Harpur, AG., Ziemiecki, A and Wilks, AF., (1992) Proc. Nail. Acad. Sci. (USA) 89: 11818-11822.

17. Partanen, J., Makela, T.P., Alitalo, H., Lehvaslaiho, H., and Alitalo, K. (1990) Proc. Nail. Acad. Sci. (USA) 87: 8913-8917.

18. Partanen, J., Armstrong, E., Makela, T.P., Korhonen, J., Sandberg, M., Renkonen, R., Knuutila, S., Huebner, K., and Alitalo, K., (1992) Mol. Cell. Biol. 12. 1698-1707.

19. Kozak, M. (1991) J. Cell Biol. 115: 887-903.

20. Gray, A, Dull, T., and Ullrich, A. (1983) Nature, 303: 722-725.

21. Ciccodicola, A, Dono, R., Obici, S., Simeone, A, Zollo, M. & Persico, M.g., (1989) EMBO J. 5: 1987-1991. 22. Kidd, S., Kelley, M.R. & Young, M.W., (1986) Mol. & Cell. Biol 6; 3094- 3108.

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24. O'Bryan, J.P., Frye, RA, Cogswell, P.C, Neubauer, A, Kitch, B., Prokop, C, Espinosa III, R., Le Beau, M.M., Shelton Earp, H. & Liu, E.T., (1991) Mol. Cell. Biol. 77: 5016-5031.

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Claims

CLAIMS:

1. 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

2. An isolated protein according to claim 1 wherein said 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.

3. An isolated protein according to claim 2 wherein said extracellular domain comprises at least three cysteine-rich EGF-like repeats.

4. An isolated protein according to claim 1 or 2 or 3 of mammalian origin.

5. An isolated protein according to claim 4 wherein the mammal is a human, livestock animal, companion animal or laboratory test animal.

6. An isolated protein according to claim 5 wherein the mammal is a human or murine animal.

7. An isolated protein according to claim 1 or 2 or 3 having:

(i) a molecular weight in the unglycosylated form as determined by SDS-PAGE of approximately 120,000-130,000 daltons; (ii) a molecular weight in the glycosylated form as determined by SDS-PAGE of approximately 135,000-145,000 daltons; and/or (iii) comprises approximately 1120-1125 amino acids in length.

8. An isolated protein according to claim 1 or 2 or 3 having an amino acid sequence substantially as set forth in Figure 1 or having at least 50-70% amino acid similarity to all or a region thereof.

9. A sub-unit, fragment, derivative, homologue or analogue of the protein according to claim 1 or 2 or 3 comprising all or part of the extracellular domain of said protein.

10. 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) 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

11. An isolated protein according to claim 10 wherein said 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.

12. An isolated protein according to claim 11, wherein said extracellular domain comprises at least three cysteine-rich EGF-like repeats.

13. An isolated polypeptide having extracellular domain-like properties of an RTK wherein said isolated polypeptide comprises three FN type III repeats, an Ig-like domain and an EGF-like domain.

14. An isolated polypeptide according to claim 13 wherein said polypeptide comprises at least three FN type III repeats and at least two Ig-like domains which flank cysteine-rich EGF-like repeats.

15. An isolated polypeptide according to claim 14 comprises at least three cysteine- rich EGF-like repeats.

16. An isolated nucleic acid molecule comprising a sequence of nucleotides which encode or which is complementary to a sequence which encodes the protein according to claim 1 or 2 or 3 or the polypeptide according to claim 13 or 14 or 15.

17. An isolated nucleic acid molecule according to claim 16 having a nucleotide sequence substantially as set forth in all or part of Figure 1 or which has at least 50% similarity thereto.

18. An isolated nucleic acid molecule according to claim 16 or 17 when present in an expression vector.

19. An isolated nucleic acid molecule:

(i) encoding a protein 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 hybridises under low stringent conditions to the nucleotide sequence set forth in Figure 1; and

(iii) which is not tie.

20. A method of inhibiting, reducing or otherwise interfering with interaction between a protein according to claim 1 or 2 or 3 and a ligand thereof in a mammal said method 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.

21. A method according to claim 20 wherein the antagonist is selected from the list consisting of a solubUised protein, a subunit, fragment, derivative or analogue carrying an extracellular domain of said protein, an antibody to said extracellular domain or a chemical molecule capable of inhibiting ligand interaction.

22. A method according to claim 20 or 21 wherein the mammal is a human.

23. A method for detecting an abnormal genomic coding sequence for the protein according to claim 1 or 2 or 3, in a human subject, said method 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 of said protein for a time and under conditions sufficient for said oligonucleotides to hybridise to said genomic sequence and then screening for said hybridisation.

24. A method according to claim 23 wherein the oligonucleotide primer is labelled with a reporter molecule selected from the list consisting of a radioactive isotope, fluorophore, biotintylated molecule or chemiluminescent molecule or fluroescent molecule.

25. A method according to claim 23 or 24 wherein the oligonucleotide is specific to a genomic sequence encoding a portion of the extracellular domain.

26. A method according to claim 25 wherein the portion of the extracellular domain is a ligand binding portion.

27. A ligand to the isolated protein of claim 1 or 2 or 3.