WO1997023629A1 - A novel receptor-type tyrosine kinase and use thereof - Google Patents

Abstract

The present invention relates generally to a novel receptor-type tyrosine kinase, to genetic sequences encoding same and to uses therefor. More particularly, the present invention contemplates a receptor-type tyrosine kinase having the following properties: (i) belongs to the Eph subfamily of RTKs as determined by conserved cysteine residues and fibronectin type III repeats; (ii) comprises protein tyrosine kinase catalytic domain motifs; and (iii) comprises an amino acid sequence substantially as set forth in SEQ ID NO: 2 or having at least about 79 % similarity thereto.

Description

A NOVEL RECEPTOR-TYPE TYROSINE KINASE AND USE THEREOF

The present invention relates generally to a novel receptor-type tyrosine kinase, to genetic sequences encoding same and to uses therefor.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ LD NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The receptor tyrosine kinases (RTKs) form an important class of molecules involved in the regulation of growth and differentiation of cells.

The RTKs are transmembrane molecules which transduce signals from the extracellular environment into the cytoplasm. They include well-studied regulators of cell proliferation and differentiation such as c-kit and the receptors for epidermal growth factor, platelet-derived growth factor and macrophage colony-stimulating factor (1). Signalling is initiated when a cognate ligand binds to the RTK extracellular domain. This triggers a sequence of events resulting in the activation of an intracellular tyrosine kinase domain. Critical to this process is ligand-mediated receptor dimerization and reciprocal tyrosine phosphorylation by the dimerized molecules (2). Once their catalytic domain is activated, RTKs can bind and phosphorylate specific intracellular proteins which act as second messengers. Eph was the first-isolated member of a new subfamily of RTKs (3). This group is distinguished by the sequential arrangement of a cysteine-rich region and two fibronectin-type-III repeats in the extracellular domain (4). At least 28 members have now been identified making this the largest subfamily of RTKs. They have been found in diverse species, including zebrafish (5), frogs (6), chickens (4,7,8), mice (7,9,10-12,13), rats (14,15) and humans (3,16,17,18,19). Certain features of the expression pattern of the Eph subfamily suggest key functions during embryonic development. First, strong expression in the embryo is characteristic (4,7,8,9,11,20). Expression is generally down-regulated later in development, but often continues in the adult at a restricted number of tissue sites. Secondly, in situ hybridization and immunolocaUzation studies have identified associations between the expression of specific Eph- subfamily molecules and particular events in morphogenesis. For example, Eck is transiently expressed in cells adjacent to the primitive streak during gastrulation and later its transcripts are found in specific rhombomeres of the developing hindbrain and in the ectoderm of the second and third branchial arches (21).

The expression of Nuk protein on growing peripheral nervous system axons, which disappears when the axons have ceased migrating, and the segment-restricted pattern of Nuk and Sek expression during hindbrain morphogenesis, are other examples (9, 11). Preferential expression at interfaces between embryonic cell populations and in intercellular junctions has led to the suggestion that Eph-subfamily molecules influence embryonic differentiation and cellular migration by interactions involving direct cell-cell contact (11,21). The recent finding that ligands for some members of this group are cell membrane bound supports this notion (22-25). Eph-subfamily kinases are also significant because of a potential role in oncogenesis. Eph and Erk are over expressed in some epithelial tumor cell lines and carcinomas (3,26), while Hek overexpression occurs sporadically in leukemia (32). Furthermore, artificial overexpression of Hek or Eph in NTH-3T3 cells resulted in a transformed phenotype, as evidenced by the ability to form colonies in agar and tumors in nude mice (27). These molecules may also be involved in tumor progression. In transgenic models of murine mammary cancer, overexpression of the Eph-subfamily members Myk-1 and Myk-2 correlated with the development of poorly- differentiated and invasive tumors (13). Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst (28). They are undifferentiated and totipotent. When grown in vitro in the absence of leukemia inhibitory factor (LIF), they develop into embryoid bodies containing cells committed to a variety of tissue lineages (29). This system provides a convenient model of very early embryonic development, which is difficult to study in vivo because of problems in harvesting preimplantation embryos and the small amounts of tissue involved.

In work leading up to the present invention, the inventors considered that RTKs expressed by ES cells and embryoid bodies are likely to be involved in the initial differentiation and organization of embryonic tissues. To analyse this further, the inventors used reverse transcriptase (RT) -mediated polymerase chain reaction (PCR) [RT-PCR] to identify Eph subfamily RTKs in ES cells. In accordance with the present invention, the inventors have identified a novel RTK member from the Eph-subfamily.

Accordingly, one aspect ofthe present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel member of the Eph subfamily of RTKs or a derivative, homologue or chemical analogue thereof.

More particularly, the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an RTK or a derivative, homologue or chemical analogue thereof having the following characteristics:

(i) belongs to the Eph subfamily of RTKs as determined by conserved cysteine residues and fibronectin type HI repeats; (ii) comprises protein tyrosine kinase catalytic domain motifs; and (iii) comprises an amino acid sequence substantially as set forth in Figure 2 [SEQ ID

NO:2] or having at least about 79% similarity to all or part thereof. Preferably, the novel RTK of the present invention is of animal or mammalian origin. Preferred mammals include but are not limited to humans, primates, livestock animals (e.g. sheep, cows, horses, pigs, donkeys), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs), companion animals (e.g. dogs, cats) or captive wild animals (e.g. foxes, kangaroos, deer). Preferred non-mammalian animals include fish and birds. The present invention is particularly exemplified herein by reference to a novel RTK of murine origin but this is done with the understanding that the present invention extends to all animal and mammalian homologues of the novel murine Eph-subfamily RTK and in particular a human form of the RTK. Hereinafter, the novel RTK ofthe present invention is referred to as "Esk" for "embryonic stem cell kinase"

The present invention extends to derivatives, homologues and chemical analogues of Esk, which Esk has an amino acid sequence set forth in Figure 2 [SEQ ID NO:2]. Derivatives include single or multiple amino acid substitutions, deletions and/or additions to the sequence and encompass mutants, part and fragments thereof. The term "derivative" also encompasses soluble or solubilized or otherwise secreted forms of the Esk molecule. Derivatives also encompass chimeric molecules comprising Esk or a derivative, homologue, or analogue thereof and at least one other molecule such as another receptor or ligand.

Homologues include novel Esks of animal or mammalian origin having at least about 79%, more preferably at least about 85%, even more preferably at least about 90% and still more preferably at least about 95% or above sequence similarity to the amino acid sequence set forth in Figure 2 [SEQ ID NO:2]. Preferred homology comparisons are done between coding regions or 3' or 5' regulatory regions or particularly conserved regions.

Analogues of Esk contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues. Such analogues may also provide stability to molecule administered in vivo or for manipulation of molecules in vitro. 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 NaBH4; 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 NaBHφ

The guanidine 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 O-acylisourea 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 the incorporation of unnatural amino acids and derivatives during polypeptide 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-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated for use in accordance with the present invention is given in Table 1.

TABLE 1

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-o-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-memylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-o-methylasparagine Dmasn α-m ethy 1-α-napthy lalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3 -aminopropy l)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-o-methylleucine Dmleu α-napthylalanine Anap

D-α-me yllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-o-memylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutyiglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-o-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropy l)glycine Narg D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmora N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg peniciilamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-o-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-a-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-o-methylhistidine Mhis L-o-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-o-methyllysine Mlys L-α-me ylmethionine Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-o-methylornithine Mora

L-α-methylphenylalanine Mphe L-o-methylproline Mpro

L-α-methylserine Mser L-o-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-o-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- l-(2,2-diphenyl- Nmbc ethylamino)cyclopropane

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=l 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 (SH) or carbodiimide (COOH). In addition, peptides can be confoπnationally constrained by, for example, incorporation of C_β and N, -methylamino acids, introduction of double bonds between and_|C 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.

Particularly useful derivatives contemplated by the present invention are soluble forms of the Esk receptor. A soluble receptor is also referred to herein as a secreted Esk protein and is most preferably in recombinant form. Soluble Esk molecules are useful reagents for ligand isolation, as antagonists of Esk-ligand interaction, as a substrate for antibody production which antibodies are in turn useful diagnostic reagents.

Accordingly, another aspect of the present invention provides a secreted recombinant Esk protein or derivative thereof comprising conserved cysteine residues and fibronectin type HI repeats and a portion ofthe amino acid sequence substantially set forth in SEQ ID NO:2 or having at least 79% homology thereto which is not part of the membrane bound region of the corresponding anchored receptor. Preferably, the amino acid sequence of the secreted recombinant Esk is encoded by a nucleotide sequence substantially as set forth in SEQ LD NO:2 or having at least 82% homology thereto.

In a particularly preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence (or a complementary form thereof) substantially as set forth in Figure 2 [SEQ LD NO: 1 ] or having at least about 82% similarity to all or part thereof or is capable of hybridising to the sequence set forth in SEQ LD NO: 1 or a complementary form thereof under low stringency conditions.

Preferred percentage nucleotide similarities include at least about 84%, more preferably at least about 90% and even more preferably at least about 95% or above. The nucleic acid molecules ofthe present invention include single or multiple nucleotide substitutions, deletions and/or additions to the nucleotide sequence set forth in Figure 2 [SEQ LD NO: 1] and mutants, parts and fragments thereof, which are all encompassed by the term "derivative" of the nucleotide sequence set forth in Figure 2 [SEQ LD NO: 2].

Reference herein to a low stringency at 42 ^PC includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.0 IM to at least about 0.15M salt for hybridisation, and at least about 0.0 IM to at least about 0.15M salt for washing conditions.

The nucleic acid molecules of the present invention are preferably carried by a vector molecule and more particularly an expression vector. Preferred expression vectors direct expression in mammalian, insect and or bacterial cells. The present invention also extends to cells carrying the recombinant nucleic acid molecules ofthe present invention.

Preferred nucleic acid molecules are DNA and more preferably cDNA.

5 The present invention is also directed to a recombinant Esk polypeptide having the following characteristics:

(i) is an RTK belonging to the Eph subfamily of RTKs as determined by cysteine residues and fibronectin type II repeats; (ii) comprises protein tyrosine kinase catalytic domain motifs; and 10 (iii) comprises an amino acid sequence substantially as set forth in Figure 2 [SEQ

LD NO: 2] or having at least about 79% similarity to all or part thereof.

The recombinant Esk is preferably in isolated form meaning that a composition comprises at least about 20%, more preferably at least about 30%, still more preferably at least about 40- 15 50%, even more preferably at least about 60-70% and yet even more preferably at least about 80-90% or above of Esk as determined by activity, molecular weight, or immunological reactivity. The preparation may also be sequencably pure or of a purity suitable for use in a pharmaceutical composition.

0 The present invention extends to the ligand(s) of Esk and to agonists and antagonists of Esk- ligand interaction. Agonists and antagonists may be, for example, antibodies or derivatives of the Esk or derivatives of the ligand. Derivatives of Esk or its ligand include soluble or solubilised forms thereof. Reference herein to "Esk" includes both anchored forms (ie. membrane bound forms) ofthe receptor as well as soluble (ie. secreted) forms ofthe receptor.

25

Modulating expression of Esk may have important potential in therapeutic regimens for the treatment or prophylaxis of cancers caused or exacerbated by aberations in Esk or aberations in Esk-ligand interaction. This will be particularly important for the treatment of mucositis. This condition remains the major adverse effect of chemotherapy and radiotherapy of

30 malignant disease. As Esk is expressed in all epithelial tissues, modulation of Esk-ligand interactions may have therapeutic applications for skin (such as following burns, abrasions, eczema and other forms of skin wounding leading to skin loss or damage); hair (hair loss or abnormal hair growth); corneal ulcers (injury, infection, Sjogrens syndrome and related autoimmune diseases; mucositis (mouth, oesophagus, stomach, small and large bowel); infective ulcerative colitis and other non-infective inflammatory enteritides; peptide ulcers; oesophageal reflux; Sjogrens syndrome and related autoimmune diseases; infection of other mucous surfaces (eg. vagi itis and vulvitis); Sjogrens syndrome and related autoimmune diseases; infection of the lung (eg. shock lung, inhalation of noxious fumes, infection); liver (eg. regeneration after viral illness or toxic damage); pancreatitis; urological disease involving tubules, pelvicalyceal system, ureters, bladder or urethra and salivary glands.

Accordingly, the present invention contemplates a method for modulating Esk-ligand interaction in an animal, said method comprising administering to said animal a modulating effective amount of an agonist or antagonist of Esk-ligand interaction. The term "modulating" includes facilitating Esk-ligand interaction or inhibiting, reducing or otherwise interfering with Esk-ligand interaction. Either form of modulation may be required depending on, for example, the type of treatment such as the treatment of cancer or the promotion or inhibition of cell apoptosis.

The present invention, therefore, contemplates a pharmaceutical composition comprising an Esk-ligand interaction modulating effective amount of an agonist or antagonist of Esk-ligand interaction and one or more pharmaceutically acceptable carriers and/or diluents.

The formation of pharmaceutical compositions is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th end., Mack Publishing Co., Easton, Pennsylvania, USA.

The active ingredients of a pharmaceutical composition comprising the Esk agonists or antagonists or their derivatives are contemplated herein to exhibit excellent therapeutic activity, for example, in modulating Esk-ligand interaction when administered to an animal in an amount which depends on the particular case. For example, from about 0.5 μg to about 20 mg per kilogram of body weight per day may be administered. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly or monthly, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compounds may be administered in any convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or by implanting (eg using slow release molecules), topical administration or following or during surgery or biopsy or other invasive procedure. Depending on the route of administration, the active ingredients which comprise the Esk agonists or antagonists or chemical analogues thereof may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. In order to administer Esk agonists or antagonists by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation. For example, homologues may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvants contemplated by the present invention include, but are not limited to, cytokines (e.g. interferons) as well as resorcinols, non-ionic surfactants such as polyoxyethelene oleyl ether and n-hexadecyl polyethylene ether.

The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and 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 superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solven with various of the ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient 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 of vacuum drying and the freeze-drying technique which yield a powder ofthe active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.

When the Esk agonists or antagonists or chemical analogues thereof are sutiably protected as described above, the active, compound may be orally administered, for example, with an inert diluent or with a assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tables, 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 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that a oral dosage unit form contains between about 0.1 μg and 2000 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 as sucrose, lactose or saccharin may be added or a flavouring 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 ofthe 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 compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. 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 incoφorated into sustained-release preparations and formulations.

A pharmaceutically acceptable carrier and/or diluent indues any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the acive ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incoφorated into the compositions.

Its is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mamallian subjects to be treated; each unit coating a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic affect to be achieved, and (b) the limiations inherent in the art of compound such a active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principial active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions coating supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

The animal to be treated is preferably a mammal such as a human, primate, livestock animal, laboratory test animal, companion animal or captive wild animal. Most preferably, the animal is human.

The present invention further extends to antibodies to the Esk molecules herein described. The antibodies may be monoclonal or polyclonal. Antibodies to the Esk molecules of the present invention are useful as therapeutic agents in modulating Esk-ligand interaction or as diagnostic agents to assay for Esk molecules or Esk-ligand intemation. Assay techniques are well known in the art and include, for example, sandwhich assays and ELISA.

Accordingly, another aspect of the present invention contemplates a method for assaying for

Esk expression on a cell, said method comprising contacting a biological sample containing cells putatively expressing Esk with an Esk-binding effective amount of an antibody thereto for a time and under conditions sufficient for said antibody to bind to said Esk and then detecting said Esk-antibody binding.

The presence of Esk on a cell can be detected using a wide range of immunoassay techniques such as those described in US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. This includes both single-site and two-site, or "sandwhich", assays ofthe non-competitive types, as well as in the traditional competitive binding assays. Sandwhich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations ofthe sandwhich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, and Esk antibody is immobilised onto a solid substrate to form a first complex and the sample containing cells to be tested b rough into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an Esk-antibody secondary complex, an antibody, labelled with a receptor molecule capable of producing a detectable signal and specific to another antigen to the cell, is then added and incubated, allowing time sufficient for the formation of a tertiary complex of Esk-antibody-labelled antibody. Any unreacted material is washed away, and the presence ofthe first antibody is determined by observation of a signal produced by the reporter molecule on the second antibody. The results may either be qualitative, by simple observation ofthe visible signal or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations of the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody.

These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. Alternatively, a labelled Esk antibody may be added directly to the sample of cells and the reporter molecule defected.

The solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier.

By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, profices an analytical identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecule in this type of assay re either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates which yield a fluorescent product.

Alternatively, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining ternary complex is then exposed to the light ofthe appropriate wavelength, the fluorescent observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radiosotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the filled technician how to vary the procedure to suite the required puφose.

The present invention further extends to genetic molecules derived from an Esk gene useful as probes, antisense or sense molecules for diagnostic or therapeutic situations.

The present invention is further described by the following non-limiting Figures and/or Examples.

Reference herein to "similarity" in relation to amino acid or nucleotide acid or nucleotide sequences has substantially the same meaning as "homology" and "identify".

In the Figures:

Figure 1 is a diagrammatic representation showing the position of the four degenerate oligonucleotide primers (PI-P4) used for RT-PCR. The relatively-conserved peptide motifs on which the primers were based are shown above a schematic representation of the basic domain structure of Eph-subfamily molecules. The particular amino acid sequences used in the figure are from eph. Pl and P2 were sense primers: P3 and P4 were antisense primers. Their sequences were as follows:

Pl, 5'-GTAGGCATGCAAGGAGAC(AC)TT(CT)AACC-3' [SEQ LD NO:3]; P2, 5 '-GCGATGATCAT(CG)AC(AGT)GA(AG)TA(CT)ATGG-3 ' [SEQ LD NO:4]; P3, 5 '-GTAGGAATTCCA(CGT)ACATC(AG)CT(AG)GC-3 ' [SEQ LD NO:5]; P4, 5'-CCA(TA)A(AG)CTCCA(CT)ACATC(AG)CT-3' [SEQ LD NO:6]. TMD denotes the transmembrane domain. Figure 2 is a reprsentation showing the nucleotide and deduced amino acid sequence of Esk. The signal peptide and transmembrance domain are boxed. Conserved cysteine residues in the extracellular domain are circled and two fibronectin type III repeats are in a striped box. In the catalytic domain, arrowheads indicate the highly-conserved Gly-X-Gly-X-X-Gly motif and a dot marks the invariant lysine residue. Two motifs assocaited with substrate specificity for tyrosine are underlined. The stop codon which terminates the coding region is indicated by an asterisk.

Figure 3 is a photographic representation of expression of Esk. Northern blots of poly(A) ⁺ RNA from day- 12 mouse embryo and the adult mouse tissues shown were hybridized to a ³²P-labelled probe derived from clone 35C15. This was then stripped from the filters and hybridization to a GAPDH probe was performed. The positions of RNA size markers are indicated to the left of the blots.

Figure 4 is a photographic representation showing Northern blot of ES cell, embyoid body and embryonic firbroblast poly (A)⁺ RNA hybridized to ³²P-labelled probes synthesized from Esk,, Mek4 and Eck cDNA. Probes were stipped from the filter between reprobingS. Hybridisaztion to a GAPDH probe was [erformed last. The sizes of transceipts are indicated to the right of the figure.

Figure 5 is a diagrammatic representation of all Eph-subfamily clones isolated from ES cells using RT-PCR aligned with the full-length cDNA clone derived from a λZAP library. Numebrs represent nucleotides in the Esk full length sequence. ECD, extracellular domain; ICD, intracellular domain.

Figure 6 is a reprsentation showing a plot of grains after scoring approximately 130 Chromosomes 6, showing probable localisation of Esk to bands B1-B2. Grains scored from C57BL and BALB/c mice are represented with solid and open dots respectively.

Figure 7 is a photographic representation of an analysis of the relationship between Esk and Eph by Southern hybridization. A mouse genomic Southern blot was prepared using DNA digested with the restriction enzymes shown. This was initially hybridized in 40% formamide to a probe derived from the catalytic domain of human Eph (A). The membrance was then stripped and rehybridized in 50% formamide to a pφbe derived from equivalent sequence in mouse Esk(B). The position of DNA size markers is indicated at left.

Figure 9 is a photographic representation showing in situ analysis of Esk expression in whole-mounted embryoid bodies (A) and day 9.5 mouse embryos (B). Embryoid bodies were differentiated in vitro from ES cells by culturing without feeder cells in LIF-deficient mediu, for 7-10 days. Sense and antisense digoxigenin-labelled riboprobes were synthesized from cDNA fragments of Esk and hybridized to the whole-mounts. Bound probe was detected using alkaline phosphate-conjugated anti-digoxigenin Fab fragments and staining to detect enzyme activity. Left panels show results of hybridization with sense-control and right panels with Esk antisense probe.

Figure 9 is a photographic representation of Esk expression in sections of embryoid bodies and selected mouse tissues. Results of hybridizations with sense-control probe are shown in the left panels and with Esk antisense probe in the middle and right panels. (A) Embryoid bodies differentiated in vitro. (B) Adult thymus. (C) Adult renal cortex. (D) Day 18 embryo skin. Abbreviations: LP, low power; HP, high power.

Figure 10 is a photographic reprsentation showing binding of ligand to Esk chip in a biosense assay for binding to a potential Erk ligands. (LI to L7) controls a FC, L3-FLAG, L7-FLAG and binding to HEK. Single and three letter abbreviations for amino acid residues are used in the specification and are defmed in Table 2.

TABLE 2

Amino Acid Three-letter One-letter

Abbreviation Symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D Cysteine Cys C

Giutamine Gin Q

Glutamic acid Glu E

Glycine Gly G

Histidine His H Isoleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Tφ w

Tyrosine Tyr Y Valine Val V

Any residue Xaa X EXAMPLE 1 ES CELL CULTURES

The murine 129/Sv-derived ES cell line, W9.5 was routinely passaged on underlays of irradiated embryonic fibroblasts in Dulbecco's modified Eagle medium supplemented with 1000 units/ml of LIF (AMRAD Operations Pty Ltd, Melbourne, Victoria, Australia), 10"⁴ M 2-mercaptoethanol and 15 % v/v fetal calf serum. Cultures were incubated in a 10% v/v CO₂ atmosphere at 37^βC. In preparation for the studies described below, ES cells were subcultured into delatinized flasks and four passages without a feeder layer were performed to deplete the embryonic fibroblasts. In some of these cultures, LIF was withdrawn 11 days prior to harvesting the cells, to allow differentiation into embryoid bodies (29). Control cultures of embryonic fibroblasts alone were also performed.

EXAMPLE 2 RT-PCR

Prior to RNA extraction, cultures of undifferentiated ES cells were disrupted with trypsin and washed in phosphate buffered saline. Cell pellets were resuspended in guanidine isothiocyanate denaturing buffer and total RNA was extracted using organic solvents (30). cDNA was then synthesized using 1 μg of total RNA, an aoligo(dT) primer and AMV reverse transcriptase (Promega). Four degenerate PCR primers were derived from three regions of sequence which are relatively conserved across the Eph subfamily (18)[Figurel}. Sense primers Plor P2 were used with antisense primer P4 to amplify the Es cell cDNA. Reactions were carried out in a 30μl volume containing 50 mM KCl, lOmM Tris. HCl (pH 8.3), 1.25 mM MgCl₂, 0.2 mM each dNTP, 2.5 units of Taq polymerase (Perkin Elmer), 30 pmol of each promer and 3μl of the ES cell cDNA synthesis reaction. In another experiment, mRNA was directly extracted from ES cells with oligo9dT)-coated magnetic beads (Dynal; Oslo, Norway) and cDNA was synthesized from 1 μg of mRNA using an oligo(dT) primer and Superscript II reverse trasnscriptase (Life Technologies). Half of the reverse transcription reaction was amplified using primers Pl and P4, with other reaction coditions identical to those described above. PCR products were electrophoretically separated, purified with the Geneclean II Kit (Bio 101 Inc) and then subjected to a second round of PCR. Products initially amplified using primers Pl and P4 were reamplified with the same primers, while those amplified with P2 and P4 were reasmplified with P2 and P3. All reactions were carried out in a PTC-100 Programmable Thermal Cotroller (MJ Research Inc) emplying programs specific to each primer combination, as follows: lmin at 95^βC, 2 min at 70^βC and 3 min at 72^βC for 30 cycles (primers Pl and P4); 1 min at 95^βC, 1 min at 5 TC and 1 min at 72^βC for 35 cycles (primers P2 and P4); 1 min at 95°C, 1 min at 41 °C and 1 min at 72°C for 35 cycles (primers P2 and P3).

EXAMPLE 3

CLONING AND SEQUENCING

Reamplified, gel-purified PCR fragments were cloned into the Sntal site of pUC18 using the

SureClone Ligation Kit (Pharmacia). A ³²P-labelled probe was synthesized from a cloned cDNA fragment of Esk. This was used to screen 8 x IO⁵ clones from a mouse liver cDNA library constructed in λZAP. (Stratagene), under high stringency conditions. Secondary screening of selected primary positive plaques was used to identify clones with the largest inserts. Recombinant clones were sequences with the Taq DyeDeoxy terminartor Cycle Sequencing Kit (Appleid Biosystems), using a Perkin Elmer GeneAmp PCR System 2400 to perform the sequencing reactions and an Applied Biosystems 373 DNA Sequencer for their subsequent analysis. Sequences were compared against sequence databank entries with the FASTA sequence analysis program. A diagrammatic representation of Esk clones is shown in Figure 5.

EXAMPLE 4

NORTHERN BLOTS

Oligo(dT)-cellulose (Pharmacia) as used to isolate poly(A)⁺ RNA from total derived from

ES cells, embryoid bodies differentiated in vitro from ES cells. Embryonic fibroblasts, day-

12 mouse embryos and adult mouse tissues. The poly(A)-selected samples (5μg of each) were electrophoresed through 1.2% w/v agarose gels containing 2.2 M formaldeyde and transferred to nylon membrances (Zeta-Probe, Bio-Rad). The resulting Northern blots were probed with cDNA inserts from selected ES cell recombinants cloned by the methods described earlier. Sequence analysis (see below) had revealed that a partial cDNA of murine Eck (20) was present in the clone designated 35C4 and that Mek4 (7) cDNA was present in clone 35C11. Furthermore, an apparently novel sequence was ocntained in clone 35C15. Inserts were digested from these three clones with the restriction enzymes EcoRI and Xbal, and used as templates for synthesizing ³²P-lebelled probes witht he Prime-It II Random Primer Labelling Kit (Stratagene). A labelled propbe was also made from glyceraldehyde-3- phosphate dehydrogenase (GAPDH) cDNA. The Northern blots were intially hybridized to the 35C15 probe. Subsequently the membrane containg RNA from ES cells, embryoid lodies and embyronic fibroblasts was rφrobed with the Eck and Mek4 probes. Between each reprobing, hybridized probe was stripped from the membrane by pouring on boiling 0.1 % w/v SDS and cooling to room temperature. Exposure to X-ray film overnight confirmed effective removal of the probe. Hybridisation to the GAPDH probe was performed last. In all cases, hybridisations took place in 50% v/v formamide at 42° C and washses were performed under stringent conditions, with the final wash at 65°C in 0.1 x SSC and 0.1 % w/v SDS (SSC is 0.15 M NaCl and 0.015 m Na citrate, pH 7.6). Autoradiograps were exposed at -70° C.

EXAMPLE 5

SOUTHERN ANALYSIS

A ³²P-labelled Esk probe was synthesised from a PCR fragment amplified from Esk cDNA using degenerate kinase-domain primers described in Example 2. The fragment included sequence from bases 2098-2437 of Esk. Probe was hybridized to a geomic Southern blot made using standard techniques with DNA extractex from the murine embryonic stem cell line W9.5 and digested with the panel of enzymes showin in Figure 7. Hybridisation was performed in 50% formamide, lOx Denhardt's solutioN (35), 50mM Tris HCl (pH7.5), 1.0 < NaCl, 2.24 mM tetrasodium pyrophosphate, 1% w/v SDS, 10% w/v dextran sulfate and 0.1 mg/ml sheared heat-denatured herring sperm DNA. The final wash was in O.lxSSC/0.1 % w/v SDS at 65°C (SSC is 0.15 M sodium chloride, 0.015 M sodium ctrate, pH 7.6). An autoradiograph was exposed at 70^βC for eight days > The filter was subsequnetly stripped using 0.4 M sodium hydroxide and removal of the probe was confirmed by exposure to film overnight.

A ³²P-labelled Eph probe was synthesised from a cloned cDNA fragment isolated by RT- PCR from human bone marrow cDNA, using the same degenerate kinase-domain primers that generated the Esk probe. The cloned cDNA included sequence from bases 2212-2553 of human Eph. Probe was hybridized to the stripped Southern blot, with hybridization conditions and washes identifical to those used with the Esk probe. An autoradiograph was exposed at -70° C for seven days.

EXAMPLE 6 In vitro DIFFERENTIATION OF EMBRYOID BODD2S

ES cells from the W9.5 line were induced to undergo differentiation by passaging them in bacterial-grade Petri dishes without an embryonic fibroblast feeder layer and in the absence of LLF. Embryoid bodies were harvested after 7-10 days of development (39).

EXAMPLE 7 PREPARAΗON OF WHOLE-MOUNTED AND SECTIONED TISSUE

CBA mouse embryos for whole-mount studies were collected 8-10 days following observation ofthe coital plug and a more accurate age was determined by reference to a standard atlas of mouse development (40). After opening the brain ventricles and heart chambers to prevent trapping of probe, the embryos were fixed in 4% v/v paraformaldehyde. Embryoid bodies for sections were set in 1% w.v agar, to which Bouin's fixative was added. Other tissues for sectioning, including day 18 CBA embryos and thymus, liver, kidney and adrenal gland from six -week-old BALB/c mice, were dissected free and fixed in 4% v/v paraformaldehyde. Sections were cut at a thickness of 5 μm after dehydration in ethanol and embedding in paraffin. EXAMPLE 9 In situ HYBRIDIZATION PROCEDURE

The hybridization of digoxigenin-labelled riboprobes to whole-mounted specimens was performed using methods described in detail elsewhere (41), except that 2mM levamisole was included in the washes prior to colour development to inactivate endogenous alkaline phosphatases. Tissue sections were first soaked in histolene (Histo Labs) to remove paraffin and then rehydrated through decreasing concentrations of methanol in PBS. Refixation in 4% v/v paraformaldehyde and 0.2% v/v glutaraldehyde was then performed. Subsequent steps were similar to those ofthe whole-mount procedure.

EXAMPLE 10 Eph-SUBFAMD Y MOLECULES EXPRESSED BY ES CELLS Primers P2 and P4 were expected to amplify approximately 350 bp from the catalytic domain of Eph-subfamily molecules. In contrast, it was anticipated that primers Pl and P4 would amplify about 2.1 kb, including much of the extracellular and intracellular domains (Figure 1). RT-PCR was initially performed on total RNA derived from ES cells. Reactions using primers P2 and P4 amplified a band of the expected size, which reamplified with primers P2 and P3. However, no PCR product ofthe anticipated size was observed when primers Pl and P4 were used. Subsequently, when magnetically-separated poly(A) RNA was substituted for total RNA, reactions with primers Pl and P4 successfully amplified a 2.1 kb product. Cloning of the 350 bp product resulted in four recombinants containing Eph-subfamily sequences (Table 3). These were highly homologous (>97%) to either Sek (9), Nuk (11) or Eck (20) - members of the Eph subfamily previously isolated from murine sources. When the 2.1 kb product was cloned, five recombinants containing sequences of this subfamily were identified. Four of these showed high levels of homology with either murine Eck or Mek4 (7), but one clone (35C15) contained 1604 bp of sequence which appeared novel after comparative databank analysis. The novel molecule was termed embryonic stem cell kinase or Esk. A diagrammative representation of Esk clones is shown in Figure 5 and the sequence ofthe Esk cDNA is shown in Figure 2.

EXAMPLE 11 ISOLATION OF A FULL-LENGTH Esk CLONE

The Esk cDNA was used to screen a mouse liver cDNA library and identified twenty-four positive clones. One clone containing the largest insert size was analysed in detail and found to include the complete coding region of Esk (Figure 2). This clone contained a single open reading frame that encoded 977 amino acids, without an initiation codon occuring in a context consistent with the Kozac rules for translation start sites (42). Amino acids 1-26 conformed to the predicted sequence for a signal peptide (43). A sequence of predominantly hydrophobic residues at positions 549-569 were consistent with a transmembrane domain, potentially dividing the mature Esk protein into extracellular and intracellular portions. Within the putative extracellular domain were 20 highly-conserved cysteine residues (indicated by circles in Figure 2) and two fibronectin type III repeats which are hallmarks of the Eph subfamily (42). In the putative intracellular domain, motifs typical of a protein-tyrosine kinase were seen (31). This included the GXGXXG ATP-binding motif (residues 632-637) and the associated invariant lysine at position 657, along with motifs indicating substrate specificity for tyrosine (DLAARN, residues 750-755; and PIRWTAPE, residues 790-797). Finally, residues 60, 339, 415 and 479 are potential sites of N-liked glycosylation. Sequence database searching confirmed that the closest known Esk homologue was human Eph, with an overall amino acid sequence identity of 84.4%.

To confirm the locaUzation of Esk, 135 grains over good-quabty Chromosomes 6 were plotted onto the accurate idiogram of Evans (38). In this plot, the two tallest peaks of grains were again over bands 6B 1 and 6B2 (Figure 6); together, these two peaks contained 56% of the total grains over Chromosome 6. The localization ofthe Esk gene to bands Bl and B2 of mouse Chromosome 6 was therefore confirmed by both sets of data. EXAMPLE 12 ANALYSIS OF THE RELATIONSHIP BETWEEN Esk AND Eph

To determine whether Esk was the closest homologue of Eph in the mouse, a Southern blot of mouse genomic DNA digested with different restriction enzymes was sequentially hybridized to Esk and Eph probes. The probes were derived from exactly corresponding regions of their respective coding sequence. As shown in Figure 7, both probes recognized fragments of identical size with all restriction enzymes tested.

EXAMPLE 13

ESK EXPRESSION

Northern analysis of Esk revealed expression of a 4.2 kb transcript in day- 12 mouse embryo and adult mouse thymus, liver, kidney, lung and placenta (Figure 3). Faint bands, approximately 6.0 kb in size, were also observed in the liver, kidney and lung samples. This may be due to alternatively spliced transcripts, although weak hybridization to related molecules cannot be excluded. No signal was detected from lymph node, spleen, heart, brain or skeletal muscle.

EXAMPLE 14

COMPARATIVE EXPRESSION OF Esk, Mek4 AND Eck

LN THE ES CELL CULTURES

To determine whether expression of Eph-subfamily molecules occurred in ES cells at a significant level and to investigate potential changes in expression as ES cells were differentiated in vitro, a Northern blot containing ES cell and embryoid body RNA was sequentially hybridized to Esk, Mek4 and Eck probes. Embryonic fibroblast RNA was included as a control on this blot, because some fibroblast contamination of the ES cell and embryoid body samples could not be excluded. As shown in Figure 4, the Esk probe hybridized to all three samples, approximately in proportion to the amount of RNA present. This result indicates significant levels of Esk expression in ES cells and embryoid bodies, and could not be accounted for by fibroblast contamination alone. In contrast, expression of Mek4 was barely detectable in the ES cell cultures. A 9.5 kb Mek4 transcript was expressed by the embryonic fibroblasts, however, suggesting that the faint bands seen in the ES cell lane may be due to contaminating fibroblast RNA. Finally, the Eck probe hybridized to all three samples, but expression was relatively greater in the undifferentiated ES cells. Transcripts were of slightly different sizes in the different lanes.

EXAMPLE 15 EXPRESSION

In situ studies with digoxigenin-labelled ESK riboprobes were used to analyse expression using manufactuers protocols (Boehringer-Mannheim In situ hybridisation manual). Whole mount preparations of early (D8-12) mouse embryos show that ESK is expressed in the developing fore-gut and branchial arches. Later embryos were sectioned and analysed by in situ hybridisation. In these stages expression was noted in the thymus, basal layer of the skin, lung and in the developing eye. Similar studies were performed in tissue sections of adult tissues. In the adult animal expression was also seen in the thymus, as expected from Northern blot analysis, but was shown to be localised in the thymic epithelial cells. There was also detectable expression in the proximal and distal tubules ofthe kidney, the adrenal cortex and in testis. While expression was expected in other sites from the Northern analysis results, no 32

signal greater than background was evident, perhaps suggesting low level expression of RNA degradation during processing.

Expression in epithelial but not in neural tissues confirms the impression that ESK is primarily involved in epithelial development. The expression in thymic epithelial cells is of particular interest as thee cells have been shown to be involved in thymocyte selection and hence in development of T cell immune competence.

EXAMPLE 16 ANALYSIS OF Esk EXPRESSION BY In situ HYBRIDIZATION

The expression of Esk was investigated with in situ hybridization in embryoid bodies differentiated in vitro, mouse embryos and selected adult mouse tissues. These experiments were performed using two pairs of antisense and sense-control riboprobes derived from non- overlapping regions of Esk sequence. Both antisense probes gave similar results. No specific staining was detected in any of the tissues hybridized to the sense-control probes with the exception of small intestine, where high levels of endogenous alkaline phosphatase activity resulted in false-positive staining of the epithelium. This occured despite the inclusion of levamisole in the post-hybridization washes.

EXAMPLE 17 EMBRYOID BODIES

The strong expression of Esk in ES cells and embryoid bodies observed with Northern analysis (Example 13) prompted investigation into whether expression was differentially regulated even at this early stage. In the current study in situ hybridization experiments were performed on embryoid bodies resulting from the differentiation of ES cells for 7-10 days in vitro. Differentiation was induced by growing the cells without a fibroblast feeder layer and in the absence of exogenous leukemia inhibitory factor (LIF), as previously described (39). Specific binding of antisense probe, indicating Esk mRNA expression, was observed in both whole-mounted and sectioned embryoid bodies. In the whole-mounts, staining appeared strongest in the central region of embryoid bodies (Figure 8A). This distribution was confirmed by the sections. As shown in Figure 9A, although Esk was expressed by most embryoid-body cells, an amoφhous layer visible near the surface at high magnification delineated an outer rind of cells that did not stain. The latter cells have been shown by others to correspond to primitive endoderm, whereas the cells within the interior are primitive ectoderm in type (45; 46). These populations of cells are separated by a layer of basement membrane-like material secreted by the endodermal cells.

EXAMPLE 18

EMBRYOS

To investigate Esk expression during later development, whole-mount preparations of embryos at days 8, 8.5, 9 and 9.5 of gestation were examined. The pattern of expression was similar in all embryos analysed and a representative specimen at day 9.5 of development is shown in Figure 8B. This demonstrates that Esk was predominantly expressed in ventral structures, including the branchial arches and the region containing the developing gut and its associated epithelial outgrowths. Expression was also observed in the region of the optic vesicle and there was lighter staining of the somites and other structures. No significant staining of the central nervous system was seen, which contrasts with the predominantly neural expression of many other Eph-subfamily receptors. Sections of day 18 embryos were also studied by in situ hybridization. These showed no specific staining for Esk mRNA in the majority of tissues. However, day 18 embryonic skin demonstrated positive staining in the deepest layers of the epidermis and in the developing hair follicles (Figure 9D). It is noteworthy that these are the regions of skin associated with active cellular proliferation and migration of cells into other layers. EXAMPLE 19 ADULT TISSUES

Northern analysis (Example 13) indicated that Esk is expressed in a variety of non-neural adult tissues. To defme the specific cells containing Esk mRNA, in situ hybridisation experiments were performed on sections of some of these tissues. In thymus, Esk expression was observed in aggregates of large cells with abundant cytoplasm, located predominantly in the medulla (Figure 9B). Moφhologically, these cells were consistent with thymic epithelia. No staining of thymocytes was observed. In kidney, Esk expression was localized to the epithelium of proximal and distal convoluted tubules (Figure 9C). Glomeruli and the interstium did not stain. Esk was also expressed in the epithelial cells of the adrenal cortex.

In sections of liver, small foci of hepatocytes stained positively, but the majority of the tissue was negative. As discussed earlier, no assessment of Esk expression in small intestine was possible, because of endogenous alkaline phosphatase activity.

EXAMPLE 20 CHROMOSOMAL LOCALIZATION

To determine the chromosomal localization of the Esk gene, a tritiated probe was made from the 1.6 kb cloned PCR fragment and hybridized in situ to preparations of mouse metaphase chromosomes. This resulted in a significant accumulation of silver grains over bands Bl and B2 of mouse Chromosome 6. Out of an initial score of 345 grains for all chromosomes, 36.8% of grains were over the proximal half of Chromosome 6. Background grains were distributed over the other chromosomes with 5 grains over distal Chromosome 15 to tallest secondary peak observed. This compared to 59 grains over band 6B2 and 28 over 6B1.

EXAMPLE 21 PREPARATION OF SECRETED RECOMBINANT Esk PROTEIN

1. MATERIALS AND METHODS Cell lines

COS cells and CHO cells were propagated in RPMI containing 10% v/v heat-inactivated FCS in a humidified 10% v/v CO₂ atmosphere at 37°C. The COS cells were stably transfected with the polyoma large T antigen, permitting replication of vectors containing an SV40 origin of replication. For experiments involving selection of CHO cells cotransfected with the pSV2neo vector, a concentration of 600 μg/ml of G418 (Gibco BRL) in RPMI 1-% v/v FCS was used.

Assembly of an Esk-IgG Fc Construct

cDNA sequence was amplified from Esk using a 5' primer derived from sequence beginning five bases upstream of the translation start site and a 3' primer from sequence immediately upstream of the transmembrane domain. Custom Bglll sites were included in both primers and a sphce donor sequence was added to the 3 ' primer. To minimize the possibility of PCR- induced errors, amplifications were performed using recombinant (Stratagene). The reaction was carried out in a 50 μl volume containing lx cloned Pfu buffer, 0.2 mM each dNTP, 2.5 units cloned Pfu polymerase, 0.2 μM each primer and approximately 250 ng of pBluescript plasmid containing the full-length Esk clone, and was performed in a Perkin Elmer GeneAmp 2400 PCR machine. PCR amplification conditions were: 30 seconds at 95 ^βC, 30 seconds at 55 ^βC and 2 minutes and 30 seconds at 72^βC for 5 cycles; 30 seconds at 95 ^βC and 3 minutes at 72°C for 15 cycles. Amplification products were digested with BgHI and ligated into the unique BamKL cloning site of the plg-BOS expression vector. This plasmid is a pEF-BOS derivative which includes a genomic fragment of human IgGI (containing exons encoding the hinge, CH2 and CH3 domains) downstream ofthe cloning site. Expression of this construct was expected to produce a polypeptide of approximately 90 kD in mass, which should become a divalent molecule of approximately 180 kD after the formation of cystine bonds between the hinge regions. The constructs were cloned in DH10 B Kcoli cells and clones containing correctly oriented inserts were identified and DNA prepared and checked by DNA sequencing. COS Cell Transfections

Five micrograms of expression plasmid prepared using a QIAGEN maxiprep kit was transiently transfected into COS cells using a standard DEAE-dextran method (35). A control was also performed using identical conditions except no DNA was added. After three days of incubation, the culture supernatant was removed for analysis. Cells lysates were also obtained using 1% v/v Triton X-100 in lOmM Tris HCl (pH 7.4) and 150 mM NaCl.

CHO Cell Transfection and Subcloning

Ten micrograms of the Esk-IgG Fc expression construct and 1 μg of pS V2neo G418-resistance plasmid were cotransfected into 2xl0⁷ CHO cells by electroporation. This was performed in 0.5 ml of PBS in a 4 mm sterile cuvette using 270 V and 960 μFD on a BioRad Gene Pulser. Cells were incubated in 15 cm tissue culture dishes for 24 hours before selection with G418 was commenced. After the development of visible clones, these were subcloned into 24-well plates and the supernatant harvested for analysis when the cells were confluent.

Western Blots

Five microlitres of supernatant or cell lysate from the transfections described above was resolved by SDS polyacrylamid gel electrophoresis and transferred to a nitrocellulose membrane using established methods (35). Samples from the transfections were electrophoresed under both reducing and non-reducing conditions. Western blots were subsequently blocked in 5% w/v skimmed milk powder and 0.5% v/v Tween-20. After blocking as both a rabbit anti-human lg antibody (HRP-conjugated) and a rabbit anti-Esk peptide polyclonal serum (see below) the antibodies were used to probe the blots with a secondary layer consisting of an HRP-conjugated donkey anti-rabbit lg antibody (Amersham) applied in the latter case. Detection was with the ECL kit (Amersham).

Production of An Anti-Esk peptide Polyclonal Serum A peptide was synthesized from the first 31 amino acids of the predicted mature protein encoded in the Esk sequence. This was coupled to keyhole limpet hemocyanin (KLH) and 200 μg of the conjugate was emulsified in complete Freund's adjuvant and injected into a New Zealand white rabbit via multiple subcutaneous sites. A second dose of antigen was administered in incomplete Freund's adjuvant after four weeks and serum was collected two weeks later. The serum was used at a dilution of 1 : 100 to probe Western blots.

Immunohistochemistry

CHO cells permanently transfected with an Esk and control CHO cells were grown in chamber slides, fixed in PBS containing l%v/v paraformaldehyde for 30 minutes and permeabilized with 100% v/v methanol. After blocking in PBS containing 2% v/v FCS and 2% v/v goat serum, the cells were incubated with either a 1 : 100 dilution ofthe rabbit anti-Esk antiserum described in the previous section or with preimmune serum as a control. In a second set of controls, 40 μg of Esk peptide coupled to KLH was added with the antiserum. A donkey anti- rabbit lg antibody conjugated to HRP was used as the secondary antibody in all cases and bound antibody was detected with diaminobenzidine (Dako).

Protein A affinity purification

The following method was used for protein A-Sepharose affinity chromatography. The column was pre-eluted with pH 3 0.1 M acetate buffer and re-equilibrated with PBS. The sample was applied and then the column washed to baseline with PBS. The sample was eluted with acetate buffer and peak fractions collected in tubes containing 1/10 vol of IM tris HCl pH8. The column was re-equilibrated with PBS/azide for storage.

Biosensor studies

The studies were carried on a BIAcore 2000 (Pharmacia) using the manufacturers protocols. Individual channels of a biosensor CM chip were derivatised with ESK-Fc or with soluble HEK (47). Samples of LERK-2, -2, 03, -4, -5, -7 as Fc fusion proteins were analysed at lOμg/ml. Single chain forms of LERK-3 and -7 expressed as FLAG™ (Kodak) constructs were produced by the inventors and were analysed at 2μg/ml. In each case a parallel sample was run in the presence of excess ESK-Fc. The results are presented at the difference between these responses (ie. the ESK-specific component) for each test sample and are shown in Figure 10.

Polyclonal anti-Esk-Fc serum

The purified ESK Fc protein (100 μg) was homogenised in complete Freund's adjuvant and administered subcutaneously at multiple sites. Animals were immunised every 14 days and bled 10 days after each injection. To purify the specific component the antiserum was exhaustively absorbed on a human immunoglobulin affinity column and then applied to an ESK Fc affinity column, the specific antibody was eluted with 0.2 M glycine HCl pH 2.3, quickly neutralised and concentrated to 0.5-1.0 mg/ml. 2. RESULTS

Analysis of the Rabbit Anti-Esk Polclonal Serum

A polyclonal serum raised against an Esk peptide was initially tested by probing a Western blot containing recombinant Esk and Hek. The antibody recognized Esk specifically, with bands ofthe expected size appearing in both the full-length and truncated Esk samples and no specific bands in the Hek samples. When the anti-Esk serum was used for immunochemistry, the cytoplasm of CHO cells transfected with the Esk construct stained strongly. The specificity of this staining for Esk was confirmed by several controls. These included incubations of Esk-transfected cells with preimmune serum and of control CHO cells with the antiserum. Only background staining was observed in either case. In addition, Esk peptide as observed to inhibit staining of the transfectants by the antiserum.

Transfection of the Esk-IgG Fc and CH48-IgG Fc Constructs

Western blots of the supernatants of COS cells transfected with the Esk-IgG construct. Specific bands ofthe expected size were detected in the transfected samples. Both the anti-Ig antibody and the anti-Esk antiserum gave positive signals, the Esk-Fc construct and a control construct being a fusion between CD48 and IgG Fc were used to generate permanently- transfected CHO cells lines. Western blots of conditioned medium from the ESK-Fc clones revealed a similar pattern to that of the COS cell transfections. The CH48 Fc band was somewhat smaller. Lines which secreted very high levels of the fusion protein were selected.

Purification of ESK & CD48 Fc proteins

High level expressing clones were expanded to 1 litre cultures in RPMI 1640 supplemented with 2.5% v/v FCS which had been absorbed on protein A-Sepharose beads to remove all protein A-binding bovine immunoglobulin. The cultures were allowed to grow to confluence and the conditioned medium removed, centrifuged to remove cellular material and concentrated 10-fold. The medium was applied to a protein A Sepharose column as above. Purified protein was analysed by SDS-PAGE to confirm purity. In each case a single band was obtained on SDS-PAGE under reducing conditions. N terminal sequencing ofthe ESK- Fc protein band gave a single un-equivocal sequence:

GluGluValThrLeuMetAspThr [SEQ ID NO 9]

which matches residues 27-34 ofthe amino acid sequence of ESK derived from the nucleotide sequence.

Binding of LERK proteins to ESK Fc

The samples show the expected pattern of binding to soluble HEK. There is also clear evidence of binding of divalent LERK-1, -3 and -4 Fc samples to the Esk channel. However, the predicted maximal binding would have given much larger responses (as seen on the HEK chip) suggesting that the binding is relatively weak. This is highlighted in the case of LERK-3 by the failure to defect any binding of monomeric ligand to Esk.

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. TABLE 3 Eph-subfamily cDNA clones isolated from ES cells by RT-PCR

Clone designation Closest homologue Homology (%)

Primers P2 and P4^*

25C5 Sek 97.8

33C1.1 Nuk 97.2

33C1.2 Eck 99.1

33C1.5 Eck 100 Primers P and P4^*

35C4 Eck 97.2

35C6 Mek4 97.3

35C10 Mek4 99.7

35C11 Mek4 99.7

35C15 Eph 83

Homologues were identified by screening nucleic acid databanks with the FASTA program. Sek (11), Nuk (15), Eck (21) and Mek (8) are murine Eph-subfamily molecules; Eph (3) is a human molecule.

Refers to the primers used in the initial amplification during RT-PCR. BLBLIOGRAPHY:

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22. Bartiey, T.D, Hunt, RW, Welcher, A.A, Boyle, W.J, Parker, V.P, Lindberg, RA, Lu, H.S., Colombero, A.M., Elliott, RL, Guthrie, B A, Hoist, P.L., Skrine, J.D, Toso, RJ, Zhang, M, Fernandez, E, Trail, G, Varnum, B, Yarden, Y, Hunter, T. & Fox, G.M. (1994) Nature 368, 558-560. 23. Beckmann, M.P, Cerretti, D.P, Baum, P, Vanden, B.T, James, L, Farrah, T, Kozlosky, C, Hollingsworth, T, Shilling, H, Maraskovsky, E, Fletcher, F A, Lhotak, V, Pawson, T. & Lyman, S.D. (1994) Embo Journal 13, 3757-3762.

24. Cheng, HJ. & Flanagan, J.G. (1994) Cell 79, 157-168.

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47 Lackmann et al (1996) Proc. Natl. Acad Sci. USA 93: 2523-2527.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: (OTHER THAN US) AMRAD OPERATIONS PTY LTD (US ONLY):- Andrew Wallace BOYD and Jason Lickliter

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

AND USE THEREOF (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DAVIES COLLISON CAVE

(B) STREET: 1 LITTLE COLLINS STREET

(C) CITY: MELBOURNE

(D) STATE: VICTORIA

(E) COUNTRY: AUSTRALIA

(F) ZLP: 3000

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: LBM PC compatible

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

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

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT INTERNATIONAL

(B) FILING DATE: 20-DEC-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: AU PN7277/95

(B) FILING DATE: 22-DEC-1995

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HUGHES DR, E JOHN L

(C) REFERENCE DOCKET NUMBER: EJH/EK

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +61 3 9254 2777

(B) TELEFAX: +61 3 9254 2770 (2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3309 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..2931

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

TATGGTGAAC CGTGTGTGTC TGCTGCATGA GAAGACCCCA GTGAGTGAGC ATGTTACCAA -295

GTGCTGTAGT GGATCCCTGG TGGAAAGGCG GCCATGCTTC TCTGCTCTGA CAGTTGATGA -235

AACATATGTC CCCAAAGAGT TTAAAGCTGA GACCTTCACC TTCCACTCTG ATATCTGCAC -175

ACTTCCAGAG AAGGAGAAGC AGATTAAGAA ACAAACGGCT CTTGCTGAGC TGGTGAAGCA -115

CAAGCCCAAG GCTACAGCGG AGCAACTGAA GACTGTCATG GATGACTTTG CACAGTTCCT -55

GGATACATGT TGCAAGGCTG CTGGGAATTC CTGGGCCAGG TCCCGGCCGG CGCC -1

ATG GAG CGG CGC TGG CCC CTG GGG CTT GCA TTG CTG CTG CTG CTG CTC 48 Met Glu Arg Arg Trp Pro Leu Gly Leu Ala Leu Leu Leu Leu Leu Leu 1 5 10 15

TGC GCC CCG CTG CCC CCG GGG GCG CGC GCC GAG GAA GTC ACT CTA ATG 96 Cys Ala Pro Leu Pro Pro Gly Ala Arg Ala Glu Glu Val Thr Leu Met 20 25 30

GAC ACA AGC ACA GCA CAA GGA GAG CTG GGC TGG CTT CTG GAT CCC CCA 144 Asp Thr Ser Thr Ala Gin Gly Glu Leu Gly Trp Leu Leu Asp Pro Pro 35 40 45 GAG ACT GGG TGG AGT GAG GTG CAA CAA ATG CTA AAC GGG ACA CCC CTG 192 Glu Thr Gly Trp Ser Glu Val Gin Gin Met Leu Asn Gly Thr Pro Leu 50 55 60

TAC ATG TAC CAA GAC TGC CCA ATA CAG GAA GGT GGG GAC ACT GAC CAC 240 Tyr Met Tyr Gin Asp Cys Pro lie Gin Glu Gly Gly Asp Thr Asp His 65 70 75 80

TGG CTT CGC TCC AAT TGG ATC TAC CGC GGA GAG GAA GCT TCA CGC ATC 288 Trp Leu Arg Ser Asn Trp lie Tyr Arg Gly Glu Glu Ala Ser Arg lie 85 90 95

TAC GTG GAG CTG CAG TTC ACC GTG CGG GAC TGT AAG AGT TTC CCA GGG 336 Tyr Val Glu Leu Gin Phe Thr Val Arg Asp Cys Lys Ser Phe Pro Gly 100 105 110

GGA GCT GGG CCT CTG GGA TGC AAA GAG ACC TTC AAC CTT TTC TAC ATG 384 Gly Ala Gly Pro Leu Gly Cys Lys Glu Thr Phe Asn Leu Phe Tyr Met 115 120 125

GAG AGT GAC CAG GAT GTG GGC ATT CAG CTC CGA CGA CCT TTG TTC CAA 432 Glu Ser Asp Gin Asp Val Gly lie Gin Leu Arg Arg Pro Leu Phe Gin 130 135 140

AAG GTA ACA ACT GTG GCA GCA GAC CAG AGC TTC ACC ATC AGA GAC CTG 480 Lys Val Thr Thr Val Ala Ala Asp Gin Ser Phe Thr lie Arg Asp Leu 145 150 155 160

GCA TCT GAC TCT GTA AAG CTG AAT GTA GAA CGC TGC TCG TTG GGC CAC 528 Ala Ser Asp Ser Val Lys Leu Asn Val Glu Arg Cys Ser Leu Gly His 165 170 175

CTC ACC CGC CGT GGC CTC TAC TTA GCT TTC CAC AAC CCG GGT TCC TGT 576 Leu Thr Arg Arg Gly Leu Tyr Leu Ala Phe His Asn Pro Gly Ser Cys 180 185 190

GTG GCG CTA GTG TCT GTA AGG GTG TTC TAC CAG CGC TGT GCC GAG ACC 624 Val Ala Leu Val Ser Val Arg Val Phe Tyr Gin Arg Cys Ala Glu Thr 195 200 205

GTG CAT GGC TTG GCC CAC TTC CCT GAC ACT CTC CCT GGA CCT GGA GGG 672 Val His Gly Leu Ala His Phe Pro Asp Thr Leu Pro Gly Pro Gly Gly 210 215 220

TTG GTT GAA GTA GCT GGA ACG TGC CTC TCC CAT GCA CAG ATC AGC TTG 720 Leu Val Glu Val Ala Gly Thr Cys Leu Ser His Ala Gin lie Ser Leu 225 230 235 24 0

GGG TCC TCA GGT ACA CCA CGA ATG CAC TGC AGC CCT GAT GGC GAG TGG 768 Gly Ser Ser Gly Thr Pro Arg Met His Cys Ser Pro Aβp Gly Glu Trp 245 250 255

CTG GTG CCT GTG GGT CAG TGC CAG TGC GAG CCT GGC TAT GAA GAA AGC 816 Leu Val Pro Val Gly Gin Cys Gin Cys Glu Pro Gly Tyr Glu Glu Ser 260 265 270

AGT GGA AAT GTG GGA TGC ACT GCC TGT CCT ACT GGT TTC TAT CGA GTG 864 Ser Gly Asn Val Gly Cys Thr Ala Cys Pro Thr Gly Phe Tyr Arg Val 275 280 285

GAC ATG AAT ACA CTC CGT TGT CTC AAG TGC CCC CAA CAT AGC ATA GCA 912 Asp Met Asn Thr Leu Arg Cys Leu Lys Cys Pro Gin His Ser lie Ala 290 295 300

GAG TCT GAG GGG TCT ACC ATC TGT ACC TGT GAG AAT GGA CAT TAT CGA 960 Glu Ser Glu Gly Ser Thr lie Cyβ Thr Cys Glu Asn Gly His Tyr Arg 305 310 315 320

GCC CCT GGG GAG GGT CCC CAG GTA GCA TGC ACA CGT CCC CCA TCG GCT 1008 Ala Pro Gly Glu Gly Pro Gin Val Ala Cyβ Thr Arg Pro Pro Ser Ala 325 330 335

CCC CAA AAT CTG AGC TTC TCC ACA TCA GGG ACT CAA CTC TCC CTG CGC 1056 Pro Gin Asn Leu Ser Phe Ser Thr Ser Gly Thr Gin Leu Ser Leu Arg 340 345 350

TGG GAG CCC CCC AGA GAT ACA GGG GGA CGC CAT GAT ATC AGA TAC AGC 1104 Trp Glu Pro Pro Arg Asp Thr Gly Gly Arg His Asp lie Arg Tyr Ser 355 360 365

GTG GAG TGC TTG CAG TGT CGG GGC ATT GCA CAG GAT GGG GGT CCC TGC 1152 Val Glu Cyβ Leu Gin Cyβ Arg Gly lie Ala Gin Asp Gly Gly Pro Cys 370 375 380

CAA CCC TGT GGA AAA GGT GTG CAC TTT TCC CCG GCT GCT TCC GGG CTC 1200 Gin Pro Cyβ Gly Lys Gly Val His Phe Ser Pro Ala Ala Ser Gly Leu 385 390 395 400

ACC ACA TCT ACC GTG CAA GTG CAA GGC CTC GAG CCT TAC GCC AAC TAC 1248 Thr Thr Ser Thr Val Gin Val Gin Gly Leu Glu Pro Tyr Ala Asn Tyr 405 410 415 ACA TTT ACC GTC AAA TCC CAA AAC AGA GTG TCA GGA CTG GAC AGT TCC 1296 Thr Phe Thr Val Lys Ser Gin Asn Arg Val Ser Gly Leu Asp Ser Ser 420 425 430

AGC CCT AGC AGC GCC TCC CTC AGT ATC AAC ATG GGG CAC GCA GAG TCA 1344 Ser Pro Ser Ser Ala Ser Leu Ser lie Aβn Met Gly His Ala Glu Ser 435 440 445

CTC TCT GGC CTG TCA CTG AAG CTG GTG AAG AAA GAA CCG AGG CAG CTG 1392 Leu Ser Gly Leu Ser Leu Lys Leu Val Lys Lys Glu Pro Arg Gin Leu 450 455 460

GAG CTG ACT TGG GCA GGG TCC CGA CCC CGA AAT CCT GGA GGG AAT CTG 1440 Glu Leu Thr Trp Ala Gly Ser Arg Pro Arg Asn Pro Gly Gly Asn Leu 465 470 475 480

AGC TAT GAG CTG CAC GTG CTG AAT CAG GAC GAA GAA TGG CAC CAG ATG 1488 Ser Tyr Glu Leu His Val Leu Asn Gin Asp Glu Glu Trp His Gin Met 485 490 495

GTG TTG GAA CCC AGG GTC TTG CTG ACA AAA CTT CAG CCA GAT ACC ACA 1536 Val Leu Glu Pro Arg Val Leu Leu Thr Lys Leu Gin Pro Asp Thr Thr 500 505 510

TAC ATT GTC AGA GTG CGA ACA CTG ACC CCA CTG GGG CCT GGC CCT TTC 1584 Tyr He Val Arg Val Arg Thr Leu Thr Pro Leu Gly Pro Gly Pro Phe 515 520 525

TCC CCT GAC CAT GAG TTT CGG ACA AGC CCA CCA GTT TCC AGA AGC CTG 1632 Ser Pro Asp His Glu Phe Arg Thr Ser Pro Pro Val Ser Arg Ser Leu 530 535 540

ACC GGA GGA GAG ATT GTG GCC GTC ATC TTT GGA TTG CTG CTT GGA ATA 1680 Thr Gly Gly Glu He Val Ala Val He Phe Gly Leu Leu Leu Gly He 545 550 555 560

GCT CTG CTG ATC GGG ATT TAT GTC TTC CGT TCA AGG AGA GGC CAG AGA 1728 Ala Leu Leu He Gly He Tyr Val Phe Arg Ser Arg Arg Gly Gin Arg 565 570 575

CAG AGA CAG CAG AGG CAG CGT GAA CGC ACC ACC AAT GTC GAT CGA GAG 1776 Gin Arg Gin Gin Arg Gin Arg Glu Arg Thr Thr Asn Val Asp Arg Glu 580 585 590

GAC AAG CTG TGG CTA AAA CCC TAT GTG GAC CTC CAG GCC TAT GAG GAC 1824 Asp Lye Leu Trp Leu Lys Pro Tyr Val Asp Leu Gin Ala Tyr Glu Aβp 595 600 605

CCT GCA CAG GGA GCC TTA GAC TTT GCC CAG GAA CTG GAC CCA GCC TGG 1872 Pro Ala Gin Gly Ala Leu Asp Phe Ala Gin Glu Leu Asp Pro Ala Trp 610 615 620

CTG ATT GTG GAC ACT GTC ATA GGA GAA GGG GAG TTT GGT GAA GTG TAT 1920 Leu He Val Asp Thr Val He Gly Glu Gly Glu Phe Gly Glu Val Tyr 625 630 635 640

CGG GGA GCC CTG AGA CTC CCC AGC CAA GAT TGC AAG ACT GTG GCC ATT 1968 Arg Gly Ala Leu Arg Leu Pro Ser Gin Asp Cys Lys Thr Val Ala He 645 650 655

AAG ACC TTG AAA GAT ACA TCC CCA GAT GGC TAC TGG TGG AAT TTC CTT 2016 Lys Thr Leu Lys Asp Thr Ser Pro Asp Gly Tyr Trp Trp Aβn Phe Leu 660 665 670

CGA GAG GCA ACT ATC ATG GGC CAG TTC AAC CAC CCA CAC ATT CTA CGC 2064 Arg Glu Ala Thr He Met Gly Gin Phe Asn His Pro His He Leu Arg 675 680 685

CTA GAA GGT GTC ATC ACA AAA AGA AAG CCC ATC ATG ATC ATC ACA GAA 2112 Leu Glu Gly Val He Thr Lys Arg Lys Pro He Met He He Thr Glu 690 695 700

TTT ATG GAA AAT GGA GCC CTG GAT GCC TTT CTG AAG GAA CGG GAG GAC 2160 Phe Met Glu Asn Gly Ala Leu Asp Ala Phe Leu Lys Glu Arg Glu Asp 705 710 715 720

CAA CTA GCT CCT GGT CAG CTA GTG GCT ATG CTA CTG GGC ATA GCA TCA 2208 Gin Leu Ala Pro Gly Gin Leu Val Ala Met Leu Leu Gly He Ala Ser 725 730 735

GGC ATG AAC TGC CTC AGT GGC CAC AAT TAT GTC CAT AGA GAC CTG GCT 2256 Gly Met Aβn Cyβ Leu Ser Gly His Asn Tyr Val Hie Arg Aβp Leu Ala 740 745 750

GCC AGG AAC ATC TTG GTG AAT CAG AAC CTG TGC TGC AAG GTG TCT GAC 2304 Ala Arg Aβn He Leu Val Asn Gin Asn Leu Cys Cys Lys Val Ser Asp 755 760 765

TTT GGC TTG ACC CGC CTC CTG GAT GAC TTT GAC GGC ACC TAT GAA ACC 2352 Phe Gly Leu Thr Arg Leu Leu Asp Asp Phe Asp Gly Thr Tyr Glu Thr 770 775 780 CAG GGA GGA AAG ATC CCC ATC CGA TGG ACA GCC CCA GAA GCT ATT GCC 2400 Gin Gly Gly Lys He Pro He Arg Trp Thr Ala Pro Glu Ala He Ala 785 790 795 800

CAT CGG AT<_ TTC ACC ACA GCC AGT GAT GTG TGG AGC TTT GGG ATT GTA 2448 His Arg He Phe Thr Thr Ala Ser Asp Val Trp Ser Phe Gly He Val 805 810 815

ACG TGG GAG GTG TTG AGT TTT GGC GAC AAA CCC TAT GGG GAG ATG AGC 2496 Thr Trp Glu Val Leu Ser Phe Gly Aβp Lys Pro Tyr Gly Glu Met Ser 820 825 830

AAC CAA GAG GTA ATG AAA AGC ATT GAA GAT GGG TAC CGG TTG CCC CCT 2544 Asn Gin Glu Val Met Lys Ser He Glu Asp Gly Tyr Arg Leu Pro Pro 835 840 845

CCT GTG GAC TGT CCT GCC CCT CTC TAT GAA CTC ATG AAG AAC TGC TGG 2592 Pro Val Asp Cys Pro Ala Pro Leu Tyr Glu Leu Met Lys Aβn Cys Trp 850 855 860

GCT TAC GAT CGT GCC CGT CGA CCC CAC TTC CTC CAG CTG CAG GCA CAT 2640 Ala Tyr Asp Arg Ala Arg Arg Pro His Phe Leu Gin Leu Gin Ala Hie 865 870 875 880

CTG GAA CAG TTG CTT ACT GAC CCC CAT TCC CTA AGG ACA ATT GCC AAC 2688 Leu Glu Gin Leu Leu Thr Asp Pro His Ser Leu Arg Thr He Ala Asn 885 890 895

TTT GAC CCT AGG GTG ACC TTA CGC CTG CCC AGC CTG AGT GGC TCT GAT 2736 Phe Aβp Pro Arg Val Thr Leu Arg Leu Pro Ser Leu Ser Gly Ser Aβp 900 905 910

GGG ATC CCT TAT CGA AGT GTC TCT GAG TGG CTT GAA TCC ATA CGC ATG 2784 Gly He Pro Tyr Arg Ser Val Ser Glu Trp Leu Glu Ser He Arg Met 915 920 925

AAG CGC TAC ATC CTG CAC TTC CGT TCG GCT GGG CTG GAC ACC ATG GAG 2832 Lys Arg Tyr He Leu Hie Phe Arg Ser Ala Gly Leu Asp Thr Met Glu 930 935 940

TGT GTG CTG GAG CTG ACG GCT GAG GAC CTG ACG CAG ATG GGA ATA ACG 2880 Cyβ Val Leu Glu Leu Thr Ala Glu Asp Leu Thr Gin Met Gly He Thr 945 950 955 960

TTG CCA GGG CAC CAG AAA CGA ATT CTC TGC AGT ATT CAA GGA TTT AAG 2928 Leu Pro Gly Hie Gin Lys Arg He Leu Cyβ Ser He Gin Gly Phe Lys 965 970 975

GAC TGAGCATCCA CTGAAAAGAT GCTCCAGCCC TCTGCCTGCC TCCATTAGCA 2981

Asp

AGGACGGGGT ACAGTCAACT CCCTGGGCCT TTCCTCAGCC TACGAAATGT AGGCTATTGG 3041

TGCTGCTCCT GCCCAGTCAA TCAGAACTCT GCCTTTGAAC CAAGGAGCCT TTGTTTATAA 3101

AGGGGGTGGA TGGGTACAAG TGAAGGGGnC TGTGGGTGGG TTCTGGGGGA GGGTTTAATA 3161

nATACACTTA CATATGCATT ATCTATTTTT GTAAATAAAC AAGAGTTGAG TTTTAAAAAA 3221

AAAAAAAAAG GAATTCCTGC AGCCCGGGGG ATCCACTAGT TCTAGAGCGG CCGCCACCGC 3281

GGTGGAGCTC CAGCTTTTGT TCCCTTTA 3309

(2) INFORMATION FOR SEQ ID Nθ:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 977 amino acidβ

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

Met Glu Arg Arg Trp Pro Leu Gly Leu Ala Leu Leu Leu Leu Leu Leu 1 5 10 15

Cys Ala Pro Leu Pro Pro Gly Ala Arg Ala Glu Glu Val Thr Leu Met 20 25 30

Asp Thr Ser Thr Ala Gin Gly Glu Leu Gly Trp Leu Leu Asp Pro Pro 35 40 45

Glu Thr Gly Trp Ser Glu Val Gin Gin Met Leu Asn Gly Thr Pro Leu 50 55 60

Tyr Met Tyr Gin Asp Cys Pro He Gin Glu Gly Gly Asp Thr Asp His 65 70 75 80

Trp Leu Arg Ser Aβn Trp He Tyr Arg Gly Glu Glu Ala Ser Arg He 85 90 95

Tyr Val Glu Leu Gin Phe Thr Val Arg Asp Cys Lys Ser Phe Pro Gly 100 105 110

Gly Ala Gly Pro Leu Gly Cys Lys Glu Thr Phe Asn Leu Phe Tyr Met 115 120 125

Glu Ser Aβp Gin Asp Val Gly He Gin Leu Arg Arg Pro Leu Phe Gin 130 135 140

Lys Val Thr Thr Val Ala Ala Asp Gin Ser Phe Thr He Arg Asp Leu 145 150 155 160

Ala Ser Asp Ser Val Lys Leu Aβn Val Glu Arg Cys Ser Leu Gly His 165 170 175

Leu Thr Arg Arg Gly Leu Tyr Leu Ala Phe His Asn Pro Gly Ser Cys 180 185 190

Val Ala Leu Val Ser Val Arg Val Phe Tyr Gin Arg Cyβ Ala Glu Thr 195 200 205

Val Hie Gly Leu Ala His Phe Pro Asp Thr Leu Pro Gly Pro Gly Gly 210 215 220

Leu Val Glu Val Ala Gly Thr Cys Leu Ser His Ala Gin He Ser Leu 225 230 235 240

Gly Ser Ser Gly Thr Pro Arg Met His Cys Ser Pro Asp Gly Glu Trp 245 250 255

Leu Val Pro Val Gly Gin Cys Gin Cys Glu Pro Gly Tyr Glu Glu Ser 260 265 270

Ser Gly Aen Val Gly Cys Thr Ala Cys Pro Thr Gly Phe Tyr Arg Val 275 280 285

Aβp Met Aβn Thr Leu Arg Cyβ Leu Lys Cys Pro Gin His Ser He Ala 290 295 300

Glu Ser Glu Gly Ser Thr He Cys Thr Cyβ Glu Aβn Gly His Tyr Arg 305 310 315 320

Ala Pro Gly Glu Gly Pro Gin Val Ala Cys Thr Arg Pro Pro Ser Ala 325 330 335 Pro Gin Asn Leu Ser Phe Ser Thr Ser Gly Thr Gin Leu Ser Leu Arg 340 345 350

Trp Glu Pro Pro Arg Asp Thr Gly Gly Arg His Aβp He Arg Tyr Ser 355 360 365

Val Glu Cyβ Leu Gin Cyβ Arg Gly He Ala Gin Aβp Gly Gly Pro Cys 370 375 380

Gin Pro Cys Gly Lys Gly Val Hie Phe Ser Pro Ala Ala Ser Gly Leu 385 390 395 400

Thr Thr Ser Thr Val Gin Val Gin Gly Leu Glu Pro Tyr Ala Asn Tyr 405 410 415

Thr Phe Thr Val Lys Ser Gin Asn Arg Val Ser Gly Leu Asp Ser Ser 420 425 430

Ser Pro Ser Ser Ala Ser Leu Ser He Aβn Met Gly Hie Ala Glu Ser 435 440 445

Leu Ser Gly Leu Ser Leu Lys Leu Val Lys Lys Glu Pro Arg Gin Leu 450 455 460

Glu Leu Thr Trp Ala Gly Ser Arg Pro Arg Asn Pro Gly Gly Asn Leu 465 470 475 480

Ser Tyr Glu Leu His Val Leu Asn Gin Asp Glu Glu Trp His Gin Met 485 490 495

Val Leu Glu Pro Arg Val Leu Leu Thr Lys Leu Gin Pro Asp Thr Thr 500 505 510

Tyr He Val Arg Val Arg Thr Leu Thr Pro Leu Gly Pro Gly Pro Phe 515 520 525

Ser Pro Asp His Glu Phe Arg Thr Ser Pro Pro Val Ser Arg Ser Leu 530 535 540

Thr Gly Gly Glu He Val Ala Val He Phe Gly Leu Leu Leu Gly He 545 550 555 560

Ala Leu Leu He Gly He Tyr Val Phe Arg Ser Arg Arg Gly Gin Arg 565 570 575

Gin Arg Gin Gin Arg Gin Arg Glu Arg Thr Thr Asn Val Asp Arg Glu 580 585 590

Asp Lys Leu Trp Leu Lys Pro Tyr Val Asp Leu Gin Ala Tyr Glu Asp 595 600 605

Pro Ala Gin Gly Ala Leu Asp Phe Ala Gin Glu Leu Asp Pro Ala Trp 610 615 620

Leu He Val Asp Thr Val He Gly Glu Gly Glu Phe Gly Glu Val Tyr 625 630 635 640

Arg Gly Ala Leu Arg Leu Pro Ser Gin Asp Cys Lys Thr Val Ala He 645 650 655

Lys Thr Leu Lys Asp Thr Ser Pro Asp Gly Tyr Trp Trp Asn Phe Leu 660 665 670

Arg Glu Ala Thr He Met Gly Gin Phe Asn His Pro His He Leu Arg 675 680 685

Leu Glu Gly Val He Thr Lye Arg Lye Pro He Met He He Thr Glu 690 695 700

Phe Met Glu Asn Gly Ala Leu Asp Ala Phe Leu Lys Glu Arg Glu Aβp 705 710 715 720

Gin Leu Ala Pro Gly Gin Leu Val Ala Met Leu Leu Gly He Ala Ser 725 730 735

Gly Met Asn Cys Leu Ser Gly His Asn Tyr Val His Arg Asp Leu Ala 740 745 750

Ala Arg Asn He Leu Val Asn Gin Asn Leu Cys Cys Lys Val Ser Aβp 755 760 765

Phe Gly Leu Thr Arg Leu Leu Asp Asp Phe Asp Gly Thr Tyr Glu Thr 770 775 780

Gin Gly Gly Lys He Pro He Arg Trp Thr Ala Pro Glu Ala He Ala 785 790 795 800

His Arg He Phe Thr Thr Ala Ser Asp Val Trp Ser Phe Gly He Val 805 810 815

Thr Trp Glu Val Leu Ser Phe Gly Asp Lys Pro Tyr Gly Glu Met Ser 820 825 830 Asn Gin Glu Val Met Lys Ser He G]u Asp Gly Tyr Arg Leu Pro Pro 835 840 845

Pro Val Asp Cys Pro Ala Pro Leu Tyr Glu Leu Met Lys Asn Cys Trp 850 855 860

Ala Tyr Asp Arg Ala Arg Arg Pro His Phe Leu Gin Leu Gin Ala His 865 870 875 880

Leu Glu Gin Leu Leu Thr Asp Pro His Ser Leu Arg Thr He Ala Asn 885 890 895

Phe Asp Pro Arg Val Thr Leu Arg Leu Pro Ser Leu Ser Gly Ser Asp 900 905 910

Gly He Pro Tyr Arg Ser Val Ser Glu Trp Leu Glu Ser He Arg Met 915 920 925

Lys Arg Tyr He Leu His Phe Arg Ser Ala Gly Leu Asp Thr Met Glu 930 935 940

Cys Val Leu Glu Leu Thr Ala Glu Asp Leu Thr Gin Met Gly He Thr 945 950 955 960

Leu Pro Gly His Gin Lye Arg He Leu Cyβ Ser He Gin Gly Phe Lys 965 970 975

Asp

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:3:

GTAGGCATGC AAGGAGAC(A/C) TT(C/T)AACC 27 (2) INFORMATION FOR SΞQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4:

GCGATGATCA T(C/G)AC(A/G/T) GA(A/G)TA (C/T)ATGG 25

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

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

GTAGGAATTC CA(C/G/T)ACATC(A/G) C T(A/G)GC 24

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CCA(T/A)A(A/G) CTCC A(C/T)ACATC(A/G) CT 20

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Asp Leu Ala Ala Arg Asn 5

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Pro He Arg Trp Thr Ala Pro 5 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Glu Glu Val Thr Leu Met Asp Thr 5

Claims

CLAIMS:

1. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a receptor tyrosine kinase (RTK) or a derivative, homologue or chemical analogue thereof, said RTK having the following characteristics: (i) belongs to the Eph subfamily of RTKs as determined by conserved cysteine residues and fibronectin type III repeats;

(ii) comprises protein tyrosine kinase catalytic domain motifs; and

(iii) comprises an amino acid sequence substantially as set forth in SEQ ID NO: 2 or having at least about 79% similarity thereto.

2. An isolated nucleic acid molecule according to claim 1 wherein the RTK is of mammalian origin.

3. An isolated nucleic acid molecule according to claim 2 wherein the RTK is of human or murine origin.

4. An isolated nucleic acid molecule according to claim 1 comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 1 or having at least about 82% similarity thereto or is capable of hybridizing to the sequence set forth in SEQ LD NO: 1 or a complementary form thereof under low stringency conditions.

5. An isolated nucleic acid molecule according to claim 1 or 2 or 3 or 4 contained in a vector.

6. An isolated nucleic acid molecule according to claim 5 wherein the vector is an expression vector.

7. An isolated nucleic acid molecule according to claim 1 or 4 encoding a soluble form of said RTK.

8. A recombinant polypeptide or a derivative, homologue or chemical analogue thereof having the folio wing characteristics:

(i) is an RTK belonging to the Eph subfamily of RTKs as determined by cysteine residues and fibronectin type II repeats;

(ii) comprises protein tyrosine kinase catalytic domain motifs; and

(iii) comprises an amino acid sequence substantially as set forth in SEQ LD NO:2 or having at least about 79% similarity thereto.

9. A recombinant polypeptide according to claim 8 wherein said RTK is of mammalian origin.

10. A recombinant polypeptide according to claim 9 wherein the RTK is of human or murine origin.

11. A recombinant polypeptide according to claim 8 encoded by a nucleotide sequence substantially as set forth in SEQ LD NO: 1 or having at least about 82% similarity thereto or a nucleotide sequence capable of hybridizing to the sequence set forth in SEQ LD NO: 1 or a complementary form thereof under low stringency conditions.

12. A recombinant polypeptide according to any one of claims 8 to 11 in soluble form.

13. A secreted recombinant polypeptide comprising conserved cysteine residues and fibronectin type III repeats and an extracellular portion of tiie amino acid sequence set forth in SEQ LD NO:2 or having at least about 79% similarity thereto.

14. A pharmaceutical composition comprising a recombinant polypeptide according to any one of claims 8 to 13 and one or more pharmaceutically acceptable carriers and/or diluents.

15. A method for modulating Esk-ligand interaction in an animal, said method comprising administering to said animal a modulating effective amount of an agonist or antagonist of Esk- ligand interaction.

16. A method according to claim 15 wherein the Esk has the following characteristics: (i) is an RTK belonging to the Eph subfamily of RTKs as determined by cysteine residues and fibronectin type II repeats;

(ii) comprises protein tyrosine kinase catalytic domain motifs; and

(iii) comprises an amino acid sequence substantially as set forth in SEQ ID NO: 2 or having at least about 79% similarity thereto.

17. An antagonist or agonist of an Esk, said Esk having the following characteristics: (i) is an RTK belonging to the Eph subfamily of RTKs as determined by cysteine residues and fibronectin type II repeats;

(ii) comprises protein tyrosine kinase catalytic domain motifs; and

(iii) comprises an amino acid sequence substantially as set forth in SEQ ID NO: 2 or having at least about 79% similarity thereto.

18. An antagonist according to claim 17 wherein said antagonist is an antibody to said Esk.

19. An antibody to the recombinant polypeptide of claim 8.

20. An antibody to the secreted recombinant polypeptide of claim 13.