AU664752B2 - Receptor-type tyrosine kinase-like molecules - Google Patents

Description

OPI DATE 13/12/93 APPLN. ID 40505/93 AOJP DATE 24/02/94 PCT NUMBER PCT/AU93/00210 AU9340505 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 International Publication Number: WO 93/23429 C07K 13/00, 15/06, C12N 9/12 Al C12N 15/12, 15/54, C12Q 1/68 (43) International Publication Date: 25 November 1993 (25.11.93) (21) International Application Number: PCT/AU93/00210 (74) Agents: SLATTERY, John, M. et al.; Davies Collison Cave, 1 Little Collins Street, Melbourne, VIC 3000 (22) International Filing Date: 10 May 1993 (10.05.93) (AU).

Priority data: (81) Designated States: AU, CA, FI, JP, KR, NO, NZ, US, Eu- PL 2358 11 May 1992 (11.05.92) AU ropean patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

(71) Applicant (for all designated States except US): LUDWIG INSTITUTE FOR CANCER RESEARCH [CH/CH]; Published Stadelhoferstrasse 22, CH-8001 Zurich With international search report.

(72) Inventors; and Inventors/Applicants (for US orny) STACKER, Steven, Alan [AU/AU]; 30 McKean Street, North Fitzroy, VIC 3068 HOVENS, Christopher, Martin [AU/CH]; Institute of Molecular Biology, University of Zurich, Winterhurerstrasse .10, CH-8057 Zurich WILKS, Andrew, Frederick [GB/AU]; 12 Rosamond Crescent, Doncaster East, VIC 3109 (AU).

(54) Title: RECEPTOR-TYPE TYROSINE KINASE-LIKE MOLECULES (57) Abstract The present invention relates to proteins having receptor-type tyrosine kinase-like properties which represent a novel family of proteins related to protein tyrosine kinases. The present invention relates to the full length proteins and to subunits, mutants, derivatives and/or analogues thereof and to nucleotide sequences encoding same. The present invention also extends to ligands for the above proteins and to pharmaceutical compositions comprising the proteins and/or mutants, derivatives and/or analogues thereof and/or ligands thereto.

WO 93/23429 PCTr/AU93/021 0 -1- RECEPTOR-TYPE TYROSINE KINASE-LIKE MOLECULES FIELD OF INVENTION The present invention relates to proteins having receptor-type tyrosine kinase-like properties which represent a novel family of proteins related to protein tyrosine kinases. The present invention relates to the full length proteins and to subunits, mutants, derivatives and/or analogues thereof and to nucleotide sequences encoding same. The present invention also extends to ligands for the above proteins and to pharmaceutical compositions comprising the proteins and/or mutants, derivatives and/or analogues thereof and/or ligands thereto.

BACKGROUND OF THE INVENTION The phosphorylation of tyrosine residues on protein substrates is a pathway whereby signals of growth and differentiation are transmitted by growth factor receptors and transforming oncogenes Evidence for this role of tyrosine phosphorylation came from the identification of receptors which bind known soluble growth factors. For example, the receptors for epidermal growth factor (EGF) platelet derived growth factor (PDGF) and colony stimulating factor-1 (CSF-1) were all shown to be transmembrane molecules with the cytoplasmic regions defining a tyrosine kinase catalytic domain The other line of evidence for a critical role played by tyrosine phosphorylation in growth control came from the study of viral oncogenes These genes were shown to be directly involved in growth dysregulation by observations of a change in cell growth following introduction of DNA encoding these genes into fibroblasts.

All oncogenes have been shown to have close cellular homologues (protooncogenes). One of the first identified oncogenes was v-src, the cellular homologue (c-src) is the prototypical representative of the family of cytoplasmic tyrosine kinases which, following myristylation, become associated with the inner leaf of the cell membrane WO 93/23429 PCFAU93/002 1 0 -2- Protein-tyrosine kinases (PTKs) represent a family of phosphotransferases related by their conserved catalytic domains (reviewed in 7 and 25). Phylogenetic analysis of this family suggests that several subfamilies of the PTKs exist based on the organisation of their non-catalytic sequences. These families include i) The Src related PTKs such as c-yes, c-lyn and hck; ii) the JAK family; and iii) at least seven subfamilies of growth factor receptors.

In particular, these previously known PTKs contain the Rossman motif (32) which is putatively associated with ATP binding. The Rossman motif has three invariant glycine residues in a six amino acid cluster as follows: Gly-X-Gly-X-X-Gly, where X is an amino acid residue.

In accordance with the present invetion, proteins having receptor-type PTK-like properties have been discovered representing a new family of proteins related to receptor-type PTKs but exhibiting one or more of the following characteristics: and/or an altered Rossman motif, a unique tri-amino acid sequence in the kinase catalytic domain and/or an extracellular region comprising leucine rich regions. The proteins having the receptor-type PTK-like properties of the present invention are designated herein "RYK" for "related to tyrosine kinases".

SUMMARY OF THE INVENTION One aspect of the present invention is directed to an isolated protein having receptor-type protein tyrosine kinase (PTK)-like properties including ATP binding, wherein said protein has an ATP binding site comprising a sequence of amino acid residues with the proviso that said amino acid sequence is not Gly-X-Gly-X-X-Gly, where X is any amino acid residue, or a subunit, fragment, derivative or analogue of said protein.

More particularly, the present invention provides an isolated protein having PTK-like properties including ATP binding wherein said protein has an ATP binding site consisting of Gln-a-Gly-b-c-Gly, where a, b and c may be the same or different and each is an amino acid residue, or a subunit, fragment, derivative or analogue of said WO 93/23429 PCT/AU93/00210 -3protein.

Another aspect of the present invention provides an isolated protein having PTK-like properties including ATP binding wherein said protein has an ATP binding site consisting of GIn-Glu-Gly-b-Phe-Gly, wherein b is Ser or Thr, or a subunit, fragment, derivative or analogue of said protein.

Still another aspect of the present invention relates to a protein having PTK-like properties including a kinase catalytic domain which contains amino acid sequence Asp-Asn-Ala or a subunit, fragment, derivative or analogue of said protein. More particularly, the protein has a kinase catalytic domain with amino acid sequence Asp- Asn-Ala in motif VII of said domain.

Yet still another aspect of the invention is directed to an isolated protein having PTK-like properties including an extracellular domain wherein said protein contains at least two leucine rich regions in said extracellular domain, or a subunit, fragment, derivative or analogue of said protein.

BRIEF DESCRIFTION OF THE FIGURES Figure 1 is a photographic representation depicting an analysis of mouse and human RYK expression by Northern blotting. PolyA mRNA (1 pg) from mouse tissues and in vitro cell lines was electrophoresed on a denaturing 1.0% w/v agarose/formaldehyde gel and the RNA transferred to Genescreen (Dupont). The transferred RNA was hybridized with either a 1.3 kb mouse RYK 3P-labelled probe and the filter autoradiographed for 16 hours at -70C. The relative mobilities of 28S rRNA and 18S rRNA are indicated.

A. The tracks from the left to right are as follows: lane 1, human breast carcinoma cell line A431; lane 2, human erythroleukaemic cell line K562; lane 3, NIH-3T3 fibroblasts; and the following mouse tissues: lane 4, 13 day old embryo; lane 5, placenta; lane 6, ovary; lane 7, testes; lane 8, thymus; lane 9, liver; lane lung; lane 11, kidney, lane 12, spleen; lane 13, brain; lane 14, salivary gland; lane heart; lana 16, skeletal muscle.

WO 93/23429 PCT/AU93/00210 -4- B. The tracks from left to right are as follows lane 1, mouse mast cell line lane 2, myelomonocytic cell line WEH13D-; lane 3, mouse mast cell line 32D; lane 4, mouse myeloid cell line B6SUTA; lane 5, mouse fibroblast cell line NIH-3T3; lane 6, mouse T cell line LB3; lane 7 and 8, myelomonocytic cell line WEHI3D lane 9, myelomonocytic cell line FDCP-1.

C. The tracks from left to right are as follows lane 1, plasma cell line PC13; lane 2, mouse embryonic cell line E30; lane 3, mouse embryonic cell line LE28; lane 4, mouse embryonic cell line LD3.3; lane 5, mouse embryonic cell-line P19; and, lane 6, mouse embryonic cell line D3.

Figure 2 shows the nucleotide and predicted amino acid sequence of the full length human RYK cDNA Schematic representation and partial restriction map of the human RYK cDNA represented by clones pBS-XYZand pBS-E. The open rectangle represents the coding region of 1896 bp, the thin line represents the 3' and untranslated regions and the transmembrane domain is a shaded box. The following restriction sites are indicated: Bgl II Hind III Pst I and Xba I (B) Nucleotide and predicted amino acid sequence of the human RYK cDNA clone compiled from the sequences of pBS-XYZ and pBS-E. The DNA sequence is numbered from the polylinker region of a 3.067 kb clone in the vector pBluescript Nucleotides numbered from 1 to 18 are the pBluescript polylinker, nucleotides thereafter represent sequence of the clones. Deduced amino acid sequence is represented by the single letter amino acid code and is found above the nucleotide sequence. The putative initiation codon is an ATG at amino acid position 1 and nucleotide position 132-134. The putative transmembrane domain is underlined, cysteine residues are circled and potential N-linked glycosylation sites (Asn-X- Ser/Thr) are indicated by an inverted bracket. Key residues of the catalytic domain are indicated in bold lettering. Two leucine-rich repeats are present in the human sequence, these are L1 (Leu-Ie-Gly-Leu-Asp-Ala-Glu-Leu-Tyr-Tyr-Val-Arg-Asn-Asp- Leu-Ile-Ser-His-Tyr-Ala-Leu-Ser-Phe) aind L2.(Leu-Met-Gln-Leu-Asn-Leu-Thr-Val- Asnc-Ser-Se-Lys-Asn-Phe-Thr-Val-Leu-Asn-Phe-Lys-Arg-Arg-Lys). An in frame termination codon is found at nucleotide position 1962.

WO 93/23429 PIT/AU9310021 0 Figure 3 is a representation of the nucleotide and predicted amino acid sequences of the mouse RYK cDNA clone. The putative initiation methionine (ATG) is located at position 1 and is immediately followed by a signal peptide sequence which is underlined. The transmembrane domain is boxed and extends from residues 184 to 211. In the cytoplasmic domain, the subdomains of the conserved PTKs are indicated under the sequence by Roman numerals Important residues of the cytoplasmic domain are boxed, th ATP binding site (Glh-Glu-Gly-Ser-Phe-Gly), conserved motif of PTK (Asp-Leu-Ala-Ala-Arg-Asn), conserved motif of RTK (Trp- Met-Ala-Leu-Glu) and the novel motif (Asp-Asn-Ala). Putative N-linked glycosylation sites are indicated with a parenthesis. Cysteine residues are circled.

The single letter amino acid code has been used throughout this diagram.

Figure 4 shows a comparison of the deduced amino acid sequences of human and mouse RYK. The amino acid sequences of human (42-606) and moue RYK (1-566) were aligned and gaps have been introduced to achieve maximum identity.

Matches are indicated by an asterisk between the sequences and conservative amino acid substitutions by a vertical line. The single letter amino acid code has been used for this figure. denotes an inframe stop codon. The putative transmembrane domain is underlined and the borders of the kinase domain indicated by square brackets.

Figure 5 shows a hydrophobicity plot of the predicted amino acid sequence of human RYK from Figure 3B (amino acids 1-606) analysed by the hydrophilicity algorithm of Kyte and Doolittle using a span length of 25 amino acids. The putative hydrophobic transmembrane domain is indicated (Tm).

Figure 6 is a schematic representation showing activity of the human RYK kinase domain.

A. Construction of the pGEX/RYK cytoplastic domain fusion proteins. Fusion proteins were generated between the cytoplastic domain of the human RYK molecule and glutathione-S-transferuse, in the pGEX-3X vector. The upper diagram represents the intact human RYK protein the lower the fusion protein. The striped 'WO 93/23429 PCT/AU93/00210 -6area represents the GST while the filled area is the catalytic domain of human RYK.

B. Demonstration of kinase activity by the human RYK kinase domain. Fusion proteins containing the human RYK kinase domain were constructed as described in Example 1 and expressed in bacteria and induced with IPTG. Bacteria were lysed in a Tx-100 lysis buffer and the solubilised proteins precipitated using glutathione- Sepharose. These immunoprecipitates were then subjected to initro labelling with gamma 3 2 p-ATP in the presence of kinase buffer. Labelled material was eluted from the beads using SDS sample buffer under reducing conditions and electrophoresed on an 8% w/v SDS-polyacrylamide gel. The two lanes are immunoprecipitates from bacteria which was either induced with IPTG, or (ii) not induced. Relative molecular weight markers are indicated.

Figure 7 is a representation of the localization of the human RYK gene on chromosomes from normal male lymphocytes using in situ hybridization. The ideogram represents the number of silver grains located on any chromosome within each metaphase.

Figure 8 shows an analysis of human RYK expression by Northern blotting. PolyA+ mRNA (0.5 pg) from in vitro cell lines MCF-7 (lane 1) and HSB-2 (lane 2) was electrophoresed on a denaturing 1.0% w/v agarose/formaldehyde gel and the RNA transferred to GeneScreen Plus (NEN). The transferred RNA was hybridised with a 2.3 kb human RYK 3P-labelled riboprobe and the filter autoradiographed for 16 hours at -70 C.

Figure 9 shows SDS-PAGE analysis of RYK. Analysis of RYK immunoprecipitated from a-S-methionine labelled MCF-7 cells. Lysates of 3 aSmethionine labelled MCF-7 cells were immunoprecipitated with a rabbit anti-RYK sera immune) or with pre-immune rabbit sera (PI; pre-immune) and analysed under reducing or non-reducing conditions as indicated (see Example Gels were dried and autoradiographed for 24 hours. Molecular weight markers are indicated.

SDS-PAGE analysis of in vitro transcription/translation products derived using T7 RNA polymerase and the techniques described in Example 1. An aliquot of the IWO 93/23429 PCT/AU93/00210 -7total reaction volume (10 pl of 75 pi) was diluted in 10p of 2x SDS-PAGE sample buffer containing 2% v/v beta-mercaptoethanol, boiled and loaded onto an 8% w/v denaturing acrylamide gel. The dried gel was exposed for 2 hours on a phosphorimager cassette (Molecular Dynamics). The following templates were used: pCDM8 (lane pCD.Human.RYK (lane' pCD.Mouse.RYK (lane 3) and pCD.Mouse.NYK (lane Molecular weight markers are indicated.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention is directed to proteins including polypeptides having receptortype PTK-like properties in isolated, recombinant and/or synthetic form including subunits, mutants, derivatives and analogues thereof. The proteins of the present invention have "receptor-type PTK-like properties" since by comparison to known receptor-type PTKs, they possess a signal peptide and extracellular, transmembrane and intracellular domains and have amino acid sequence homology to a kinase catalytic domain within the intracellular domain. By convention, the kinase catalytic domain is divided into motifs identified by Roman numerals I-XI.

However, the proteins of the present invention differ in one or more important respects thus defining a new family of tyrosine kinases. The differences are in one or more of the ATP binding site, motif VII of the kinase catalytic domain and/or the extracellular region. For convenience, the proteins of the present invention having receptor-type PTK-like properties are referred to herein as "RYK". When reference is made to extracellular, transmembrane and intracellular domains, this applies whether or not the RYK molecule is in isolated, recombinant or synthetic form.

Preferably, the RYK is of human or murine origin. Preferably, the murine RYK is mouse RYK. However, the present invention extends to RYK homologues from non-human and non-murine sources such as livestock animals sheep, goats, cows, horses or pigs), companion animals cats or dogs), laboratory test animals rabbits or guinea pigs) and captive and free wild animals. The present invention also encompasses peptide and polypeptide fragments derived from the above mentioned RYK proteins including subunits, mutants, derivatives or analogues WO 93/23429 PCT/AU93/00210 -8thereto.

The RYK molecule of the present invention comprises an amino acid sequence with ap- ATP binding site which is not Gly-X-Gly-X-X-Gly (Rossman motif) wherein X is any amino acid residue. More particularly, the ATP binding site is defined by the amino acid sequence Gln-a-Gly-b-c-Gly where a, b and c may be the same or different and each is an amino acid residue. Preferably, the amino acid sequence is Gln-Glu-Gly-b-Phe-Glywhere b is Ser or Thr. The term "ATP binding site".inuiades a putative ATP binding site such as when determined by amino acid sequence similarity rather than direct AT? binding data.

The present invention extends to a portion of the RYK molecule without an ATP binding site or putative ATP binding site provided that in its full length or near full length form, the molecule comprises an AT? binding site as defined above.

Advantageously, the mammalian RYK is a biologically pure or isolated preparation meaning that it has undergone some purification away from other proteins and/or nor,-proteinacous material. The purity of the preparation may be represented as at least 40% RYK, preferably at least 60% RYK, more preferably at least 75% RYK, even more preferably at least 85% RYK and still more preferably at least 95% RYK relative to non-RYK material as determined byweight, activity, amino acid similarity, antibody reactivity or other convenient means.

The mammalian RYK of the present invention may be naturally occurring or may be synthetic meaning that it is prepared by recombinant DNA or chemical synthetic techniques. In any event, the present invention encompasses RYK molecules having the naturally occurring amino acid sequence as well as molecules having single or multiple amino acid substitutions, deletions and/or additions.

Amino acid insertional derivatives of the mammalian RYK of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those ,WO 93/23429 PCT/AU93/00210 -9in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table 1: TABLE 1 Suitable residues for amino acid substitutions Original Residue Ala Arg Asn Asp Cys Gin Glu Gly His Ile Leu Lys Met Phe Ser Thr Trp Tyr Val Exemplary Substitutions Ser Lys Gin; His Glu Ser Asn Asp Ala Asn; Gin Leu; Val Le; Val Arg; Gin; Glu Leu; Ile Met; Leu; Tyr Thr Set Tyr Trp; Phe Ile; Leu W093/23429 PCT/AU93/00210 10 Where mammalian RYK is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.

Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions Will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.

The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield synthesis) and the like, or by recombinant DNA manipulations.

Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, oligonucleotide directed mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently elsewhere described (for example see Sambrook et aL Other examples of recombinant or synthetic mutants and derivatives of the mammalian RYK of the present invention include single or multiple substitutions, deletions and/or additions of any molecule naturally or artificially associated with the molecule such as carbohydrates, lipids and/or proteins or polypeptides. For example, different glycosylation patterns or elimination of glycosylation can result from expressing the mammalian RYK in different host cells. It should be noted that reference herein to "mammalian RYK" includes RYK produced by recombinant means in bacteria or in animals cells or produced by chemical synthetic menas.

Accordingly, "mnammalian RYK" is a RYK of mammalian origin but not necessarily produced in mammalian cells.

Particularly useful mutants include truncated mutants, i.e. RYK molecule absent Nterminal and/or C-terminal portions conveniently made using cDNA molecules truncated at the 5' and/or 3' ends, respectively. Furthermore, the present invention extends to subunits or fragments of mammalian RYK carrying one or more of the WO 93/23429 PCTf/AU93/00210 11 extracellular domain, transmembrane domain and/or cytoplasmic domain (also referred to as kinase catalytic domain or intracellular domain). A subunit or fragment containing the extracellular domain is particularly useful for screening for ligands of RYK or antagonists to RYK-ligand binding or may be useful as an antagonist itself. Accordingly, the present invention extends to a subunit or fragment of mammalian RYK containing the extracellular domain or portion or derivative thereof. By "subunit" or "f'agment" is meant a non-full length RYK molecule.

Preferably, the subunit or fragment is the extracellular domain portion of RYK.

More particularly, the present invention extends to an isolated extracellular domain or part or derivative thereof, said domain characterised in that it is isolatable from mammalian RYK and comprises at least one leucine rich region. Preferably, the mammalian RYK is human RYK. Preferably, the extracellular domain comprises at least two leucine rich regions. Preferably, the leucine rich regions comprise the amino acid sequences: Leu-Ile-Gly-Leu-Asp-Ala-Glu-Leu-Tyr-Tyr-Val-Arg-Asn-Asp-Leu-Ile-Ser-His-Tyr- Ala-Leu-Ser-Phe; and/or Leu-Met-Gln-Leu-Asn-Leu-Thr-Val-Asn-Ser-Ser-Lys-Asn-Phe-Thr-Val-Leu-Asn- Phe-Lys-Arg-Arg-Lys, or have at least 70-80% amino acid similarity thereto.

Other useful mutants include hybrid molecules and fusion molecules. A hybrid RYK molecule includes a molecule with at least part of one domain from a RYK from a first species of mammal fused or otherwise associated with at least part of another domain from a RYK from a second different species of mammal. For example, the extracellular domain or part thereof of human RYK maybe fused or associated with other domains of mouse RYK. Alternatively, the RYK hybrid or fusion molecules may be with regions of growth factor receptors such as Epidermal Growth Factor Receptor (EGFR).

I WO 3/23429 PCI'/AU93/00210 -12 In a further alternative embodiment, the hybrid occurs within a single domain and hence, for example, thd hybrid molecule may comprise a hybrid extracellular domain.

The present invention also extends to subunits, functional chemical equivalents or analogues of mammalian RYK herein described.

Analogues of the mammalian RYK protein contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatising the molecule and the use of crosslinkers and other methods which impose conformational constraints on the molecule. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation vf 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 NaBH4.

The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyozal.

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 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 WVO 93/23429 PCT/AU93/00210 -13chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with Nbromosuccinimide or alkylation of the indole ring with 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 iododcetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use or norleucine, 4-amino butyric acid, 4acid, 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.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homobifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n 1 to n= 6, glutaraldehyde, N hydroxysuccinimide esters and heterobifunctional reagents which usually contain an amino-reactive moiety such as Nhydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety or carbodiimide. In addition, peptides could be conformationally constrained by, for example, incorporation of C, and N,-methylamino acids, introduction of double bonds between C, and Cp atoms of amino acids and the formation of cyclic pepTides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention also extends to subunits, fragments, derivatives, homologues, analogues and immunological, functional and/or structural relatives of the mammalian RYK contemplated herein. Accordingly, reference herein to mammalian WO 93/23429 PC/AU93/00210 -14- RYK, RYK molecules or RYK-like molecules is to be taken as covering the full length molecule or any subunits, fragments, derivatives, analogues, homologues and/or relatives thereof. In its most preferred form, the RYK of the present invention is human RYK having an amino acid sequence substantially as set forth in Figure 2B, or having similarity thereto such as in the order of at least 50-70%, preferably at least 80% and most preferably at least 90% to all or a region or part thereof or is mouse RYK having an amino acid sequence substantially set forth in Figure 3 or having similarity thereto as defined above.

In a most preferred embodiment, the mammalian RYK of the present invention is human RYK and comprises a signal peptide, and extracellular, transmembrane and cytoplasmic (kinase) domains. The human RYK molecule is approximately 500-700 amino acids in length; preferably 550-650 amino acids in length, more preferably 590 to 620 amino acids in length and most preferably about 606 amino acids in length with a molecular weight of the unglycosylated form of about 60,000 72,000 daltons as determined by SDS-PAGE relative standard molecular weight markers.

The glycosylated form of the molecule has a molecular weight in the range 70,000- 120,000 as determined by SDS-PAGE relative standard molecular weight markers.

The glycosylated form of the molecule includes both natural glycosylation patterns and altered glycosylation patterns.

The present invention extends to an isolated protein having RYK properties including a catalytic kinase domain or a putative catayltic kinase domain wherein said catalytic kinase domain contains amino acid sequence Asp-d-e wherein d and e may be the same or different and each is an amino acid residue. Preferably, d is Asn. Preferably, e is Ala. Most preferably, the sequence is Asp-Asn-Ala and the RYK protein is as hereinbefore defined or a subunit, fragment, derivative or analogue thereof.

Another aspect of the present invention provides an isolated protein having receptortype PTK-like properties including an extracellular domain wherein said extracellular domain contains two leucine rich regions, or a subunit, fragment, derivative or ,WO 93/23429 PCT/AU93/00210 analogue of said protein. Preferably, th? -ine rich regions are: Leu-le-Gly-Leu-Asp-Ala-Glu-Leu-Tyr-Tyr-Val-Arg-Asn-Asp-Leu-Ie-Ser-His-Tyr- Ala-Leu-Ser-Phe; and/or Leu-Met-Gln-Leu-Asn-Leu-Thr-Val-Asn-Ser-Ser-Lys-Asn-Phe-Thr-Val-Leu-Asn- Phe-Lys-g-A-Arg-Lys or having at least 70-80% amino acid similarity thereto.

Preferably, the protein is RYK as hereinbefore described or a subunit, fragment, derivative or analogue thereof.

Yet another aspect of the present invention contemplates an isolated protein having receptor-type PTK-like properties including ATP binding, a kinase catalytic domain and/or an extracellular domain wherein said protein has one or more and preferably at least two of the following characteristics: i) it has an ATP binding site consisting of Gln-Glu-Gly-b-Phe-Gly, where b is Ser or Thr; ii) it has a kinase catalytic domain which includes amino acid sequence Asp-Asn-Ala in motif VII of said kinase catalytic domain; and/or iii) it has an extracellular domain comprising two leucine rich repeats, or subunits, fragments or derivatives or analogues of said protein.

Preferably, the RYK protein is as hereinbefore defined including subunits, fragments, dertivatives or analogues thereof.

The mammalian RYK of the present invention may be of normal cell origin or may be from a genetically modified cell such as, for example, tumour cells. Types of cells carrying the human RYK molecule include but are not limited to cells from one or more of the following sources: kidney, brain, placenta, ovary, lung, thymus and spleen. Most preferably, the RYK is from cytokine IL-1) induced human %WO 93/23429 PCT/AU93/00210 -16hepatoma cells.

The present invention is also directed to nucleic acid molecules encoding mammalian such as human or m'ise RYK including its fragments, derivatives, analogues, homologues and/or relatives. The nucleic acid molecules may be RNA or DNA (e.g.

cDNA), single or double stranded, linear or a covalently closed circle. The nucleic acid molecules may also be genomic DNA. The nucleotide sequence may correspond to the naturally occurring sequence or may contain single ormultiple nucleotide substitutions, deletions and/or additions. The nucleic acid molecules may also be part of a vector stich as an expression and/or cloning vector and may contain extraneous nucleic acid material encoding a signal peptide, fusion peptide, purification peptide and/or marker peptide. The preferred nucleotide sequence is set forth in Figure 2B (human) or Figure 3 (mouse) and includes molecules having at least 50-70%, preferably at least 80% and most preferably at least 90% similarity to all or a region thereof.

The mammalian RYK and/or nucleic acid molecules encoding same of the present invention have important utility in modulating growth and differentiation of cells.

Accordingly, the present invelation extends to ligands for the mammalian RYK and in partieuiar ligand(s) to human and mouse RYK and to any agonists and antagonists of RYK-ligand interaction.

Accordingly, another aspect of the present invention contemplates a method for identifying a ligand for mammalian RYK said method comprises labelling an extracellular portion of a RYK molecule with a reporter molecule and using said labelled molecule to screen a cDNA expression library. The labelled molecule may be a full length RYK molecule or may be a fragment or part thereof. The labelled molecule must still be able to bind or otherwise associate with a putative ligand.

In an alternative embodiment, a RYK extracellular domain is fused to a truncated growth factor receptor lacking its extracellular domain to form a hybrid molecule and then transfecting the hybrid molecule into a cell line dependent on that particular WO 93/23429 PC/AU93/00210 -17growth factor. The cell line is then contacted with a range of molecules including proteinaceous molecules and non-proteinaceous chemicals. Putative ligands are identified by screening for survival, maintenance or proliferation of said cell line.

An example of a suitable growth factor is Epidermal Growth Factor (EGF).

Since the RYK or a genetically modified form thereof may be an oncogenic protein, antagonists to the RYK are of particular relevance. Such antagonists include antibodies (monoclonal or polyclonal), the receptor itself in soluble form, specific peptides or proteins and/or carbohydrates amongst others the fragments, derivatives, analogues, homologues and relatives of RYK as contemplated above).

These types of antagonists are useful in developing anti-tumour agents where the growth or maintenance of the tumour itself is supported by the RYK of the present invention. Accordingly, the addition of an effective amount of an antagonist to the tumour-associated RYK will inhibit, reduce or otherwise interfere with RYK activity and thus prevent, reduce and/or inhibit tumour growth. The present invention, therefore, also extends to pharmaceutical compositions comprising one or more antagonists to RYK. The pre.ent invention is particularly directed to the treatment of carcinomas and tumours in epithelial tissues and haemopoietic tumours and sarcomas.

In other circumstances, however, it may be useful to promote ligand-RYK binding ad/or interaction. Accordingly, the present invention extends to agonists of RYK whica facilitate ligand-RYK interaction and to pharmaceutical compositions compriing s-me.

Accordingly, the present invention contemplates a pharmaceutical composition comprising as active ingredient, RYK or fragments, parts or derivatives thereof, RYK fusion or hybrid molecules, RYK ligands, RYK-ligand antagonists and/or RYKligand agonists, depending on the condition to be treated. For example, a RYKligand antagonist or a RYK ligand may be useful as an anti-cancer agent such as for treatment of carcinomas. For convenience, and as a short hand notation for the following description of pharmaceutical compositions, all of the above molecules and WO 3/23429 PCT/AU93/00210 -18referred to herein after as "active molecules". The use of the term "active molecule(s)" therefore should be read as one or more of the above molecules depending on the condition to be treated.

The active molecules of the pharmaceutical composition are contemplated to exhibit therapeutic activitywhen administered in an amount which depends on the particular case. The variation depends, for example, on the animal and the active molecule.

For example, from about 0.05 pg to about 20 mg of RYK ligand or RYK-ligand antagonist may be administered per kilogram of body weight per day to distrupt RYK-ligand interaction. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The active molecules may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active molecules may be required to be coated in a material to protect said molecules from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, a low lipophilicity of RYK or its ligands may allow these to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. Accordingly, in order to administer the pharmaceutical composition by other than parenteral administration, the active molecules may be coated by, or administered with, a material to prevent its inactivation.

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

NWO 93/23429PC/U3021 PCr/AU93/00210 19 The pharmaceutical forms suitable for injectable ixe 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 fluiiity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example,, parabens, chiorobutanol, phenol, sorbic acid, thormerosal, and the like. In many cases, it -will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, or example.

Sterile injectable solutions are prepared by incorporating the active molecules in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterle injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterilefiltered solution thereof.

I WO 93/23429 WO 9323429PCT/AU93/00210 20 When the active molecules are suitably protected as described above, the pharmaceutical composition may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,_and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be betweeni about 5 to about 80% of the, weight of the unit. The amount of active compound in the pharmaceutical compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are preparedso that an oral dosage unit form contains between about 0.05 ug and 20 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalciumn phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavomuing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

Various other materials may be present as coatings or to otherwise modify the physical formi of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active molecule, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

WO 93/23429 PC~/AU93/00210 -21 Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical compositions is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions is contemplated. Supplementary active molecules can also be incorporated into the compositions.

Another aspect of the present invention contemplates a method of inhibiting, reducing or otherwise interfering with interaction between a protein having receptortype PTK-like properties and a ligand thereof in a mammal said method comprising the administration of a ligand binding interfering effective amount of an antagonist to said ligand interaction for a time and under conditions sufficient to inhibit, reduce or otherwise interfere with said interaction. More particularly, the present invention contemplates a method of inhibiting, reducing or otherwise interfering with RYKligand interaction in a mammal said method comprising the administration of a RYK-ligand interfering effective amount of an antagonist to RYK-ligand interaction for a time and under conditions sufficient to inhibit, reduce or otherwise intefere with said interaction. Generally, such inhibition, reduction or interference will be useful in preventing, reducing and/or inhibiting tumour growth. Generally, the mammal is a human, mouse, livestock animal, laboratory test animal, captive or free wild animal. More particularly, the mammal is a human. The antagonist may be a chemcical compound or polypeptide or protein. The antagonist may also be the ligand itself or the extracellular region of RYK. The tumour is generally a carcinoma or sarcoma or epithelial or haemopoietic tissue.

Yet another embodiment of the present invention extends to the use of RYK to phosphorylate tyrosine residues on a protein substrate. This will be useful for in vitro labelling.

.WO 93/23429 PCT/AU93/00210 -22- Still yet another aspect of the present invention is directed to the use of mammalian RYK in the manufacture of a medicament for the treatment against cancer or tumour growth.

The present invention also extends to nucleic acid molecules in the form of oligonucleotide probes or primers useful for detecting, genomic sequences encoding a mammalian RYK molecule and in particular human RYK. More particularly, the oligonucleotide probes will be specific to particular regions of the genomic sequence such as those sequences encoding the extracellular domain, transmembrane domain or intracellular domain (including kinase catalytic domain) of RYK. Even more particularly, the oligonucleotide probes will be useful in screening a genomic sequence for abnormalities in relation to the RYK coding sequence which result in an abnormal or mutant RYK which might in turn result in or facilitate RYK related tumours or sarcomas.

Another aspect of the present invention contemplates an assay for identifying or otherwise diagnosing abnormalities in RYK or for identifying or otherwise screening for a normal RYK molecule in a human. In accordance with this aspect of the invention, a source of genetic material is isolated from a human subject to be tested and subjected to Southern blot analysis, Northern blot analysis, Western blot analysis, radioimmunoassay (RIA) and other immunological techniques or variations or combinations of such analyses.

In one embodiment, there is provided a method for detecting an abnormal genomic coding sequence for a protein having receptor-type PTK-like properties in a human subject said method comprising contacting a genetic sample from said human subject with one or more oligonucleotide primers specific for a part of the naturally occurring genomic sequence for said protein orfor an abnormal coding sequence for said protein for a time and under conditions sufficient for said oligonucleotides to hybridise to said genomic sequence and then screening for said hybridisation.

.WO 93/23429 PCT/AU93/00210 -23- In a more particular embodiment, a human subject is screened for a normal or abnormal RYK gene by isolating a genetic sample including genomic DNA from said human subject, subjecting said genetic sample to restriction endonuclease digestion to produce digested or partially digested DNA, subjecting said digested DNA to electrophoresis to separate the digested DNA based on length of fragments in the DNA digestion and screening the separated DNA digest to Southern blot analysis to screen for the presence or absence of particular regions of the RYK gene. For example, an oligonucleotide probe can be generated capable of screening for a nucleotide sequence corresponding to a "normal" extracellular region of RYK such as one or both of the leucine rich regions. In an abnormal RYK, the restriction pattern of this region may alter or contain deleted or duplicated sequences. Such an assay will screen for these modifications.

An "abnormal RYK" is defined inter alia at the genetic level as an alteration in the nucleotide sequence encoding normal RYK such as to result in a RYK molecule with an altered amino acid sequence such as an insertion, deletion and/or substitution. The altered RYK may also have a different glycosylation pattern relative to the naturally occurring normal) RYK molecule. Such a change in glycosylation patterns can result from a change in a single amino acid residue. An "abnormal RYK" can be defined inter alia at the functional level as a molecule having altered ligand binding characteristics. Frequently, this can result in a tumour or sarcoma or a predisposition thereto.

The human subject may be an adult, adolescent, child, infant or a foetus.

The assay may be particularly useful in screening members of a family with a pre- "isposition to cancer based on a defective or modified RYK molecule.

WO93/23429 rcr/AU93/o210 -24- Yet another aspect of the present invention provides a fragment of mammalian RYK, said fragment containing a proteolytic cleavage site. The amino acid sequence constituting this site consists of Lys-Arg-Arg-Lys and such a cleavage site would be useful in engineered proteins, polypeptides or peptides to render these capable of enzymatic modification. For example, a diagnostic agent could be prepared comprising a reporter molecule connected to otherwise fused to a protein, polypeptide or peptide via the above-mentioned proteolytic cleavage site. The reporter molecule can then be cleaved off as part of the diagnostic assay.

The proteolytic cleavage site may also be useful in generating, truncated or cleaved molecules and/or fusion molecules.

WO 93/23429 PTAJ3021 PCY/AU93/00210 25 The following single and three letter abbreviations. for amino acid residues are used in the specification: Amino Acid TIhree-letter One-letter Abbreviation Symbol Alanine Ala

A

Arginine Arg

R

Asparagine Asn

N

Aspartic acid Asp

D

Cysteine Gys

C

Glutamine, Gin Q Glutamic acid Glu

E

Glycine Gly

G

Histidine His

H

Isoleucine Ile

I

Leucine Leu,

L

Lysine LYS

K

Methionine Met

M

Phenylalanine Phe

F

Proline Pro

P

Serine Ser

S

Threonine Thbr

T

Tryptophan Trp

W

Tyrosine Tyr

Y

Valine Val

V

WO 93/23429 PCrAU93/00210 -26- EXAMPLE 1 MATERIALS AND METHODS The following materials and methods are used in subsequent examples.

Ce Crul re Human cell lines were grown at 37 "C in a humidified atmosphere of 5% v/v CO 2 and maintained in RPMI-1640 medium plus the following supplements: 10% v/v foetal bovine serum, 10 mM glutamine, 100 units/ml penicillin and 12.5 pg/ml streptomycin (Commonwealth Serum Laboratories, Melbourne, Australia). The following human cell lines were used in this study: MCF-7 (adenocarcinoma of the breast; CCRF-HSB-2 (T lymphoblastoid; Screenig of cDNA libraries cDNA libraries were screened according to the methods elsewhere described (11).

Clones of human RYK were isolated from an oligo dT primed Interleukin-1 stimulated human hepatoma eDNA library in lambda ZAP (Stratagene, La Jolla, CA, cat#935202) using a 90 bp PCR fragment derived from the extracellular region of the mouse RYK cDNA sequence as a probe To facilitate sequencing, the human clones described herein (pBS-XYZ and pBS-E; see Figure 2) were subcloned into the EcoRV site of pBluescript II (Stratagene) using EcoRV linkers. cDNA libraries used were from murine NFS TPA activated spleen (Clontech cat.#ML1018), murine Swiss-albina 3T3 fibroblast (Clontech cat.#1023b) and murine ICR linoleic acid activated pleural macrophage (Clontech cat.#ML1005b) generated in .gt1l.

cDNA libraries from murine BALB/c testis (Clontech cat.#ML1020b) and murine day 10 embryonic neuro-epithelium (27) were generated in Xgt0O. Around 106 recombinants of each of these libraries were screened on each occasion.

WO 93/23429 PCT/AU93/00210 27 SDNA Sequencing A range of RYK specific oligonucleotide primers were used to sequence the RYK cDNA clones in addition to the universal primers of M13 (forward and reverse). To sequence the GC-rich 5' region, a number of smaller (150-300 base pair) fragments were generated and subcloned into the plasmid Bluescript II. DNA was sequenced by the dideoxynr-dectide chain termination method Sequence data were derived from complete analysis of both strands of the DNA.

Northern Motting A 2.6 kb Pst I fragment from the Bluescript-XYZ clone was subcloned into the PstI site of Bluescript in order to transcribe an antisense RNA probe. This fragment represents the complete insert without the GC-rich 5 region, but includes the 3' untranslated region. In vivo synthesis of the 2P-dUTP (100 pCi) labelled 2.6 kb message was performed using T3 RNA polymerase and the Message Maker Kit (Bresatec, Adelaide, Australia), PolyA+ mRNA samples were prepared as described elsewhere Aliquots (0.5 pg) were electrophoresed on a 1% w/v agarose gel containing 2.2 M formaldehyde; 20 mM MOPS, pH 6.8;1 mM EDTA; 5 mM sodium acetate, and transferred to GeneScreen Plus (cat# NEF-976; DuPont, NEN, Boston, MA). Filters were prehybridised for 16 hours in 50% v/v formamide containing 3xSSC; 5x Denharts; 10 mM Hepes pH 7.0; 100 pg/ml poly C, 100 pg/ml denatured herring sperm DNA; 10 pg/ml E.coliDNA; 0.1% w/v SDS, then hybridised with the IP-labelled human riboprobe in the same solution for 18 hours at 42 Filters were washed at a final stringency of 0.IxSSC/0.1% w/v SDS at 65"C, followed by treatment with a solution of 1 pg/ml RNase A (Boehringer Mannheim GmbH, Mannheim, Germany)/2 x SSC at room temperature for 15 minutes before exposure to Kodak XAR-5 X-ray film (Eastman-Kodak Company, Rochester, NY) with two intensifying screens. Prior to transfer, gels were observed under UV illumination to ensure equivalent amounts of RNA were loaded in each lane.

I WO 93/23429 WO 9323429PCT/AU93/002 -28- Immnopmedptaton of the RYpcij.ipid MCF-7 cells (5 x 106) were grown in methionine/cysteine free medium (Flow Laboratories) coiiiain-1iiig I mCi 35 S-methionine/cysteine (Translabel, XCN) for 6 hours prior to har-vesting with PBS/20 mM EDTAL Cells were lysed with 5 ml of lysis buffer (10 mM Tris-HCI pH 8.0, 1% w/v Tx-lOG, 150 mM NaCI, 0.05% NaN 3 0.2 mM PMSF, 5 mM Na2V0 3 10 pg/mI leupeptin, 0.2 TJ.U/ml aprotinin) and nuclei and debris removed by centrifugation for 15 min in a microfuge at 4 0 C. The lysate was then precleared with 500 p1 of protein A-Sepharose beads and 250 p 1 of preimmune sera for 16 hours at 4*C. The beads were removed by centrifugation at 40,000 rpm in a Beckman ultracentrifuge using a Ti 50 rotor. Equal amounts of lysates (1 ml) were then incubated with 5 p1 of either preinimune rabbit sera or rabbit anti-Keyhole Limpet Heamocyanin (KLH)-RYK peptide for 2 hours at 4 0

C

followed by the addition of 10 p~l of protein-A-sepharose beads for a further 45 mins.' Lysates were washed three times with lysis buffer followed by three washes with mM Tris-HCI pH 8.0,150 mM NaCI, 0.1% v/v Tx-100. Immunoprecipiti'es were then eluted with 20 iii of SDS sample buffer in the presence or absence of 2% Pmercaptoethanol as the reducing agent SDS-PAC3E was performed according to the methods of Laeminli After drying, the gels were autoradiographed using X-ray film (KODAK XAR-5) or using a Phosphorimage analyser and Imagequant software (Molecular Dynamics, Sunnyvale, CA). Relative molecular weight markers were 200,000 (Myosin), 97,400 (Phosphorylase 69,000 (Bovine Serum Albumin), 46,000 (Ovalbumin), 30,000 (Carbonic anhydrase), 21,500 (Trypsin Inhibitor) and 14,300 (Lysozyme) (Rainbow markers, Amersham).

ID iErau ftrwicqmzntw~ afion Inl vifro translation of cD)NA clones was performed using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). Human and mouse RYK cDNAs wer.. subcloned into the expression vector pCDM8 using Bst XI Iinkers (Invitrogen; pCD.H.RYK and pCD.M.RYK). pCD.M.R7K, pCD.H.RYK, pCDM8 or control cDNAs (1 pg) were used as templates for the in vitro synthesis of protein products using 'f7 RNA Polymerase in the presence of 5 S-methio-,une (1097 WO 93/23429 PCT/AU93/00210 -29- Ci/mmol, Translation grade, Amersham). Proteins were then treated with reducing SDS-PAGE sample buffer and analysed by SDS-PAGE.

Chromosomal localisation Lymphocyte cultures from two apparently normal males were used to prepare chromosomes using standard methods. Synchronization of cultures and G-banding were performed by the method of Zabel et al A 1.6kb fragment containing 3' untranslated regions of the RYK cDNA clone containing the intracellular domain and 550 bp of 3' untranslated region was labelled to a specific activity of 7.45 x 107 cpm/ug with 3 H]dATP, 3 H]dCTP and 3 H]dGTP and used to probe the chromosomal preparations. Hybridisation to the chromosomes was performed at a concentration of 10.05-0.1 ug of probe per ml of fluid. The slides were developed using a 2:1 dilution of KODAK NTB-2 nuclear research emulsion with water after a period of 13 days. All individual silver grains that touched any chromosome within each metaphase were plotted on an ideogram (see Figure 7).

Gluathione unmsfese fusion preinsr To demonstrate the kinase activity of the RYK molecule, fusion proteins between the cytoplasmic domain of RYK and glutathione transferase were constructed (Fig.

7A). This was achieved by ligating a PCR product representing the entire ryk cytoplasmic domain (from position 820 to the in frame stop of Figure 2B) in frame to the sequence encoding glutathione-S-transferase in pGEX-3X (between the BamH1 and EcoRI sites; (17) to produce the construct pGEX-CYT-RYK. The fusion protein was induced by addition of 0.1 mM IPTG to the culture two hours prior to harvesting as elsewhere described (17).

Ptein kinase assra The fusion protein of the RYK cytoplasmic domain and the gluthathione-Stransferase was extracted from the bacterial cells using 10 mM Tris-HC1 pH 7.5/5 mg/ml lysozyme/100 uM Na 2

VO

4 and solubilized with 1% Tx-100. Soluble proteins were immunoprecipitated with glutathione-Seharose beads. Immunoprecipitates were then placed in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCI 2 10 mM WO 93/23429 PCT/AU93/00210 MnC12). Each immunoprecipitate (approx 10 ul of packed beads) was then incubated with xCi of carrier-free gamma- 32 P-ATP (Amersham cat#101691) for minutes at room temperature. After incubation, the excess label was removed by aspiration with a 30 gauge needle and the beads resuspended in SDS sample buffer, boiled and loaded onto an 8% w/v polyacrylamide gel for electrophoresis. SDS- PAGE was performed by the methods of Laemmli, After drying the gels were autoradiographed using X-ray film (KODAK XAR-5). Relative molecular weight markers were 200,000, 97,000, 67,000, 45,000, 30,000 and 20,000 (Amersham, Rainbow markers).

omputAided Seuence Anabs Comparisons of the amino acid sequences of mouse and human RYK were performed using an alignment programme from the Staden group of programmes on a VAX VMS 5.2. Phylogenetic analysis of the RYK protein/kinase domain (Leucine 329-Arginine 588) were performed using the procedure of Feng and Doolittle (18), based on the tree building concept of Fitch and Margoliash using a micro VAXII-vms computer system.

EXAMPLE 2 ISOLATION AND SEQUENCE OF THE MOUSE RYK cDNA Thirteen mouse cDNA libraries were screened in order to isolate a full-length cDNA clone. Two RYK mRNAs were identified and were 3.5kb and 2.5kb, respectively.

One clone XZ represented an almost full-length copy of the 2.8 kb mRNA. The sequence of the RYK mRNA is presented in Figure 4. The 5' end of this clone was extremely GC rich (88% in the region 5' of the putative initiation codon), a feature which perhaps explains the relative rarity of clones bearing this region of the RYK mRNA in any of the libraries screened.

There are two potential initiation methionine codons at the 5' end of the XZ clone (located at positibo 1 and -27 in Figure The ATG at position 1 is considered the most likely candidate for the start codon of RYK based on the following .WO 93/23429 PCT/AU93/00210 -31 observations: i. there is a better candidate leader peptide following this methionine; ii. it is positioned immediately after the highly GC rich region of the XZ clone, (and not buried within it, "as is the case for the methionine at position iii.

comparison of the mouse and human RYK coding sequences (see Example 3) shows a degeneration of the otherwise extremely high degree of homology between these two sequences immediately prior to this methionine. Initiation at position 1 would lead to an open reading frame of 1698 bases. The prediced RYK protein is therefore 566 amino acids long, with a predicted molecular mass of 63,598 daltons.

The predicted RYK protein bears all the features usually associated with membership of the growth factor receptor family of PTKs. Accordingly, the kinase domain is located towards the C-terminus of the protein (between the putative ATP binding site, -Gln-Glu-Gly-Ser-Phe-Gly- [located at amino acid position 295] and the most C-terminal conserved element [motif XI, (-CysTrp.,.ArgPro...Leu-) beginning at amino acid position 537 in Figure 3].

The mouse RYK gene is expressed widely as two mRNAs of 2.8 kb and 3.5 kb in both mouse tissue tnRNAs and in vitro cell lines. Highest levels of RYK are found in ovary, lung and pla<enta poly-(A+) RNA, although all of the tissues examined express detectable ]R:YK mRNA. It s noteworthy that RYK mRNA is also expressed in the human mamm ary carcinoma ell line A431, but that only the larger mRNA species appears to be present.

EXAMPLE 3 CLONING AND SEQUENCING OF HUMAN RYK A 90 bp PCR fragment corresponding to the 5' end of the mouse RYK cDNA clone was generated and used as a probe to isolate a number of clones from a human hepatoma cDNA library which were subsequently subcloned into the vector pBluescript for sequencing (see above). The clone pBS-XYZ (3.0 kb) was full length whilst pBS-E (2.7 kb) was missing sequence at the 5' end.

WO 93/23429 PCT/AU93/00210 -32- Nothem blotting anal-fs RYK mRNA appears to be broadly distributed on a range of normal tissues and in vitro cell lines (Figure 1A, B and Two species are detected in the mouse of and 2.8 kb whereas in the human only the larger of the two species is present (lane 1, A431, Figure The two mRNA transcripts found in the mouse are likely to have been derived from the use of different polyadenylation signals. A tissue Northern demonstrated high levels of expression on the placenta, ovaries and lung while the 13 day embryo, kidneys and brain expressed significant amounts.- Thymus and spleen expressed low levels indicating a tendency not to be expressed on cells of haematopoietic origin. Figures 1B and C show a wide distribution of RYK expression on in vitro cell lines including NIH-3T3, embryonic stem cell and the FDCP-1 cell line.

Ogaistion, _the RYK gene produc The complete cDNA sequence of human RYK is contained within a 3067 bp clone (see Figure 2B) isolated from an Interleu!'dn-1 stimulated human hepatoma cDNA library. An open reading frame extends from nuctotide 54 for approximately 1.9 kb to an in frame stop codon (TGA) at position 1950. The methionine codon located at position 132-134 is designated herein as the translation initiation codon as it is preceded by an inframe stop codon at position 51 of the clone and followed by a hydrophobic stretch of amino acids typical of a leader sequence. In addition, the predicted size of human RYK translated from this start site corresponds with the size of the protein generated by in viro transcription/translation of pCD.H.RYK (see Figure 9B). It should be noted that a leucine (CTG) at position 252-254 (amino acid position 41) could also potentially be a site for translation initiation. The sequence surrounding CTG 41 is consistent with the CC(A/G)CCATGG consensus sequence defined for mammalian initiation codons described by Kozak By comparison, the sequence surrounding the ATG at position 1 differs from the "Kozak" consensus at the critical +4 and -3 positions. Although ATG is the most commonly used translation initiation codon, initiation of translation from CTG have been reported for a number of molecules including the tyrosine kinase hck (19).

,WO 93/23429 PCr/AU93/0021 0 -33- It is noteworthy that a highly GC-rich region (nucleotides 19-250; 70-90% G+C content) characteristic of 5' untranslated regions of other receptor-type tyrosine kinases (growth factor receptors), transcription factors, DNA-binding proteins and proto-oncogenes (20) was found in the human RYK sequence. These regions are thought to be involved in the regulation of protein translation Therefore, given that RYK is related to the family of growth factor receptors (see below) the poor "Kozak" sequence and the highly GC-rich 5' region may represent two potential mechanisms for translational control. The 3' untranslated region extends from the inframe stop codon over 1.1 kb to the poly A tail. A consensus signal sequence for polyadenylation (ATTAAA) lies just prior to this at position 3036-3041.

Domain stuctue f RYK The amino acid sequence deduced from the human RYK cDNA contains all the key features of a typical transmembrarie receptor (Figure that of a signal peptide, extracellular, transmembrane and cytoplasmic domains. Unprocessed RYK is a protein of 606 amino acids with a predicted molecular weight of 67,659 Daltons; slightly larger than the predicted size for mouse RYK (63,598 Daltons). Hydropathy analysis of the deduced sequence by the methods of Kyte and Doolittle (21) show a 28 amino acid hydrophobic domain which subdivides the molecule (Figure The C-terminal side of this sequence is bordered by a stretch of basic residues (Lys-Arg) typical of stop transfer sequences. This sequence, therefore, most likely represents a transmembrane domain. The cytoplasmic domain contains a large juxtamembrane region (84 amino acids) containing many serine residues. The extracellular domain of RYK contains 224 amino acids, which by comparison to other RTK members is relatively small. Contained within this domain are five potential N-linked glycosylation sites (Asn-X-Ser/Thr); their presence is further evidence that this region of the cDNA encodes for an extracellular domain. Based on the predictions of von Heijne the cleavage site for the signal peptidase is N-terminal to Alas producing a mature protein of some 582 amino acids. Comparison of the puvative human RYK extracellular domain with extracellular domains of other known RTKs demonstrated no significant homology that would allow it to be placed into one of the six structurally related clusters of RTKs (23; 24). Two leucine-rich repeats are WO 93/23429 PCr/AU93/00210 -34present in the extracellular domain of the human sequence (Figure 2B). The leucine-rich regions are: Leu-Ile-Gly-Leu-Asp-Ala-Glu-Leu-Tyr-Tyr-ValArg-A-Asp-Leu-lle-Ser-His-Tyr- Ala-Leu-Ser-Phe; and/or Leu-Met-Gln-Leu-Asn-Leu-Thr-Val-Asn-Ser-Ser-Lys-Asn-Phe-Thr-Val-Leu-Asn- Phe-Lys-Arg-Arg-Lys.

RYK, therefore, possesses an extracellular domain which is unique in structure compared to other RTKs.

Human RYK kiaselike domain The cytoplasmic portion of human RYK extends for 354 amino acids and contains all of the conserved motifs found in the protein tyrosine kinase family (Figure 2; subdomains I-XI, 25). This begins with the putative ATP-binding site (Gln-Glu-Gly- Thr-Phe-Gly) located at amino acids 334-339 and ends with motif XI (-Cys-Trp..Arg- Pro...Leu) at position 577-591. There are alterations to several of the most highly conserved motifs held in common by members of the PTK and RTK families. In subdomain I of PTKs, the Rossman motif thought to be associated with ATP binding, has three invariant glycine residues in a six amino acid cluster (Gly-X-Gly- X-X-Gly); human RYK has the sequence Gln-Glu-Gly-Thr-Phe-Gly which is similar to mouse RYK and unique within the kinase family. The Ala-X-Lys sequence characteristic of subdomain II cannot be found but two sequences Phe-X-Lys and Thr-X-Lys are both present close to the correct location. Subdomains III to V of human RYK all contain the conserved motifs of the tyrosine kinase family as previously defined. Subdomain VIb of human RYK contains the conservative amino acid substitution of a lysine foi a arginine in the motif (-His-Arg-Asp-Leu-Ala-Ala- Arg-Asn-; subdomain Vlb). Although conservative this is, however, a highly unusual change to a motif which is one of the most conserved in the PTK/RTK family. It is especially unusual given that the mouse sequence also has the conserved arginine W 93/23429 PCT/AU93/00210 residue. This lysine for arginine substitution was found in two independently derived clones (pBS-XYZ and pBS-E) so it is unlikely to represent a sequencing artefact.

Further, the highly conserved Asp-Phe-Gly sequence of subdomain VII is altered to Asp-Asn-Ala in human RYK and is identical to the change to this redon in the mouse sequence This is the most highly conserved motif within the protein kinase family. The only published exception to this is kIlg containing the sequence Ala-Leu-Ser- (26).

In RYK, the -Gly-X-Gly-X-X-Gly- motif has a glutamine at the first position. These changes to the catalytic'domain of human RYIY may reflect a unique nucleotide and/or substrate specificity for RYK. It is also possible that the substitutions are compensate for each other and thus allow kinase function to proceed as with mouse

RYK.

The remaining subdomains (VIII-XI) of human RYK follow the conserved motifs of the tyrosine kinase family. It is noteworthy that the RYK sequence also contains residues characteristic of receptor-type tyrosine kinases such as the methionine in the sequence Arg-Trp-Met-Ala-Leu-Glu-Ser found in subdomain VII. Furthermore, between subdomains VII and VIII of human RYK is a tyrosine residue which is frequently used as an autophosphorylation site in the tyrosine kinase family, the sequence is -Met-Asp-Tyr-His and is identical to mouse RYK.

Comparin of mouse and hwnan RYK Comparison of human and mouse RYK cDNA sequences demonstrates a high degree of conservation over almost their entire length with homology of 92% and 97% at the nucleotide and amino acid levels, respectively (Figure Both open reading frames terminate at the identical portion of their deduced amino acid sequence; after the sequence -His-Ala-Ala-Leu-Gly-Ala-Tyr-Val-*. The most pronounced difference, however, is at the 5' end of the cDNA where the two sequences diverge from each other. After amino acid 55 of the human sequence, mouse and human amino acid sequences are greater than 95% identical; the sequences are only 15% identical prior to this point. Prior to amino add 55, the WO 93/23429 PCT/AU93/00210 -36human and mouse nucleotide sequences also become less similar even though both sequences remain high in GC content with some smaller areas of homology. The kinase domains of human and mouse RYK are more related to each other than any kinase domain found in the database (95% compared to 35-53% for other kinases).

The human and mouse RYK sequences have unexpected differences at key regions within the conserved PTK motifs, for example the highly conserved argirine residue in the subdomain VIb is a lysine in the human sequence. Comparison of the kinase domains (Figure 4) show only twelve anmino acid changes in the PTK subdomains (I- XI) with the majority being conservative substitutions. The other amino acid changes between the mouse and human sequences are also, in general, highly conservative substitutions. In addition to conservative substitutions, there is also a deletion of sequences between mouse and human RYK The human sequence has a deletion of three amino acids [-Ser-Ser-Gly-] in the region between the transmembrane domain and the putative ATP binding site compared to the mouse sequence. Furthermore, the human sequence contains an additional asparagine residue in subdomain IX not found in the deduced mouse sequence. In the human, this region is [-Val-Asn Ser- Leu-Trp-Glu-Leu-Met-] and in the mouse [-Val-Thr-Leu-Trp-Glu-Leu-Met-]. All of these changes were found in both clones pBS-XYZ and pBS-E and therefore unlikely to be sequencing errors The additional asparagine residue is highly unusual given the conserved spacing of residues seen in this motif RYK mRNA Exprssion in Human Cel Lines In the mouse, RYK mRNA appears to be broadly distributed on, a range of normal tissues and in vitro cell lines (Example Northern blotting demonstrates the expression of a single species of mRNA found in the MCF-7 cell line; CCRF-HSB-2 showed no expression of RYK. Interestingly, two mRNA species (3.5 and 2.7 kb) are detected in the mouse whereas in the human only the larger message is present (Figure These differences are presumably due to use of alternative polyadenylation sites. Using the polymerase chain reaction, it was demonstrated that RYK is expressed in the epithelial cell line LIM-1863 of Whitehead et al (28) which is derived from normal colonic crypts.

WO 93/23429 P(-7AU93/0021 0 -37- Analysis of the hwnan RYKpolypeptide In order to determine the size of the mature RYK protein, SDS-PAGE analysis of RYK was performed. Immunoprecipitation was performed from 35 S-methionine labelled lysates of the human, adenocarcinoma of the breast cell line MCF-7 which by Northern blotting was shown to express RYK mRNA (Figure The antisera used was raised against a C-terminal peptide of mouse RYK (Lys-Phe-Gln-Gln-Leu- Val-Gln-Cys-Leu-Thr-Glu-Phe-His-Ala-Ala-Leu-Gly-Ala-Tyr-Val) which is highly conserved in the human RYK sequence. Using this antisera a species of Mr 85,000-90,000 was detected under both reducing and non-reducing conditions (Figure 10A; I, reduced non-reduced). This species was not precipitated using a pre-immune rabbit sera (Figure 9A; PI, reduced non-reduced). This result is consistent with the size of the polypeptide predicted from the human RYK cDNA given use of the five potential N-linked glycosylation sites. It is also consistent with the size of mouse RYK immunoprecipitated from NIH-3T3 cells. Under both reducing and non-reducing conditions, a band of Mr=35,000 was seen, which appeared specifically in the immunopr ~.,pitates using the RYK antisera. Its association with RYK is unclear. Non-r.ducing conditions produced a smear of >200,000 in addition to the 85,000-90,3 0 Mr species which could potentially represent aggregated receptor.

In vitro transcription/translation of the human RYK cDNA using T7 RNA Polymerase has demonstrated a protein product of around 65,000-70,000 by SDS- PAGE (Figure 9B, pCD.H RYK). This is similar to the size of human RYK predicted from the human cDNA sequence. In comparison, the mouse cDNA gave a slightly smaller protein product (Figure 9B, pCD.M.RYK) which is consistent with the human clone having a more 5' translational start site than the mouse. Further evidence to support this comes from the comparison of RYK immunoprecipitated from MCF-7 (human) and NIH-3T3 (mouse) which shows that human RYK is marginally larger by about 2-4 kd. The quantitative ratio of products produced by both plasmids in the in vitro transcription/translation assay was also different and could be due to the higher GC-rich content of the human RYK 5' end. The protein product of both the mouse and human RYK cDNA clones also contained two ,WO 93/23429 PCT/AU93/00210 38 identical smaller species of Mr= 40,000-45,000 which are RYK specific, possibly due to false translational start sites. Control DNA produced no specific bands whereas the positive control plasmid pCD-NYK yielded a 170,000 species plus a number of smaller species; none of which correlated with the species seen with the RYK cDNAs (Figure 9B). The immunoprecipitation studies correlate well with the cDNA cloning and suggest that the human RYK cDNA encodes the sequence of the entire RYK polypeptide.

Chrmasomal Mapping of RYK The in situstaining of chromosomes from normal male lymphocytes show that of 160 silver grains on 38 metaphases, 22 (13.75% of all grains) were on chromosome 3 in the region 3q13.3 to 3q25, and 24 (15% of all grains) were on the short arm of chromosome 17 (see Fig.7). Similar results were obtained from hybridization of the probe to the chromosomes of the second normal male. A second series of experiments demonstrated a preference for staining of chromosome 3 with less pronounced staining on chromosome 17. These results indicate that the gene encoding RYK is found on chromosome 3 in the human.

EXAMPLE 4 TRUNCATED AND FUSION RYK MOLECULES: METHODS A series of mutants, truncated forms and fusion hybrids of human and mouse RYK are obtainable using oligonucleotide directed mutagenesis. The RYK cDNA clones were subcloned into the expression vector pCDM8 (30) and single stranded (ss) UTP+ DNA of RYK was made. This ssUTP DNA is then used as a template for annealing a mutagenic oligonucleotide which ecodes a region or part of RYK together with the change or changes to be introduced to that region or part. A second strand of DNA is then generated by transformation into the appropriate strain of bacteria which provides a selection against the wild type strand. Mutations are then selected by preparing DNA from these bacteria and analysing same by restriction enzyme digestion or nucleotide sequencing.

IWO 93/23429 PCT/AU93/00210 39 Alternatively, using standard restriction digestion and ligation proceedures, a series of hybrid cDNA molecules are prepared carrying, for example, the 5' end of human RYK cDNA and the 3' end of murine RYK cDNA or the 3' end of murine RYK cDNA or the 3' end of growth factor receptor cDNA EGFR). A vast array of hybrid or fusion molecules are obtainable by such a procedure.

EXAMPLE TRUNCATED AND FUSION RYK MOLECULES I The extracellular portion of RYK was fused to the kinase domain of the EGF receptor. A mutant form of mouse RYK cDNA was produced which contained a KpnI restriction site (GGTACC) within the transmembrane domain of RYK (glycine 190 and valine 191). A KpnI site was also introduced into the transmembrane domain of the human EGF receptor at glycine 628 and alanine 629. Utilising the KpnI site located in the expression vector pCDM8 the kinase domains of the two receptors were switched.

EXAMPLE 6 TRUNCATED AND FUSION RYK MOLECULES II The extracellular portion of the EGFR was fused to the kinase domain of RYK as described in Example EXAMPLE 7 TRUNCATED AND FUSION RYK MOLECULES m Secreted forms of mouse and human RYK extracellular domains were produced. An inframe stop codon was introduced at valine 184 in the mouse sequence and valine 225 in the human sequence. This valine residue lies at the N-terminal border of the transmembrane domain. These mutations were introduced by site-directed mutagenesis and selected through the addition of a diagnostic restriction site.

WO 93/23429 PCT/AU93/00210 EXAMPLE 8 TRUNCATED AND FUSION RYK MOLECULES TV A chimera between the RYK extracellular domain and the immunogenic peptide FLAG (IBI/Kodak) was made to allow purification of the presumed ligand binding domain of RYK using the FLAG biosystem (M2 antibody). To achieve this, a mutant of the RYK cDNA was made which contained an inframe BglII site at the junction between the extracellular domain and the transmembrane domain.of RYK.

The BglII site (AGATCT) was introduced in place of the sequence GTGTIT which encodes valine 184 and phenylalanine 185 in the mouse sequence, The sequence encoding the FLAG peptide is provided by synthetic oligonucleotide linkers which also contain preformed BglII sites on either end for ligation to the BglII site on the extracellular domain of RYK. The end result is the fusion of RYK extracellular domain inframe to the FLAG peptide.

EXAMPLE 9 TRUNCATED AND FUSION RYK MOLECULES V The mouse RYK kinase domain was ligated to the FLAG peptide in the pFLAG expression vector (IBI). The FLAG peptide is ligated inframe at the N-terminus of the kinase domain. The human RYK kinase domain was ligated to GST as a fusion protein in the vector pGEX A BamHI (5')-EcoRI PCR fragment representing the cytoplasmic domain of RYK from the C-terminal border of the transmembrane domain to the in frame stop codon of the mature sequence ligated into the BamHI/EcoRI sites of pGEX.

.WO 93/23429 PCT/AU93/00210 -41 EXAMPLE TRUNCATED AND FUSION RYK MOLECULES VI The RYK extracellular domain was fused in frame to secreted alkaline phosphatase, using the vector AP-tag-1 A Hind III -BglII fragment of pCD-M- RYK-BglII was subcloned into Hind III-BglII digested.AP-tag-1. This achieves the inframe ligation of the mouse RYK extracellular domain and secreted-alkaline phosphatase.

EXAMPLE 11 IDENTIFICATION OF LIGANDS TO RYK MOLECULES Two approaches were used to identify ligands for RYK. These involve: i) transfection of RYK into the factor-dependent cell line FDCP-1 (33) with subsequent rescue using a RYK-ligand; and ii)i tsing a tagged version of the RYK extracellular domain (FLAG, AP, 2 I) to screen an expression library.

Constructs for these expression studies were made using methods based on oligonucleotide directed mutagenesis to introduce unique restriction sites or terminatiu- codons and thus allow the precise subcloning of appropriate DNA fragments.

Initially, the full length clones of human RYK, mouse RYK and human EGFR were subcloned into the expression vector pCDM8 (30) to enable the generation of single stranded UTP +DNA for site-directed mutagenesis. In addition, this vector permits in vitro transcription/translation, transient expression in COS cells as well as cotransfection with pgKNEO for expression in FDCP-1 cells.

To produce secreted forms of RYK and the EGFR, termination codons were introduced at the predicted border of the extracellular and transmnembrane domain of the receptors.

WO 93/23429 PCT/AU93/00210 42 To study the function of RYK, transfected into FDCP-1 cells, a series of chimeric molecules between RYK and the EGFR were made. These constructs consist of the EGFR cytoplasmic domain ligated to the putative ligand-binding domain of RYK plus the converse, RYK cytoplasmic domaii coupled to the EGFR extracellular region. These chimeras were made using a KpnI site which was introduced into the transmembrane domain of the three receptors by mutagenesis. Using a KpnI site already in the vector pCDM8 (position 3572) fragments of the receptors can be readily exchanged. The chimeras are useful for examining whether the RYK catalytic domain can transmit a mitogenic signal through stimulation of the EGFR extracellular domain with EGF. In addition, the RYK-EGFR chimera is usable in case the RYK cytoplasmic domain is unable to generate a mitogenic response.

Another approach involves the ligation of the extracellular domain of RYK to secreted alkaline phosphatase using the vector AP-TAG-1 BglII sites were introduced into mouse and human RYK at the junction of extracellular domain and the transmembrane domain to allow inframe ligation to secreted alkaline phosphatase. These constructs have been produced and will be co-transfected with pBS-NEO into NIH-3T3 cells for production of a tagged affinity reagent.

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.

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Claims (39)

1. An isolated protein having receptor-type protein tyrosine kinase (PTK)-like properties including ATP binding wherein said protein has an ATP binding site comprising Gln-a-Gly-b-c-Gly where a, b and c may be the same or different and each is an amino acid residue or a subunit, fragment, derivative or analogue of said protein.

2. An isolated protein according to claim 1 wherein amino acid residue a is Glu.

3. An isolated protein according to claim 2 wherein amino acid residue b is Ser or Thr.

4. An isolated protein according to claim 3 wherein amino acid residue c is Phe. i

5. An isolated protein according to claim 1 having a molecular weight as determined by SDS-PAGE of approximately 60,000 to 72,000 in the non-glycosylated form.

6. An isolated protein according to claim 1 or 4 of mouse origin.

7. An isolated protein rclated to tyrosine kinases (RYK) according to claim 1 or 4 of human origin.

8. An isolated protein according to claim 7 having an amino acid sequence substantially as set forth in Figure 2B or having at least 50-70% amino acid similarity to all or a region thereof.

9. An isolated protein according to claim 6 having an amino acid sequence substantially as set forth in Figure 3 or having at least 50-70% amino acid similarity to all or a region thereof. A subunit, fragment, derivative or analogue of the protein according to claim 8 951004,q'o per'j h,40505.276,46 47 or 9 comprising all or part of the extracellular domain of said protein.

11. A subunit, fragment, derivative or analogue of the protein according to claim 8 or 9 comprising all or part of the kinase catalytic domain of said protein.

12. An isolated protein having receptor-type PTK-like properties including a kinase catalytic domain, wherein said protein contains amino acid sequence Asp-Asn-Ala in said kinase catalytic domain or a subunit, fragment, derivative or analogue of said protein and said protein has a molecular weight as determined by SDS-PAGE of approximately 60,000 to 72,000 in the non-glycosylated form.

13. An isolated protein according to claim 12 wherein amino acid sequence Asp-Asn- Ala is in motif VII of said kinase catalytic domain.

14. An isolated protein according to claim 12 or 13 of mouse origin. An isolated protein according to claim 12 or 13 of human origin.

16. An isolated protein according to claim 15 having an amino acid sequence substantially as set forth in Figure 2B or having at least 50-70% amino acid similarity *e to all or a region thereof.

17. An isolated protein according to claim 14 having an amino acid sequence substantially as set forth in Figure 3 or having at least 50-70% amino acid similarity to all or a region thereof.

18. A subunit, fragment, derivative or analogue of the protein according to claim 16 or 17 comprising all or part of the extracellular domain of said protein. 19, A subunit, fragment, derivative or analogue of thie protein according to claim 16 or 17 comprising all or part of the kinase catalytic domain of said protein. -o95 ,q\doper\eJh,0505.276,47 NT O~9S 104,q-:ptr-',4O5O5.276,47 -48- An isolated protein having receptor-type PTK-like properties including an extracellular domain wherein said extracellular domain contains two leucine rich regions.

21. An isolated protein according to claim 20 wherein said leucine rich regions consist of amino acid sequences: Leu-Ile;-Gly-Leu-Asp-Ala-Glu-Leu-Tyr-Tyr-Val-Arg-Asn-Asp-Leu-Ile- Ser-His-Tyr-Ala-Leu-Ser-Phe; and Leu-Met-Gln-Leu-Asn-Leu-Thr-Val-Asn-Ser-Ser-Lys-Asn-Phe-Thr-Val- Leu-Asn-Phe-Lys-Arg-Arg-Lys. S Ol 22. An isolated protein according to claim 20 or 21 having a molecular weight as determined by SDS-PAGE of approximately 60,000 to 72,000 in the non-glycosylated form.

23. An isolated protein according to claim 21 of mouse origin.

24. An isolated protein according to claim 21 of human origin. 9 An isolated protein according to claim 24 having an amino acid sequence substantially as set forth in Figure 2B or having at least 50-70% amino acid similarity to all or a region thereof.

26. An isolated protein according to claim 23 having an amino acid sequence substantially as set forth in Figure 3 or having at least 50-70% amino acid similarity to all or a region thereof.

27. A subunit, fragment, derivative or analogue of the protein according to claim 23 or 24 comprising all or part of the extracellular domain of said protein. R 28. A subunit, fragment, derivative or analogue of the protein according to claim 23 951 OW,1q ;operejh,405.276,48 -49- or 24 comprising all or part of the kinase catalytic domain of said protein.

29. An isolated protein having receptor-type PTK-like properties including ATP binding, a kinase catalytic domain and/or an extracellular domain wherein said protein has one or more of the following characteristics: i) it has an ATP binding site consisting of Gln-Glu-Gly-b-Phe-Gly, where b is Ser or Thr; ii) it has a kinase catalytic domain which includes amino acid sequence Asp- Asn-Ala in motif VII of said kinase catalytic domain; and/or iii) it has an extracellular domain comprising two leucine rich repeats, or subunits, fragments or derivatives or analogues of said protein.

30. An isolated protein according to claim 29 of mouse origin where b is Ser and is of mouse origin.

31. An isolated protein according to claim 29 wherein b is Thr and is of human origin. 0* f

32. An isolated protein according to claim 30 having an amino acid sequence substantially as set forth in Figure 2B or having at least 50-70% amino acid similarity to all or a region thereof.

33. An isolated protein according to claim 30 having an amino acid sequence substantially as set forth in Figure 3 or having at least 50-70% amino acid similarity to all or a region thereof.

34. A subunit, fragment, derivative or analogue of the protein according to claim 32 or 33 comprising all or part of the extracellular domain of said protein. A subunit, fragment, derivative or analogue of the protein according to claim 32 or 33 comprising all or part of the kinase catalytic domain of said protein. 951O04,q p cr\ej h,4050S.276,49

36. A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the isolated protein defined by claim 1 or 12 or or 29.

37. A nucleic acid molecule according to claim 36 having a nucleotide sequence substantially as set forth in Figure 2B or having 50-70% similarity to all or a region thereof.

38. A nucleic acid molecule according to claim 36 having a nucleotide sequence substantially as set forth in Figure 3 or having 50-70% similarity to all or a region thereof.

39. An expression vector comprising the nucleic acid molecule according to claim 37 or 38 operably linked to a promoter. 4*.

40. An expression vector according to claim 39 wherein the promoter is a prokaryotic qpromoter.

41. An expression vector according to claim 39 wherein the promoter is a eukaryotic promoter.

42. A. method of inhibiting, reducing or otherwise interfering with interaction between a protein according to claim 1 or 12 or 20 or 29 and a ligand thereof in a mammal said method comprising the administration of a ligand binding interfering effective amount of an antagonist to said ligand interaction for a time and under conditions sufficient to inhibit, reduce or otherwise interfere with said interaction.

43. A method according to claih 42 wherein the antagonist is selected from the list consisting of a solubilised protein, a subunit, fragment, derivative or analogue carrying an extracellular domain of said protein, an antibody to said exti acellular domain or a chemical molecule capable of inhibiting ligand interaction. 9510 4,q.optrekh,40S01I76,SO -51-

44. A method according to claim 42 or 43 wherein the mammal is a human, A method for detecting an abnormal genomic coding sequence for the protein according to claim 1 or 12 or 20 or 29, in a human subject said method comprising contacting a genetic sample from said human subject with one or more oligonucleotide primers specific for a part of the naturally occurring genomic sequence for said protein or for an abnormal coding sequence of said protein for a time and under conditions sufficient for said oligonucleotides to hybridise to said genomic sequence and then screening for said hybridisation.

46. A method according to claim 45 wherein the oligonucleotide primer is labelled S. with an reporter molecule selected from the list consisting of a radioactive isotope, fluorophore, biotinTylated molecule or chemiluminescent molecule or fluorescent molecule.

47. A method according to claim 45 or 46 wherein the oligonucleotide is specific to a genomic sequence encoding a portion of the extracellular domain.

48. A method according to claim 47 wherein the portion of the extracellular domain is a ligand binding portion. *i An isolated protein according to any one of claims 1 to 9 or 11 to 17 or 20 to 26 or 29 to 33 or a subunit thereof according to claim 10 or 11 or 18 or 19 or 27 or 28 or 34 or 35 or a nucleic acid molecule according to claim 37 to 38 or a method according to any one of claims 42 to 48 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 4th day of October, 1995 LUDWIG INSTITUTE FOR CANCER RESEARCH By Its Patent Attorneys DAVIES COLLISON CAVE 9SIO0,q1 cptrtJh,405..276,51