AU711646B2 - Novel receptor ligands and genetic sequences encoding same - Google Patents

Description

i /1 WO 97/04091 PCT/AU96/00460 -1- NOVEL RECEPTOR LIGANDS AND GENETIC SEQUENCES ENCODING SAME The present invention relates generally to novel receptor ligands and to genetic sequences encoding same. The novel receptor ligands and their encoding genetic sequences are useful in the development of a wide range of agonist, antagonist, therapeutic and diagnostic reagents based on ligand-receptor interaction. The present invention further provides a means for identifying ligands without knowledge of their receptor and a means for identifying receptors.

Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined at the end of the description.

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

The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research into the medical and allied health fields. Research into cellular growth factors such as cytokines is of particular importance due to their involvement in proliferation, differentiation and function of a wide variety of cells. Administration of recombinant growth factors or of molecules capable of regulating growth factor function and/or synthesis is becoming increasingly the focus of medical research into the treatment of a range of conditions.

Many cellular growth factors and other extracellular mediators of cell function exert their actions by interacting with cell surface receptor protein tyrosine kinases (RPTKs). Ligand binding to such receptors usually results in receptor aggregation, cross-phosphorylation on tyrosine residues of the receptor cytoplasmic domains, activation of the intrinsic kinase activity of the cytoplasmic domains and phosphorylation of down-stream cell signalling molecules WO 97/04091 PCT/AU96/00460 -2- (Schlessinger and Ullrich Neuron 9: 383-391, 1990; Flanti et al Ann. Rev. Biochem. 61: 453-481, 1993; van der Greer and Hunter Ann. Rev. Cell. Biol. 10: 251-337, 1994).

RPTKs can be subdivided into several classes dependent primarily on the predicted structural features of their extracellular domains and whether or not they contain an uninterrupted tyrosine kinase domain in the cytoplasmic portion of the receptor (Hilton In "Guidebook to Cytokines and Their Receptors" N.A. Nicola ed, Oxford University Press, Oxford pp 8-16, 1994). The largest class of RPTKs is known as the eph-related kinases (ERKs) and include the tyrosine kinases eph, elk, cck, eek, erk, cek 4-10, mek 4, hek, tk2, tyro 5 and sek. They are characterised by an extracellular sequence that includes a single cysteine-rich repeat domain followed by two fibronectin type III domains and an uninterrupted tyrosine kinase domain in the cytoplasmic region.

Despite the fact that ERKs comprise the largest subclass of RPTKs, most of the ERKs were identified without prior knowledge of their biological function nor of the ligands with which they interact. Nevertheless, the expression patterns of the ERKs suggest that they might have important roles in early vertebrate development and in brain function and hemopoietic cell function as well as functions on other organs in the adult (Cheng and Flanagan Cell 79: 157- 168, 1994; Shao et al J. Biol. Chem. 270: 3467-3470, 1995; Beckman et al EMBO J. 13: 3757-3762, 1994).

There is clearly a need, therefore, to identify and characterise ERKs. There is also an important need to identify ligands for ERKs with or without prior knowledge of the specific ERK to which a ligand might interact.

Recently, some ligands that can bind to ERKs have been identified and are referred to as "ligands for ERKs" (LERKs) amongst other names. The first of these, LERK1, is equivalent to the B61 gene product previously identified as an interleukin-1 or tumour necrosis factorinducible product of human umbilical vein endothelial cells (Holzman et al Mol. Biol. 5830-5838, 1990; Beckmann et al EMBO J. 13: 3757-3762, 1994). LERK2 appears to be WO 97/04091 PCT/AU96/00460 -3identical to human ELK ligand (ELK-L) and the human homologue of the mouse proteins elg 2 (eph-ligand 2) and cek5 L (cek5-ligand) (Beckmann et al EMBO J. 13: 3757-3762, 1994; Fletcher et al Genomics 24: 127-132, 1994; Shao et al J Biol. Chem. 269: 26602-26609, 1994; Davis et al Science 266: 816-819, 1994). LERK3 is nearly identical to EHK -L (ehk1ligand) Kozlosky et al Oncogene 10: 299-306, 1995; Davis et al Science 266: 816-819, 1994), LERK4 is unique (Kozlosky et al Oncogene 10: 299-306, 1995) and the cek7 ligand is identical to elf-1 (eph ligand family-1) (Shao et al J Biol. Chem. 270: 3467-3470, 1995); Cheng and Flanagan Cell 79: 157-168, 1994). All of these ligands appear to be cell surfaceassociated either through a C-terminal glycosyl-phosphatidyl inositol (GPI)-linkage (LERKs 1, 3 and 4) or through a traditional transmembrane domain (LERK2). Most appear to require cell surface expression or soluble dimer forms in order to activate the tyrosine kinase activity of the appropriate ERKs (Davis et al Science 266: 816-819, 1994). Moreover, none of the LERKs described so far show an absolute specificity for one ERK. For example, LERK 1 binds to hek, elk and eck; LERKs 2, 3 and 4 bind to hek and elk and cek 7-L binds to sek and mek 4 with equilibrium dissociation constants varying from 1 nM to 500 nM.

In accordance with the present invention, the inventors have identified a new LERK which will assist in the development of a range of new therapeutics and diagnostics and in the identification of new ERKs.

Accordingly, one aspect of the present invention contemplates an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a LERK or part thereof, said nucleotide sequence or its complementary form encoding at least one of the following amino acid sequences: VXWXSXN [SEQ ID NO:1]; (ii) DXXDIXCP [SEQ ID NO:2]; (iii) EXYXLYXVXXXXXXXC [SEQ ID NO:3]; and/or (iv) KFQXXXXXXXGXEFXXXHXYYXI [SEQ ID NO:4]; wherein X is any amino acid residue.

WO 97/04091 PCT/AU96/00460 -4- Preferably, the nucleotide sequence or its complementary form encodes a product comprising at least two of, more preferably at least three of and even more preferably all four of amino acid sequences SEQ ID NO: 1 to SEQ ID NO:4.

In this regard, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a LERK or part thereof, said nucleotide sequence or its complementary form encoding an amino acid sequence selected from the list consisting of: VXWXSXN [SEQ ID NO:1]; (ii) DXXDIXCP [SEQ ID NO:2]; (iii) EXYXLYXVXXXXXXXC [SEQ ID NO:3]; and (iv) KFQXXXXXXXGXEFXXXHXYYXI [SEQ ID NO:4]; wherein X is any amino acid residue.

Preferably, the nucleic acid molecule of the present invention encodes a LERK referred to herein as "NLERK2", said nucleic acid molecule selected from the list consisting of: a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID (ii) a nucleic acid molecule comprising a sequence of nucleotides having at least about 70% similarity to the nucleotide sequence set forth in SEQ ID NO:5; and (iii) a nucleic acid molecule capable of hybridising under low stringency conditions to the nucleotide sequence set forth in SEQ ID The nucleotide molecule is preferably derivable from the human genome but genomes and nucleotide sequences from non-human animals are also encompassed by the present invention.

Non-human animals contemplated by the present invention include livestock animals (e.g.

sheep, cows, pigs, goats, horses, donkeys), laboratory test animals mice, rats, guinea pigs, hamsters, rabbits), domestic companion animals dogs, cats), birds chickens, geese, ducks and other poultry birds, game birds, emus, ostriches) and captive wild or tamed animals WO 97/04091 PCT/AU96/00460 foxes, kangaroos, dingoes).

Preferred human genomic sequences include sequences from brain, liver, kidney, neonatal, embryonic, cancer or tumour-derived tissues.

Once a novel nucleotide sequence is obtained as indicated above encoding a LERK, oligonucleotides may be designed which bind cDNA clones with high stringency. Direct colony hybridisation may be employed or PCR amplication may be used. The use of oligonucleotide primers which bind under conditions of high stringency ensures rapid cloning of a molecule encoding the novel LERK and less time is required in screening out cloning artefacts. However, depending on the primers used, low or medium stringency conditions may also be employed.

Reference herein to a low stringency at 42 0 C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions.

Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.

Accordingly, another aspect of the present invention contemplates a method for cloning a nucleotide sequence encoding a novel LERK, said method comprising searching a nucleotide data base for a sequence which encodes at least one of amino acid sequences SEQ ID NO: 1 to SEQ ID NO:4, designing one or more oligonucleotide primers based on a nucleotide sequence located in the search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefrom which encodes said novel LERK or part thereof.

WO 97/04091 PCT/AU96/00460 -6- Preferably, the nucleic acid library is a cDNA, genomic or mRNA library.

Preferably, the nucleic acid library is a cDNA expression library.

Preferably, the nucleotide data base is of human origin such as from brain, liver, kidney, neonatal tissue, embryonic tissue, tumour or cancer tissue.

In one preferred embodiment, the oligonucleotide primers are selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12 and SEQ ID NO:13.

In a particularly preferred embodiment the LERK is selected from the list consisting of: a nucleotide sequence substantially as set forth in SEQ ID (ii) a nucleotide sequence having at least about 75% similarity to the nucleotide sequence set forth in SEQ ID NO:5; and (iii) a nucleotide sequence capable of hybridising under low stringency conditions to the nucleotide sequence as set forth in SEQ ID In an alternative aspect of this embodiment, medium stringency conditions are employed. In another alternative aspect, high stringency conditions are employed.

Another aspect of the invention is directed to the genomic gene and to 3' and 5' regions thereof.

The 3' region of the NLERK2 gene is represented in SEQ ID NO: 14 and the 5' region is shown in SEQ ID NO: Accordingly, another aspect of the present invention is directed to a nucleic acid molecule corresponding to or derived from the 3' region of the genomic gene encoding NLERK2 or its derivatives, said nucleic acid molecule or its complementary form having a nucleotide sequence substantially as set forth in SEQ ID NO: 14 or having at least 75% similarity thereto or capable of hybridising thereto under low stringency conditions. Other levels of stringency such as WO 97/04091 PCT/AU96/00460 -7medium and high may also be used.

A further embodiment is directed to a nucleic acid molecule corresponding to or derived from the 5' region of the genomic gene encoding NLERK2 or its derivatives, said nucleic acid molecule or its complementary form having a nucleotide sequence substantially as set forth in SEQ ID NO: 15 or having at least 75% similarity thereto or capable of hybridising thereto under low stringency conditions. Other levels of stringency such as medium and high may also be used.

The present invention also extends to hybrid genetic sequences comprising a coding region of a structural gene flanked or fused to one or both of SEQ ID NO: 14 and/or SEQ ID NO: 15 or derivatives thereof.

Still another embodiment contemplates the promoter or a functional part thereof of the genomic gene encoding NLERK2. The promoter may readily be obtained by, for example, "chromosome walking".

Another aspect of the present invention is directed to NLERK2 including a recombinant polypeptide having an ability to interact with an ERK wherein said NLERK2 or said polypeptide is encoded by a nucleotide sequence translatable to at least one of amino acid sequences SEQ ID NO: 1 to SEQ ID NO:4 and subsequently cloned by designing at least one oligonucleotide probe based on said nucleotide sequence and using same to clone a nucleotide sequence encoding said LERK or polypeptide from a DNA or RNA library.

This aspect of the present invention extends to recombinant NLERK2 or a polypeptide having NLERK2-like properties but does not extend to known LERKs such as LERKI, LERK2, LERK3 or LERK4.

WO 97/04091 PCT/AU96/00460 -8- In a particularly preferred embodiment, the present invention provides NLERK2 which comprises an amino acid sequence substantially as set forth in SEQ ID NO:6 or having at least about 70% similarity, preferably at least about 80% similarity, more preferably at least about similarity or most preferably at least about 95 to 100% similarity to all or part of the sequence set forth in SEQ ID NO:6. This aspect encompasses NLERK2 and derivatives thereof such as fragments, parts, portions, mutants and homologues.

In a related embodiment, the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID NO:6 or to an amino acid sequence having at least about 70% or above similarity to all or part of the amino acid sequence set forth in SEQ ID NO:6.

As stated above, the present invention further contemplates a range of derivatives of NLERK2.

Derivatives include fragments, parts, portions, mutants, homologues and analogues of the NLERK2 polypeptide and corresponding genetic sequence. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to NLERK2 or single or multiple nucleotide substitutions, deletions and/or additions to the genetic sequence encoding NLERK2.

"Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences. Reference herein to "NLERK2" includes reference to all derivatives thereof including functional derivatives or NLERK2 immunologically interactive derivatives.

Analogues of NLERK2 contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

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 WO 97/04091 PCT/AU96/00460 -9anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with phosphate followed by reduction with NaBH4.

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

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.

Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, orithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D- WO 97/04091 PCT/AU96/00460 isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH).

In addition, peptides can be 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 further contemplates chemical analogues of NLERK2 capable of acting as antagonists or agonists of NLERK2 or which can act as functional analogues of NLERK2.

Chemical analogues may not necessarily be derived from NLERK2 but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of NLERK2. Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening.

The identification of NLERK2 permits the generation of a range of therapeutic molecules capable of modulating expression of NLERK2 or modulating the activity of NLERK2.

Modulators contemplated by the present invention includes agonists and antagonists of NLERK2 expression. Antagonists of NLERK2 expression include antisense molecules, ribozymes and co-suppression molecules. Agonists include molecules which increase promoter ability or interfere with negative regulatory mechanisms. Agonists of NLERK2 include molecules which overcome any negative regulatory mechanism. Antagonists of NLERK2 include antibodies and inhibitor peptide fragments.

WO 97/04091 WO 9704091PCT/AU96/00460 TABLE 1 Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid a-amino-a-methylbutyrate amninocyclopropanecarboxylate amninoisobutyric acid aminonorbomylcarboxylate cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamnine D-glutamnic acid D-histidine D-isoleucine D-leucine D-lysine D-methionine D-ornithine D-phenylalanine D-proline D-serine D-threonine Abu Mgabu Cpro Aib Norb Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Dile Dieu Dlys Dmet Dorm Dphe Dpro Dser Dthr L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid Chexa L-N-methylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylornithine L-N-methylphenylalanine L-N-methylprolmne L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosmne L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmnhis Nmile Nmleu Nmlys Nmnmet Nmnle Nmnva Nmorn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe WO 97/04091 WO 9704091PCT/AU96/00460 12 D-tryptophan D-tyrosine D-valine D--methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-u-methylglutamine D-ct-methylhistidine D-a-methylisoleucine D-iz-methylleucine D-a-methyllysine D-a-methylmethionine D-a-methylornithine D-ix-methylphenylalanine D-a-methylproline D-iz-methylserine D-a-methylthreonine D-ix-methyltryptophan D-a-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginie D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-methylglutamine D-N-methylglutaxnate D-N-methylliistidine Dtrp Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dmleu Dmlys Dmmet Dmom Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Dnmala Dnmarg Dnmasn Dnmasp Dnmcys Dnmgln Dninglu Dnmhis L-norvaline ra-methyl-aminoisobutyrate e-methyl-y-aminobutyrate a-methylcyclohexylalanine a-methylcylcopentylalanine a-methyl-rL-napthylalanine a-methylpenicillainine N-(4-aminobutyl)glycine N-(2-aminoethyl)glycine N-(3 -aminopropyl)glycine N-amino-ez-methylbutyrate iz-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine N-(carbamnylmethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2,2-diphenylethyl)glycine N-(3 ,3 -diphenylpropyl)glycine N-(3-guanidinopropyl)glycine 1-hydroxyethyl)glycine N-(hydroxyethyl))glycine Nva Maib Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Nomn Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe Narg Nthr Nser WO 97/04091 WO 9704091PCT/AU96/00460 13 D-N-methylisoleucine D-N-methylleucine D-N-methyllysine N-methylcyclohexylalanine D-N-methylomithine N-methylglycine N-methylarninoisobutyrate I-methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyltryptophan D-N-methyltyrosine D-N-methylvaline y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homnophenylalanine L-a-methylarginine L-a-methylaspartate L-a-methylcysteine L-a-methylglutamnine L-a-methylliistidine L-a-methylisoleucine L-rz-methylleucine L-rz-methylmethionine L-ea-methylnorvaline L-E-methylphenylalanine L-c-methylserine L-a-methyltryptophan Dnmile Dnmleu Dnmlys Nmchexa Dnmorn Nala Nmaib Nile Nleu Dnmtrp Dnmtyr Dnmval Gabu Tbug Etg Hphe Marg Masp Mcys Mgln Mfhis Mile Mleu Mmet Mnva Mphe Mser Mtrp N-(imidazolylethyl))glycine N-(3 -indolylyethyl)glycine N-methyl-y-aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine 1 -methylethyl)glycine N-methyla-napthylalanine N-methylpenicillamnine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamnine L-a-methylalanine L-a-methylasparagine L-rz-methyl-t-butylglycine L-methylethylglycine L-iz-methylglutamate L-rz-methylhomophenylalanine N-(2-methylthioethyl)glycine L-rt-methyllysine L-ra-methyinorleucine L-a-methylomithine L-a-methylproline L-a-methylthreonine L-a-methyltyrosine Nhis Nhtrp Nmgabu Dnmmet Nmcpen Dnmphe Dnmpro Dnmser Dnmthr Nval Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys Mnle Mom Mpro Mthr Mtyr WO 97/04091 PCT/AU96/00460 -14- L-a-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1-carboxy- 1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane These types of modifications may be important to stabilise NLERK2 if administered to an individual or for use as a diagnostic reagent.

Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule.

Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

Another embodiment of the present invention contemplates a method for modulating expression of NLERK2 in a human, said method comprising contacting the NLERK2 gene encoding NLERK2 with an effective amount of a modulator of NLERK2 expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of NLERK2. For example, a nucleic acid molecule encoding NLERK2 or a derivative thereof may be introduced into a cell to enhance the ability of that cell to survive, conversely, NLERK2 antisense sequences such as oligonucleotides may be introduced to decrease the survival capacity of any cell expressing the endogenous NLERK2 gene.

Another aspect of the present invention contemplates a method of modulating activity of NLERK2 in a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease NLERK2 activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of NLERK2 or its receptor or a chemical analogue or truncation mutant of NLERK2 or its receptor.

WO 97/04091 PCT/AU96/00460 Accordingly, the present invention contemplates a pharmaceutical composition comprising NLERK2 or a derivative thereof or a modulator of NLERK2 expression or NLERK2 activity and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the active ingredients.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds 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 into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

WO 97/04091 PCT/AU96/00460 -16- When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ug and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

WO 97/04091 PCT/AU96/00460 -17- The present invention also extends to forms suitable for topical application such as creams, lotions and gels.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

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

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 gg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 glg to about 2000 mg/ml of carrier.

In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

WO 97/04091 PCT/AU96/00460 18- The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating NLERK2 expression or NLERK2 activity. The vector may, for example, be a viral vector.

Still another aspect of the present invention is directed to antibodies to NLERK2 and its derivatives. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to NLERK2 or may be specifically raised to NLERK2 or derivatives thereof. In the case of the latter, NLERK2 or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant NLERK2 or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.

For example, NLERK2 and its derivatives can be used to screen for naturally occurring antibodies to NLERK2. These may occur, for example in some autoimmune diseases.

Alternatively, specific antibodies can be used to screen for NLERK2. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA.

Knowledge of NLERK2 levels may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.

Antibodies to NLERK2 of the present invention may be monoclonal or polyclonal.

Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.

A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.

The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimin.

For example, specific antibodies can be used to screen for NLERK2 proteins. The latter would be important, for example, as a means for screening for levels of NLERK2 in a cell extract or other biological fluid or purifying NLERK2 made by recombinant means from WO 97/04091 PCT/AU96/00460 -19culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of NLERK2.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of NLERK2, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates a method for detecting NLERK2 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for NLERK2 or its derivatives or homologues for a time and under conditions sufficient for an antibody-NLERK2 complex to form, and then detecting said complex.

WO 97/04091 PCT/AU96/00460 The presence of NLERK2 may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain NLERK2 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.

WO 97/04091 PCT/AU96/00460 -21- In the typical forward sandwich assay, a first antibody having specificity for the NLERK2 or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient 2- 40 minutes) and under suitable conditions 25" C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.

Alternatively, a second labelled antibody, specific to the first antibody is exposed to the targetfirst antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, WO 97/04091 PCT/AU96/00460 22 a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

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

The present invention also contemplates genetic assays such as involving PCR analysis to detect NLERK2 gene or its derivatives. Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded WO 97/04091 PCT/AU96/00460 -23conformation polymorphoms analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.

The nucleic acid molecules of the present invention may be DNA or RNA. When the nucleic acid molecule is in DNA form, it may be genomic DNA or cDNA. RNA forms of the nucleic acid molecules of the present invention are generally mRNA.

Although the nucleic acid molecules of the present invention are generally in isolated form, they may be integrated into or ligated to or otherwise fused or associated with other genetic molecules such as vector molecules and in particular expression vector molecules. Vectors and expression vectors are generally capable of replication and, if applicable, expression in one or both of a prokaryotic cell or a eukaryotic cell. Preferably, prokaryotic cells include E.

coli, Bacillus sp and Pseudomonas sp. Preferred eukaryotic cells include yeast, fungal, mammalian and insect cells.

Accordingly, another aspect of the present invention contemplates a genetic construct comprising a vector portion and a mammalian and more particularly a human NLERK2 gene portion, which NLERK2 gene portion is capable of encoding an NLERK2 polypeptide or a functional or immunologically interactive derivative thereof.

Preferably, the NLERK2 gene portion of the genetic construct is operably linked to a promoter on the vector such that said promoter is capable of directing expression of said NLERK2 gene portion in an appropriate cell.

In addition, the NLERK2 gene portion of the genetic construct may comprise all or part of the gene fused to another genetic sequence such as a nucleotide sequence encoding glutathione-Stransferase or part thereof.

The present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.

WO 97/04091 PCT/AU96/00460 -24- The present invention also extends to any or all derivatives of NLERK2 including mutants, part, fragments, portions, homologues and analogues or their encoding genetic sequence including single or multiple nucleotide or amino acid substitutions, additions and/or deletions to the naturally occurring nucleotide or amino acid sequence.

The NLERK2 and its genetic sequence of the present invention will be useful in the generation of a range of therapeutic and diagnostic reagents and will be especially useful in the detection of a corresponding ERK. For example, recombinant NLERK2 may be bound or fused to a reporter molecule capable of producing an identifiable signal, contacted with a cell or group of cells putatively carrying ERKs and screening for binding of the labelled LERK to the ERK.

Alternatively, labelled NLERK2 may be used to screen expression libraries of putative ERK genes or functional parts thereof.

LERKs are important for the proliferation, differentiation and survival of a diverse array of cell types. Accordingly, it is proposed that NLERK2 or its functional derivatives be used to regulate development, maintenance or regeneration in an array of different cells and tissues in vitro and in vivo. For example, NLERK2 is contemplated to be useful in modulating neuronal proliferation, differentation and survival.

Soluble NLERK2 polypeptides are also contemplated to be useful in the treatment of disease, injury or abnormality in the nervous system, e.g. in relation to central or peripheral nervous system to treat Cerebral Palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neuronal tumours, motoneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, Multiple Sclerosis, peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and infectious diseases such as herpes, rubella, measles, chicken pox, HIV or HTLV-1.

Membrane bound NLERK2 may be used in vitro on nerve cells or tissues to modulate proliferation, differentiation or survival, for example, in grafting procedures or transplantation.

WO 97/04091 PCT/AU96/00460 As stated above, the NLERK2 of the present invention or its functional derivatives may be provided in a pharmaceutical composition comprising the LERK together with one or more pharmaceutically acceptable carriers and/or diluents. In addition, the present invention contemplates a method of treatment comprising the administration of an effective amount of a LERK of the present invention. The present invention also extends to antagonists and agonists of LERKs and their use in therapeutic compositions and methodologies.

A further aspect of the present invention contemplates the use of NLERK2 or its functional derivatives in the manufacture of a medicament for the treatment of NLERK2 mediated conditions defective or deficient.

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

In the Figures: Figure 1 is a representation of the amino acid sequence of NLERK2 [SEQ ID NO: 6] of the present invention in comparison to known LERKs. LERK-1/B61 is described in International Patent Publication No. WO 95/06085; MCEK7/ELF1 in WO 96/10911 and NLERK1/ELF2 in WO 96/01839. The putative leader sequences, transmembrane sequences and GPI anchor sequences are shaded and boxed. Areas of amino acid sequence conservation are boxed, dashes signify gaps introduced to produce the alignment and potential N-glycosylation sites are underlined.

Figure 2 is a photographic representation showing expression of NLERK2. Panel A represents SDS-PAGE of extracellular medium from COS cells transfected with pEF sol NLERK2 cDNA (tracks a and b) or pEF fl NLERK2 cDNA (tracks c and Panel B represent SDS-PAGE of whole cell lysates from COS cells transfected with pEF sol NLERK2 cDNA (tracks a and b) or pEF fl NLERK2 cDNA (tracks c and b are from two independent pEF sol NLERK2 clones and c, d are from two independent pEF fl NLERK2 WO 97/04091 PCT/AU96/00460 -26clones). SDS-PAGE gels were transferred to PVDF filters and blotted with M2 anti-FLAG

M

antibody (IBI/Kodak, CT, USA) followed by HRP-conjugated rabbit anti-mouse immunoglobulin (Dako code P0260, Denmark) and binding detected by enhanced chemiluminescence as described for Amersham ECL reagent (Amersham, Buckinghamshire, England).

Figure 3 is a schematic representation showing structure of NLERK2 cDNA and location of NLERK2 clones.

Figure 4 is a representation of the nucleotide sequence [SEQ ID NO:5] and corresponding amino acid sequence [SEQ ID NO: 14] of full length NLERK2 cDNA. The leader sequence is lightly underlined, the transmembrane domain is heavily underlined and the potential Nglycosylation site is indicated by a bold N.

Figure 5 is a representation of the N-terminal amino acid sequence of pEF solNLERK2. The underlined portion is the C-terminal portion of the IL-3 signal sequence. The dotted underlined sequence is the sequence of the FLAG tag used to detect the expressed molecule.

The double underlined sequence is the 5' cloning site for the NLERK2 cDNA. The nonunderlined residues are the mature 5' coding region.

WO 97/04091 WO 9704091PCT/AU96/00460 27 A summary of SEQ ID Nos used in this specification is given in Table 2.

TABLE 2 SUMMARY OF SEQ ED) NOs SEQUENCE SEQ ID NO.- VXWXSXN 1 DXXDIXCP 2 EXYXLYXVXOXX C 3 KFQXXXXXXGXEFXXCXHXYYXI 4 Nucleotide sequence of full length NLERK2 Amino acid sequence of NLERK2 6 Oligonucleotide 7 Oligonucleotide 8 Oligonucleotide 9 Oligonucleotide Oligonucleotide I1I Oligonucleotide 12 Oligonucleotide 13 3' end of NLERK2 gene 14 end of NLERK2 gene Signal sequence of murine 11-3 16 FLAG epitope 17 WO 97/04091 WO 9704091PCT/AU96/00460 -28- Single and triple letter abbreviations for amino acid residues are used in the subject specification, as defined in Table 3.

TABLE 3 AMINO ACID ABBREVIATIONS Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine le I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X WO 97/04091 PCT/AU96/00460 -29- EXAMPLE 1

RATIONALE

An approach was used based on conserved amino acid sequence elements in the LERKs to seek additional members of the family, reasoning that the large number of ERKs (at least 12) implied an equally large number of LERKs. The known LERK sequences were aligned and are shown in Figure 1. It was noted that there were small conserved amino acid sequence blocks, namely VXWXSXN [SEQ ID NO:1]; (ii) DXXDIXCP [SEQ ID NO:2]; (iii) EXYXLYXVXXXXXXXC [SEQ ID NO:3]; and/or (iv) KFQXXXXXXXGXEFXXXHXYYXI [SEQ ID NO:4].

These amino acid sequence blocks were used to individually search the NCBI-Genbank data bank for any nucleotide sequences that, when translated in any of the six possible ways into amino acids (forward in each of three reading frames and backward in each of three reading frames), could encode a peptide corresponding to the above sequence blocks. For this purpose, the Pearson TFASTA search routine (Pearson WR and Lipman DJ Proc. Natl. Acad Sci. USA 85: 2444-2448, 1988; and Pearson WRMethods in Enzymology 183: 63-98, 1990) was used. Using SEQ ID NO:4 resulted in highest scores for human LERK2 (Gene Bank Code No. HSU09304), rat ELK-L (Gene Bank Code No. RSU07560), human ELK-L (Gene Bank Code No. HUMEFL3), human EHK1-L (Gene Bank Code No. HUMEFL2), mouse LERK2 (Gene Bank Code No. MLERK2), human B61 (Gene Bank Code No. HUMB61), mouse ERF-1 (Gene Bank Code No. MMU14941), mouse cek 5-L (Gene Bank Code No.

MMU12983).

The next highest score was for deposited Gene Bank Code No. 269886NCBI which represents a human-expressed sequence tag (EST) derived from a 73 day post-natal female whole brain cDNA library. Alignment and translation of an amino acid sequence predicted to be encoded by the EST (Gene Bank Code No. 269886NCBI) demonstrates it to be related to the known LERKs (Figure The sequence does diverge, however, and hence encodes a novel human WO 97/04091 PCT/AU96/00460 LERK, referred to herein as "NLERK2" (Figures 1 and 4).

EXAMPLE 2 CLONING FULL LENGTH NLERK2 cDNA Pooled oligos were used to probe library 53 which is a human fetal brain cDNA library. The oligos were synthesized according to the cDNA sequence of the expressed sequence tag [EST tag no. 269886NCBI]. The oligonucleotide probes comprise the following sequences: 5' GGCAGGTTCTCCTTCCCCAGGCTCCCA 3' [SEQ ID NO:7] (ii) 5' GTAGTAATCGTGGTGCGAGCG 3' [SEQ ID NO:8] (iii) 5' GGCATGAAGGTGCTTCTCCGA 3' [SEQ ID NO:9] A number of duplicate positive hybridizing plaques were picked and purified on CsCI gradient and sequenced on both strands. Overlapping cDNA sequences from 5 independent clones were obtained encompassing the entire coding region and were sequenced on both strands (see Figure 4).

EXAMPLE 3 GENERATION OF EXPRESSION CONSTRUCTS Constructs were generated to express full length and soluble forms of the protein. A derivative of the mammalian expression vector pEF-BOS (Mizushima et al, Nucl. Acids. Res. 18: 5322, 1990) was engineered to contain DNA encoding the signal sequence of murine IL-3 (MVLASSTTSIHTMLLLLLMLFHLGLQASIS [SEQ ID NO:16]) and the FLAG epitope (DYKDDDDK [SEQ ID NO:17]) followed by a unique Xba 1 cloning site. This vector was named pEF/IL3SIG/FLAG.

i WO 97/04091 PCT/AU96/00460 -31 PCR was performed using oligos to amplify DNA fragments encoding the entire mature coding region from L28 to V340 using clones 53.1 and 53.8. The oligonucleotide probes comprise the following sequences: 5' AGCTTCTAGACTCAACCTGGAGCCTGTCTAC 3' [SEQ ID (ii) 5' AGCTTCTAGATCATACCTTGTAGTAGATGTTTGG 3' [SEQ ID NO:11] PCR was used to amplify the extracellular domain without the transmembrane or cytoplasmic region. The sequences of the oligonucleotides used in this PCR are as follows: 5' AGCTTCTAGACTCAACCTGGAGCCTGTCTAC 3' [SEQ ID NO:12] (ii) 5' AGCTTCTAGATCAGCTGGGAGGGGGCAGGGGGCC 3' [SEQ ID NO:13] Full length and soluble forms were digested with XbaI. Both products were cloned in frame into the XbaI site of pEF/IL3SIG/FLAG to yield pEF fl NLERK2 and pEF sol NLERK2. The vectors are expressed in COS cells as described below.

In order to confirm that full-length and soluble forms of NLERK2 could be produced using the expression vectors pEF-flNLERK-2 and pEF-solNERK-2, COS cells were transiently transfected with these constructs. Briefly, COS cells from a confluent 175 cm 2 tissue culture flask were resuspended in PBS and electroporated (BioRad Gene pulser; 500 AF, 300 V) with /g of uncut expression vector in a 0.4 cm cuvette (BioRad). After 2 to 3 days at 37"C in a fully humidified incubator containing 10% v/v CO 2 in air, cells were used for analyses of protein expression. Conditioned medium was collected by centrifugation, concentrated tenfold, and stored sterile at 4"C. Cells were also harvested and lysed for 5 min in 500 lI of mM Tris Hcl pH7.4 containing 150 mM NaC1, 2 mM EDTA and 1% v/v Triton X-100. The ~11 WO 97/04091 PCT/AU96/00460 -32intact nuclei were removed by centrifugation at 10,000g for 5 min. The cell lysate and conditioned medium were electrophoresed on pre-cast 12% w/v polyacrylamide gels (BioRad).

The resolved proteins were then electroblotted onto PVDF membranes, which were then blocked with 5% skim milk, 0.1% v/v Tween 20 in PBS, rinsed and incubated with 5 fl of anti-FLAG M2 antibody (IBI) in 2.5 ml of PBS containing 5% skim milk and 0.1% v/v Tween The membrane was then rinksed and incubated with peroxidase-conjugated human antimouse Ig in 5% skim milk, 0.1% v/v Tween in PBS, rinsed and incubated with ECL reagent (Amersham, Buckinghamshire, UK) or 1 min. Filters were then blotted dry and exposed to autoradiographic film for 1 min.

Figure 2 shows expression ofNLERK2 in COS cells. The apparent molecular mass of soluble and full length NLERK2 were 32 and 50 kDa, respectively, suggesting glycosylation.

Figure 5 shows the N-terminal amino acid sequence of anti-FLAG M2 antibody purified soluble NLERK2 from pEF solNLERK2. This protein is transiently expressed in COS cells (Figure Single underlined sequence is the C-terminal portion of the IL-3 signal sequence which was cleaved before the Alanine. The dotted underlined sequence is sequence of the FLAG tag used to detect the expressed molecule. The double underlined sequence is the cloning site for the NLERK2 cDNA. The non-underlined residues are the mature 5' coding region (lacking the leader sequence). The italicised Asparagine was modified for cloning reasons from the Serine residue of the sequence given in Figure 4.

The full length nucleotide sequence [SEQ ID NO:5] and corresponding amino acid sequence [SEQ ID NO:6] is shown in Figure 4.

M

WO 97/04091 PCT/AU96/00460 -33 EXAMPLE 4 FLANKING REGIONS OF NLERK2 GENE The 5' and 3' portions of the NLERK2 genomic gene were determined and are shown in SEQ ID NO: 15 and SEQ ID NO:14, respectively.

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.

WO 97/04091 PCT/AU96/00460 -34- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT (countries other than US): AMRAD OPERATIONS PTY. LTD.

(US only): NICOS ANTHONY NICOLA (ii) TITLE OF INVENTION: NOVEL RECEPTOR LIGANDS AND GENETIC SEQUENCES ENCODING SAME (iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT INTERNATIONAL FILING DATE: 19-JUL-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PN4263 FILING DATE: 20-JUL-1995 APPLICATION NUMBER: PN6847 FILING DATE: 27-NOV-1995 WO 97/04091 PCT/AU96/00460 APPLICATION NUMBER: PN7299 FILING DATE: 22-DEC-1995 APPLICATION NUMBER: PN7890 FILING DATE: 05-FEB-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: HUGHES DR, E JOHN L REFERENCE/DOCKET NUMBER: EJH/EK (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +61 3 9254 2777 TELEFAX: +61 3 9254 2770 WO 97/04091 PCT/AU96/00460 -36- INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Val Xaa Trp Xaa Ser Xaa Asn 1 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Asp Xaa Xaa Asp Ile Xaa Cys Pro 1 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Glu Xaa Tyr Xaa Leu Tyr Xaa Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 WO 97/04091 PCT/AU96/00460 -37- INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Lys Phe Gin Xaa Xaa Xaa Xaa Xaa Xaa 1 5 Xaa His Xaa Tyr Tyr Xaa Xaa Ile Xaa Gly Xaa Glu Phe Xaa Xaa 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1021 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GGC CCC CCC CAT TCT GGG CCG GGG GGC GTG Met Gly Pro Pro His Ser Gly Pro Gly Gly Val 1 5 10 CTC CTA CTG GGG GTT TTG GGG CTG GTG TCT GGG Leu Leu Leu Gly Val Leu Gly Leu Val Ser Gly 25 CGA GTC GGG GCC CTC Arg Val Gly Ala Leu CTC AGC CTG GAG CCT Leu Ser Leu Glu Pro GTC TAC TGG AAC TCG GCG AAT AAG AGG TTC CAG GCA GAG Val Tyr Trp Asn Ser Ala Asn Lys Arg Phe Gin Ala Glu 40 GGT GGT TAT 144 Gly Gly Tyr WO 97/04091 PCT/AU96/00460 -38- TAC CCT CAG ATC GGG GAC CGG CTA GAC CTA CTC TGC CCC CGG Tyr Pro Gin Ile Gly Asp Arg Leu Asp Leu Leu Cys Pro Arg GTG CTG Vai Leu

GCC

Ala CGG CCT CCT GGC Arg Pro Pro Gly CAC TCC TCT CCT His Ser Ser Pro TAT GAG TTC TAC AAG Tyr Giu Phe Tyr Lys TGT GAG GCA CCC CCT Cys Glu Ala Pro Pro CTG TAC CTG GTA GGG GGT GCT CAG GGC Leu Tyr Leu Val Gly Gly Ala Gin Gly CGA CGC Arg Arg 90 GCC CCA AAC Ala Pro Asn TTC ACC ATC Phe Thr Ile 115

CTC

Leu 100 CTT CTC ACT TGT Leu Leu Thr Cys CGC CCA GAC Arg Pro Asp CTG GAT CTC CGC Leu Asp Leu Arg 110 TGG GGC CAC GAG Trp Gly His Glu 125 AAG TTC CAG GAG Lye Phe Gin Glu ACC CCT AAT CTC Ser Pro Asn Leu TTC CGC Phe Arg 130 TCG CAC CAC GAT TAC TAC ATC ATT GCC ACA TCG CAT GGG ACC Ser His His Asp Tyr Tyr Ile Ile Ala Thr Ser Asp Guy Thr 135 140

CGG

Arg 145 GAG GGC CTG GAG Glu Gly Leu Clu

AGC

Ser 150 CTA CAG GGA GGT Leu Gin Gly Gly

GTG

Val 155 TGC CTA ACC AGA Cys Leu Thr Arg

GGC

Gly 160 ATG AAG GTG CTT Met Lye Val Leu CGA GTG GGA CAA Arg Val Guy Gin CCC CGA GGA GGG Pro Arg Cly Gly CCT GTC Ala Val 175 CCC CGA AAA Pro Arg Lye GCC CAC AGC Ala His Ser 195 GTG TCT GAA ATG CCC ATG GAA AGA GAC Val Ser Glu Met Pro Met Giu Arg Asp 185 CGA GGG GCA Arg Gly Ala 190 CTG GAG CCT GGG Leu Glu Pro Gly

AAG

Lye 200 GAG AAC CTG CCA GGT GAC CCC ACC Glu Asn Leu Pro Guy Asp Pro Thr 205 AGC AAT GCA ACC TCC CGG Ser Asn Ala Thr Ser Arg GGT GCT GAA GGC CCC CTG CCC CCT CCC AGC Gly Ala Clu Gly Pro Leu Pro Pro Pro Ser 215 220 ;ij lil _Ij I WO 97/04091 PCT/AU96/00460 -39- CCT GCA GTG GCT GGG GCA GCA GGG GGG Pro Ala Val Ala Gly Ala Ala Gly Gly 230

CTG

Leu 235 GCG CTG CTC TTG Ala Leu Leu Leu GGC GTG GCA GGG Gly Val Ala Gly GGG GGT GCC ATG TGT Gly Gly Ala Met Cys 250 TGG CGG AGA CGG Trp Arg Arg Arg CGG GCC Arg Ala 255

AAG

Lys CCT TCG GAG Pro Ser Glu 260 AGT CGC CAC CCT Ser Arg His Pro CCT GGC TCC TTC Pro Gly Ser Phe GGG AGG GGA Gly Arg Gly 270 CGG GAG GCT Arg Glu Ala GGG TCT Gly Ser GAG CCT Glu Pro 290 GGC CTG GGG GGT Gly Leu Gly Gly

GGA

Gly 280 GGT GGG ATG GGA Gly Gly Met Gly

CCT

Pro 285 GGG GAG CTA GGG Gly Glu Leu Gly

ATA

Ile 295 GCT CTG CGG GGT Ala Leu Arg Gly

GGC

Gly 300 GGG GCT GCA GAT Gly Ala Ala Asp CCC TTC TGC CCC Pro Phe Cys Pro TAT GAG AAG GTG Tyr Glu Lys Val

AGT

Ser 315 GGT GAC TAT GGG Gly Asp Tyr Gly CCT GTG TAT ATC Pro Val Tyr Ile CAG GAT GGG CCC Gin Asp Gly Pro CAG AGC CCT CCA Gin Ser Pro Pro AAC ATC Asn Ile 335 1008 TAC TAC AAG GTA TGA Tyr Tyr Lys Val 340 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 340 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Gly Pro Pro His Ser Gly Pro Gly Gly Val Arg Val Gly Ala Leu 1 5 10 E7 WO 97/04091 PCT/AU96/00460 40 Leu Leu Leu Gly Val Leu Gly Leu Val Ser Gly Leu Ser Leu Glu Pro Val. Tyr Trp Val Leu Tyr Ser Ala Asn Lys Asp Phe Gin Ala Gly Gly Tyr Cys Pro Arg Pro Gin Ile Ala Arg Gly His Arg Leu Asp Pro Pro Gly Ser Ser Pro Giu Phe Tyr Leu Lys Tyr Leu Val Ala Gin Gly Cys Giu Ala Pro Pro Ala Pro Asn Phe Thr Ile 115 Phe Arg Ser Leu Thr Cys Pro Asp Leu Phe Gin Giu Tyr 120 Tyr Pro Asn Leu Asp Leu Arg 110 Gly His Giu Asp Gly Thr His His Asp 130 Ara Glu Tyr 135 Leu Ile Ile Ala Gly Leu Giu Gin Gly Gly Leu Thr Arg 145 Met Lys Val Leu Val Gly Gin Arg Gly Gly Ala Val 175 Pro Arg Lys Ala His Ser 195 Ser Asn Ala Ser Glu Met Glu Arg Asp Arg Gly Ala 190 Asp Pro Thr Giu Pro Gly Asn Leu Pro Thr Ser Arg Giu Gly Pro 210 Met Pro 225 Leu 220 Al a Pro Pro Ser Ala Val Ala Ala Gly Gly Leu 235 Leu Leu Leu Gly Val Ala Gly Ala Gly Gly Ala Met Cys Trp, Arg Arg Arg Arg Ala 245S 250 255 F- 1i i-l-iil--~ WO 97/04091 PCT/AU96/00460 -41- Lys Pro Ser Glu Ser Arg His Pro Gly Pro Gly Ser Phe Gly Arg Gly 260 265 270 Gly Ser Leu Gly Leu Gly Gly Gly Gly Gly Met Gly Pro Arg Glu Ala 275 280 285 Glu Pro Gly Glu Leu Gly Ile Ala Leu Arg Gly Gly Gly Ala Ala Asp 290 295 300 Pro Pro Phe Cys Pro His Tyr Glu Lys Val Ser Gly Asp Tyr Gly His 305 310 315 320 Pro Val Tyr Ile Val Gln Asp Gly Pro Pro Gln Ser Pro Pro Asn Ile 325 330 335 Tyr Tyr Lys Val 340 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGCAGGTTCT CCTTCCCCAG GCTCCCA 27 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GTAGTAATCG TGGTGCGAGC G 21 -I~iif(l-i I WO 97/04091 PCT/AU96/00460 -42- INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGCATGAAGG TGCTTCTCCG A 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID AGCTTCTAGA CTCAACCTGG AGCCTGTCTA C 31 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AGCTTCTAGA TCATACCTTG TAGTAGATGT TTGG 34 WO 97/04091 WO 97/04091 PCT/AU96/00460 -43- INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: AGCTTCTAGA CTCAACCTGG AGCCTGTCTA C 31 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AGCTTCTAGA TCAGCTGGGA GGGGGCAGGG GGCC 34 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 1692 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TGAGGGCTCC TCTCACGTGG CTATCCTGAA TCCAGCCCTT CCTGGGGTGC TCCTCCAGTT TAATTCCTGG TTTGAGGGAC ACCTCTAACA TCTCGGCCCC 100 WO 97/04091 PCT/AU96/00460 44 CTGTGCCCCC CCAGCCCCT' TCCTTAGGAT TCCCACTGC( TGTCTCAGTG TCCCTGGATC GACCATGAMc CAGGGATCCI CCACCTTTTG CTTGGCACCG TTATTCTTTC CCTCTCTTCC ACATCTCCTT

TCACCCTCTT

SATCAAAGCA TTTCTCCCCT CCCCTCAGTC TSYCAAAAAT CAGGCCATGG GCAGCAGGGC CAGCCATTTG GGGTGGTTGG r' CACTCCTCCC GGGTGCTGTC CTCGTCTCC 7CCACTTCCTG CCCTCCCGTT TGGCCATGGC 7CTTTTTCCTT GGGGAGGGGC ACAGGCTCAG TGTCCCCCTC ANCCACCCAG AGCTAGGGGG CCTTCTTTCT GCCTCTCACT GGTTTTCTCT GTCTCTAGGT CTGTTCTTCT TCCCTAGCAT GGCTTCTTAT CCTGTGCCTC TCCCATCTCC TAGCTTTCAS CCCCCCTTCT GACCTCTCAT GGGGGCCTTA TGGGGAAGGC

TCTGACACTC

TCCATTCTCT GGCCTGGCCC AGGCCTCTAC GTCATGACAG CTACCATGAG AAGAAGTGTC kCTTTTAGGAT

TGCCCCCCTC

CCTCCTCTCT

CGGGAACAGC

TCTCTATCTC

CCTCCTCCCC

TGGGTGGGGS

ACCAACCACT

CACCCCAGCT

ATACTTACTC

CCGTTTTGTC

160 220 280 340 400 460 520 580 640 700 760 CAGTGGCCAA TAGCAAGATA TGAACCGGTC GGGACATGTA TGGACTTGGT CTGATGCTGA ATGGGCCACT TGGGACCGGA AGTGACTTGc TCCAGACAAG AGGTGACCAG AAATGGCCTG GGAAGTAGCA GAAGCAGTGC AGCAGGAACT GGAAGTGCCC

ACAGGAAGTA

CTGGGGGATC

GAACTTCCTT

GAAGGACTTT

GCTGATTTTA

TCTTTCCTTT

ATGTGCTCTC

GCACTTCTGA

AGGAGGTGGG

TCAGGAGGAA

TCCCAGTCTT

GGACAGCTAA

CCATATCCCA

CCGAGTAACC

WRCNAGGAAG

AGGTGGATGG

GCTGGAACTT

CAGTGGCACT

GATGACTGCC

GTATAATCTC

CAGATGGCTG

TGRTCTGTCT

TTCTTATTCT

ACTGACTGTA

TCCCAAGATC

ATGTGCTGTG

RGAACACCAA

GTGGAGAAGA

AGAGGTTAGA

TCCCTTCCCT

GCAGGCCTAA

GCCCGGACAG

TTCATCCAGG

GTGGCWTART

AGGGCGGGAA

GGTGGACCGA

TGTGCTCTGT

TTTGTCTTGT

880 940 1000 1060 1120 1180 1240 1300 1360 1420 1480 TGTTAATCAA CAGGACTACC CCAAGAACCC TCTTGTTCAT TCCATCCTAC ATTTCTGACT CCTTTCAGAC TCAACACAGT TCCCTTCTTA GTGACCACAA TGGTGGCCTA CTGCCTGGTC TAGCTGACAG TGGTACTTAG CAMAGGCCAC TGTTTCCATA GTGACCAGCT GATACCTCTT WO 97/04091 PCT/AU96/00460 CCTGCCCTCT AGTGTGCAAT TGGGTGTTGC CTCAGTTTCC TCCCAGCTCA GTTTTATTAG ATCAAACCTG TTGTTGGGCA. CCAGGTTGTC CACCTCAATC ACCAGCCAAG ATGGTTGCTT TGTCCACCAG AGGTCAAGTT CACCTCTCTG GTGCTGTAGT TCCCAGCTCC TTCCTGATTT TTCTAATGGA ATTCGATATC AAGCTTATCG

AT

INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 399 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID 1540 1600 1660 1692 GTGGAGCCCA CGCGGTGGCG GTCGCTCTAG CAGCCCCTTC CTCGCTCCCT

GGTCCGGCGC

CCCCAGTCCC CCACTTAGGC

GGGATCACAG

CGCGTAGGGC GCCTGCAGAC

GGCCCCTGGA

CGGGGGCGCG GGCACAGCAG GAAGCAGGTC GGTGGT-CCGG GCTGAAGAGC CAGGCASCAA GGGGAGTTGG TGCGCCCCCC

CCCAGACCTT

AATAGTGGAT

CCCATGCCGC

ATCCCGGGGT

AGGGCTCTGG

CGCGTGGGCG

GGCAGCCACC

GACGGGGTC

CCCCCGGGCT

CCCCGCCCGG

GCTGGCGCGT

TGGGGCTGAG

CTGGGGGCAT

CCGGGGGGTG

GCAGGAATTC

TCCCCGGYTC

GGGCCGGGGG

CGCTCTGCCG

CAGCTACCGG

GGCGACTTTG

120 180 240 300 360 399 WO 97/04091 PCT/AU96/00460 -46- INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Val Leu Ala Ser Ser Thr Thr Ser Ile His Thr Met Leu Leu Leu 10 Leu Leu Met Leu Phe His Leu Gly Leu Gln Ala Ser Ile Ser INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Asp Tyr Lys Asp Asp Asp Asp Lys

Claims (15)

1. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a NLERK 2 or part thereof, said nucleotide sequence or its complementary form capable of encoding at least one of the following amino acid sequences: VXWXSXN [SEQ ID NO:1]; (ii) DXXDIXCP [SEQ ID NO:2]; (iii) EXYXLYXVXXXXXXXC [SEQ ID NO:3]; and/or (iv) KFQXXXXXXXGXEFXXXHXYYXI [SEQ ID NO:4], wherein X is any amino acid residue.

2. An isolated nucleic acid molecule according to claim 1 encoding at least two of SEQ ID NO:1 to SEQ ID NO:4.

3. An isolated nucleic acid molecule according to claim 1 encoding at least three of SEQ ID NO:1 to SEQ ID NO:4.

4. An isolated nucleic acid molecule according to claim 1 selected from the group consisting of: a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID (ii) a nucleic acid molecule having a nucleotide sequence having at least similarity to the sequence set forth in SEQ ID NO:5; and (iii) a nucleic acid molecule capable of hybridising under low stringency conditions to the nucleotide sequence or its complementary form set forth in SEQ ID An isolated nucleic acid molecule according to claim 4 further comprising a sequence of nucleotides as set forth in SEQ ID NO: 14 and/or SEQ ID NO: 15 or having at least P similarity thereto and/or is capable of hybridising thereto under low stringency conditions. P:\OPER\FJH\64098-96.CLM 19/8/99 -48-

6. An isolated nucleic acid molecule according to claim 4 or 5 obtainable from the human genome.

7. An isolated nucleic acid molecule according to claim 4 or 5 wherein the human genome is in brain, liver, kidney, neonatal, embryonic, cancer or tumour-derived tissue.

8. A method for cloning a nucleotide sequence encoding a novel NLERK 2 said method comprising searching a nucleotide database for a sequence which encodes at least one of amino acid sequences SEQ ID NO:1 to SEQ ID NO:4, designing one or more oligonucleotide S* primers based on a nucleotide sequence located in the search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefrom which encodes said novel NLERK 2.

9. A method according to claim 8 wherein the nucleic acid library is a cDNA expression library.

10. A method according to claim 9 wherein the cDNA expression library is of human origin derived from brain, liver, kidney, neonatal, embryonic, tumour or cancer tissue. from the listing consisting of: a nucleotide sequence as set forth in SEQ ID (ii) a nucleotide sequence having at least 75 similarity to the sequence set forth in SEQ ID NO:5; and (iii) a nucleotide sequence capable of hybridising under low stringency conditions to the nucleotide sequence or its complementary form set forth in SEQ ID P:\OPER\EJH\64098-96CLM 19/8/99 -49-

13. A recombinant polypeptide having the ability to interact with an ERK wherein said recombinant polypeptide comprises at least one of amino acid sequence SEQ ID NO: 1 to SEQ ID NO:4.

14. A recombinant polypeptide according to claim 13 having an amino acid sequence as set forth in SEQ ID NO:6 or a functional part thereof. A pharmaceutical composition comprising a recombinant polypeptide as defined in claim 12 and one or more pharmaceutically acceptable carriers and/or diluents. C *09*

16. A pharmaceutical composition according to claim 15 wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:6 or a functional part thereof.

17. A use of a recombinant polypeptide having an amino acid sequence as set forth in SEQ ID NO:6 or a functional part thereof in the manufacture of a medicament for the treatment of disease conditions resulting from NLERK 2-deficiency or NLERK2-defectiveness.

18. A nucleic acid corresponding to the 3' end of an NLERK 2 gene comprising a sequence S.0: of nucleotides as set forth in SEQ ID NO:14 or having at least 75% similarity thereto or is capable of hybridising thereto under low stringency conditions.

19. A nucleic acid corresponding to the 5' end of an NLERK 2 gene comprising a sequence of nucleotides as set forth in SEQ ID NO:15 or having at least 75% similarity thereto or is capable of hybridising thereto under low stringency conditions. M P:\OPER\mH\4098-96.CLM 19/8/99 An isolated nucleic acid molecule according to any one of claims 1 to 7 or 18 or 19 or a method according to any one of claims 8 to 12 or a polypeptide according to claim 13 or 14 or a composition according to claim 15 or 16 substantially as herein described with reference to the Figures and/or Examples. DATED this 19th day of August 1999 AMRAD Operations Pty Ltd By DAVIES COLLISON CAVE Patent Attorneys for the Applicants *9* t w 50 9