AU710221B2 - Therapeutic compositions - Google Patents

Description

I WO 97/23501 PCT/AU96/00827 -1- THERAPEUTIC COMPOSITIONS The present invention relates generally to molecules capable of modulating apoptosis of animal cells. More particularly, the present invention provides cell homologues of viral-derived, apoptotic-inhibiting molecules which are useful in modulating apoptosis of animal cells. The molecules contemplated by the present invention may be used to promote or inhibit cell apoptosis depending on the exigencies of the therapeutic situation.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence Identity numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the Examples.

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 inte ge r r group of elements or integers.

Apoptosis is a cellular death program that may be initiated by a variety of stimuli and typically leads to characteristic changes in cells, frequently resulting in activation of a endonuclease which catalyses chromatin fragmentation and condensation, membrane blebbing and collapse of the nucleus.

The mechanisms for apoptosis have been strongly conserved during evolution (Vaux et al, 1994). For example, proteins resembling Bel-2 can protect nematode, insect and vertebrate cells from apoptosis (Vaux et al, 1992; Alnemri et al, 1992), and cysteine proteases resembling interleukin-lp converting enzyme (ICE) are required for apoptosis in both C. elgans and mammals (Ellis and Horvitz, 1986; Miura et al, 1993; Kuida et al, 1995). These apoptosis WO 97/23501 PCT/AU96/00827 -2effector proteases exist as precursors in many cells, but must be cleaved and assembled into tetramers before they are active (Thornberry et al, 1992; Wilson et al, 1994; Walker et al, 1994; Munday et al, 1995).

Although many apoptosis effector proteases have been discovered, and numerous stimuli that induce apoptosis have been found, little is known about the signalling and activation pathways that connect the cell death stimuli to the apoptosis effector mechanisms. For example, ligation of the cell surface receptor CD95 (Fas/Apo-1) can induce apoptosis in many cell types (Nagata and Golstein, 1995; Krammer et al, 1994), and in T lineage cells, ICE is required for induced cell death (Kuida et al, 1995), but the molecular pathway(s) that lead(s) from to ICE activation is/are largely uncharacterised. The cytoplasmic domain of CD95, which bears a motif termed the "death domain" is known to associate with other death domain bearing proteins such as FADD/MORT1 (Boldin et al, 1995; Chinnaiyan et al, 1995) and RIP (Stager etal, 1995) which are believed to participate in signalling from CD95. The downstream targets of FADD and RIP are unknown.

Tumour Necrosis Factor (TNF) can also trigger apoptosis, and its receptor resemble (Beutler and van-Huffel, 1994). The TNF-receptor 1 (p55) is known to bind to TRADD (Hsu et al 1995) and the TNF-receptor 2 (p75) is known to bind to TNF receptors associated factors (TRAFs) 1 and 2 (Rothe et al, 1994), but how these proteins work has not yet been determined.

In work leading up to the present invention, the inventors studied viral anti-apoptosis proteins and the intermediate steps of apoptosis signalling. Apoptosis can be used as a defence against viruses, but many viruses carry genes for anti-apoptosis proteins, presumably to keep the host cell alive while they replicate. Some viral anti-apoptosis proteins resemble known cellular proteins such as Bcl-2 (reviewed in Vaux, 1993). Others, such as the baculovirus p35 proteins, have unknown cellular counterparts, but can function in heterologous systems such as nematodes and mammals where they are thought to act as competitive inhibitors of ICE-like cysteine proteases (Clem et al, 1991; Hay et al, 1994; Rabizadeh et al, 1993; Xue and Horvitz, 1995; Bump etal, 1995).

WO 97/23501 PCT/AU96/00827 -3- Miller and coworkers identified a family of proteins in baculoviruses they designated "IAPs" because they could inhibit the apoptotic response of insect cells to viral infection (Birnbaum et al, 1994; Crook et al, 1993). Viral IAP proteins typically have two terminal repeats designated baculovirus IAP repeats (BIRs), and a carboxy terminal RING finger domain.

Autographa californica nuclear polyhedrosis virus (AcNPW) encodes a IAP protein that does not inhibit apoptosis, so it is possible that IAP proteins also have other functions, for example, regulatory cytokine function.

The present inventors sought cellular IAP homologues and chemical analogues including derivatives which function in cell death pathways by screening for gene segments potentially encoding novel IAP proteins. Full length cDNA clones were then obtained and tested for their ability to mediate apoptosis by ICE and by FADD.

Accordingly, one aspect of the present invention is directed to a homologue or chemical analogue of a viral derived peptide, polypeptide or protein or a derivative thereof which viralderived molecule is capable of inhibiting an apoptotic response of cells to viral infection.

More particularly, the present invention provides an isolated proteinaceous molecule or derivative of chemical analogue thereof capable of inhibiting an apoptotc response in cells_ lv vl t 'J xs t, ll dL, o11 IC ,LJLlr "14al U011 S to viral infection, said proteinaceous molecule comprising a cell-derived homologue of a viral inhibitor of apoptosis (IAP).

Viral derived IAPSs are polypeptides or proteins. The IAPs represent a family of proteinaceous molecules which inhibit the apoptotic response of cells to viral infection. More particularly, the IAPs inhibit the apoptotic response of insect cells to infection by baculoviruses. The present invention provides, therefore, homologues or chemical analogues of baculovirus IAPs which are useful in modulating apoptosis of animal cells. Preferably, the apoptosis is mediated by ICE or by FADD.

Accordingly, another aspect of the present invention contemplates a homologue or chemical WO 97/23501 PCT/AU96/00827 -4analogue of a baculovirus IAP peptide, polypeptide or protein or a derivative thereof.

Preferably, the homologue is a animal, cell derived molecule. Examples of particularly preferred animal cells are those from humans, livestock animals sheep, pigs, horses, donkeys, cows), companion animals dogs, cats), laboratory test animals rabbits, mice, rats, guinea pigs), captive wild animals foxes, deer, kangaroos), insects mosquitoes) and nematodes. Homologous may also be from yeast or fungi.

Particularly preferred homologues are for human, murine, insect, yeast or nematode cells. An example of a nematode is Caenorhabditis elegans.

In a most preferred embodiment, the mammalian homologues are derived from murine cells and are designated herein MIHA, MIHB, MIHC and MIHE. In a alterative embodiment, the homologue is an insect homologue from Drosophila designated herein DIHA. In a further alternative embodiment the homologue is a C. elegans homologue designated herein CIA-1 and CIA-2. In yet a further alternative embodiment the homologue is a yeast homologue designated herein YIA-1. However, the present invention extends to human and other mammalian homologues of baculovirus IAPs or homologues or chemical analogues of MIHA, MIHB, MIHC and/or DIHA.

According to another aspect of the present invention, there is provided an isolated molecule having an amino acid sequence comprising: Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro [Xaa]m [Xaal Ala Xaa Ala Gly Phe [Xaa]o Asp Xaa [Xaa]p Xaa Cys Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys Xaa [Xaa], [Xaa], wherein .WO 97/23501 PCT/AU96/00827 Xaa is an amino acid residue; [Xaa]m is a series of at least 5 and preferably at least 9 amino acids; [Xaa], is Met or Leu; [Xaa]o is a series of at least 3 and preferably at least 5 amino acids; [Xaa], is Val or Ala; [Xaa]q is Phe or Tyr; [Xaa], is Leu or Val; or a homologue, chemical analogue or derivative thereof.

More particularly there is provided a isolated molecule designated MIHA having a amino acid sequence substantially as set forth in SEQ ID NO:2 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

A related aspect of the present invention is directed to a isolated molecule designated MIHB having an amino acid sequence substantially as set forth in SEQ ID NO:4 or having at least similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

Yet a further related aspect of the present invention relates to a isolated molecule designated MIHC having an amino acid sequence substantially as set forth in SEQ ID NO:6 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

Still a further related aspect of the present invention provides a isolated molecule designated DIHA having an amino acid sequence substantially as set forth in SEQ ID NO:8 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

Another related aspect of the present invention provides a isolated molecule designated DIHA WO 97/23501 PCT/AU96/00827 -6having an amino acid sequence substantially as set forth in SEQ ID NO:11 or having at least similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

Still another related aspect of the present invention provides a isolated molecule designated DIHA having an amino acid sequence substantially as set forth in SEQ ID NO: 13 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

A further related aspect of the present invention provides a isolated molecule designated DIHA having an amino acid sequence substantially as set forth in SEQ ID NO: 15 or having at least similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

Yet another related aspect of the present invention provides a isolated molecule designated DIHA having an amino acid sequence substantially as set forth in SEQ ID NO: 17 or having at least 40% similarity to all or part thereof or a homologue, chemical analogue or derivative thereof.

The present invention also encompasses nucleic acid molecules encoding IAP homologues such as MIHA, MIHB, MIHC, MIHE, CIA-1, CIA-2, YIA-1 and DIHA.

The IAP homologues may or may not bear RING finger domains, for example, MIHA, MHB and MIHC bear the domain whereas MIHE, CIA-1, CIA-2 or YIAY do not bear the domain.

The present invention further contemplates a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence comprising: Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro [Xaa]m [Xaa] Ala Xaa Ala Gly Phe [Xaa]o Asp Xaa [Xaa]p Xaa Cys Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys Xaa [Xaa], [Xaa]r .1 WO 97/23501 PCT/AU96/00827 -7wherein Xaa is an amino acid residue; [Xaa]m is a series of at least 5 and preferably at least 9 amino acids; [Xaa], is Met or Leu; [Xaa]o is a series of at least 3 and preferably at least 5 amino acids; [Xaa], is Val or Ala; [Xaa]q is Phe or Tyr; [Xaa]r is Leu or Val; or having at least 40% similarity to a nucleotide sequence encoding the above-mentioned amino acid sequence.

In particular, the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:2 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:2.

In a related embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:4 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:4.

In another related embodiment, there is contemplated a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:6 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:6.

A further embodiment is directed to a nucleic acid molecule comprising a sequence of IWO 97/23501 PCT/AU96/00827 -8nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:8 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:8.

Another related aspect is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO: 11 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO: 11.

Still another related aspect is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO: 13 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO: 13.

A further related aspect is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:15 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID Yet another related aspect is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence substantially set forth in SEQ ID NO:17 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO: 17.

In yet another embodiment of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 1 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO: 1 under low stringency conditions.

In a related embodiment of the present invention, there is provided a nucleic acid molecule SWO 97/23501 PCT/AU96/00827 -9comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO:3 under low stringency conditions.

In a further related embodiment of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:5 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO:5 under low stringency conditions.

In a still a further related embodiment of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:7 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO:7 under low stringency conditions.

In another further related embodiment of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 10 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO: 10 under low stringency conditions.

In a further related embodiment of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:12 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO: 12 under low stringency conditions.

In a still a further related embodiment of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 14 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO: 14 under low stringency conditions.

In yet another related embodiment of the present invention, there is provided a nucleic acid I WO 97/23501 PCT/AU96/00827 molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:16 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO: 16 under low stringency conditions.

Reference herein to a low stringency at 42"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.

The nucleic acid molecules are preferably in isolated form and/or carried by a vector molecule such as an expression vector.

The IAP homologues and their derivatives and chemical analogues of the present invention are useful in inhibiting apoptosis and are useful in the treatment of diseases including but not limited to those characterised by apoptosis such as degenerative diseases including Alzheimer's disease, motor neuron disease, neuropathies; ischemic vascular disease including stroke and myocardial infarction; infectious diseases including Acquired Immuno-Deficiency Syndrome caused by HIV. Certain derivatives of the homologues contemplated herein may promote contemplated herein may promote apoptosis or inhibit anti-apoptotic processes and are potentially useful in the treatment of diseases characterised by failure of apoptosis of certain cells including but not limited to cancer or autoimmune disease, alzheimer's and motor neuron diseases. Derivatives of the homologues contemplated herein many promote apoptosis or inhibit anti-apoptotic processes and are useful in the treatment of, for example, certain cancers or in the promotion of the death of malignant cells.

WO 97/23501 PCT/AU96/00827 11 Derivatives of the homologues of the present invention include mutants, parts and fragments of the homologue molecules as well as single or multiple amino acid substitutions, deletions and/or additions to the homologue molecules. Nucleic acid derivatives including single or multiple nucleotide substitutions, deletions and/or additions to the nucleic acid molecules as defined in SEQ ID NO: 1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.

Chemical analogues of the baculovirus IAPs or of their homologues include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with a aldehyde followed by reduction with aBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; triitrobezylation of amino groups with 2, 4, 6, -trinitrobezene sulphonic acid (TBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with phosphate followed by reduction with aBH 4 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-chloromercuribezoate, 4- WO 97/23501 PCT/AU96/00827 -12chloromercuripheylsulphonic 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 -bromosuccinimide or alkylation of the idole 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 hisitidine 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, norithine, sarcosine, 4 -amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or Disomers of amino acids. A list of unnatural amino acids is provided in Table 1.

WO 97/23501 PTA9/02 PCT/AU96/00827 13 TABLE 1 Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid ca-ammno-a-methylbutyrate aminocyclopropanecarboxylate aminoisobutyric acid aminonorbomylcarboxylate cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamine D-glutamic acid D-histidine D-isoleucine D-leucine D-lysine D-metbionine D-ornithine D-phenylalanine D-proline D-serine D-threonine Abu Mgabu Cpro Aib Norb Cpen Dal Darg Dasp Dcys Dgln.

Dglu Dhis Dile Dleu Dlys Dmet Domn 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-methylnorieucine L-N-methylnorvaline L-N-methylomithine L-N-methylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine Nmala Nmarg Nmasn Nma~p Nmcys Nmgln Nmglu.

Nmhis Nmile Nmleu Nmlys Nmmet Nmnle Nmnva Nmorn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe WO 97/23501 W097/3501PCT/AU96/00827 14 D-tryptophan D-tyrosine D-valine D-ix-methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-ra-methylglutamine D-a-methylhistidine D-a-methylisoleucine D-a-methylleucine D-a-methyllysine D-a-methylnethionine D-a-methylornithine D-ez-methylphenylalanine D-a-methylproline D-a-methylserine D-a-methylthreonine D-ci-methyltryptophan D-ra-methyltyrosine D-ea-methylvaaie D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-methylglutamine D-N-methylglutamate D-N-methylhistidine Dtrp Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dmleu Dmlys Dmmet Dmorn Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Dnmala Dnmarg Dnmasn Dnmasp Dnmcys Dnmgkn Dnmglu Dnnihis L-norvaline ra-methy1-aminoisobutyrate ix-methyl-y-amninobutyrate a-methylcyclohexylalanine ra-methylcylcopentylalanine a-methy1-a-napthylalanine a-methylpenicillamine N-(4-aminobutyl)glycine N-(2-aminoethyl)glycine N-(3 -aminopropyl)glycine N-amino-a-methylbutyrate e-napthylalanine N-b enzylglycine N-(2-carbamylethyl)glycine N-(carbaniylmethyl)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-hydroxyetliyl)glycine N-(hydroxyethyl))glycine Nva Maib Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Norn Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe Narg Nthr Nser WO 97/23501 WO 9723501PCT/AU96/00827 15 D-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu D-N-methyllysine Dnmlys N-methylcyclohexylalanine Nmchexa D-N-methylornithine Dnmorn N-methylglycine Nala N-methylaminoisobutyrate Nmaib 1-methylpropyl)glycine Nile N-(2-methylpropyl)glycine Nleu D-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr D-N-methylvaline Dnmval y-aminobutyric acid Gabu L-t-butylglycine Thug L-ethylglycine Etg L-homophenylalanine Hphe L-a-methylarginine Marg L-ra-methylaspartate Masp L-e-methylcysteine Mcys L-a-methylglutainine Mgln L-a-methylhistidine Mhis L-ca-methylisoleucine Mile L-a-methylleucine Mleu L-a-methylmethionine Mmet L-a-methylnorvaline Mnva L-a-methylphenylalanine Mphe L-a-methylserine Mser L-rL-methyltryptophan Mtrp N-(imidazolylethyl))glycine N-(3 -indolylyethyl)glycine N-methyl-y-aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-mnethylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine 1-methylethyl)glycine N-methyla-napthylaianine N-methylpenicillamine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillaniine L-ra-methylalanine L-a-methylasparagine L-a-methyl-f-butylglycine L-methylethylglycine L-a-methylglutamate L-a-methylhomophenylalanine N-(2-methylthioethyl)glycine L-a-methyllysine L-a-methylnorieucine L-a-methylornithine L-a-methylproline L-ra-methylthreonine L-a-methyltyrosine Nbis Nhtrp Nmgabu Dnnimet Nmcpen Dnmphe Dnmpro Dnmser Dnmthr Nval Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhfphe Nmet Mlys Mnle Morn Mpro Mthr Mtyr WO 97/23501 PCT/AU96/00827 -16- 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-l-(2,2-diphenyl- Nmbc ethylamino)cyclopropane Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifuctional crosslinkers such as the bifunctional imido esters having (CH 2 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 specificreactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and Na-methylamine acids, introduction of double bonds between C a and Cp atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming a amide bond between the and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention further contemplates a method for modulating cell apoptosis in a animal, said method comprising administering to said animal a cell apoptosis modulating effective amount of a homologue or chemical analogue of a baculovirus IAP or a derivative thereof for a time and under conditions sufficient for said cell apoptosis to be modulated.

The terms "modulating" or "modulated" refer to the promotion or enhancement of cell apoptosis or the deminuation, inhibition or reduction of cell apoptosis. Enhancing cell apoptosis may be important in treating certain cancers and cell malignancies; reducing cell apoptosis may be important in the treatment of degenerative disorders such as neuropathies and alzheimer's and motor neuron diseases.

Accordingly, the present invention is also directed to therapeutic and pharmaceutical WO 97/23501 PCT/AU96/00827 -17compositions useful for modulating cell apoptosis in a animal.

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

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

WO 97/23501 PCT/AU96/00827 -18- The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.

Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

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

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously .WO 97/23501 PCT/AU96/00827 -19sterile-filtered solution thereof.

When the IAP homologues or chemical analogues or derivatives are suitably protected as described above, the active, compound may be orally administered, for example, with an inert diluent or with a assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into 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 of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that a oral dosage unit form contains between about 0.1 /ig and 2000 mng of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a 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 may be incorporated into sustained-release preparations and formulations.

WO 97/23501 PCT/AU96/00827 A pharmaceutically acceptable carrier and/or diluent includes 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 coating a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such a active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The 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 jig to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 jzg to about 2000 mg/ml of carrier.

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

The 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 IAP homologue expression or IAP homologue activity. The vector may, for WO 97/23501 PCT/AU96/00827 -21example, be a viral vector.

Another aspect of the present invention contemplates the use of an animal cell homologue of an baculovirus IAP to modulate apoptosis in animals suffering from a degenerative disease, an infectious disease, cancer or an autoimmune disease.

Preferably, the cell homologue is as defined by one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or is a derivative or chemical analogue thereof.

The present invention further contemplates antibodies to the IAP homologues and the derivatives of the present invention. Antibodies are useful in diagnostic assays for the homologues as well as purifying the homologues or isolating the homologues from biological fluid or culture medium. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to an IAP homologue or may be specifically raised to an IAP homologue or a derivative thereof. In the case of the latter, an IAP homologue or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant IAP homologue or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.

For example, an IAP homologue and its derivatives can be used to screen for naturally occurring antibodies to the homologue. These may occur, for example in some autoimmune diseases. Alternatively, specific antibodies can be used to screen for an IAP homologue.

Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of IAP homologue levels may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.

Antibodies to an IAP homologue 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.

WO 97/23501 PCT/AU96/00827 -22- 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 regimen.

For example, specific antibodies can be used to screen for an IAP homologues. The latter would be important, for example, as a means for screening for levels of an IAP homologue in a cell extract or other biological fluid or purifying IAP homologue made by recombinant means from culture 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 an IAP homologue. An antibody may also be directed to an amino acid sequence such as set forth in SEQ ID NO:9.

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 IAP homologue, 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 WO 97/23501 PCT/AU96/00827 -23preparation ofhybridoma 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 an IAP homologue in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for the IAP homologue or its derivatives or homologues for a time and under conditions sufficient for an antibody-IAP homologue complex to form, and then detecting said complex.

The presence of IAP homologue 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 noncompetitive 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 ofhapten. Variations on the forward assay include a simultaneous assay, in which WO 97/23501 PCT/AU96/00827 -24both 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 an IAP homologue including a 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.

In the typical forward sandwich assay, a first antibody having specificity for the IAP homologue 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 ofmicroplates, 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 polymerantibody 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 25C) 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.

WO 97/23501 PCT/AU96/00827 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, 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 WO 97/23501 PCT/AU96/00827 -26 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 IAP homolgoue gene or its derivatives. Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphoms analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.

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

In the Figures: Figure 1 is a comparison of deduced peptide sequences of IAP proteins.

Comparison of MIHA (SEQ ID NO:2), MIHB (SEQ ID NO:4), MIHC (SEQ ID NO:6), and DIHA (SEQ ID NO:8). Amino acids shared by three or more of the proteins are highlighted. Arrows indicate the three BIRs. The RING finger domain is indicated by a dashed arrow.

Comparison of BIRs of IAP proteins. Strongly conserved residues are highlighted.

CPI derives from Cydia pomonella granulosis virus; OpIAP derives from Orgyia pseudotsugata PV; CilAP derives from Chilo iridescent virus; AIP is a candidate gene for SMA; AcIAP derives from Autographa Californica PV; ASV derives from IAPlike sequences from African swine fever virus.

Comparison of RING finger motifs of IAPs and other proteins. Strongly conserved residues are highlighted. CRAF (TRAF3) and TRAF2 are TRAF family members; BCRA1 and RAG1 are two mammalian proteins with RING finger motifs; c-CBL is a cellular oncogene and SLI-1 is its C. elgans homologue; cpi derives from Cydia pomonella granulosis virus; OpIAP derives from Orgyia pseudotsugata PV; CilAP .IWO 97/23501 PCT/AU96/00827 -27derives from Chilo iridescent virus; AclAP derives from Autographa califorica PV; UKMT derives from the Drosophila Unkempt gene; EctV derives from the p28 protein of Ectromelia Virus.

Figure 2 is a photographic representation showing mammalian IAP homologues are expressed in a variety of tissues.

An adult mouse tissue total RNA Northern blot was probed with the mMIHA cDNA coding region at high stringency. This Northern was also probed with GAPDH as a indicator of each lane's loading.

A adult mouse tissue poly RNA Northern blot (CLONTECH) was probed with the hMHB CDNA coding region and the hMIHC CDNA coding region at low stringency.

Figure 3 is a graphical representation showing that MIHA and MIHB protect against death induced by overexpression of ICE, but not FADD.

Induction of apoptosis by transfection with ICE. Columns 1-6 (In black) indicate the percentage of dead cells cotransfected with p32ICE-lacZ fusion plasmid and the plasmids bearing either the LAP homologues or controls. Death of cells cotransfected with lacZ only together with the same test plasmids is show in columns 7-12 (in white) and indicates the amount of cell death due to the transfection procedure itself.

Induction of apoptosis by transfection with FADD. Plasmids encoding the MIH proteins were cotransfected with a lacZ vector and a construct bearing the FADD coding region (columns As with the ICE experiment, background death was monitored in a parallel set of cultures (columns 7-12).

Figure 4 is a schematic representation showing a hypothetical and non-limiting cell death model showing the proposed site of action of IAP proteins. Direct interactions are indicated by solid lines, and indirect actions are show as dotted lines. Uncertain interactions are indicated by a question mark. Spontaneous self-association of molecules such as TRAFs and death-domain bearing proteins may generate a intrinsic activation signal that in normal WO 97/23501 PCT/AU96/00827 -28circumstances is insufficient to activate enough cell death proteases to cause apoptosis.

Increased amounts of ICE precursor due to transfection could allow activation of enough enzyme to induce apoptosis. IAP proteins may act to decrease the spontaneous activation signals to various extents depending on their affinity for their target(s). According to this model, OpIAP may be able to offer greater protection against FADD than the mammalian IAP homologues because it has greater affinity for its target, but it is not as effective against FADD as it is against pro-ICE because it is required to block a larger intrinsic activation signal in FADD transfected cells. CrmA and p35, which directly inhibit active ICE, are effective inhibitors of ICE mediated apoptosis on matter how ICE is activated.

Figure 5 is a representation of the nucleotide sequence and corresponding amino acid sequence of MIHA.

Figure 6 is a representation of the nucleotide sequence and corresponding amino acid sequence of MIHB.

Figure 7 is a representation of the nucleotide sequence and corresponding amino acid sequence of MIHC.

Figure 8 is a representation of the nucleotide sequence and corresponding amino acid sequence of DIHA.

Figure 9A and 9B show protection by MIHA, MIHB and MIHC against death induced by over expression of ICH-1 protease.

Figure 10 is a photographic representation of a Western blot of 293T cells transfected with MIHA constructs.

WO 97/23501 PCT/AU96/00827 29 The following single and three letter abbreviations are used for amino acid residues: Amino Acid Three-letti Abbreviati One-letter Symbol Alanine Ala

A

Arginine Arg

R

Asparagine Asn

N

Aspartic acid Asp

D

Cysteine, Cys

C

Glutamine Gln

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 Thr

T

Tryptophan Trp

W

Tyrosine Tyr

Y

Valine Val

V

Any residue Xaa x WO 97/23501 PCT/AU96/00827 A summary of SEQ ID NOs is as follows: SEQ ID NO:

DESCRIPTION

1 Nucleotide sequence of MIHA 2 Amino acid sequence of MIHA 3 Nucleotide sequence of MIHB 4 Amino acid sequence of MIHB Nucleotide sequence of MIHC 6 Amino acid sequence of MIHC 7 Nucleotide sequence of DIHA 8 Amino acid sequence of DIHA 9 Concensus amino acid sequence Nucleotide sequence of MIHE 11 Amino acid sequence of MIHE 12 Nucleotide sequence of CIA-1 13 Amino acid sequence of CIA-1 14 Nucleotide sequence of CIA-2 Amino acid sequence of CIA-2 16 Nucleotide sequence of YIA-1 17 Amino acid sequence of YIA-1 18 Amino acid sequence of MIHA peptide I. WO 97/23501 PCT/AU96/00827 -31- EXAMPLE 1 cDNA CLONING The GenBank database was searched using the GCG Sequence Analysis Software Package (Madison, WI) for translated sequences resembling the OpIAP BIR and RING finger amino acid motifs. A human X chromosome genomic STS thus identified (GenBank:L24579) was used to design PCR primers flanking the putative RING finger motif. This region was amplified and used to scree a human genomic DNA library (Stratagene). A fragment isolated from this library was used to probe a mouse liver cDNA library (Stratagene) at low stringency yielding three murine cDNA clones that were designated mammalian IAP homologueA (MIHA). Translations of the human EST sequences GenBank:R19628 and GenBank:T96284 were found to resemble the BIR repeats of OpIAP. These sequences were used to design PCR primers within their putative BIR domains which were used to generate probes that were used to scree a human fetal liver cDNA library (Stratagene). The hybridising cDNA clones were designated MIB and MIHC, respectively.

The Drosophila genomic sequence (GenBank:DROCCAAT) was used to design primers to amplify a 900 bp product from Drosophila CDNA. This fragment was subcloned and used to screen an oligo(dT) primed Drosophila larval cDNA library constructed in lambda ZAP (Stratagene). A 2kb cDNA (DIHA) clone encoding all but the 8 -terminal amino acids was isolated. For peptide sequence comparisons (Figure 1) these residues deduced from the genomic sequence GenBank:DROCCAAT were added.

EXAMPLE 2 RNA ANALYSIS Radiolabelled mMIHA and GAPDH were hybridised at high stringency to a mouse tissue Northern blot bearing 5 /zg/lane of total RNA. A mouse multiple tissue Northern blot (CLONTECH) bearing 2 /g poly(A)+ RNA was probed with radiolabelled hMIHC at low stringency, stripped, probed with hMHB at low stringency, stripped again and probed at high stringency to a P-actin probe according to the manufacturer's instructions.

I WO 97/23501 PCT/AU96/00827 -32- EXAMPLE 3 YEAST TWO-HYBRID SYSTEM The coding regions of MIHA, MIHB and MIHC were amplified by PCR using primers incorporating an EcoRI site at the 5' end and a BamHI site at the 3' end (or a BglII site in the case of MIHB), such that the proteins would be expressed as in frame fusions with the GAL4 DNA binding domain. The PCR products were the ligated into the pGBT9 vector (CLONTECH). The OpIAP gene from the HindIII site 17 codons upstream of the initiating ATG was cloned into the pGBT9 vector such that a in frame fusion would result. PCRderived inserts were sequenced to check for misincorporations by Taq polymerase. TRAF1, TRAF2 and TRAF3 expression vectors have been previously described (Rothe et al, 1994; Hu et al, 1994b). Vectors with the coding regions of c-jun in GBT9 andfos were used as controls for the detection of interacting proteins. The yeast strain HF7c was transformed with these plasmids using the lithium acetate protocol (Gietz et al, 1992).

EXAMPLE 4 TRANSIENT TRANSFECTION

ASSAYS

A 1.8 kb SacI-EcoRI fragment from a MIHA cDNA clone in pBluescript (Stratagene) was subcloned into pSP72 (Promega) to generate pSPMIHA, and recloned into pEF, a derivative of the pEFBOS vector (Mizushima and Nagata, 1990) as a 1.8 kb BamHI-EcoRV fragment.

A 1.64 kb BamHI-ScaI fragment from the DIHA cDNA was cloned in pEF. Pfu polymerase (Stratagene) was used to amplify the coding regions of MIHB and MIHC. Unique restriction sites were included at the ends of the primers for the cloning of these products into pEF.

The p32-lacZ fusion plasmid ppactM11Z was as described by Miura et al (1993). The FADD expression construct FADD-AU1 was as described by Chinnaiyan et al (1995). The coding regions of bcl-2, crmA, p35 from AcPV, and IAP from OpPV were inserted into the pEF vector. The truncated OplAP plasmid was constructed by digestion of the pEF vector coating full length IAP with rul and Smal, and re-ligating. This deleted sequences 3' of the rul site in the OplAP gene which encode the RING finger domain.

WO 97/23501 PCT/AU96/00827 -33- EXAMPLE CLONING OF DIHA, MIHA, MIHB and MIHC Searches of GenBank revealed a Drosophila genomic sequence (GenBank:DROCCAAT) that resembled baculoviral IAP genes. A Drosophila cDNA library was screened using PCR generated probes and clones isolated which encodes this protein, designated herein Drosophila IAP homologue A (DIHA). The GenBank searches also revealed a number of mammalian sequences that resembled either the BIRs or the RING finger domains of viral IAPs. The first mammalian IAP homologue identified (MIHA), was cloned using a PCR generated probe corresponding to a STS sequence on the X chromosome, HUMSWX595, which when translated encoded a peptide resembling a baculoviral IAP RING finger motif. The inventors isolated partial length human genomic clones and mouse MIHA cDNA clones compassing the entire coding region. A further pair of closely related human IAP homologues (MIHB and MIHC) were isolated using PCR generated probes corresponding to GenBank EST sequences R19628 and T96284, which encode BIR-like motifs.

Figure 1A compares the predicted amino acid sequences for DIHA (predicted molecular weight 55 kD), MIHA (56 kD), MIHB (70 kD) and MIHC (68 kD). Start codons were chose as the most 5' methionine with upstream, in frame stop codons. All four proteins bear three BIR repeats in the amino terminal half and a single RING finger domain close to the carboxy terminus. MIHB and MIHC are the most closely related, with 73% amino acid identity.

MIHA shares 43% identity and 62% similarity with MIHB and MIHC. DIHA has 36% identity and 57% similarity to MIHC.

The BIR repeats of these proteins are compared to each other, the BIR repeats of AIP and the viral IAPs in Figure lB. Among the mammalian IAPs some of the BIRs resemble the analogous BIR from another IAP more closely than they resemble the remaining BIRs in the same protein, indicating that duplication of a primordial BIR was a early event. Four of the most highly conserved residues in the BIRs are three cysteines and a histidine in the pattern

CX

2 CX16HX6C. These may bind metal ions (Birnbaum et al, 1994).

WO 97/23501 PCT/AU96/00827 -34- Figure 1C shows a comparison of the C3HC4 RING finger domains of the IAP molecules with RING fingers from other selected proteins. The RING fingers from IAP proteins resemble each other more than they resemble the RING fingers from other proteins, such as TRAF2 (Rothe et al, 1994) and CRAF (Hu etal, 1994b; Cheng et al, 1995; Sato et al, 1995), that do not bear BIRs. The most similar RING fingers in o-BIR bearing proteins are in the mammalian oncogene, c-Cbl (Blake et al, 1991) and its C. elgans homologue, Sli-1 (Yoon et al, 1995). Like the baculoviral IAP proteins, the RING finger bearing protein p28 of Ectromelia virus is not required for growth, but is essential for virulence (Senkevich et al, 1994). However, p28 does not have BIRs and its function is unknown.

EXAMPLE 6 EXPRESSION OF MAMMALIAN HOMOLOGUES The message for murine MIHA is about 7.5 kb, and is expressed in most mouse tissues with the except of skeletal and cardiac muscle. The highest level detected was in the lung, with intermediate levels detected in the brain and kidney, and lower levels in the thymus, liver, bone marrow, skin and testes (Figure 2A).

A cDNA probe spaning the coding region of human MIHB hybridised at low stringency to two messages of-4.0 kb and -5.5 kb on a mouse multiple tissue Northern blot (Figure 2B). The upper, less abundant transcript was expressed least in the spleen and skeletal muscle and at higher levels in all other tissues analysed. The more abundant -4.0 kb transcript was expressed at lowest levels in the spleen, intermediate levels in the heart, brain, lung, liver, skeletal muscle and kidney and at highest levels in the testes. A additional transcript of kb was detected in the testes but not see in other tissues.

A full length human MIHC cDNA probe hybridised at low stringency to two messages of and -4.0 kb (Figure 2C). on 4.0 kb message was detectable in the spleen, high levels were detected in the testes and intermediate levels were detected in all other tissues. The -3.0 kb transcript could be see in the lung and skeletal muscle and very faintly in the spleen, liver, kidney and testes. As the hMIHB and hMIHC probes were hybridised at low stringency to WO 97/23501 PCT/AU96/00827 mouse RNA, it is possible the upper (4.0 kb) transcript is the same as that detected by the MIHB probe. Some of the transcripts may also represent other closely related mammalian IAP homologues.

EXAMPLE 7 PREVENTION OF ICE OR FADD INDUCED APOPTOSIS The mammalian IAP homologues were tested for their ability to prevent apoptosis due to two stimuli, overexpression ofp32ICE and overexpression of FADD. Transfection with constructs expressing the Ced-3-like cysteine protease ICE have previously been show to cause apoptosis (Miura et al, 1993). BaculovirallAP can prevent this death, as can other anti-apoptosis genes such as bcl-2, crmA and p35 (Miura et al, 1993; Xue et al, 1995). The inventors tested MIHA, MIHB and MIHC to determine whether they too could block apoptosis caused by ICE overexpression. HeLa cells were cotransfected with a plasmid bearing a ICE-lacZ fusion construct together with plasmids encoding the IAP homologues or controls. The cells were stained with X-gal to identify those that hand been transfected, and these were assessed visually for viability. As show in Figure 3A, MIHA, MIHB and OpIAP significantly reduced the amount of death caused by ICE, whereas MIHC did not provide detectable protection.

Enforced expression of the Cd95 associated protein FADD also causes cell death (Chinnaiyan et al, 1995; Boldin et al, 1995). HeLa cells were cotransfected with three plasmids: a FADD expression construct, a plasmid carrying the lacZ gene and vectors encoding the mammalian IAP homologues or OpIAP. OpIAP provided partial protection against FADD, but it was not as effective as it was against ICE (compare Figure 3A, lane 2 with Figure 3B, lane The inventors could not detect any reduction in the amount of FADD induced cell death by MIHA, MIHB orMIHC.

EXAMPLE 8 INTERACTIONS WITH TRAF FAMILY MEMBERS Two of the mammalian IAP homologues, MIHB and MIHC, were isolated as part of a protein complex that binds to the cytoplasmic domain of the TNF-R2 (p 7 5 together with the TNF-R2 WO 97/23501 PCT/AU96/00827 -36associated molecules TRAF1 and TRAF2 (Rothe et al, 1994). Using the yeast two hybrid system (Fields and Song, 1989), the three mammalian IAP homologues and viral OpIAP were tested for their ability to bind TRAF1, TRAF2 and TRAF3, a related protein also known as CRAF-1 CAP (Hu et al, 1994b; Cheng et al, 1995; Sato et al, 1995) As show in Table 2, yeast cotransfected with MIHB or MIHC and TRAF1 or TRAF2 but not TRAF3 were rendered his+ and lacZ+, indicating that interactions can occur between these proteins within yeast. In contrast, on interactions were detectable in this system between any of the TRAFs tested and OpIAP or MIHA. These results show that MIHB and MIHC bind to TRAF1 and TRAF2, but suggest that OpIAP and MIHA interact with other proteins.

EXAMPLE 9 IDENTIFICATION OF MHE, CIA-1, CIA-2 and YIA-1 A consensus sequence of baculovirus IAP repeats (BIR) was deduced as follows: Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro [Xaa]m [Xaa], Ala Xaa Ala Gly Phe [Xaa]o Asp Xaa [Xaa]p Xaa Cys Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys Xaa [Xaa], [Xaa], wherein Xaa is an amino acid residue; [Xaa]m is a series of at least 5 and preferably at least 9 amino acids; [Xaa], is Met or Leu; [Xaa]o is a series of at least 3 and preferably at least 5 amino acids; [Xaa]p is Val or Ala; [Xaa]q is Phe or Tyr; [Xaa]r is Leu or Val; WO 97/23501 PCT/AU96/00827 -37or a homologue, chemical analogue or derivative thereof.

A database search using this consensus sequence resulted in identification of IAP homologes from mouse (MIHE), yeast (YIA-1) and C. elegans (CIA-1, CIA-2). The nucleotide and corresponding amino acid sequence from these IAP homologes is given in SEQ ID NOS: to 17. Interestingly, MIHE, YIA-1, CIA-1 and CIA-2 do not bear RING finger domains.

EXAMPLE PROTECTION BY MIHA, MIHB, and MIHC Figures 9A and B shows the protection from MIHA, MIHB and MIHC against death induced by over expression of the ICH-1 protease.

EXAMPLE 11 WESTERN BLOT A polyclonal rabbit antibody was prepared and affinity purified against the MIHA peptide KDIKKTMEEKIQTSG (SEQ ID NO: 18). This antibody was used in a western blot of 293T cells transfected inter alia MIHA. The results are shown in Figure 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.

.IWO 97/23501 PCT/AU96/00827 38 TABLE 2 Yeast Two Hybrid Assays for Binding Between TRARi, TRAF2, TRAF3 and Mammalian TAP Homologues Transformant jGrowth on Trp DNA binding Activation hybrid Leu- His- Colony hybrid I jmedium Colour OpIAP TRAM1______ NIIIA TRAF 1 NM TRAF 1 1mIC TRAF1 c-Jun TRAFi OpIAP TRAF2 MI1HA TRAF2 Nm~h TRAF2 NMC TRAF2 c-jun TRAF2 OpIAP TRAF3 MIfHA TRAF3 MIHB TRAF3 NMC TRAF3 c-jun TRAF3 OpIAP fo s MIIH4A fos c-JAP1 fos NMHC fos c-jun fos WO 97/23501 PCT/AU96/00827 -39- The yeast strain HF7c was cotransformed with constructs which express fusion proteins between the GAL4 DNA-binding domain and the IAP family members or controls, and vectors which encode fusions between the TRAF proteins or controls and the GAL4 activation domain. Expression from the his and lacZ reporter genes (which indicates interactions) was analysed by growth of double transformants on medium lacking histidine (Column and blue staining of colonies, with X-gal (Column c-jun andfos were used as control genes encoding interacting proteins in the DNA binding vector and activation vector, respectively.

WO 97/23501 WO 9723501PCT/AU96/oo827 40

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P:\OPER\EJH\10891-97.SPE 7/7/99 -43- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: AMRAD OPERATIONS PTY LTD (US ONLY): DAVID LAWRENCE VAUX, CHRISTINE JOY HAWKINS, ANTHONY GERALD UREN, MIHA PAKUSCH and KIRSTEN LOUISE PULS (ii) TITLE OF INVENTION: THERAPEUTIC

COMPOSITIONS

(iii) NUMBER OF SEQUENCES: 18 (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: 20-DEC-1996 a.

(vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: [AU] PN 7275 FILING DATE: 22-DEC-1995 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: HUGHES DR, E JOHN L REFERENCE/DOCKET NUMBER: EJH/EK WO 97/23501 PCT/AU96/00827 -44- (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +61 3 9254 2777 TELEFAX: +61 3 9254 2770 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1988 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 212..1702 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAATTCCTGC AGTTAGTTGT ATTGCTGTGG TAGGGGGTCT AAAGATTCTA CATACAGTGA TACACATTTG GAATTTTTGT ACTTGGCAAA TTGAGGCAGG AAGCTAACGT TTTCCAGAAC 120 GGCCTGGGCT GCTTTTGAGA GCTCCGATCT CAGAGAAGAA GCACTAAAAG ATGAATATGA 180 AAAGGTGGAC AAGTCCTATT TTCCAGAGAA G ATG ACT TTT AAC AGT TTT GAA 232 Met Thr Phe Asn Ser Phe Glu 1 GGA ACT AGA ACT TTT GTA CTT GCA GAC ACC AAT AAG GAT GAA GAA TTT 280 Gly Thr Arg Thr Phe Val Leu Ala Asp Thr Asn Lys Asp Glu Glu Phe 15 GTA GAA GAG TTT AAT AGA TTA AAA ACA TTT GCT AAC TTC CCA AGC AGT 328 Val Glu Glu Phe Asn Arg Leu Lys Thr Phe Ala Asn Phe Pro Ser Ser 30 AGT CCT GTT TCA GCA TCA ACA TTG GCG CGA GCT GGG TTT CTT TAT ACC 376 Ser Pro Val Ser Ala Ser Thr Leu Ala Arg Ala Gly Phe Leu Tyr Thr 45 50 WO 97/23501 PCT/AU96/00827 GGT GAA GGA GAO Gly Glu Gly Asp AGA TGG CAG TAT Arg Trp Gin Tyr ACC GTG Thr Val CAA TGT TTC Gin Cys Phe TGT CAT GCG GCA Cys His Ala Ala ATA GAT Ile Asp GGA GAO TCA GOT GTT GGA AGA CAC AGG Gly Asp Ser Ala Vai Gly Arg His Arg 80 AGA ATA TCC Arg Ile Ser CCA AAT TGC AGA TTT ATC AAT GGT TTT TAT TTC GAA Pro Asn Cys Arg Phe Ile Asn Gly Phe Tyr Phe Glu AAT GGT OCT OCA Asn Gly Ala Ala 100 AAA TCT GAA AAC Lys Ser Giu Asn CAG TCT Gin Ser 105 ACA AAT COT GGT ATC CAA AAT GGC CAG Thr Asn Pro Gly Ile Gin Asn Gly Gin 110

TAO

Tyr 115

TGT

Cys 120 GTG GGA AAT AGA Val Oly Asn Arg

AAT

Asn 125 CCT TTT GCC CCT Pro Phe Ala Pro AGG CCA OCT GAG Arg Pro Pro Glu CAT GOT GAT TAT His Ala Asp Tyr

CTC

Leu 140 TTO AGA ACT OGA Leu Arg Thr Gly OTT GTA OAT ATT Val Val Asp Ile TCA GAO Ser Asp 150 ACC ATA TAO Thr Ile Tyr AAG TOA TTT Lys Ser Phe 170

CCG

Pro 155 AGG AAC COT GCC Arg Asn Pro Ala TGT AGT GAA GAA Cys Ser Olu Glu GCC AGA TTG Ala Arg Leu 165 CAG AAO TGG CCG Gin Asn Trp Pro TAT GOT OAT TTA ACC CCC AGA GAG Tyr Ala His Leu Thr Pro Arg Glu 180 TTA GCT Leu Ala 185 AGT GOT GGC CTC TAO TAC ACA GGG GCT Ser Ala Gly Leu Tyr Tyr Thr Gly Ala 190

OAT

Asp 195 GAT CAA GTG CAA Asp Gin Val Gin

TGC

Cys 200 TTT TOT TGT GGG Phe Cys Cys Gly AAA CTG GAA AAT Lys Leu Olu Asn

TGG

Trp 210 GAA CCC TOT GAT Glu Pro Cys Asp GCC TGG TCA OAA Ala Trp Ser Glu AGO AGA CAC TTT CCC AAT TGO TTT TTT Ary Arg His Phe Pro Asn Cys Phe Phe 225 GTT TTG Val Leu 230 GGC CGG AAC OTT AAT OTT OGA AGT GAA TOT GOT GTG AGT TOT OAT AGG Oly Arg Asn Val Asn Val Arg Ser Glu Ser Oly Val Ser Ser Asp Arg WO 97/23501 WO 9723501PCT/AU96/00827 -46 AAT TTC CCA Asn Phe Pro 250 AAT TCA ACA AAC Asn Ser Thr Asn

TCT

Ser 255 CCA AGA AAT CCA Pro Arg Asn Pro

GCC

Al a 260 ATG GCA GAA Met Ala Glu 1000 TAT GAA GCA CGG ATC GTT Tyr Glu Ala Arg Ile Val 265

ACT

Thr 270 TTT GGA ACA TG Phe Gly Thr Trp

ACA

Thr 275 TOC TCA OTT AAC Ser Ser Val Asn 1048 1096

AAG

Lys 280 GAG CAG CTT GCA Glu Gin Leu Ala

AGA

Arg 285 GCT GGA TTT TAT Ala Gly Phe Tyr

OCT

Ala 290 TTA GGT GAA GGC Leu Gly Glu Gly AAA GTG AAG TGC Lys Val Lye Cys

TTT

Phe 300 CAC TGT GGA GGA His Cys Gly Gly

GGG

Gly 305 CTC ACG GAT TGG Leu Thr Asp Trp AAG CCA Lys Pro 310 1144 AGT GAA GAC Ser Glu Asp TAC CTA TTG Tyr Leu Leu 330 TGG GAA CAG CAT Trp Giu Gin His AAG TGG TAC CCA GGG TGC AAA Lys Trp Tyr Pro Gly Cys Lys 325 1192 GAT GAG AAG GGG Asp Glu Lys Gly

CAA

Gin 335 GAA TAT ATA AAT Giu Tyr Ile Asn

AAT

Aen 340 ATT CAT TTA Ile His Leu 1240 ACC CAT Thr His 345 TCA CTT GAG GAA TCT TTG GGA AGA ACT Ser Leu Giu Glu Ser Leu Gly Arg Thr 350

OT

Ala 355 GAA AAA ACA CCA Glu Lys Thr Pro 1288 1336

TOG

Ser 360 CTA ACT AAA AAA ATO GAT GAT ACC ATO TTC CAG AAT COT ATG Leu Thr Lye Lye Ile Asp Asp Thr Ile Phe Gin Asn Pro Met CAA GAA GCT ATA Gin Giu Ala Ile

CGA

Arg 380 ATG GGA TTT AGC Met Giy Phe Ser

TTC

Phe 385 AAG GAC ATT AAG Lye Asp Ile Lye AAA ACA Lys Thr 390 1384 ATG GAA GAA Met Glu Giu GTC CTG ATT Val Leu Ile 410

AAA

Lys 395 ATO CAA ACA TCC Ile Gin Thr Ser

GGG

Gly 400 AGC AGC TAT OTA Ser Ser Tyr Leu TCA OTT GAG Ser Leu Glu 405 ACO GAG GAT Thr Giu Asp 1432 GOA GAT CTT GTG AGT GCT CAG AAA GAT Ala Asp Leu Val Ser Ala Gin Lye Asp 415

AAT

Aen 420 1480 WO 97/23501 WO 9723501PCT/AU96/00827 47 GAG TCA AGT CAA ACT Giu Ser Ser Gin Thr 425 CTA AGG CGC CTA CAA Leu Arg Arg Leu Gin TCA TTG CAG AAA GAC ATT AGT ACT GAA GAG CAG Ser Leu Gin Lys Asp Ile Ser Thr Giu Giu Gin 430 435 GAG GAG AAG CTT TGC AAA ATC TGT ATG GAT AGA Glu Glu Lys Leu Cys Lys Ile Cys Met Asp Arg 445 450 455 TTT GTT CCT TGT GGA CAT CTG GTC ACT TGT AAA Phe Val Pro Cys Gly His Leu Vai Thr Cys Lys 440

AAT

Asn 1528 1576 1624 1672 ATT GCT ATC Ile Ala Ile 465 4 TC ATT al Ile CAG TGT GCA Gin Cys Ala GTT GAC AAA Vai Asp Lys

TGT

Cys 480

TCT

Ser CCC ATG TGC TAC ACC G Pro Met Cys Tyr Thr V 485 TAGTGGGGCA CCACATGTTA ACG TTC AAG CAA AAA ATT TTT ATG Thr Phe Lys Gin Lys Ile Phe Met 490 495 TGTTCTTCTT GCTCTAATTG AATGTGTGAT TTCCATTAGC ATCTGCTGTT TCCAAATGGA AACATTCAAT TTCTGGATCT TTCGAGTTAT GATTGAAACC TTAGACAGAG AAGCATTTTA TGACTTGATT TCTATATGTA AGTGAATTC 1719

GGGAGCGAAC

GACCAATGCT

CAGCTGTATC

TAGCTTTTCA

TTTAAGTAAT CTGCATTGCA AACAGCACTG TTTCCGTCTA ATTTAGCCAG TGTTTTACTC CATGTATATT GGTAGTACAC 1779 1839 1899 1959 1988 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 496 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Thr Phe Asn Ser Phe Glu Gly Thr Arg Thr Phe Val Leu Ala Asp 1 5 10 is WO 97/23501 WO 9723501PCT/AU96/00827 48 Thr Asn Lys Phe Ala Asn Arg Ala Gly Glu Giu Phe Val Giu Phe Asn Arg Leu Lys Thr Pro Ser Ser Ser 40 Gly Val Ser Ala Ser Val Thr Leu Ala Gin Cys Phe Phe Leu Tyr so Ser Cys Thr Asp Glu Gly Asp His Ala Ala Gly Ile Ile Arg Trp Gin Asp Ser Ala Arg His Arg Arg Gly Ser Pro Asn Phe Ile Asn Gly Phe Tyr Phe Giu Giy Gin Tyr 115 Pro Asp Arg Ala Ala Gin Asn Pro Gly Ser Giu Asn Cys 120 His Gly Asn Arg Asn 125 Leu Ile Gin Asn 110 Pro Phe Ala Arg Thr Gly Pro Pro Giu 130 Gin Val Thr 135 Asp Ala Asp Tyr Val Asp Ile 145 Cys Ser 150 Arg Thr Ile Tyr Asn Pro Ala Ser Giu Giu Ala 165 Pro Leu Lys Ser Asn Trp Pro Asp Tyr 175 Ala His Leu Gly Ala Asp 195 Asn Trp Giu Arg Giu Leu Ala Gly Leu Gin Val Gin Cys Cys Gly Gly 205 Arg Tyr Tyr Thr 190 Lys Leu Giu Arg His Phe Pro Cys Asp Trp Ser Glu 210 Pro Asn Cys Phe Phe 225 Ser Val 230 Asp Gly Arg Asn Val Arg Ser Gly Val Ser Ser 245 Arg Asn Phe Pro 250 Ser Thr Asn Arg Asn Pro Ala Met Ala Giu Tyr Giu Ala Arg Ile Val Thr Phe Gly WO 97/23501 WO 9723501PCT/AU96/00827 -49 265 Thr Trp Thr 275 Ser Ser Val Asn Glu Gin Leu Ala Arg 285 Ala Gly Phe Tyr Ala 290 Leu Gly Giu Gly Asp 295 Lys Val Lys Cys His Cys Gly Gly Leu Thr Asp Trp Lys 310 Pro Ser Giu Asp Pro 315 Trp Glu Gin His Al a 320 Lys Trp Tyr Pro Gly 325 Cys Lys Tyr Leu Leu 330 Asp Giu Lys Gly Gin Glu 335 Tyr Ile Asn Arg Thr Ala 355 Asn 340 Ile His Leu Thr His 345 Ser Leu Glu Giu Ser Leu Gly 350 Asp Asp Thr Giu Lys Thr Pro Leu Thr Lys Lys Ile Phe 370 Gin Asn Pro Met Val 375 Gin Giu Aia Ile Met Gly Phe Ser Lys Asp Ile Lys Lys 390 Thr Met Giu Giu Lys 395 Ile Gin Thr Ser Ser Ser Tyr Leu Ser 405 Leu Giu Val Leu Ile 410 Ala Asp Leu Val Ser Ala 415 Gin Lys Asp Asp Ile Ser 435 Thr Giu Asp Giu Ser 425 Ser Gin Thr Ser Leu Gin Lys 430 Giu Lys Leu Thr Giu Glu Gin Leu 440 Arg Arg Leu Gin Cys Lys 450 Ile Cys Met Asp Arg 455 Asn Ile Ala Ile Phe Val Pro Cys Gly 465 His Leu Val Thr Lys Gin Cys Ala Giu Ala 475 Val Asp Lys Pro Met Cys Tyr Thr Val 485 Ile Thr Phe Lys Gin Lys Ile Phe Met Ser WO 97/23501 WO 9723501PCT/AU96/00827 50 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 3532 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1160. .3016 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GAATTCTATG

AGAAAGAAGG

GTACAATATT

AAATTGATTA

ACAGGTTATT

CTAAGTTTAT

GAAGAAAGTC

ATAGAGTTTA

GCACAGGTTC

TTTAGCCCAC

GTGACCATAT

TGTCATGTTA

AATCAGTAAT

ATAATTTAGG

GAGTGTAATT

CTAGTAGAGT

TTGTTAGGCG

ATGTTTACAA

TAGTTTTTAA

GGAGAAAATA

AACATGATGC

CAGTTTTTGA

AACAAAGCTT

TTAAGTAAAT

GAAGGTTTTA

AAGTGCTTAT

TGGTCTGTTA

TATTCTATAG

TTGTGTATGA ATTATATTTT

TGATTACTGA

TTTCTGATAA

CATGACTGAT

ATGCAGTGTA

CCTTCAGTTG

TGCAGGAAAT

ACTTTAAGCC

GTGGGTATTG

ACTATGATGA

ATTACTTTTG

AGGGAAAGAA

TTCAGGCTTC

TTGCTTAGAA

TACTTTATGC

CACTAGAAAG

AATTATAGCT

AAAAGTGTGC

ATCAAGAATA

GGAAACAAAT

AAATTCATTT

ACTTCCCCCA

TAAGCTGTGT

TTTATTGGAA

GCCTGCATAT

ATAGCTTGTA

TTTTGTTAAT

TAAAACATTG

TAAGCAGTAC

GACAAGTTTT

GAATAGTCCT

TGTGGAAATT

ATAGTGGTAT

ACAAATGATA

GACATCAAGC

AAAGTTGTCA

GAACTTAGCT

TAAAATGAGA

AATTTTTTAC

ACCAAATATA

ATAAATCTCT

AAGAGTTTTC

TTTTTTGGTA

ATCTTGTGAT

TAAATGATGA

TTATGGCTAA

ACAAAGTTAG

TTTAACAAAG

ACTATAGCAG

GCTGAAGTAA

TTTAAATAGT

TTTTTTGGGT

CTTGTGGCAT

AATAAAAGGC

GTGAAAAATC

120 180 240 300 360 420 480 540 600 660 720 780 840 I WO 97/23501 WO 9723501PCT/AU96/00827 51 AAGGAGTTTT AATATTTTCA GAAGTGCATC CACCTTTCAG GGCTTTAAGT TAGTATTACT CAAGATTATG AACAAATAGC ACTTAGGTTA CCTGAAAGAG TTACTACAAC CCCAAAGAGT TGTGTTCTAA GTAGTATCTT GGTAATTCAG AGAGATACTC ATCCTACCTG AATATAAACT GAGATAAATC CAGTAAAGAA AGTGTAGTAA ATTCTACATA AGAGTCTATC ATTGATTTCT TTTTGTGGTA AAAATCTTAG TTCATGTGAA GAAATTTCAT GTGAATGTTT TAGCTATCAA ACAGTACTGT CACCTACTC ATG CAC AAA ACT GCC TCC CAA AGA CTT TTC CCA Met His Lys Thr Ala Ser Gin Arg Leu Phe Pro 900 960 1020 1080 1140 1192 GGT CCC TCG TAT Gly Pro Ser Tyr TTG TCA GAT TGG Leu Ser Asp Trp CAA AAC ATT AAG AGT ATA ATG GAA GAT Gin Asn Ile Lys Ser Ile Met Giu Asp 20 ACA AAC AGC AAC AAA CAA AAA ATG AAG Thr Asn Ser Asn Lys Gin Lys Met Lys 35 AGC ACG ATC Ser Thr Ile TAT GAC TTT Tyr Asp Phe 1240 1288 TCC TGT Ser Cys GAA CTC TAC AGA ATG TCT ACA TAT TCA Glu Leu Tyr Arg Met Ser Thr Tyr Ser so

ACT

Thr TTC CCC GCC GGG Phe Pro Ala Gly 1336

GTG

Val CCT GTC TCA GAA Pro Val Ser Glu

AGG

Arg AGT CTT GCT CGT Ser Leu Ala Arg GGT TTT TAT TAT Gly Phe Tyr Tyr 1384 1432 GGT GTG AAT GAC Gly Val Asn Asp GTC AAA TGC TTC Val Lys Cys Phe TGT GGC CTG ATG Cys Gly Leu Met CTG GAT Leu Asp AAC TGG AAA Asn Trp Lys CCT AGC TGT Pro Ser Cys 110 GGA GAC AGT CCT Gly Asp Ser Pro CAA AAG CAT AAA Gin Lys His Lys CAG CTA TAT Gin Leu Tyr 105 CTG GGA TCC Leu Giy Ser 1480 1528 AGC TTT ATT CAG Ser Phe Ile Gin CTG GTT TCA GCT Leu Val Ser Ala ACC TCT Thr Ser 125 AAG AAT ACG TOT Lys Asn Thr Ser ATG AGA AAC AGT Met Arg Asn Ser GCA CAT TCA TTA Ala His Ser Leu 1576 WO 97/23501 PCT/AU96/00827 -52- TCT CCC ACC TTG GAA CAT AGT AGC TTG TTC AGT GGT TCT TAC TCC AGC 1624 Ser Pro 140 Thr Leu Glu Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser CTT TCT CCA AAC Leu Ser Pro Asn CTT AAT TCT AGA Leu Asn Ser Arg GCA GTT Ala Val 165 GAA GAC ATC Glu Asp Ile TCT TCA Ser Ser 170 1672 TCG AGG ACT Ser Arg Thr TTT CTT ACC Phe Leu Thr 190

AAC

Asn 175 CCC TAC AGT TAT Pro Tyr Ser Tyr

GCA

Ala 180 ATG AGT ACT GAA Met Ser Thr Glu GAA GCC AGA Glu Ala Arg 185 CCA TCA GAA Pro Ser Glu 1720 1768 TAC CAT ATG TGG Tyr His Met Trp

CCA

Pro 195 TTA ACT TTT TTG Leu Thr Phe Leu TTG GCA Leu Ala 205 AGA OCT GOT TTT Arg Ala Gly Phe TAT ATA OGA CCT Tyr Ile Gly Pro GGA OAT AGO GTA GCC Gly Asp Arg Val Ala 215

TGC

Cys 220 TTT GCC TOT GGT Phe Ala Cys Gly

GGG

Gly 225 AAO CTC ACT AAC Lys Leu Ser Asn

TGG

Trp 230 GAA CCA AAG GAT Glu Pro Lys Asp 1816 1864 1912 OCT ATO TCA GAA Ala Met Ser Olu CGG AGO CAT TTT Arg Arg His Phe AAC TOT CCA TTT Asn Cys Pro Phe TTO GAA Leu Glu 250 AAT TCT CTA OAA ACT CTO AGO TTT Asn Ser Leu Glu Thr Leu Arg Phe 255 ATT TCA AAT CTO Ile Ser Asn Leu AGC ATO CAG Ser Met Gin 265 TCT AGT OTT Ser Ser Val 1960 ACA CAT OCA Thr His Ala 270 OCT CGA ATO AGA Ala Arg Met Arg

ACA

Thr 275 TTT ATO TAC TGG Phe Met Tyr Trp

CCA

Pro 280 2008 CCA OTT Pro Val 285 CAG CCT GAO CAG Gin Pro Giu Gin

CTT

Leu 290 OCA AGT OCT GGT Ala Ser Ala Oly TAT TAT GTG GGT Tyr Tyr Val Gly 2056 2104

CGC

Arg 300 AAT OAT OAT GTC Asn Asp Asp Val

AAA

Lye 305 TGC TTT TOT TGT Cys Phe Cys Cys GGT GGC TTG AGO Oly Oly Leu Arg

TGT

Cys 315 TGG GAA TCT GGA OAT OAT Trp Olu Ser Gly Asp Asp CCA TOG OTA OAA CAT GCC AAC TGG TTT CCA Pro Trp Val Olu His Ala Lys Trp Phe Pro 2152 IIWO 97/23501 PCT/AU96/00827 53 AGG TGT GAG TTC TTG ATA CGA ATG AAA Arg Cys Giu Phe Leu Ile Arg Met Lys 335 340 ATT CAA GGT AGA TAT CCT CAT CTT CTT Ile Gin Gly Arg Tyr Pro His Leu Leu 350 355 GGC CAA GAG TTT GTT GAT GAG 2200 Giy Gin Glu Phe Val Asp Glu 345 TCA ACT TCA Ser Thr Ser GAA CAG CTG Giu Gin Leu

TTG

Leu 360 GAT ACC Asp Thr 365 ACT GGA GAA GAA Thr Gly Giu Glu GCT GAC CCA CCA Ala Asp Pro Pro

ATT

Ile 375 ATT CAT TTT GGA Ile His Phe Gly 2248 2296 2344 2392

CCT

Pro 380 GGA GAA AGT TCT Gly Giu Ser Ser GAA GAT GCT GTC Giu Asp Ala Val ATG AAT ACA CCT Met Asn Thr Pro GTT AAA TCT GCC Vai Lys Ser Ala

TTG

Leu 400 GAA ATG GGC TTT Giu Met Gly Phe

AAT

Asn 405 AGA GAC CTG GTG Arg Asp Leu Val AAA CAA Lys Gin 410 ACA GTT CAA Thr Val Gin

AGT

Ser 415 AAA ATC CTG ACA Lys Ile Leu Thr ACT GGA Thr Gly 420 GAG AAC TAT Giu Asn Tyr AAA ACA GTT Lys Thr Val 425 2440 AAT GAT Asn Asp GAG GAG Giu Giu 445

ATT

Ile 430 GTG TCA GCA CTT Val Ser Ala Leu AAT GCT GAA GAT Asn Ala Giu Asp GAA AAA AGA GAA Giu Lys Arg Glu 440 2488 AAG GAA AAA CAA Lys Glu Lys Gin

GCT

Al a 450 GAA GAA ATG GCA TCA GAT GAT TTG TCA Glu Glu Met Ala Ser Asp Asp Leu Ser 455 2536

TTA

Leu 460 ATT CGG AAG AAC Ile Arg Lys Asn

AGA

Arg 465 ATG GCT CTC TTT Met Ala Leu Phe

CAA

Gin 470 CAA TTG ACA TGT Gin Leu Thr Cys 2584 2632 CTT CCT ATC CTG Leu Pro Ile Leu GAT AAT CTT TTA Asp Asn Leu Leu 480 AAG GCC Lys Ala 485 AAT GTA ATT AAT Asn Val Ile Asn AAA CAG Lys Gin 490 GCG AGA Ala Arg GAA CAT GAT ATT ATT AAA CAA AAA Glu His Asp Ile Ile Lys Gin Lys 495 ACA CAG ATA Thr Gin Ile 500 CCT TTA CAA Pro Leu Gin 505 2680 WO 97/23501 WO 9723501PCT/AU96/00827 54 GAA CTG ATT Giu Leu Ile 510 TTC AAA AAC Phe Lys Asn 525 TTT GTG GAT Phe Vai Asp GAT ACC ATT TTG Asp Thr Ile Leu

GTT

Val 515

ATT

Ile 540

CTG

Leu TGT CTA AAA Cys Leu Lys AAO AAT ATG Lys Asn Met 545 GAA GAA CAA Giu Giu Gin 560 ATO GAC AAA Met Asp Lys

GAA

Olu 530 AAG TAT Lys Tyr AAA OGA AAT GCT Lys Gly Asn Ala GAC TCT ACA TTG Asp Ser Thr Leu 535 ATT CCA ACA GAA Ile Pro Thr Giu GCG 0CC AAC ATC Ala Ala Asn Ile 520 TAT AAO AAC TTA Tyr Lys Asn Leu GAT GTT TCA Asp Val Ser 2728 2776 2824 2872 2920 550

CAA

Gin TCA CTG Ser Leu AAA GTG TOT Lys Val Cys CAT CTG GTA H-is Leu Val TTO AGO AGO Leu Arg Arg GAA OTT TCT Giu Val Ser 580

TTG

Leu 565

OTT

Val OAA GAA COA Oiu Giu Arg ACT TOT Thr eye 570 TOT GOT Cys Oly OTA TTT ATT Val Phe Ile 575

OTA

Val

CCT

Pro 585 TOC CAG Cys Gin GAA TOT G Oiu Cys A ATT TOC AGO GOT ATA ATC AAO GOT A( Ile Cys Arg Gly Ile Ile Lys Gly T 605 610 TAOTCTATAT TTTAACCTGC ATAAAAAGGT TCTAAAOTAA AAAGGGAATT ATGAOTTTTT TTGGTACTAA TAATCTTGTT TCTGAAAAGA ATTTACAAGG GAAOATTTAT GTTTGGTGAA GTAGTOTCAC TGCTTOTTAT GCATCATTTC AAGCTTTGAA TACTAAATTA TAGTGTAGAA ATCAGAOTTA TGGTGCCGAA TTOTCTTTGG TTTTCTCTTA. TTTCTCCCCC TAOTTTGTGA AAAAAAAAAO TCOACGCGOC CGCGAATTC CC CCT TCT CTA AGA AAA TOC CCT 2968 ia Pro Ser Leu Arg Lys Cys Pro 600 CT OTT COT ACA TTT CTC TCT TAAAOAAAAA 3023 hr Val Arg Thr Phe Leu Ser 615 CTTTAAAATA TTGTTGAACA CTTGAAGCCA 3083 CAATTAGTAA CATTCATGTT CTAGTCTGCT 3i43 TOGTATCATA TATTTAATCT TAATCTGTTT 3203 CTATATTAGT ATOTATOTOT ACCTAAOOGA 3263 AOOAGTTACT OGATTTGTTG TTCTTTCAOA 3323 AAGAACTOGA AACCAOOAAC TCTOOAOTTC 3383 TOCTTTTCAC TTOTOTTTTA AAATAAOGAT 3443 GAAACATCTC AATAAAGTOC TTTCCAAAAA 3503 3532 WO 97/23501 WO 9723501PCT/AU96/00827 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 618 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met 1 Asn His Lys Thr Ile Lys Ser Ala 5 I le Ser Gin Arg Leu Met Glu Asp Ser 25 Tyr Phe Pro Gly 10 Thr Ile Leu Asp Phe Ser Pro Ser Tyr Gin Ser Asn Ser Asn Arg Met Ser Lys Gin Lys Met Asp Trp Thr Glu Leu Tyr Vai Ser Giu Thr Tyr Ser Arq Ser Leu Ala Arg Thr Gly Gly Pro Ala Giy Phe Tyr Tyr Leu Met Leu Val Asn Asp Lys Cys Phe Asp Asn Trp Lys Leu Gly Asp Ser Pro Ile Gin Asn 115 Ser Pro Met 130 Gin Lys His Lys Leu Tyr Pro Ser Leu Val Ser Ala Leu Giy Ser Thr Ser 125 Pro Cys Ser Phe 110 Lys Asn Thr Thr Leu Giu Arg Asn Ser Ala His Ser Leu Ser 140 His 145 Ser Ser Leu Phe Ser 150 Val Ser Tyr Ser Ser 155 Leu Ser Pro Asn Leu Asn Ser Arg Al a 165 Giu Asp Ile Ser 170 Ser Ser Arg Thr Asn 175 WO 97/23501 PCT/AU96/00827 56 Tyr Ser Tyr Ala Met Ser Thr Glu Giu Ala Arq Phe Leu Thr Tyr His Met Trp Pro 195 Leu Thr Phe Leu Ser 200 Pro Ser Glu Leu Al a 205 Arg Ala Gly Phe Tyr 210 Tyr Ile Gly Pro Asp Arg Val Ala Phe Ala Cys Gly Gly 225 Lys Leu Ser Asn Giu Pro Lys Asp Ala Met Ser Glu Arg Arg His Phe Pro 245 Asn Cys Pro Phe Giu Asn Ser Leu Glu Thr 255 Leu Arg Phe Met Arg Thr 275 Ser 260 Ile Ser Asn Leu Ser 265 Met Gin Thr His Ala Ala Arg 270 Gin Pro Giu Phe Met Tyr Trp Ser Ser Val Pro Gin Leu 290 Ala Ser Ala Gly Phe 295 Tyr Tyr Val Gly Arg 300 Asn Asp Asp Val Lys 305 Cys Phe Cys Cys Asp 310 Giy Gly Leu Arg Trp Glu Ser Gly Asp Pro Trp Val His Ala Lys Trp Pro Arg Cys Giu Phe Leu 335 Ile Arg Met Pro His Leu 355 Lys 340 Gly Gin Glu Phe Asp Glu Ile Gin Gly Arg Tyr 350 Thr Gly Glu Leu Glu Gin Leu Leu Ser Thr Ser Asp 360 Thr 365 Glu Asn 370 Ala Asp Pro Pro Ile 375 Ile His Phe Gly Pro 380 Gly Giu Ser Ser Ser 385 Glu Asp Ala Val Met Met Asn Thr Pro 390 Val Val 395 Lys Ser Ala Glu Met Gly Phe Asn Arg Asp Leu 405 Val Lys Gin Thr Val Gin Ser Lys Ile Leu Thr Thr Gly Giu Asn Tyr Lys Thr Val Asn Asp Ile Val Ser WO 97/23501 PCT/AU96/00827 57 430 Ala Leu Leu Asn Ala Giu Asp 435 Giu 440 Lys Arg Giu Giu Giu Lys Giu Lys 445 Gin Ala 450 Giu Glu Met Ala Ser 455 Asp Asp Leu Ser Ile Arg Lys Asn Met Ala Leu Phe Gin Leu Thr Cys Val 475 Leu Pro Ile Leu Asp 480 Asn Leu Leu Lys Ala Asn Vai Ile Asn 485 Lys 490 Gin Giu His Asp Ile Ile 495 Lys Gin Lys Ile Leu Val 515 Thr 500 Gin Ile Pro Leu Gin 505 Ala Arg Giu Leti Ile Asp Thr 510 Aen Cys Leu Lye Gly Asn Ala Ala Aen Ile Phe Lye 525 Lye Giu 530 Ile Asp Ser Thr Tyr Lye Aen Leti Val Asp Lye Aen Met 545 Lye Tyr Ile Pro Thr 550 Giu Asp Val Ser Leti Ser Leu Giu Gin Leu Arg Arg Gin Giti Giti Arg Cys Lye Val Cye Met Asp 575 Lye Giti Val Gin Giti Cys 595 Val Vai Phe Ile Cys Gly His Leu Vai Val Cys 590 Arg Gly Ile Ala Pro Ser Leti Arg 600 Lye Cys Pro Ile Ile Lye 610 Gly Thr Vai Arg Thr 615 Phe Leti Ser INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 3076 base pairs TYPE: nuclic acid CC) STRANDEDNESS: single I WO 97/23501 WO 9723501PCT/AU96/00827 -58- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 725. .2539 (xi) SEQUENCE DESCRIPTION: SEQ ID GAATTCAAAA TGTCTTCAGT TGTAAATCTT ACCATTATTT

AAGTGCTTCT

TTTAAAATAG

TTAGGTCTTG

AACATATTCT

GAAGCTTCAT

GTAGCCTGGA.

CTTAATACAC

ATCATGTTTA

AAAATGTATC

AAATAATAAA

AATTAAAATA

CCATCTTAGA

TGCTTTTTTT

GAATATTTTT

GAGTCACACA

GAAGTTGACC

ATCACTCTTC

CATTGTATGT

AGTATAGGAT

AAATTTTTCA

TGATGTCATT

ATCAGTGAAA

TCCTGGCCAC

GCTGTGAAAC!

TTACATCTTT

TACCTGTGGA

TGTGAAGGGT

ATAAAGATTA

TTAGAATCTC

TTTTGGCTTT

AATTATGAAA

TATGGTAATG

TAAATTTCAC

ACTTGACAGC

GGGTTGATTG

GATGCCTGCC

TTTAATTTTC

TACAAAGGTG

CATGTTGAAA

TCAGCCTAGT

TACGTACCTC

TACTTCTTGA

TATTATTTTC

AATTTCCAAA

AGAGCTTTCC

AATGCCACTG

ATTAAATGGC

AACACAGCTT

CAATTGTGTA

CTCTAAATGC

ATTAAAACTG

TAAGAAATAA

TAACAGAAGT

CTCCTTTGAG

AAGCAAAATA

ACCATGAAAA

AAACATTCTA

ATCCTGATGG

ACTCTGTAGC

TTTCTTCCTT

ATAGAAATAA

ATAAAAGCAA

120 180 240 300 360 420 480 540 600 660 720 769 AGCCATGCAC AAAACTACCT CCCTAGAGAA AGGCTAGTCC CTTTTCTTCC CCATTCATTT CATT ATG AAC ATA OTA GAA AAC AGC ATA TTC TTA TCA AAT TTG ATG AAA Met Asn Ile Val Glu Asn Ser Ile Phe Leu Ser Asn Leu Met Lys 1 5 10 AGC GCC AAC ACG TTT GAA CTG AAA TAC GAC TTG TCA TGT GAA CTG TAC Ser Ala Asn Thr Phe Glu Leu Lys Tyr Asp Leu Ser Cys Glu Leu Tyr 25 CGA ATG TCT ACG TAT TCC ACT TTT CCT OCT G OTT CCT GTC TCA GAA Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu 40 817 865 WO 97/23501 WO 9723501PCT/AU96/00827 59 AGG AGT CTT GCT CGT GCT GGT Arg Ser Leu Ala Arg Ala Cly TAT TAC ACT GGT GTG AAT GAC AAG, Tyr Tyr Thr Gly Val Asn Asp Lys GTC AAA Val Lys TGC TTC TGT TGT Cys Phe Cys Cys

GGC

Gly CTG ATG CTG CAT Leu Met Leu Asp TGG AAA AGA GCA Trp Lys Arg Gly

CAC

Asp ACT CCT ACT CAA Ser Pro Thr Glu CAT AAA AAG TTG His Lys Lys Leu

TAT

Tyr CCT AGC TCC AGA Pro Ser Cys Arg GTT CAG ACT CTA Val Gin Ser Leu

AAT

Asn 100 TCC GTT AAC AAC Ser Val Asn Asn GAA GCT ACC TCT Glu Ala Thr Ser CAG CCT Gin Pro 110 CCG GGT Pro Gly 961 1009 1057 1105 1153 ACT TTT CCT Thr Phe Pro ACA GAA AAC Thr Glu Asn 130

TCT

Ser 115 TCA GTA ACA AAT Ser Val Thr Asn ACA CAC TCA TTA Thr His Ser Leu ACT GGA TAT TTC Ser Gly Tyr Phe CGT GGC TCT TAT TCA AAC TCT CCA TCA Arg Cly Ser Tyr Ser Asn Ser Pro Ser 135 140 AAT CAA GAT TTT TCT GCC TTG ATG AGA Asn Gin Asp Phe Ser Ala Leu Met Arg 155 AAT CCT Asn Pro 145 GTA AAC TCC AGA Val Asn Ser Arg 1201 TCC TAC CAC TGT Ser Tyr His Cys ATG AAT AAC GAA Met Asn Asn Glu GCC AGA TTA CTT Ala Arg Leu Leu

ACT

Thr 175 1249 TTT CAG ACA TG Phe Gin Thr Trp TTC ACT TTT CTC TCC CCA ACA CAT CTG Leu Thr Phe Leu Ser Pro Thr Asp Leu 185 GCA AAA Ala Lys 190 1297 CCA CCC TTT Ala Cly Phe TCT COT CCA Cys Cly Gly 210 TAC ATA GCA CCT Tyr Ile Cly Pro

CCA

Cly 200 GAC ACA CTC OCT Asp Arg Val Ala TOC TTT CC Cys Phe Ala 205 CCT ATG TCA Ala Met Ser 1345 1393 AAA TTG AGC AAT Lys Leu Ser Asn

TG

Trp 215 GAA CCC AAG GAT Clu Pro Lys Asp

AAT

Asn 220 CAA CAC CTC ACA CAT TTT CCC AAA TCC CCA TTT ATA CAA AAT CAC CTT Clu His Leu Arg His Phe Pro Lys Cys Pro Phe Ile Clu Asn Gin Leu 1441 WO 97/23501 WO 9723501PCT/AU96/00827 60 235

CAA

Gin 240 GAC ACT TCA AGA Asp Thr Ser Arg

TAC

Tyr 245 ACA GTT TCT AAT Thr Val Ser Asn AGC ATG CAG ACA Ser Met Gin Thr

CAT

His 255 1489 1537 GCA GCC CGC TTT Ala Ala Arg Phe

AAA

Lys 260 ACA TTC TTT AAC Thr Phe Phe Asn CCC TCT AGT GTT Pro Ser Ser Val CTA GTT Leu Val 270 AAT CCT GAG Asn Pro Giu GAT GAT GTC Asp Asp Val 290 CTT GCA AGT C Leu Ala Ser Ala TTT TAT TAT GTC Phe Tyr Tyr Val GGT AAC ACT Gly Asn Ser 285 1585 AAA TGC TTT TOC Lys Cys Phe Cys

TGT

Cys 295 CAT GGT GGA CTC AGG TCT TGG GAA Asp Gly Gly Leu Arg Cys Trp Glu 300 1633 TCT GGA GAT GAT CCA TGG GTT Ser Gly Asp Asp Pro Trp Val 305 310 CAA CAT GCC AAG Gin His Ala Lys TTT CCA AGG TGT Phe Pro Arg Cys TAC TTG ATA AGA Tyr Leu Ile Arg

ATT

Ile 325 AAA GGA CAG GAG Lys Gly Gin Ciu ATC CGT CAA GTT Ile Arg Gin Val

CAA

Gin 335 1681 1729 1777 1825 GCC ACT TAC CCT Ala Ser Tyr Pro CTA CTT CAA CAG Leu Leu Glu Gin CTA TCC ACA TCA Leu Ser Thr Ser GAC AGC Asp Ser 350 CCT GGA Pro Gly CCA GGA CAT Pro Cly Asp GAA GAC CAT Glu Asp His 370 AAT GCA GAG TCA Asn Ala Giu Ser

TCA

Ser 360 ATT ATC CAT TTT Ile Ile His Phe

CAA

Giu 365 TCA GAA CAT GCA Ser Clu Asp Ala

ATC

Ile 375 ATG ATC AAT ACT Met Met Asn Thr

CCT

Pro 380 GTC ATT AAT Val Ile Asn CAG ACA GTT Gin Thr Val 1873 GCT CCC GTG GAA ATG GCC Ala Ala Val Giu Met Gly 385

TTT

Phe 390 ACT AGA AGC CTG Ser Arg Ser Leu GTA AAA Val Lys 395 1921

CAA

Gin 400 AGA AAA ATC CTA CCA ACT GGA GAG AAT TAT AGA CTA CTC AAT Arg Lys Ile Leu Ala Thr Gly Giu Asn Tyr Arg Leu Val Asn

CAT

Asp 415 1969 WO 97/23501 WO 9723501PCT/AU96/00827 61 CTT GTG TTA GAC TTA CTC AAT GCA Leu Val Leu Asp Leu Leu Asn Ala 420 GAA GAT Giu Asp 425 GAA ATA AGG GAA Giu Ile Arg Giu GAG GAG Giu Giu 430 2017 2065 AGA GAA AGA GCA ACT GAG GAA AAA GAA TCA AAT GAT TTA Arg Giu Arg Ala Thr Giu Giu Lys Giu Ser Asn Asp Leu TTA TTA ATC Leu Leu Ile 445 GTA ATT CCA Val Ile Pro CGG AAG AAT Arg Lys Asn 450 AGA ATG GCA CTT Arg Met Ala Leu CAA CAT TTG ACT Gin His Leu Thr 2113 ATC CTG Ile Leu 465 GAT AGT OTA CTA Asp Ser Leu Leu

ACT

Thr 470 GCC GGA ATT ATT AAT GAA CAA GAA CAT Ala Gly Ile Ile Asn Giu Gin Giu His 475 2161

GAT

Asp 480 GTT ATT AAA CAG AAG ACA CAG ACG TCT Val Ile Lys Gin Lys Thr Gin Thr Ser 485

TTA

Leu 490 CAA GCA AGA GAA Gin Ala Arg Giu 2209 ATT GAT ACG ATT Ile Asp Thr Ile

TTA

Leu 500 OTA AAA GGA AAT Val Lys Giy Asn

ATT

Ile 505 GCA GCC ACT GTA Ala Ala Thr Val TTC AGA Phe Arg 510 2257 AAC TCT CTG Asn Ser Leu CAA CAG GAC Gin Gin Asp 530

CAA

Gin 515 GAA GCT GAA GCT Giu Ala Giu Ala TTA TAT GAG CAT Leu Tyr Giu His TTA TTT GTG Leu Phe Val 525 GAT CTA CCA Asp Leu Pro 2305 ATA AAA TAT ATT CCC ACA GAA GAT GTT Ile Lys Tyr Ile Pro Thr Giu Asp Val 535

TCA

Ser 540 2353 GTG GAA Val Giu 545 GAA CAA TTG CGG Giu Gin Leu Arg

AGA

Arg 550 CTA CAA GAA GAA Leu Gin Giu Glu

AGA

Arg 555 ACA TGT AAA GTG Thr Cys Lys Vai

TGT

Cys 560 ATG GAC AAA GAA Met Asp Lys Giu TCC ATA GTG TTT Ser Ile Val Phe CCT TGT GGT CAT Pro Cys Giy Hi-s

CTA

Leu 575 2401 2449 2497 GTA GTA TGC Val Val Cys AAA GAT Lys Asp 580 TGT GCT CCT TCT Cys Ala Pro Ser AGA AAG TGT CCT Arg Lys Cys Pro ATT TGT Ile Cys 590 AGG AGT ACA ATC AAG GGT ACA GTT CGT ACA TTT CTT TCA TGAAGAAGAA Arg Ser Thr Ile Lys Gly Thr Val Arg Thr Phe Leu Ser 2546

I

WO 97/23501 PTA9/02 PCT/AU96/00827 62

CCAAAACATC

TCCTAATTTG

TAACATATTT

TGATAGGCTT

AAAATTTCAG

TTAAGAATTT

TTTGTACAGA

AAACACCAGG

CATATATGTT

595

ATCTAAACTT

GTTTCCTTAA

ATATATGTAT

TTGTTCTTAT

CATTATTGAA

TAAATATTTT

CAGGGCAGCA

GACACATTTC

GAATGACATT

TAGAATTAAT

AATTTTTATT

CTAAACCATA

GAACGAAAAA

ATTGTAAGTG

GGCATTGTAC

TACTGAGACC

TCTGTCTTTT

TTAGGGACAT

600

TTATTAAATG

TATTTACAAC

TGAACATATA

GAGGTAGCAC

AAGTAAAACT

TAATACCTGG

CTGCCTTTAA.

TTGATCAGTG

GGTGTTTTTA

TATTATAACT

TCAAAAAACA

TTTTTTAGAA

TACAAACACA

TAAGATATTT

TTTTTTTTTT

AAACAAACAG

TCCTATACAT

TAAAGAATTC

TTAACTTTTA

TTGTTTTGTG

ACTAAGAGAA

ATATTCAATC

GAGTTAACCT

GTTTTGTTTT

AACAAAAACA

CGAAGGTGTG

2606 2666 2726 2786 2846 2906 2966 3026 3076 INFORMATION FOR SEQ ID NO:6: i)SEQUENCE CHARACTERISTICS: LENGTH: 604 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met 1 Al a Asn Ile Val Glu 5 Asn Thr Phe Glu Met Ser Thr Tyr Ser Ser Leu Ala Arg Ala Asn Ser Ile Phe Leu Ser Asn 10 Leu Lys Tyr Asp Leu Ser Cys 25 Thr Phe Pro Ala Gly Val Pro 40 Gly Phe Tyr Tyr Thr Gly Val 55 Leu Met Lys Ser is Giu Leu Tyr Arg Val Ser Giu Arg Asn Asp Lys Val WO 97/23501 WO 9723501PCT/AU96/00827 63 Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Trp Lys Arg Gly Ser Pro Thr Glu His Lys Lys Leu Tyr Pro Ser Cys Arg Phe Val Gin Ser Leu Phe Pro Ser 115 Asn 100 Ser Val Asn Asn Leu 105 Giu Ala Thr Ser Gin Pro Thr 110 Pro Gly Thr Ser Val Thr Asn Ser 120 Thr His Ser Leu Giu Asn 130 Ser Gly Tyr Phe Gly Ser Tyr Ser Ser Pro Ser Asn Val Asn Ser Arg Asn Gin Asp Phe Ala Leu Met Arg Ser 160 Ser Tyr His Cys Met Asn Asn Giu Ala Arg Leu Leu Thr Phe 175 Gin Thr Trp Gly Phe Tyr 195 Pro 180 Leu Thr Phe Leu Ser Pro Thr Asp Leu 185 Ala Lys Ala 190 Phe Ala Cys Tyr Ile Gly Pro Gly 200 Asp Arg Val Ala Gly Gly 210 Lys Leu Ser Asn Giu Pro Lys Asp Ala Met Ser Glu Leu Arg His Phe Lys Cys Pro Phe Glu Asn Gin Leu Asp Thr Ser Arg Thr Val Ser Asn Ser Met Gin Thr His Ala 255 Ala Arg Phe Pro Glu Gin 275 Thr Phe Phe Asn Pro Ser Ser Val Leu Val Asn 270 Asn Ser Asp Leu Ala Ser Ala Gly 280 Phe Tyr Tyr Val Gly 285 Asp Vai 290 Lys Cys Phe Cys Cys 295 Asp Gly Giy Leu Arg 300 Cys Trp Glu Ser Gly Asp Asp Pro Trp Val Gin His Ala Lys Trp Phe Pro Arg Cys Glu WO 97/23501 PCT/AU96/00827 64 Tyr Leu Ile Arg 320 Gin Ala 335 Lys Gly Gin Giu Phe Ile Arg Gin Val 330 Ser Tyr Pro Giy Asp Giu 355 His 340 Leu Leu Giu Gin Leu Leu 345 Ser Thr Ser Asp Ser Pro 350 Pro Gly Giu Asn Aia Giu Ser Ser 360 Ile Ile His Phe Asp His 370 Ser Giu Asp Aia Met Met Asn Thr Vai Ile Asn Ala Val Giu Met Giy Phe Ser Arg Ser Leu 390 Vai 395 Lys Gin Thr Vai Gin 400 Arg Lys Ile Leu Ala 405 Thr Gly Giu Asn Tyr 410 Arg Leu Vai Asn Asp Leu 415 Val Leu Asp Giu Arg Aia 435 Leu Asn Ala Giu Giu Ile Arg Glu Giu Giu Arg 430 Thr Giu Giu Lys Giu 440 Ser Asn Asp Leu Leu Leu Ile Arg 445 Lys Asn 450 Arg Met Ala Leu Phe 455 Gin His Leu Thr Cys 460 Val Ile Pro Ile Leu 465 Asp Ser Leu Leu Thr 470 Ala Gly Ile Ile Asn 475 Giu Gin Giu His Vai Ile Lys Gin Thr Gin Thr Ser Gin Ala Arg Giu Leu Ile 495 Asp Thr Ile Ser Leu Gin 515 Val Lys Giy Asn Ala Ala Thr Val Phe Arg Asn 510 Phe Val Gin Giu Ala Glu Ala Val Leu Tyr Giu His 520 Leu 525 Gin Asp 530 Ile Lys Tyr Ile Gin Leu Arg Arg 550 Pro 535 Thr Giu Asp Vai Ser 540 Asp Leu Pro Vai Giu 545 Giu Leu Gin Giu Gilu Arg 555 Thr Cys Lys Val SWO 97/23501 PCT/AU96/00827 Met Asp Lys Glu Val Cys Lys Asp 580 Ser Thr Ile Lys 595 Ser Ile Val Phe Ile Pro Cys Gly His Leu Val 570 575 Cys Ala Pro Ser Leu 585 Gly Thr Val Arg Thr 600 Arg Lys Cys Pro Ile Cys Arg 590 Phe Leu Ser INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1552 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 6..1502 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCACG ATG ACG GAG CTG GGC ATG GAG CTG Met Thr Glu Leu Gly Met Glu Leu GAG AGC GTT CGC CTG GCG Glu Ser Val Arg Leu Ala ACA TTT GGG GAA TGG CCC CTG AAT GCC CCA GTT Thr Phe Gly Glu Trp Pro Leu Asn Ala Pro Val 20 GTC GCC AAT GGT TTC TTT GCC ACG GGA AAC TGG Val Ala Asn Gly Phe Phe Ala Thr Gly Asn Trp 40 TCC GCG GAG GAT Ser Ala Glu Asp CTG GAG GCC Leu Glu Ala GAG TGC Glu Cys CAT TTC TGC CAC GTG CGC ATC GAC CGC TGG GAA TAC GGC GAT CAA GTG His Phe Cys His Val Arg Ile Asp Arg Trp Glu Tyr Gly Asp Gin Val 55 GCG GAG CGC CAT CGC CGC TCC TCG CCC ATC TGC TCC ATG GTT CTG GCT Ala Glu Arg His Arg Arg Ser Ser Pro Ile Cys Ser Met Val Leu Ala 70 143 191 239 WO 97/23501 PCT/AU96/00827 -66- CCC AAT Pro Asn CAC TGC GGC AAT His Cys Gly Asn GTT CCC AGG AGC CAG GAG Val Pro Arg Ser Gin Glu 85 AGC GAC AAC GAG Ser Asp Aen Glu GGA AAC AGC GTA GTG GAC AGC CCG GAG TCC TGC TCT Asn Ser Val Val Asp Ser Pro 100 Glu Ser Cys 105 Ser TGT CCC GAT Cys Pro Asp TTG TTG GAG GCC AAT CGA TTG GTA ACT Leu Leu Glu Ala Asn Arg Leu Val Thr 115 AAG GAC TGG CCG Lys Asp Trp Pro AAT CCC Asn Pro 125 AAC ATC ACG Asn Ile Thr CGT CTG GAT Arg Leu Asp 145

CCG

Pro 130 CAG GCT CTG GCA Gin Ala Leu Ala

AAG

Lys 135 GCA GGT TTC TAC Ala Gly Phe Tyr TAC CTG AAC Tyr Leu Asn 140 ATT GCC AAG Ile Ala Lye CAC GTG AAG TGT His Val Lye Cys TGG TGC AAC GGA Trp Cys Asn Gly TGG GAG Trp Glu 160 AAG AAC GAC AAT Lye Asn Asp Asn

GCC

Ala 165 TTT GAA GAG CAC Phe Giu Glu His

AAG

Lye 170 CGC TTT TTT CCC Arg Phe Phe Pro

CAA

Gin 175 TGT CCT CGT GTG Cys Pro Arg Val ATG GGC CCC CTT Met Gly Pro Leu

ATA

Ile 185 GAG TTT GCC ACC Glu Phe Ala Thr AAG AAC CTG GAT Lye Asn Leu Asp

GAG

Glu 195 CTG GGC ATC CAG Leu Gly Ile Gin

CCC

Pro 200 ACC ACT CTG CCA Thr Thr Leu Pro CTG CGT Leu Arg 205 CCC AAG TAC Pro Lye Tyr CCC ATT TCC Pro Ile Ser 225

GCC

Ala 210 TGC GTG GAC GCC Cys Val Asp Ala

AGA

Arg 215 TTG AGA ACC TTC Leu Arg Thr Phe ACC GAT TGG Thr Asp Trp 220 AAT ATT CAG CCC Asn Ile Gin Pro

GCT

Ala 230 TCG GCT TTG GCG CAG GCT GGC CTA Ser Ala Leu Ala Gin Ala Giy Leu 235 TAT TAT CAG AAA ATA GGC Tyr Tyr Gin Lye Ile Gly 240

GAC

Asp 245 CAG GTG CGC TGT Gin Val Arg Cys

TTC

Phe 250 CAC TGC AAC ATT His Cys Asn Ile GGA TTG CGC Gly Leu Arg TCC TGG CAG AAG GAG Ser Trp Gin Lye Glu GAC GAG CCA TGG TTC GAA CAC GCC Asp Giu Pro Trp Phe Giu His Ala .WO 97/23501 PCTAU96/00827 -67- AAG TCG TCG CCA AAG Lys Trp Ser Pro Lys 275 TGT CAG TTC GTT Cys Gin Phe Val CTC GAC AAG GGT Leu Asp Lys Gly CCA GCC Pro Ala 285 TAT GTA AGC Tyr Val Ser ACA GCA ACT Thr Ala Thr 305

GAA

Glu 290 GTG CTC CC ACA Val Leu Ala Thr

ACA

Thr 295 GCT GCC AAC GCC Ala Ala Asn Ala AGT TCC CCG Ser Ser Pro 300 CTG ATG GAT Leu Met Asp CCT CCG GCT CCA Ala Pro Ala Pro

ACG

Thr 310 CTC CAA GCA GAC Leu Gin Ala Asp

GTA

Val 315 GAA GCT Glu Ala 320 CCG GCC AAG GAG Pro Ala Lys Clu CTA GCC TTG GGT Leu Ala Leu Gly CAT GCA GGA GTT Asp Gly Cly Val 1007

GTG

Val 335 CGC AAT GCC ATC Arg Asn Ala Ile CGC AAC CTC TTG ACT TCC GGC TCC CCT Arg Lys Leu Leu Ser Ser Gly Cys Ala 345

TTC

Phe 350 1055 TCT ACG TTC GAC Ser Thr Leu Asp

GAA

Glu 355 CTA TTG CAT CAC Leu Leu His Asp

ATC

Ile 360 TTT GAC CAT GCC Phe Asp Asp Ala GGC CCA Gly Ala 365 1103 GGA CCT CCA Cly Ala Ala ATT GAA CCA Ile Glu Pro 385

CTC

Leu 370 GAG CTG CCC GAG Glu Val Arg Glu

CCC

Pro 375 CCC GAG CCA AGT Pro Glu Pro Ser GCT CCT TTT Ala Pro Phe 380 GTG CCA ATA Vai Pro Ile 1151 1199 TGT CAG GCC ACC Cys Gin Ala Thr

ACC

Thr 390 AGC AAG GCG GCA Ser Lys Ala Ala

TCT

Ser 395 CCG GTG Pro Val 400 CCC CAT TCC ATC Ala Asp Ser Ile CCC AAA CCA GAG Ala Lys Pro Gin

CCA

Ala 410 GCT CAA GCA GTC Ala Glu Ala Val 1247

GCG

Ala 415 AAT ATA TCG AAA ATC ACA GAC CAA ATA Asn Ile Ser Lys Ile Thr Asp Glu Ile 420

CAA

Gin 425 AAG ATC TCG GTG Lys Met Ser Val 1295 ACC CCC AAC GGA Thr Pro Asn Gly

AAC

Asn 435 CTC TCG CTG GAG GAG CAA AAC CCC CAG Leu Ser Leu Glu Glu Giu Asn Arg Gin 440 CTG AAG Leu Lys 445 1343 WO 97/23501 PTA9/02 PCT/AU96/00827 68 GAT GCA CGC TTA TGC AAG GTA TGC Asp Ala Arg TTC CTG CCC Phe Leu Pro 465 Cys Lys Val Cys TTG GAC Leu Asp 455 GAG GAG GTT Glu Glu Val GGC GTA GTG Gly Val Val 460 GCC CCC ACT Ala Pro Ser 1391 1439 TGC GGC CAC TTG GCC Cys Gly His Leu Ala 470 ACC TGC AAT CAG Thr Cys Asn Gin

TGC

Cys 475 GTA CC Val Ala 480 AAT TGT CCC ATG Asn Cys Pro Met TGC CC Cys Arg 485 GCA GAC ATC Ala Asp Ile

AAG

Lys 490 CGA TTC GTG CC Cly Phe Val Arg 1487

ACG

Thr 495 TTC CTT TCC Phe Leu Ser TGAAGTGCAT ATCGACATCA TTTAGAGTTC ACCAGCTAGT 1539 TAGCAGTTAA GCA INFORMATION FOR SEQ ID NO:8: Wi SEQUENCE CHARACTERISTICS: LENGTH: 498 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Thr Ciu Leu Cly Met Glu Leu Glu Ser Val Arg Leu Ala 1552 Thr Phe is Gly GlU Trp Leu Asn Ala Pro Val Ser Ala Giu Asp Leu Val Ala Cys His Phe Asn Gly Phe Phe Ala Thr Gly Asn Trp Leu Giu Ala Cys His so Val Arg Ile Asp Arg Trp Clii Tyr Gly Asp Cln Val Ala Glu Arg His Arg Arg Ser Ser Pro Ile Cys Ser Met Val Leu Ala Pro Asn WO 97/23501 PCT/AU96/00827 69 His Cys Gly Ser Val Val Asn Val Pro Arg Ser Gin Glu Ser 90 Asp Ser Pro Giu Ser Cys Ser Cys 100 105 Asp Asn Glu Gly Asn Pro Asp Leu Leu Leu 110 Giu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Pro Asn Ile Asn Arg Leu Thr Pro 130 Asp His Gin Ala Leu Ala Lys Ala Gly Phe Tyr Tyr 140 Ile Val Lys Cys Trp Cys Asn Gly 145 Lys Ala Lys Trp Asn Asp Asn Glu Glu His Phe Phe Pro Gin Cys 175 Pro Arg Val Leu Asp Giu 195 Tyr Ala Cys Gin 180 Leu Gly Pro Leu Ile 185 Thr Phe Ala Thr Gly Ile Gin Thr Leu Pro Leu 205 Asp Gly Lys Asn 190 Arg Pro Lys Trp Pro Ile Val Asp Ala Arg Thr Phe 210 Ser Asn Ile Gin Pro 225 Gin Al a 230 Gin Ala Leu Ala Gin 235 His Gly Leu Tyr Lys Ile Gly Vai Arg Cys Cys Asn Ile Gly Leu 255 Arg Ser Trp Ser Pro Lys 275 Ser Giu Val Gin 260 Cys Giu Asp Giu Phe Glu His Gin Phe Val Leu 280 Al a Asp Lys Gly Pro 285 Ser Ala Lys Trp 270 Ala Tyr Val Pro Thr Ala Leu Ala Thr 290 Thr Ala Pro Ala Pro Thr Thr 295 Leu Ala Asn Ala Ser 300 Leu Gin Ala Asp Val Met Asp Giu Ala 320 Pro Ala Lys Giu Ala Leu Ala Leu Gly Ile Asp Gly Gly Val Val Arg WO 97/23501 PCT/AU96/00827 70 Asn Ala Ile Leu Asp Giu 355 Ala Leu Giu Gin 340 Leu Lys Leu Leu Ser 345 Phe 330 Ser Gly Cys Asp Asp Ala 335 Ala Phe Ser Thr 350 Gly Ala Gly Ala Leu His Asp 365 Pro Val Arg Giu 370 Pro Cys Pro 375 Ser Giu Pro Ser Phe Ile Glu Gin Ala Thr 385 Al a Thr 390 Ala Lys Ala Ala Ser 395 Ala Pro Ile pro Val 400 Asp Ser Ile Pro 405 Thr Lys Pro Gin Al a 410 Lys Giu Ala Val Ala Asn 415 Ile Ser Lys Asn Gly Asn 435 Arg Leu Cys Asp Giu Ile Gin 425 Glu Met Ser Val Ser Leu Giu Glu 440 Asp Asn Arg Gin Ala Thr Pro 430 Lys Asp Ala Val Phe Leu Lys Val Cys Giu Glu Val 450 Pro Cys Gly 460 Gly His Leu 465 Asn Al a 470 Cys Asn Gin Cys Ala Pro Ser Val 475 Asp Ile Lys Gly Phe Val Arg Thr 490 495 Cys Pro Met Cys 485 Arg Al a Leu Ser WO 97/23501 PCT/AU96/00827 -71- INFORMATION FOR SEQ ID NO:9 SEQUENCE CHARACTERISTICS: LENGTH: 55 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro Xaam Xaa, Ala Xaa Ala Gly Phe Xaa o Asp Xaa Xaa, Xaa Cys Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys Xaa Xaaq Xaar INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 500 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 48..467 (xi) SEQUENCE DESCRIPTION: SEQ ID TGAATCCGCG TTTGAGTCGT CTTGGCGGAG GTTGTGGTGA CGCCATC ATG GGA GCT Met Gly Ala 1 TAC CGC ATC Tyr Arg Ile CCG GCG CTG CCC CAG ATC TGG CAG CTG TAC CTC AAG AAC Pro Ala Leu Pro Gin Ile Trp Gln Leu Tyr Leu Lys Asn GCC ACC TTC AAG AAC TGG CCC TTC CTG GAG GAC TGC GCC TGC ACC CCA Ala Thr Phe Lye Asn Trp Pro Phe Leu Glu Asp Cys Ala Cys Thr Pro 25 30 WO097/23501 PCT/AU96/oo827 72 GAG CGA ATG GCG GAG GOT GGC TTC ATC CAC Glu Arg Met Ala Ala Gly Phe Ile His TGC CCT ACC GAG AAO GAG Cys Pro Thr Giu Asn Glu OCT GAT TTG Pro Asp Leu GAA COO GAT Glu Pro Asp CAG TGT TTT TTO Gin Cys Phe Phe TGO TTT AAG Cys Phe Lys GAA TTG GAA GGC TGG Glu Leu Glu Gly Trp GAO AAO OOG ATA Asp Asn Pro Ile GAG OAT AGA AAG CAC TOO OOT GGO Giu His Arg Lys His Ser Pro Gly TGO GC Cys Al a TTO OTO ACT GTO Phe Leu Thr Val

AAG

Lys AAG OAG ATG GAA Lys Gin Met Glu OTA ACC GTO AGT Leu Thr Val Ser

GAA

Glu 100 TTO TTG AAA OTG Phe LeU Lys Leu

GAO

Asp 105 AGA OAG AGA GOO Arg Gin Arg Ala

AAG

Lys 110 AAO AAA ATT GOA Asn Lys Ile Ala

AAG

Lys 115 344 392 440 GAG ACC AAO AAO Giu Thr Asn Asn CAA AAA GAG TTT Gin Lys Glu Phe GAA GAG ACT GOA AAG ACT ACC Glu Glu Thr Ala Lys Thr Thr 125 130 CGT CAG TCA ATT GAG Arg Gin Ser Ile Glu 135 CAG OTG GOT GOC TAATGOTGAG OGTTTGOTGA

G

Gin Leu Ala Ala* 140 ATA ACT TGG AOG INFORMATION FOR SEQ ID NO:ll: SEQUENCE

CHARACTERISTICS:

LENGTH: 140 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:il: Met Gly Ala Pro Ala Leu Pro Gin Ile Trp Gin Leu Tyr Leu Lys Asn 1 5 10 Tyr Arg Ile Ala Thr Phe Lys Asn Trp Pro Phe Leu GlU Asp Cys Ala 25 WO 97/23501 PCT/AU96/00827 -73- Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr Glu Asn Glu Pro Asp Leu Gin Cys Phe Phe Cys Phe Lys Glu Leu Gly Trp Glu Pro Asp Asn Pro Ile Glu His Arg Lys His Glu Leu Ser Pro Gly Cys Phe Leu Thr Val Lys Gin Met Glu Thr Val Ser Ile Ala Lys 115 Glu 100 Phe Leu Lys Leu Arg Gin Arg Ala Lys Asn Lys 110 Glu Thr Ala Glu Thr Asn Asn Lys 120 Gin Lys Glu Phe Glu 125 Lys Thr 130 Thr Arg Gin Ser Ile 135 Glu Gin Leu Ala Ala 140 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 1075 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 18..941 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GAATTCGGCA CGAGAGA ATG CCA TAT ACA Met Pro Tyr Thr 1 TTC GAG AAC TCA GAA GCT CTG Phe Glu Asn Ser Glu Ala Leu CTC AAA AAC TTG AAA GAC GCG GCT CCG TAC ATA TCA GCT GCC GAA CGC WO 97/23501 PCT/AU96/00827 -74- Leu Lye Asn Leu Lye Asp Ala Ala Pro Tyr Ile Ser Ala Ala Giu Arg TTT GCA TCT TTC Phe Ala Ser Phe AAA GGA TTC GTT TAT GAT AAG Lye Gly Phe Tyr Asp Lys AGA ATT AAC ATC GCC Arg Ile Asn Ile Ala TGT ACT Cys Thr TCC GAA AAG CTT GCA CGT GCA GGA TTC Ser Glu Lye Leu Ala Arg Ala Gly Phe TCG ACA GCC TCA Ser Thr Ala Ser

CCA

Pro GAA TTC CCC GCT Glu Phe Pro Ala GCC AAA TGC Ala Lye Cys CCG TTT Pro Phe TGT ATG CTG GAA Cys Met Leu Glu AAT TTC GAA CAG Asn Phe Glu Gin GAC GAT CCA TGG GAG AAA CAC AAA TCA Asp Asp Pro Trp Glu Lye His Lye Ser GGA AGC Gly Ser CCA CAC TGT Pro His Cys TCT TTC CGA Ser Phe Arg 110

GAA

Glu TTT GTC ATG ATT Phe Val Met Ile

OGA

Gly 100 GAA ATC GAA GAA Glu Ile Glu Glu AGT GAA CTT Ser Glu Lu 105 ACA GTG AGA Thr Val Arg 338 386 ATC ATA TCC AAT Ile Ile Ser Asn

CTT

Lou 115 GCG ATA CGA CAC Ala Ile Arg His

GCA

Ala 120 CTT TAC Leu Tyr 125 GAA GAG CTA CTT Giu Giu Leu Leu ATC GTA GCG ACA Ile Val Ala Thr GAA AAT GGA GAC Glu Asn Gly Asp 434 482

ATT

Ile 140 GCT AAT GAA AAC Ala Asn Giu Asn

CCG

Pro 145 ATT ACC CGC GCT Ile Thr Arg Ala

GAC

Asp 150 GCC ACC AGG AAA Ala Thr Arg Lye

TTG

Lou 155 ATT TCA TTC CGA Ile Ser Phe Arg

TCT

Ser 160 TCT AGC AAG CTT Ser Ser Lye Leu

CTC

Leu 165 ACT TTT GAT Thr Phe Asp CAT CGA TT His Arg Lou 170 GCC ACA TTC CAA AAT TTC ATT TTO GAC AAA AAA AGG AAT GTA AAA TGC Ala Thr Phe Gin Asn Phe Ile Phe Asp Lye Lye Arg Asn Val Lye Cys 175 180 185 ACT TCA AAG AAG CTC GCC AAA GCT GGA TGG TTT TOT ATT GCC AAC AAA Thr Ser Lye Lye Lou Ala Lye Ala Gly Trp Phe Ser Ile Ala Aen Lye 190 195 3fn WO 97/23501 PCT/AU96/00827 75 AAA GAT AAA ACT TCT GCA AAA TOT CCA TTC TGC CTT GTT GAG CTG GAC Lys Asp 205 Lys Thr Ser Ala Cys Pro Phe Cys Val Giu Leu Asp GAC GAA TCA GAT Asp Glu Ser Asp CCG TOO GAA GAG Pro Trp Giu Giu CAG AAG TTC AGT Gin Lys Phe Ser

GCA

Ala 235 TCC TGC GAT TTT Ser Cys Asp Phe

ATC

Ile 240 AAA CTA GGC AAG Lys Leu Gly Lys

CTT

Leu 245 GAT GAG AAA AAA Asp Giu Lye Lys TGG ACT Trp, Thr 250 CAA AAA Gin Lys GAA AAT GAA Glu Asn Glu TAC GAA AAA Tyr Giu Lye 270

GCA

Al a 255 TTA ATG CTG GGA Leu Met Leu Gly

GCA

Al a 260 AGA ATC ACG ATT Arg Ile Thr Ile GGA ACT TOG TTG Gly Ser Trp Leu

ATT

Ile 275 GAC GAA CTC GAG Asp Glu Leu Giu

AAA

Lys 280 GAA AAT AGA Glu Asn Arg ATT GAT Ile Asp 285 GAA ATA ATC AAG Glu Ile Ile Lys

ATT

Ile 290 COA AAA ATT ATG Arg Lye Ile Met AAA CCA AAT CAT Lys Pro Aen His 914 944 OTT CTC AAA CGA CGT Val Leu Lys Arg Arg CGA TGC ACT ATT TAA Arg Cys Ser Ile TTTATATTTC GACTTTTTAT CTGTAAACTT TTGCCTCCCT CCCCGACCTG TTTAACATCC GTAATTCATT CCAAAAATGT TCTAATTCGA ATGAATATGT TTTCCTAAAA AAAAAA AAAAACTCGA G INFORMATION FOR SEQ ID NO:i3: SEQUENCE CHARA~CTERISTICS: LENGTH: 308 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Pro Tyr Thr Phe Giu Asn Ser Giu Ala Leu Leu Lye Aen Leu Lye 1004 1055 WO 97/23501 PCT/AU96/00827 76 Asp Ala Ala Pro Tyr Ile Ser Ala Ala Glu Arg Phe Ala Ser Phe Lys Gly Phe Val Tyr Asp Lys Arg Asn Ile Ala Cys Thr Ser Glu Lys Leu Ala s0 Arq Ala Gly Phe Ser Thr Ala Ser Glu Phe Pro Ala Ser Ala Lys Cys Pro Cys Met Leu Glu Asn Phe Glu Gln Asp Asp Pro Trp Lys His Lys Ser Ser Pro His Cys Glu Phe Val Met Ile Ser Asn Leu 115 Gly 100 Glu Ile Glu Glu Ser 105 Glu Leu Ser Phe Arg Ile Ile 110 Ala Ile Arg His Thr Val Arg Leu Tyr Glu Glu Leu 125 Leu Gly 130 Ile Val Ala Thr Leu 135 Glu Asn Gly Asp Ile 140 Ala Asn Glu Asn Pro 145 Ile Thr Arg Ala Asp 150 Ala Thr Arg Lys Ile Ser Phe Arg Ser Ser Lys Leu Thr Phe Asp His Leu Ala Thr Phe Gln Asn 175 Phe Ile Phe Ala Lys Ala 195 Lys Lys Arg Asn Lys Cys Thr Ser Lys Lys Leu 190 Lys Thr Ser Gly Trp Phe Ser Ile 200 Ala Asn Lys Lys Asp 205 Ala Lys 210 Cys Pro Phe Cys Leu 215 Val Glu Leu Asp Phe 220 Asp Glu Ser Asp Asp 225 Pro Trp Glu Glu His 230 Gln Lys Phe Ser Ala 235 Ser Cys Asp Phe Lys Leu Gly Lys Leu 245 Asp Glu Lys Lys Trp 250 Thr Glu Asn Glu Ala Leu 255 WO 97/23501 PCT/AU96/00827 -77- Met Leu Gly Trp Leu Ile 275 Ala 260 Arg Ile Thr Ile Met Gin Lys Tyr Glu Lys Gly Ser 265 270 Asp Glu Leu Glu Lye 280 Ile Lye 295 Glu Asn Arg Pro Asn His Ile Asp Glu Ile Ile 285 Val Leu Lys Arg Arg 300 Lys Ile 290 Arg Lys Ile Met Cys Ser Ile INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 512 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

1..123 (ix) FEATURE: NAME/KEY: CDS LOCATION: 168..512 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATG GCA CCC GGG ACC AAA AAA AAG TCG GAT ATG GCA AAA TTC ACA TTC Met Ala Pro Gly Thr Lys Lys Lys Ser Asp Met Ala Lys Phe Thr Phe 1 5 10 TAC AAA GAT CGC TTG ATG ACA TTC AAA AAT TTC GAA TAT GAT AGA GAC Tyr Lys Asp Arg Leu Met Thr Phe Lys Asn Phe Glu Tyr Asp Arg Asp 25 CCG GAT GCA AAA TGC ACG TCT CAA GCG GTTTGTCCAT TTTCGTGTTT Pro Asp Ala Lys Cys Thr Ser Gin Ala WO 97/23501 PCT/AU96,00827 -78- TAAATAAAAT TATTCTTGTT TTAG GTT GCT CAA GCC GGA TTT TAC TGC ACC Val Ala Gin Ala Gly Phe Tyr Cys Thr CCT CAG TCT GGC AAA TGT GCA TTT TGC AAC Pro Gin Ser Gly Lys Cys Ala Phe Cys Asn AAG GAA CTT GAT TTT Lys Glu Leu Asp Phe GAC CCA GAA GAC GAT CCG Asp Pro Glu Asp Asp Pro TGG TAC GAG CAC ACG AAA CGT GAT GAA CCG Trp Tyr Glu His Thr Lye Arg Asp Glu Pro 35 TGC GAG TTT GTA CGG Cys Glu Phe Val Arg ATT GGA AAG Ile Gly Lys GAT GAC TCG GAA TTA ACT ATT Asp Asp Ser Glu Leu Thr Ile AAC GAT ACC Asn Asp Thr GTT CGT CTC TCA Val Arg Leu Ser ACC GCC ATG ATT Thr Ala Met Ile

ATG

Met ACT AAA CTC Thr Lys Leu TTT GAG Phe Glu CAT GAG ATG ATG His Glu Met Met AAT AAT TTG TCT Asn Asn Leu Ser CAT TCT TCT TCT His Ser Ser Ser 434 482 GAT GCT CTC TTC Asp Ala Leu Phe ACA AAA TCT AAC Thr Lys Ser Asn GAT CAG CTG AAA AAA GTA Asp Gin Leu Lye Lye Val 95 AGC CGC CGC GGC AAA TA Ser Arg Arg Gly Lys

CCG

Pro 100 AAC ACA GCA TCG Asn Thr Ala Ser

ACA

Thr 105 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID WO 97/23501 PCT/AU96/00827 79 Met Ala Pro Gly Thr Lys Lys Lys Ser Asp Met Ala Lys Phe Thr Phe Tyr Lys Asp Arg Leu Met Thr Phe Lys Asn Phe Glu Tyr Asp Arg Asp 25 Pro Asp Ala Lys Cys Thr Ser Ala Val. Ala Gin Gly Phe Tyr Cys Thr so Gly Pro Gin Ser Lys Cys Ala Phe Asn Lys Glu Leu Asp Phe Asp Pro Glu Asp Pro Trp Tyr Glu 75 His Thr Lys Arg Asp so Glu Pro Cys Glu Val Arg Ile Gly Leu Asp Asp Ser Glu Leu Thr Ile Asn Lys Leu Phe 115 Thr Val Arg Leu Gin Thr Ala Met Ile Met Thr 110 Asn His Ser Glu His Glu Met Met 120 Ile Asn Asn Leu Ser 125 Ser Ser 130 Asp Ala Leu Phe Asp 135 Gin Leu Lys Lys Pro Asn Thr Ala Ser 145 Thr Thr Lys Ser Asn 150 Ser Arg Arg Gly INFORMVATION FOR SEQ ID NO:16: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2866 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS (B3) LOCATION: 1. .2863 WO 97/23501 WO 9723501PCT/AU96/00827 80 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i6: ATG GAT GOT CAA Met Asp Gly Gin ATA GAT AAA ATG GAG Ile Asp Lys Met Giu AAG OGA TAT ACC Lys Arg Tyr Ser ATG ACC AAA Met Thr Lys CTG GAG AAT Leu Giu Asn AAA AAG TTA Lye Lys Leu

AGO

Arg TTA AGO ACT TTC Leu Arg Thr Phe

CAA

Gin GAT GOT OTT OCA CTT GAA AAA Asp Oiy Vai Aia Leu Giu Lys AAG TGG AOT TTT Lys Trp Ser Phe OTT ATT CCO TAT Vai Ile Pro Tyr GCA ATO GCC Aia Met Ala AAG CTT Lys Leu so GGA TTT TAC TTT Oiy Phe Tyr Phe CCA GTG ATC GAT Pro Vai Ile Asp AAO ACA TCT AAA Lys Thr Ser Lys

CTG

Leu AAA AAA GAC TCT GTA AGA TGT TGC TAT Lys Lys Asp Ser Vai Arg Cys Cys Tyr CAT CGT CAA ACA His Arg Gin Thr AAT GTG AGA GAC Asn Vai Arg Asp AGA TCC AAA AGA Arg Ser Lys Arg GAT GTT CTT GAG Asp Vai Leu Giu ACT CTA Thr Leu 288 336 AGC AAT ATA Ser Asn Ile TGC CTC CTT Cys Leu Leu 115

ATO

Met 100 AGO CAA CAT CTG Arg Gin His Leu

ACT

Thr 105 OTT ACT GAT AAC AAA CAA GTT Vai Thr Asp Asn Lys Gin Vai 110 ATT TAC TTG COC Ile Tyr Leu Arg AAG CTA TTO ACO Lys Leu Leu Thr

GAC

Asp 12 TAT AGC TTC Tyr Ser Phe CAT ATO His Met 130 000 OTT TCO GAC Giy Vai Ser Asp AAA AAT GAC AAG TAC TTC AOC AAT CCA Lys Asn Asp Lys Tyr Phe Ser Asn Pro OAT OAT GAA AAC OTG ATA AAC CTA AGA AAA TTT ACT TTT CAA OAT Asp Asp Giu Asn Val Ile Asn Leu Arg Lys Phe Thr Phe Gin Asp TOG CCT CAC ACT Trp Pro His Ser GOT TCT Oiy Ser 165 CAA AAT GAA Gin Asn Oiu CCG CTG GOT ATA Pro Leu Giy Ile GAA AO Oiu Lys 175 WO 97/23501 WO 9723501PCT/AU96/00827 -81 ATG GTG AAT GCA GGA CTC ATG CGA Met Vai Asn Ala Cly Leu Met Arg 180 GAC TCC AGC ATA GAA GGC TTA Asp Ser Ser Ile Giu Gly Leu GGC GAT CCA Gly Asp Pro 195 AGC ATG GAT AAA ACA CTT ATG AAC GAT Ser Met Asp Lys Thr Leu Met Asn Asp 200

ACC

Thr 205 TGT TAT TGT Cye Tyr Cys ATT TAT Ile Tyr 210 TGC AAA CAG TTG Cye Lys Gin Leu

TTA

Leu 215 CAA OCT TGG TCA Gin Gly Trp, Ser

ATA

Ile 220 AAT CAC GAT CCG Aen Asp Asp Pro AGC CGA CAT TAT Ser Arg His Tyr GTT TCT CAA AAT Val Ser Gin Asn

GGG

Gly 235 AAC TCT TAT TTC Asn Cys Tyr Phe

TTT

Phe 240 CAG ACA CCT AAT Gin Thr Arg Asn

CGA

Arg 245 TTT GAG AGA ATA Phe Giu Ar Ile AAT GAC AAT CAC Asn Asp Asn Asp ACT ATC Ser Ile 255 AAA AGA Lye Arg ACG AAA AAT Thr Lys Asn GAA CTG ATC Giu Vai Ile 275

TGC

Cys 260 GAA GTC TCT CCA ACT TTA GCC GAA AAT Ciu Val Ser Pro Thr Leu Gly Ciu Asn 265

GGG

Gly 270 AAC ACG AAA ACC Asn Thr Lys Thr

GCT

Ala 280 TCT CAA AGC CAC Ser Gin Arg Gin

TGT

Cys 285 CCT TTA TTT Pro Leu Phe GAG TCA Ciu Ser 290 CCT CCA TCT ACT Pro Pro Ser Ser GGT CCT CAA CTT Gly Pro Gin Leu GAT TAT AAT GAG Asp Tyr Asn Glu 912 960 ACA CAT ATA TCT Thr Asp Ile Ser ATT CAA CAT AAT Ile Gin His Asn ACT GTC CTT CAT Ser Val Leu Asp

GGA

Cly 320 CCA CAA CCC CAA AAT CTA AAA CCT AAT ACT GTG GAA CAA AAA Ala Gin Cly Clu Asn Val Lys Arg Aen Ser Val Giu Giu Lys CAA CAA Ciu Gin 335 1008 ATC AAC ATG Ile Asn Met

GAA

Ciu 340 AAT GGA ACC ACT ACA TTA CAA GAG GGC Asn Gly Ser Thr Thr Leu Ciu Giu Gly 345 AAT ATA AAT Asn Ile Asn 350 1056 COT CAT CTT TTA CCA GAT AAC AAA GAA GTT ATT TCG ACA CCA ACT CCA Arg Asp Val Leu Ala Asp Lye Lys Ciu Val Ile Ser Thr Pro Thr Ala 1104 WO 97/23501 WO 9723501PCT/AU96/00827 82 AAA GAA Lys Giu 370 ATC AAA CGT Ile Lys Arg CCA AAT Pro Asn 375 GTC CAC CTA ACC Vai Gin Leu Thr TCA TCG TCA CCT Ser Ser Ser Pro

ATA

Ile 385

GAT

Asp AAA AAG AAA AGA AAC Lys Lys Lys Arg Lys 390 GAG GAA GAT AGC GAA Giu Giu Asp Ser Giu 405 TTT AAG AGA ATA Phe Lys Arg Ile

TCG

Ser 395 CCT AGA AAA ATC Pro Arg Lys Ile

TTT

Phe 400 1152 i2 00 1248 CAC TCC CTT His Ser Leu AAT AAT TCT CC Aen Asn Ser Ala AAC GCA Asn Gly 415 GAC AAC AAA Asp Asn Lys AAA AAT AGA Lye Asn Arg 435

GAT

Asp 420 AAC GAT TTC GTT ATA Lye Asp Leu Vai Ile 425 GAT TTT ACA AC Asp Phe Thr Ser CAT ATC ATA His Ile Ile 430 GAC ACC ACA Asp Ser Thr 1296 1344 CAT GTA G AGA Asp Vai Cly Arg

AAG

Lye 440 AAT GCT ATT TTA Aen Ala Ile Leu

CAT

Asp 445 CAT GAA Asp Ciu 450 TTC ACT TTC ACT Phe Ser Phe Ser CAA GGA CAT AAC Gin Cly His Asn

ACA

Thr 460 TTT GAC ATA CCT Phe Asp Ile Pro

ATA

Ile 465 CCA ACC TCA TCA Pro Thr Ser Ser

CAT

His 470 TTC CTC AAA CCT Leu Leu Lye Cly

ATA

Ile 475 GAT TCA CAT AAT Asp Ser Asp Aen

CAC

Asp 480 1392 1440 1488 AAT CTT ATA CCA Asn Val Ile Arg

CAA

Ciu 485 CAT CAT ACT GC Asp Asp Thr Gly

ATA

Ile 490 AAT ACA GAC ACA Asn Thr Asp Thr AAA CGA Lye Cly 495 C TCT TCT Ala Ser Ser AAT TTC ACT Asn Phe Ser 515

AAA

Lys 500 CAC GAA AAA TTT His Clu Lys Phe CTT AAT TOT CAA Val Asn Ser Giu CAA GTC AAA CTC Clu Val Lye Leu

ACA

Thr 520 CGT AGA CAC ACT Cly Arg Asp Ser

ACT

Ser 525 CAA CAT TTA Ciu Asp Leu 510 ACT AAT ATT Thr Aen Ile ATC GAC ACA Ile Asp Arg 1536 1584 CTT ATA ACA ACC CAA ATT Leu Ile Arg Thr Gin Ile 530

GTA

Val 535 CAT CAC AAT TTG Asp Gin Asn Leu COT CAT Cly Asp 540 1632 WO 97/23501 PCT/AU96/00827 -83

GAT

Asp 545 AAG GTC CCC AAT Lye Val Pro Asn GGA TCC CCA GAA Gly Ser Pro Glu GTT CCA AAG ACA CAT GAG Val Pro Lys Thr His Glu 1680 TTA ATT AGA GAT Leu Ile Arg Asp

AAC

Asn 565 TCT GAA AAG AGG Ser Glu Lys Arg

GAA

Glu 570 GCA CAA AAC GGG Ala Gin Asn Gly GAA TTT Glu Phe 575 1728 CGA CAT CAA Arg His Gin TCT AAT AAA Ser Asn Lys 595

AAA

Lys 580 GAT TCA ACT GTA Asp Ser Thr Val CAA TCT CCA GAT Gin Ser Pro Asp ATA TTA CAT Ile Leu His 590 ATA CCC AAA Ile Pro Lys 1776 1824 AGT GGT GAT AAT Ser Gly Asp Asn

TCC

Ser 600 AGT AAT ATT ACC Ser Asn Ile Thr

GCA

Ala 605 GAG GAA Glu Glu 610 CAA AGG AGA GGC Gin Arg Arg Gly

AAT

Asn 615 AGC AAA ACA TCC Ser Lys Thr Ser

AGC

Ser 620 ATT CCC GCT GAT Ile Pro Ala Asp 1872 1920

ATT

Ile 625 CAT CCT AAA CCA His Pro Lys Pro

AGG

Arg 630 AAA AAC TTG CAA Lys Asn Leu Gin CCA AGA AGC CTA TCA Pro Arg Ser Leu Ser 640 ATA AGT GGA AAA Ile Ser Gly Lys GTT CCA ACA GAA Val Pro Thr Glu AAA TTA GAT AAC Lys Leu Asp Asn ATC AAT Ile Asn 655 1968 ATA GAT CTA Ile Asp Leu GAG CAA TCA Glu Gin Ser 675 TTT TCG GCG TCC Phe Ser Ala Ser TTT TCA CCA TCA Phe Ser Pro Ser TCA CAA TCT Ser Gin Ser 670 GTA GCG AGC Val Ala Ser 2016 2064 TCA AAA AGC TCA Ser Lys Ser Ser

AGC

Ser 680 GTT ATT TCA ACG Val Ile Ser Thr CCT AAA Pro Lys 690 ATT AAT CTC ACA Ile Asn Leu Thr

CGC

Arg 695 AGT TTG CAT Ser Leu His GCA GTC Ala Val 700 AAA GAG CTT TCT Lys Glu Leu Ser 2112 2160

GGC

Gly 705 CTC AAA AAA GAA Leu Lys Lys Glu GAC GAT GGT AAG Asp Asp Gly Lys TTT ACT AAT AAG Phe Thr Asn Lys

CAG

Gin 720 GAA ACT ATA AAA ATA CTG GAA GAT GTG TCT GTG AAG AAT GAG ACC CCA Glu Thr Ile Lys Ile Leu Glu Asp Val Ser Val Lys Asn Glu Thr Pro 2208 .1 WO 97/23501 PCT/AU96/00827 -84- AAT AAT GAA Asn Aen Glu GAA AAC AAG Glu Asn Lye 755 ATG TTA Met Leu 740 CTT TTT GAG ACT GGA ACC CCG ATC Thr Pro Ile Leu Phe Glu Thr Gly 745 GCA TCT CAA Ala Ser Gin 750 2256 2304 TCA AGA AAA TTA Ser Arg Lye Leu GAT GAA GAG TTT Asp Giu Giu Phe TCT GGG AAG GAA Ser Giy Lye Glu 765 AAA AAG GTT ATT Lye Lye Val Ile TTG GAT Leu Asp 770 ATA CCT ATA GAT Ile Pro Ile Asp TCA ACT GTA GAA Ser Thr Val Glu

AAA

Lye 785 CCC GAA TTT GAG Pro Giu Phe Glu

CCA

Pro 790 GTC CCA TCA GTT Vai Pro Ser Vai

GCA

Ala 795 AGG AAT TTG GTG Arg Aen Leu Vai 2352 2400 2448 OGA ACC TCT TCA Gly Thr Ser Ser

TAT

Tyr 805 CCA CGG AAT TCA Pro Arg Asn Ser

CGT

Arg 810 TTA GAA GAA CAA Leu Giu Giu Gin AGA AAG Arg Lye 815 GAA ACA AGC Glu Thr Ser AAT GAG GGC Asn Giu Gly 835

ACA

Thr 820 AGT TTG GCT GAT Ser Leu Ala Asp

AAT

Asn 825 TCT AAG AAA GGA Ser Lye Lye Gly AGT TCA TTT Ser Ser Phe 830 TGG TTT AAA Trp Phe Lye 2496 2544 AAC AAC GAA AAA Asn Aen Giu Lye CCG AAT GCA GCA Pro Asn Ala Ala ATT GAT Ile Asp 850 GAA AAT AGA CAC CTT GTA AAA AAT TAC TTC CAT GAT TTG TTA Glu Aen Arg His Leu Val Lye Asn Tyr Phe His Asp Leu Leu 2592

AAA

Lye 865 TAT ATA AAT AAT AAT GAT GCT ACG CTA GCT AAT GAC AAA GAC Tyr Ile Asn Asn Asn Asp Ala Thr Leu Ala Asn Asp Lye Asp 2640 GAT CTG GCT TTT Asp Leu Ala Phe

TTG

Leu 885 ATT AAG CAA ATG Ile Lye Gin Met

CCT

Pro 890 GCA GAA GAG TTG Ala Glu Giu Leu GAC ATG Asp Met 895 2688 ACA TTC AAC Thr Phe Asn

AAT

Asn 900 TGG GTG AAC CTA Trp Val Asn Leu

AAA

Lys 905 GTG CAA TCT ATT Val Gin Ser Ile AAG CCC GAA Lye Arg Giu 910 2736 .WO 97/23501 PCT/AU96/00827 85 TTT ATA GAT Phe Ile Asp 915 GAC TGT GAT AAG AAA Asp Cys Asp Lys Lys 920 TTG GAC ATA TTG Leu Asp Ile Leu

AGA

Arg 925 AGG GAT TAC Arg Asp Tyr 2784 TAT ACT Tyr Thr 930 GCC ACA AAT TTT Ala Thr Asn Phe GAA ACT TTG Giu Thr Leu GAA GAT Glu Asp 940 GAC AAT CAA TTG Asp Asn Gin Leu 2832 ATC GAT ATT GCT AAG AAA ATG GGC ATT TTA TAG Ile Asp Ile Ala Lys Lys Met Gly Ile Leu 945 950 955 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 954 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 2865 Met Asp Gly Gin Ile Asp Lys Met Glu Arg Tyr Ser Met Thr Lys Leu Glu Asn Arg Lys Lys Leu Lys Leu Arg Thr Phe Gin Asp 25 Gly Val Ala Leu Giu Lys Ala Met Ala Trp Ser Phe Val Ile Pro Tyr Gin Lys Leu Gly Phe Tyr Phe Asp Pro Val Ile Asp Lys Thr Ser Lys Lys Lys Asp Ser Arg Cys Cys Tyr His Arg Gln Thr Tyr Asn Val Arg Asp Cys Arg Ser Lys Arg Lys Asp Val Leu Giu Thr Leu Ser Asn Ile Met Arg 100 Gin His Leu Thr 105 Val Thr Asp Asn Lys Gin Val 110 Cys Leu Leu Ile Tyr Leu Arg Asn Lys Leu Leu Thr Asp Tyr Ser Phe WO 97/23501 WO 9723501PCT/AU96/00827 86 His Met 130 Asp Asp Gly Val Ser Asp Lys Asn Asp Lys Tyr Phe Ser Asn Pro 140 Giu Asn Val Thr Leu Arg Lys Phe Gin Asp Pro His Ser Gly 165 Gly Gin Asn Giu Leu Gly Ile Giu Lys 175 Met Val Asn Gly Asp Pro 195 Ile Tyr Cys Leu Met Arg Ser Ser Ile Met Asp Lys Thr 200 Met Asn Asp Thr 205 Asn Giu Gly Leu 190 Cys Tyr Cys Asp Asp Pro Lys Gin Leu Gin Gly Trp Ser 210 Ser Ile 220 Asn Arg His Tyr Ser Gin Asn Gly 235 Asn Cys Tyr Phe Thr Arg Asn Giu Arg Ile Asp Asn Asp Ser Ile 255 Thr Lys Asn Giu Val Ile 275 Giu Ser Pro 290 Val Ser Pro Gly Giu Asn Thr Lys Thr Aia 280 Gly Gin Arg Gin Gly Lys Arg 270 Pro Leu Phe Tyr Asn Giu Pro Ser Ser Thr 295 Ile Pro Gin Leu Asp 300 Lys 305 Ala Thr Asp Ile Ser Val 310 Val Gin His Asn Ser Val Leu Asp Gly 320 Gin Giy Giu Asn 325 Asn Lys Arg Asn Ser 330 Leu Giu Giu Lys Giu Gin 335 Ile Asn Met Arg Asp Val 355 Giu 340 Leu Giy Ser Thr Thr 345 Glu Giu Giu Giy Asn Ile Asn 350 Pro Thr Ala Ala Asp Lys Lys 360 Vai Ile Ser Thr 365 WO 97/23501 WO 9723501PCT/AU96/00827 87- LYS Giu Ile Lys Arg Pro Asn Val Gin Leu Thr Gin Ser Ser Ser Pro Lys Lys Lys Arg Phe Lys Arg Ile Pro Arg Lys Ile Asp Giu Giu Asp Glu His Ser Leu Asn 410 Asn Asn Ser Aia Asn Gly 415 Asp Asn Lys Lys Asn Arg 435 Asp 420 Lys Asp Leu Vai Ile Asp Phe Thr Ser 425 His Ile Ile 430 Asp Ser Thr Asp Val Giy Arg Asn Aia Ile Leu Asp Giu 450 Phe Ser Phe Ser Gin Gly His Asn Thr 460 Phe Asp Ile Pro Pro Thr Ser Ser Leu Leu Lys Giy Asp Ser Asp Asn Asn Val Ile Arg Giu 485 Asp Asp Thr Giy Ile 490 Asn Thr Asp Thr Lys Gly 495 Ala Ser Ser Lys His Giu Lys Phe 500 Ser 505 Vai Asn Ser Glu Giu Asp Leu 510 Thr Asn Ile Asn Phe Ser 515 Giu Vai Lys Leu Thr 520 Giy Arg Asp Ser Ser 525 Leu Ile 530 Arg Thr Gin Ile Asp Gin Asn Leu Gly 540 Asp Ile Asp Arg Asp Lys Val Pro Asn 545 Leu Ile Arg Asp Asn 565 Gly 550 Gly Ser Pro Giu Pro Lys Thr His Ser Giu Lys Arg Ala Gin Asn Gly Giu Phe 575 Arg His Gin Ser Asn Lys 595 Asp Ser Thr Val Gin Ser Pro Asp Ile Leu His 590 Ile Pro Lys Ser Gly Asp Asn Ser Ser Asn Ile Thr 600 Al a 605 Giu Glu Gin Arg Arq Giy Asn Ser Lys Thr Ser Ser Ile Pro Ala Asp WO 97/23501 PCT/AU96/00827 88 His Pro Lys Pro Arg 630 Lys Asn Leu Gin Pro Arg Ser Leu Ile Ser Gly Lys Val Pro Thr Giu Lys Leu Asp Asn Ile Asn 655 Ile Asp Leu Giu Gin Ser 675 Asn 660 Phe Ser Ala Ser Asp 665 Phe Ser Pro Ser Ser Gin Ser 670 Val Ala Ser Ser Lys Ser Ser Ser 680 Vai Ile Ser Thr Pro Lys 690 Ile Asn Leu Thr Ser Leu His Ala Lys Giu Leu Ser Leu Lye Lys Giu Asp Asp Gly Lys Phe Thr Asn Lys Gin* 720 Giu Thr Ile Lys Ile 725 Leu Giu Asp Vai Ser 730 Val Lys Asn Giu Thr Pro 735 Asn Asn Giu Glu Asn Lys 755 Leu Leu Phe Giu Thr 745 Giy Thr Pro Ile Ala Ser Gin 750 Gly Lye Giu Ser Arg Lye Leu Asp Giu Giu Phe Ser 765 Leu Asp 770 Ile Pro Ile Asp Ser Thr Val Giu Lys Lys Vai Ile Lye 785 Pro Giu Phe Giu Pro 790 Val Pro Ser Val Arg Asn Leu Vai Gly Thr Ser Ser Tyr 805 Ser Pro Arg Aen Ser Leu Ala Asp Aen 825 Leu Giu Giu Gin Arg Lye 815 Giu Thr Ser Asn Giu Gly 835 Thr 820 Ser Lye Lye Gly Ser Ser Phe 830 Asn Asn Giu Lye Giu Pro Asn Ala Ala Giu Trp Phe Lye Ile Asp Glu Aen Arg His Leu Val Lye Aen Tyr Phe 860 His Asp Leu Leu WO 97/23501 PCT/AU96/00827 89 Lys Tyr Ile Asn Asn Asn Asp Ala Thr Leu 865 870 Ala Asn Asp Lys Asp Gly 875 880 Ala Giu Giu Leu Asp Met 895 Asp Leu Ala Phe Leu Ile 885 Lys Gin Met Pro 890 Thr Phe Asn Phe Ile Asp 915 Trp Val Asn Leu Val Gin Ser Ile Lys Arg Giu 910 Asp Cys Asp Lys Leu Asp Ile Leu Arg Arg Asp Tyr 925 Tyr Thr 930 Ala Thr Asn Phe Ile 935 Glu Thr Leu Giu Asp 940 Asp Asn Gin Leu Asp Ile Ala Lys Lys 950 Met Gly Ile Leu INFORMATION FOR SEQ ID NO:18: Wi SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Lys Asp Ile Lys Lys Thr Met Giu Glu Lys Ile Gin Thr Ser Gly 10

Claims (34)

1. An isolated proteinaceous molecule or derivative of chemical analogue thereof capable of inhibiting an apoptotic response in cells to viral infection, said proteinaceous molecule comprising a cell-derived homologue of a viral inhibitor of apoptosis (IAP).

2. An isolated proteinaceous molecule according to claim 1 wherein said molecule is a homologue of baculovirus IAP.

3. An isolated proteinaceous molecule according to claim 2 wherein the baculovirus IAP inhibits apoptosis mediated by an interleukin 13 converting enzyme (ICE) protease or death domain bearing protein FADD.

5. An isolated proteinaceous molecule according to claim 1 wherein the cell is from a *mammal, nematode, yeast or insect.

6. An isolated proteinaceous molecule according to claim 5 having the amino acid rm sequence: Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro [Xaa] [Xaa] Ala Xaa Ala Gly Phe [Xaa] Asp Xaa [Xaa] Xaa Cys Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys Xaa [Xaa], [Xaa]r wherein wherein Xaa is an amino acid residue; WO 97/23501 PCT/AU96/00827 -91- [Xaa]m is a series of at least 5 and preferably at least 9 amino acids; [Xaa], is Met or Leu; [Xaa]o is a series of at least 3 and preferably at least 5 amino acids; [Xaa], is Val or Ala; [Xaa]q is Phe or Tyr; [Xaa]r is Leu or Val; or a homologue, chemical analogue or derivative thereof.

7. An isolated proteinaceous molecule according to claim 6 having an amino acid sequence substantially as set forth in SEQ ID NO: 2 or having at least 40% similarly thereto.

8. An isolated proteinaceous molecule according to claim 6 having an amino acid sequence substantially as set forth in SEQ ID NO: 4 or having at least 40% similarly thereto.

9. An isolated proteinaceous molecule according to claim 6 having an amino acid sequence substantially as set forth in SEQ ID NO: 6 or having at least 40% similarly thereto. An isolated proteinaceous molecule according to claim 6 having an amino acid sequence substantially as set forth in SEQ ID NO: 8 or having at least 40% similarly thereto.

11. An isolated proteinaceous molecule according to claim 6 having an amino acid sequence substantially as set forth in SEQ ID NO: 11 or having at least 40% similarly thereto.

12. An isolated proteinaceous molecule according to claim 6 having an amino acid sequence substantially as set forth in SEQ ID NO: 13 or having at least 40% similarly thereto.

13. An isolated proteinaceous molecule according to claim 6 having an amino acid WO 97/23501 PCT/AU96/00827 -92- sequence substantially as set forth in SEQ ID NO: 15 or having at least 40% similarly thereto.

14. An isolated proteinaceous molecule according to claim 6 having an amino acid sequence substantially as set forth in SEQ ID NO: 17 or having at least 40% similarly thereto. An isolated nucleic acid molecule encoding the proteinaceous molecule according to any one of claims 1 to 14.

16. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO: 2.

17. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID NO: 4 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO: 4.

18. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID No: 6 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO: 6.

19. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID No: 8 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO: 8. WO 97/23501 PCT/AU96/00827 -93- An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID No: 11 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO: 11.

21. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID No: 13 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO: 13.

22. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID No: 15 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO:

23. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID No: 17 or having at least 40% similarity to a nucleotide sequence encoding to amino acid sequence substantially set forth in SEQ ID NO: 17.

24. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 1 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 1 under low stringency conditions. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 3 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 3 under low stringency conditions. WO 97/23501 PCT/AU96/00827 -94-

26. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 5 or having at least 40 similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 5 under low stringency conditions.

27. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 7 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 7 under low stringency conditions.

28. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 10 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 10 under low stringency conditions.

29. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 12 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 12 under low stringency conditions. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 14 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 14 under low stringency conditions.

31. An isolated nucleic acid molecule according to claim 15 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 16 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybridizing to SEQ ID NO: 16 under low stringency conditions. WO 97/23501 PCT/AU96/00827

32. A method for modulating cell apoptosis in an animal said method comprising administering to said animal a cell apoptosis modulating effective amount of a proteinaceous molecule or derivative or chemical analogue thereof according to any one of claims 1 to 14 for a time and under conditions sufficient for said cell apoptosis to be modulated.

33. A method according to claim 32 in the treatment of a degenerative disease, an infectious disease, cancer or an autoimmune disease.

34. A pharmaceutical composition comprising a proteinaceous molecule or a derivative or chemical analogue thereof according to any one of claims 1 to 14 and one or more pharmaceutical carriers and/or diluents. Use of an animal cell homologue of an baculovirus IAP to modulate apoptosis in animals suffering from a degenerative disease, an infectious disease, cancer or an autoimmune disease.

36. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 2 or has at least about 40% similarity thereto.

37. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 4 or has at least about 40% similarity thereto.

38. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 6 or has at least about 40% similarity thereto.

39. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 8 or has at least about 40% similarity P:\OPER\EJH\10891-97.SPE 7/7/99 -96- thereto. 39. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 8 or has at least about 40% similarity thereto. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 11 or has at least about 40% similarity thereto.

41. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 13 or has at least about 40% similarity thereto. sequence substantially as set forth in SEQ ID NO: 15 or has at least about 40% similarity thereto. 4 0. 43. Use according to claim 35 wherein the cell homologue comprises an amino acid sequence substantially as set forth in SEQ ID NO: 17 or has at least about 40% similarity thereto. P- P:\OPER\EJH\10891-97.SPE 9/7/99 -97-

44. An isolated proteinaceous molecule according to any one of claims 1 to 14 or an isolated nucleic acid according to any one of claims 15 to 31 or a method according to claim 32 or 33 or a composition of claim 34 or a use according to any one of claims 35 to 43 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 9th day of June, 1999 AMRAD Operations Pty. Ltd. By DAVIES COLLISON CAVE Patent Attorneys for the Applicants S S S. *5 S 9 ol *ft S C. S S *S. S S S.. C S C S 5 7,