CA2241129A1 - Therapeutic compositions - Google Patents

Abstract

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.

Description

WO 97/23401 PCT/AU~6~003~7 TE~ERAPEUTIC COMPOSITIONS

The present invention relates generally to m~ lP~ capable of mod~ tin~ apoptosis of animal cells. More particularly, the present invention provides cell homologues of viral-derived, a~o~L~Lic-inhibiting molecules which are useful in mocl-ll~tin~ apoptosis of animal cells. The molecules contemplated by the present invention may be used to promote or inhibit cell 10 apoptosis depending on the Pxi~enciçs of the therapeutic si~l~tinn Bibliographic details of the public~tirn.e referred to by author in this speçific~tif n are collected at the end of the description. Sequence Identity u~ el., (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the sperific~tif)n are def'med following the ~xamples.
Throughout this specification, unless the context re~luilt;s otherwise, the word "comprise", or variations such as "col~ ises" or "comrri~ing", will be understood to imply the incl~ ion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or in~ege Apoplosi, is a cellular death program that may be initi~tecl by a variety of stimuli and typically leads to characteristic ~h~ngeq in cells, frequently r.qsllltin~ in activation of a ~ncloml~le~e which catalyses chromatin fr~gm~nt~tion and c~n-len~tion, membrane blebbing and collapse of the nucleus.
The mech~ni~m.~ for apoptosis have been strongly conserved during evolution (Vaux et al, 1994). For ç~mrle~ ~loLeills resembling Bc1-2 can protect nematode, insect and ve~
cells from a~op~sis (Vaux et al, 1992; Alnemri et al, 1992), and cysteine proteases resembling interleu3~in-1~ converting enzyme (ICE) are required for apoptosis in both C. elgans and 30 m~mm~l~ (Ellis and Horvi~, 1986; Miura et al, 1993; Kuida et al, 1995). These apoptosis W O 97/23501 PCT/~U96/00827 effector proteases exist as prewrsors in many cells, but must be cleaved and assembled into tetramers before they are active (Thornberry ef al, 1992; Wilson ef al, 1994; Walker et al, 1994; Munday ef al, 1995).
-5 Although many apoptosis effector proteases have been discovered, and numerous stimuli thatinduce ap~L~,sis have been found, little is known about the ~i~n~lling and activation pall,w~y~
that connect the cell death stimuli to the a~op~usis effector merh~ni-~m-c For example, ligslt-r,n of the cell surface l~cepLor CD95 (Fas/Apo-l) can induce apoptosis in many cell types (Nagata and Golstein, 1995; Krammer ef al, 1994), and in T lineage cells, IC~ is required for CD95 10 induced cell death (Kuida ef al, 1995), but the molecular paL~w~y(s) that lead(s) from CD95 to ICE aclivaliol~ is/are largely unchar~ct~ri.~ed The cytoplasmic domain of CD95, which bears a motif termed the "death domain" is l~nown to associate with other death domain bearing plol~illS such as FADD/~IORTl (Boldin et al, 1995; Chinnaiyan ef al, 1995) and RIP (Stager ef al, 1995) which are believed to participate in ~ign~lling from CD95. The downstream targets 15 of FADD and RIP are unknown.

Tumour Necrosis Factor (TNF) can also trigger apoptosis, and its ~~;;c~or resemble CD95 ~3eutler and van-Huffel, 1994). The TNF-rec~or 1 (p55) is known to bind to TRADD (Hsu ef al, 1995) and the TN~ .;e~ r 2 (p75) is known to bind to TNF receptors associated factors 20 CI~AFs) 1 and 2 (Rothe ef al, 1994), but how these proteins work has not yet been detPrmin~

In work leading up to the present invention, the illV~ studied viral anti-apoplosis prc lt;i~s and the intp.rmp~ te steps of apoptosis ~ign~lling Apoptosis can be used as a defence against viruses, but many viruses carry genes for anti-al)o~osis p,. ~ei~ s, presumably to keep the host 25 cell alive while they replicate. Some viral anti-apo~3~osis proteins resemble known cellular pl~ s such as Bc1-2 (reviewed in Vaux, 1993). Others, such as the baculovirus p35 proteins, have unknown cellular counte p~ls, but can function in heterologous systems such as nematodes and m~mm~l~ where they are thought to act as competitive inhibitors of ICE-like cysteine proteases (Clem et al, 1991; Hay et al, 1994; Rabizadeh el al, 1993; Xue and 30 Horvitz, 1995; Burnpetal, 199~).

WO 97/23501 PCT/AU96,'00~27 Miller and cowu,Le~ .ntifiç(l a family of proteins in baculoviruses they dP~i~n~ted "L~Ps"
because they could inhibit the apol,LoLic response of insect cells to viral infection (13irnbaum et al, 1994; Crook e~ al, 1993). Viral IAP proteins typically have two t~orrnin~l repeats dçsi~n~ted baculovirus IAP repeats (13IRs), and a c~uxy t~rmin~l RING finger ~I~m~in 5 ~l1to~r~ph~ californica nuclear polyhedrosis virus (AcNPW) encodes a IAP protein that does not inhibit a~o~lusis, so it is possible that L~P proteins also have other fimction~, for example, regulatory cytokine function.

The present inventors sought cellular L~P homologues and ~hemic~l analogues incl~ in~
10 d~iv~iYes which function in cell death pal~lw~y~ by screening for gene segments pûtentially encoding novel L~P proteins. Full leng~ cDNA clones were then obtained and tested for their ability to mer~i~te apoptosis by ICE and by FADD.

Accordingly, one aspect of the present invention is directed to a homologue or çhemic~l 15 ~n~ln~le of a viral derived peptide, polypeptide or protein or a de,iv~live thereofwhich viral-derived molecule is capable of inhibiting an a~o~lic response of cells to viral infection.

More particularly, the present invention provides an isolated L,rolei,laceous molecule or derivative of chemical analogue thereof capable of inhibiting an apoptotic response in cells 20 to viral il~fec~iol-, said pl~olPi~ eoll~ m~ PClllPCo~ g a cell-derived homologue of a viral inhibitor of apoptosis (IAP).

~Iral derived IAPSs are polypeptides or p~oleins. The IAPs represent a family of prot~in~r.eQus molecules which inhibit the apo~ic response of cells to viral infection. More particularly, 25 the IAPs inhibit the apop~lic response of insect cells to infection by baculoviruses. The present invention provides, therefore, homologues or chemical analogues of baculovirus IAPs which are useful in mocl~ tin~ a~ usis of animal cells. Preferably, the apoptosis is mecii~ted by ICE
or by FADD.

30 Accordingly, another aspect of the present invention contemplates a homologue or çhemic.~l W O 97n3501 PCT/AU96/00827 analogue of a baculovirus L~P peptide, polypeptide or protein or a de~ivalive thereof.

Preferably, the homologue is a animal, cell derived molecule. Examples of particularly - prefellt;d animal cells are those from hllm~nc, livestock ~tnim~lq (e.g. sheep, pigs, horses, 5 donkeys, cows), co.-lr~l~inn animals (e.g. dogs, cats), laboratory test ~nim~l~ (e.g. rabbits, mice, rats, guineapigs), captivewild ~nim~1.q (e.g. foxes, deer, kangaroos), insects (e.g. mos~uitoes) and nematodes. Homologous may also be from yeast or fungi.

Particularly p~ ed homologues are for human, murine, insect, yeast or nematode cells. An 10 example of a nematode is Caenorhabditis elegans.

In a most p~ d embodiment, the m~ntm~ n homologues are derived from murine cells and are dçsi~n~t~d herein M~IA, MIHB, MIHC and MIHE. In a al~e,~live embodiment, thehomologue is an insect homologue from Drosophila dPqi n~tecl herein DIHA. In a further 15 ~tltP.rn~tive embodimentthe hnmolo~le is a C elegans homologue dPqign~tPd herein CIA-l and CIA-2. In yet a filrther ~ ;vt; embodiment the homologue is a yeast homologue design~ted herein YIA-l. However, the present invention extends to human and other m~mm~ n homologues of baculovirus IAPs or homologues or chemical analogues of ~HA, MIHB,MIHC and/or DIHA.
According to another aspect of the present invention, there is provided an isolated molecule having an amino acid se~luence comrri~in~

Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro [Xaa]m [Xaal, Ala Xaa Ala Gly Phe 25 ~aa]O Asp Xaa [XaaJp 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]q [Xaa]r wherem WO 97/23501 PCT~AU96/00827 Xaa is an amino acid residue;

[Xaa]m is a series of at least S a~d prerel~bly at least 9 amino acids;
[Xaa~n is Met or Leu;
S [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;
[Xaa3r is Leu or Val;

10 or a homologue, chemical analogue or de iv~Liv~ thereof.

More particularly there is provided a isolated molecule desi n~ted MIEIA having a amino acid seqll~nre subst~nti~lly as set forth in SEQ ID NO:2 or having at least 40% similarity to all or part thereof or a homologue, Ghemical analogue or deliv~ive thereof.
A related aspect of the present invention is directed to a isolated molecule ~ign~ted MIHB
having an amino acid sequence substantially as set forth in SEQ ID NO:4 or having at least 40% ~imil~rity to all or part thereof or a homologue, chemical analogue or deliv~tive thereof.

20 Yet a further related aspect of the present invention relates to a isolated molecule d~ignz~ted MIHC having an amino acid sequ~nce ~ul~s~ tly as set forth in SEQ ID NO:6 or having at least 40% simil~rity to all or part thereof or a homologue, chemical analogue or deliv~live thereof.

25 Still a further related aspect of the present invention provides a isolated molecule d~ci~n~te(l DIHA having an amino acid seq~l~nce ~ul, ~ ly as set forth in SEQ ID NO:8 or having at least 40% simil~rity to all or part thereof or a homologue, chemical analogue or de~ivalive thereof.
-30 Another related aspect of the present invention provides a isolated molecule d~:sign~ted DIHA

CA 02241129 1998-06-l9 having an amino acid sequence ~ul,sl~Lially as set forth in SEQ rD NO: 11 or having at least 40% .eimil~rity 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 d~iEl-~ted DIHA having an ~mino acid sequence ~bsLi~lially 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 deliv~liv~
thereof.

A further related aspect of the present invention provides a isolated molecule ~esign~ted DIHA
10 having an amino acid sequence substantially as set forth in SEQ ID NO:15 or having at least 40% .eimil~rity to all or part thereof or a homologue, chemical analogue or derivative thereof.

Yet another related aspect of the present invention provides a i.eol~ted molecule d~.ei~n~ted DIHA having an amino acid seq~lence subst~nti~lly as set forth in SEQ ID NO: 17 or having at 15 least 40% similarity to all or part thereof or a homologue, chemical analogue or deliv~Live thereof.

The present invention also ~~n~" "rA~ .e nucleic acid molecules encoding IAP homologues such as MIHA, MIHB, MIHC, ~, CIA-l, CIA-2, YIA-l and DIHA.
The LAP homologues may or may not bear RrNG finger dom~ine, for example, MIHA, MHB
and MIHC bear the domain whereas MIHE, CIA-l, CIA-2 or YIAY do not bear the dom~in The present invention further contemplates a nucleic acid molecule compri.ein~ a seq~l~nce of 25 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 [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 30 Xaa Pro Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys Xaa rXaa]q [Xaa]r CA 02241129 1998-06-l9 WO 97/23501 PCT/AU96~W~27 wherein Xaa is an amino acid residue;

[Xaa]m is a series of at least 5 and preferably at least 9 amino acids;
[Xaa]n is Met or Leu;
~Xaa]0 is a series of at least 3 and ~l~fel~bly at least 5 amino acids;
[Xaa]p is Val or Ala;
~Xaa]q is Phe or Tyr;
~Xaa]r is Leu or Val;

or having at least 40% ~imil~nty to a nllrleotitle seqllPnre encoding the above-mentioned amino acid sequence.

15 ID particular, the present invention co~ AtPc a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence subst~nti~lly set forth in SEQ ID NO:2 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence subst~nti~lly set forth in SEQ ID NO:2.

20 In a related embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence P.nco-lin~ the amino acid sequPnce substantially set forth in SEQ ID NO:4 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence subst~nti~ly set forth in SEQ ID NO:4.

2~ In another related embodiment, there is contPmp!~ted 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 seqllPnce Pnro-lin~ the amino acid sequPnce substantially set forth in SEQ ID NO:6.

30 A further embodiment is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence ~ub~k~Lially set forth in SEQ ID NO:8 or having at least 40% similarity to a nucleotide sequence encoding the amino acid sequence subst~nti~lly set forth in SEQ ID NO:8.

5 Another related aspect is di~ ;ted 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.

10 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 IO NO:13 or having at least 40% ~imil~rity to a nucleotide sequence encoding the amino acid sequence substantially set forth in SEQ ID NO: 13, 15 A fu,rther related aspect is directed to a nucleic acid molecule comprieinp: 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% sirnilarity to a nucleotide sequence encoding the amino acid sequence subst~nti~lly set forth in SEQ ID NO: 15.

20 Yet another related aspect is directed to a nucleic acid molecule co...r~ g 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% ~eimil~ity to a nucleotide sequence encoding the amino acid sequence ~ub~lially set forth in SEQ ID NO: 17.
25 In yet another embodiment of the present invention, there is provided a nucleic acid molecule coml-ri~ing 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 lD
NO: 1 under low stringency conditions.

30 In a related embodiment of the present invention, there is provided a nucleic acid molecule CA 02241129 1998-06-l9 c~mrricing a sequPnce of nucleotides sul,s~lially 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 conllition~

5 In a further related embodiment of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides sul~s~ Li~lly as set forth in SEQ ID NO:5 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybri-licin~ to SEQ D~ NO:5 under low stringency cont1ition~.

10 In a still a fur~er related embodiment of the present i~v~"lion, there is provided a nucleic acid molecule coml ri.qing a seqllence of nucleotides subst~nti~lly as set forth in SEQ ID NO:7 or having at least 40% simil~rity thereto or is a nucleic acid molecule capable of hybritiicing to SEQ ID NO:7 under low strin~ency cl n-liti~mc 15 In another further related embodiment of the present invention, there is provided a nucleic acid molecule comrricin~: a seql~n~e of nucleotides subs~ lly as set forth in SEQ ID NO: 10 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybri~ ing to SEQ ID NO:lO under low stringency c~ ntlition~

20 In a further related embodiment of ~e present invention, there is provided a nucleic acid molecule comrri.~in~ a seqll~n~e of nucleotides subst~nti~lly as set forth in SEQ ID NO: 12 or having at least 40% ~imil~rity thereto or is a nucleic acid molecule capable of hybri~ in~ to SEQ ID NO: 12 under low s~rin~ncy contlitit~n~

25 In a still a filrther related e_bodiment of the present invention, there is provided a nucleic acid molecule comrri~in~ a sequence of nucleotides substantially as set forth in SEQ ~ NO: 14 or having at least 40% similarity thereto or is a nucleic acid molecule capable of hybri~ in~ to SEQ ID NO: 14 under low stringency con~ition~
-30 In yet another related embodiment of the present invention, there is provided a nucleic acid molecule c~ mprixqin~ a sequence of nucleotides substantially as set forth in SEQ rD NO: 16 or having at least 40% simil~rity thereto or is a nucleic acid molecule capable of hybridising to SEQ ID NO:16 under low stringency conAitionq 5 Reference herein to a low strin~ncy at 42nC inrll-d~q and encomp~x.ses from at least about 1%
v/v to at least about 15% v/v form~mide and from at least about lM to at least about 2M salt for hybridisation, and at least about lM to at least about 2M salt for washing conAhinnq~
AlL~lualive strin~oncy conAitinnq may be applied where necess~y, such as medium stringency, which inchld~$ and encompasses from at least about 16% v/v to at least about 30% v/v lO formamide and from at least about 0.5M to at least about O.9M salt for hybridisation, and at least about 0.5M to at least about O.9M salt for washing conditions, or high stringency, which inr.l~lAes and ~.nr~...p~qx~$ from at least about 31% v/v to at least about 50% v/v fnrm~miAe and from at least about O.OlM to at least about O.l5M salt for hybritliq~tion and at least about O.OlM to at least about 0.15M salt for washing conAitions.
The nucleic acid mc~lçc~ q are preferably in isolated form and/or carried by a vector molecule such as an ~ ession vector.

The IAP homologues and their d~livaLiv~q and chemical analogues of the present invention are 20 useful in inhibiting apoptosis and are useful in the treatment of diseases incluAin~ but not limited to those char~ct~ri~ed by apoplosis such as degenerative di~ç~q~q inchlAin~ ~17h~imer's disease~ motor neuron disease, n~u,upalhies; iqr.hemic vascular disease inclnAin~ stroke and myocardial infarction; infectious Aiqe~çs including Acquired Tmmllnn-Deficiency Syndrome caused by E~V. Certain deliv~ives of the homologues cont~mplated herein may promote 25 contPmpl~ted herein may promote apoptosis or inhibit anti-apoptotic processes and are potentially useful in the treatment of Ai.qç~q~q characterised by failure of apoptosis of certain cells incl~lAing but not limited to cancer or ~uloi,~ ..e disease, ~l7h~imer's and motor neuron diseases. Deliv~L~ives of the homologues cont~mplated herein many promote apoptosis or inhibit anti-a~ lic processes and are useful in the treatment of, for example, certain cancers 30 or in the promotion of the death of mali~n~nt cells.

CA 02241129 1998-06-l9 W O 97/235~1 PC~AU9Ci~a~?7 Deliv~ives of the homologues of the present invention include ~ , parts and fr~gment~
of the homologue molecules as well as single or multiple amino acid ~ul~ 1;one, deletions and/or addition~ to the homologue molecules. Nucleic acid de~iv~ives including single or multiple nucleotide substitutions, deletions and/or ad~iiti~n~ to the nucleic acid molecules as 5 defined in SEQ ID NO: 1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ IDNO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.

Chemical ~n~ln~lP.~ of the baculovin~s IAPs or of their homologues incllldP; but are not limited to, mo-lific~ti()n~ to side chains, incol~ ;nn of Datural amino acids and/or their deliv~iv~s 10 during peptide synthesis and the use of cro.~slink~r.~ and other methods which impose collÇo""~tionAl col~Ll~ on the peptides or their analogues.

Examples of side chain mo-lificAtinns contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with a aldehyde 15 followed by reducti--n with aBH4; ~miclinAtiQn with methyl~cetimi~Ate; 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 pyridoxal-5 '-phosphate followed by reduction with aB~4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic con~n~tion products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The c~l,u~yl group may be modified by carbodiimide activation via O-acylisourea form~ti~m 25 followed by subsequent delivi~ AI;on, for example, to a collespollding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodo~cet~mide; p~lrolmic acid o~ tion to cysteic acid; f~rm~tion of a mixed - dislllphi~ with other thiol compounds; reaction with m~l~imitl~, maleic anhydride or other 30 substituted maleimide; form~ti~n of mercurial deliv~Lives using 4-chloromercuribezoate, 4-W O 97~3501 PCT/AU96/00827 chlorome~ heyl~ llrh~)nic acid, phenylmercury ~l~lori~i~, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at ~Ik~line pH.

Tryptophan residues may be modif1ed by, for example, oxidation with -brnmosllccinimide or 5 alkylation of the idole ring with 2-hydlu~y-5-nitrobenzyl bromide or sulph~nyl halides.
Tyrosine residues on the other hand, may be altered by nitration with t~LI~iLlvlllethane to form a 3-niLIvly~vsine delivaLive.

Modification of the imidazole ring of a hisitidine residue may be accomplished by aLkylation 10 with iodoacetic acid dt;liva~ives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incol~vl~ g unnatural amino acids and deliv~ives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-~minnh~xanoic acid, t-butylglycine, norv-aline, phenylglycine, 15 nnrithinP; sarcosine, 4-amino-3-hy-Lv~y-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids is provided in Table 1.

W O 97/23501 P~TJAU96/~0827 Non-conventional Code Non-co.,v~ ion~l Code amino acid amino acid oc-aminobutyric acid Abu L-N-methyl~l~nine Nmala a-amino-a-methyll~ulyl~le Mgabu L-N-methylarginine Nmarg aminocyclopl~palle- Cpro L-N-methylasparagine Nmasn carboxylate L-N-met_ylaspartic acid Nmasp 10 aminoisobutyric acid Aib L-N-methylcysteine Nmcys ~min~-n~rbornyl- Norb L-N-methyl~h~ P, Nmgln carboxylate L-N-methylglut~mic acid Nmglu cyclohexyl~l~nine Chexa L-N-methylhi~tit1ine Nmhis cyclopentyl~l~nine Cpen L-N-methylisolleucine Nmile 15 D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmetllir)nine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-~hlt~mine Dgln L-N-methylnorvaline Nmnva 20 D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine D_is L-N-methylphenyl~l~nine Nmphe D-isoleucine Dile L-N-m~;lhylpl~line Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-me~ yl~leol~ine Nmthr 25 D-methionine Dmet L-N-me~ylLIy~ophan Nmtrp D-ornithine Dorn L-N-methyl~ylusine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug 30 D-threonine Dthr L-norl~ cine Nle D-llyp~ophan Dtrp L-norvaline Nva D-tyrosine Dtyr a-methyl-aminoiso~uLy~ale Maib D-valine Dval oc-methyl-y-amino~ulyl~le Mgabu D-~c-methylala~ne Dmala a-methylcyclohexylalanine Mchexa 5 D-oc-methylarginine Dmarg oc-methylcylcopentyl~l~nine Mcpen D-a-methylasparagine Dmasn oc-methyl-a-napthyl~l~nine Manap D-oc-methylaspal~e Dmasp a-mc;~ylpel~icill~mine Mpen D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-a-methylgl~lt~mine Dmgln N-(2-aminoethyl)glycine Naeg 10 D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-a-methylisoleucine Dmile N-amino-~c-methyll)uLyl~le Nmaabu D-a-methylleucine Dmleu cc-napthylalanine Anap D-a-methyllysine Dmlys N-benzylglycine Nphe D-a-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln 15 D-a-methy!o, .,;~ e Dmorn N-(carbamylmethyl)glycine Nasn D-a-methylphenyl~lAnine. Dmphe N-(2-carboxyethyl)glycine Nglu D-a-m~ ylpl~line Dmpro N-(carboxymethyl)glycine Nasp D-oc-methylserine Dmser N-cyclobutylglycine Ncbut D-a-mellyltLr~ol-il-e Dmthr N-cycloheptylglycine Nchep 20 D-a-m~ ylllyplophan Dmtrp N-cyclohexylglycine Nchex D-sc-me:Lhyllylusille Dmty N-cyclodecylglycine Ncdec D-a-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methyl~l~nine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cycloplopylglycine Ncpro 25 D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylasp~ ~le Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphel,yl~lu~3yl)glycine Nbhe D-N-methy!gll1t~mine Dnmgln N-(3-~ni~ pr~pyl)glycine Narg D-N-methyl~ t~m~te Dnmglu N-(l-hyL~xy~lyl)glycine Nthr 30 D-N-methylhi~ti~ine Dnmhis N-(hydluxy~ yl))glycine Nser WO 97~3501 PCT/AU96/00827 D-N-methylisoleucine Dnmile N-(imid~ lylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolyly~yl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobuLyl~Le Nmgabu N-methylcyclohexyl~l~nine Nmchexa D-N-methylmetllionine Dnmmet 5 D-N-methylollli l~e Dnmorn N-methylcyclopentyl~l~nine Nmcpen N-methylglycine Nala D-N-methylphenyl~l~nine Dnmphe N-methylaminoisolw~yl~Le Nmaib D-N-m~yl~)loline Dnmpro N-(l-m~l~ylplupyl~glycine Nile D-N-methylserine Dnmser N-(2-me~llyl~ yl)glycine Nleu D-N-m~yl~l~;onil:le Dnmthr 10 D-N-m~ llylLIy~l~Jphan Dnmtrp N-(l-methylethyl)glycine Nval D-N-melllyllylosLlle Dnmtyr N-methyla-napthyl~l~nine Nmanap D-N-metnylvaline Dnmval N-methylpenicill~mine Nmpen y-aminobutyric acid Gabu N-~-hydr~yphenyl)glycine Nhtyr L-f-butylglycine Tbug N-(thiomethyl)glycine Ncys 15 L-ethylglycine Etg p~.nicill~rnine Pen L-homophenyl~l~nine Hphe L-a-methyl~l~nine Mala L-a-methylarginine Marg L-a-methylasparagine Masn L-a-methylasp~ k~le Masp L-a-methyl-t-butylglycine Mtbug L-a-methylcysteine Mcys L-methylethylglycine Metg 20 L-a-methylglllt~mine Mgln L-a-methylglllt~m~te Mglu L-a-methylhistidine Mhis L-a-methylhomophenylalanine Mhphe L-a-m~ ylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-a-methylleucine Mleu L-a-methyllysine Mlys L-a-methylmethionine Mmet L-a-methylnorleucine ~le 25 L-a-methylnorvaline Mnva L-a-methylornithine Morn L-a-methylphenyl~l~nine Mphe L-a-me llylproline Mpro L-a-methylserine Mser L-a-melllyl~llreolline Mthr L-a-m~llylllyl,lo~han Mtrp L-a-methyltyrosine Mtyr CA 02241129 1998-06-l9 L-cc-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-~3,3-diphe~yl~lopyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine l-carboxy-1-(2,2-diphenyl- Nmbc 5 ethylarnino)cyclopl()paL~e Crnselink~re can be used, for example, to stabilise 3D co~-ro, ~ "~tion.e~ using homo-bifuctional croeelink~rs such as the bifimrtinn~l imido esters having (CH2) spacer groups with n=l to n~6, 10 glutaraldehyde, N-Ly~ y~uccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydrv~yxl~cçinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In lition, peptides can be cOllro~ tion~lly constrained by, for example, incol~ol~ion of C~
and N~-methylamine acids, introduction of double bonds between C~ and C~ atoms of amino 15 acids and the form~tion of cyclic peptides or ~n~lo~l~,e by introducing covalent bonds such as forming a amide bond between the and C t~rrnini~ between two side chains or between a side chain and the N or C trrmin~le The present invention further cc ~ plalt;s a method for mo~ tin~ cell apop~sis in a animal, 20 said method co...l,,;xi~ lmh~ix~ g to said animal a cell apoptosis modlll~tin~ effective amount of a hnmolo~le or rhrmir,~l~n~ e of a baculovirus LAP or a d~liv~live thereof for a time and under cnnClitinnx sufficient for said cell apoptosis to be modlll~ted The terms ''modlll~tingll or "modulated" refer to the promotion or enhancement of cell 25 apoptosis or the drmiml~ti(-n, inhibition or re~llctinn of cell apoptosis. Pnh~ncing cell apoptosis may be i.l.polL~l~ in treating certain cancers and cell m~lign~ncies; recl~lr,in~ cell ~o~osis may be important in the treatment of degenerative disorders such as n~;;ulopa 3~ies and ~ hPimer's and motor neuron diseases.

30 Accordingly, the present invention is also directed to th~ ulic and ph~rm~ce~ltic~

WO 97/23501 PCT/AU96/'~0~ 7 compositions useful for mot~ Ating cell apoptosis in a animal.

The form~ti~ n of phArmA~fil.Al composiLions is generally known in the art and reference can collv~iellLly be made to P~e~mington~s PhArmAce~tical Sciences, 17th end., Mack Publishing S Co., Easton, P~n~yl~ia, USA.

The present invention, therefore, contemplates a ~hArmAce~Ttical co~ o~ilion compri~in~ an apoptopic mo~ tin~ effective arnount of an IAP homologue as hereinbefore defined or chemical analogues or deiivaLives thereof and optionally inçlut1in~; one or more other active 10 moleclllp~ and one or more rhArmAcelltically acceptable carriers and/or r~ lPnti The active ingredients of a phArmAce~ltical composition co~ in~ the IAP homologues or theird~livalives are contemplated herein to exhibit excellent the ~pelllic activity, for example, in mo~l..lAtin~ ap~Losis of animal cells when a~lminict~red in an amount which depends on the particular case. For example, from about 0.5 ~g to about 20 mg per kilogram of body weight 15 per day may be ~Atlmini~tPred. Dosage regime may be adjusted to provide the opLi~ulll therapeutic l~ollse. For example, several divided doses may be A.-lmini.~tP.red daily, weekly or monthly, or the dose may be pl.,polLionally reduced as indicated by the eYig~-nçies of the therapeutic .ci~l~Ation The active compounds may be A.-lmini~t~red in any convenient manner such as by the oral, i~.Ll~v~i~ous (where water soluble), intr~ml-~cllAr, subcutaneous, 20 intranasal, intradlo.rm~l or ~uppoSi~uly routes or by implanting (eg using slow release molecules), topical Arlmini~tration or following or during surgery or biopsy or other hlv~ive procedure. Depending on the route of ~rlmini~fration, the active ingredients which comrri~e the IAP homologues or chemical analogues or dtlivaLives may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural 25 c~ntlition~ which may inactivate said ingredients. In order to arlmini.~Pr IAP homologues by other than p~t;nLel~l A-lmini~tration, they will be coated by, or al1mini.ctered with, a material to prevent its inactivation. For ~ plr; homologues may be a~lmini.~t~red in an adjuvant, co-A~ ed with enzyme inhibitors or in liposomes. Adjuvants contemplated by the present invention incllldr, but are not limited to, cytokines (e.g. interferons) as well as resorcinols, 30 non-ionic s~ ctAnt~ such as polyo~y~hrl~nr oleyl ether and n-h~YA-lecyl polyethylene ether.

The active colll~uul~ds may also be a~iminictlo.red pal~Lt;l~lly or inLIap~ ul~e~lly. Dispersions can also be ple~ d in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary con~itinnC of storage and use, these prep~lions contain a pres~,v~livd to plt;velll the grovvth of microorganicm.c.
s The ph~rm~r~ltical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the ci~Lt;.,.l)ol~eous pl~;pa~Lion of sterile injectable sollltionc or dispersion. In all cases the form must be sterile and must be fluid to the e~tent that easy syringability exists. It must be stable under the contiitinns of m~mlf~ct lre 10 and storage and must be preserved against the cont~min~ting action of microorg~nicmc such as bacteria and fungi. The carrier can be a solvent or dispersion medium co,.~ for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liq~lid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be m~int~ined, for example, ~y the use of a coating such as lecithin, by the 15 maintenance of the required particle size in the case of dispersion and by the use of superf~ t~nt~. The plt;v~ ions of the action of microor~ni~m~ 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 ~hlnride Prolonged absorption of the injectable 20 compositions can be brought about by the use in the compositions of agents delaying absorption, for example, ~ mini~lm monosleaL~le and gelatin.

Sterile injectable sol~ltinn~ are prepared by il~col~u~ g the active compounds in the required amount in the a~~ .;ale solvent with various of the other ingredients enumerated above, as 25 required, followed by filtered sterili~tit)n Generally, dispersions are prepaled by incul~ul~ing the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the l~.lired other ingredients from those enumerated above. In the case of sterile powders for the prepa ~lion of sterile injectable solutions, the preferred methods of p,~Lion are vacuum drying and the freeze-drying technique which yield a 30 powder of the active ingredient plus any additional desired ingredient from previously WO 97/23501 PCT~AU96~00827 sterile-filtered solution thereof.

When the IAP homologues or çhP.mir.~l analogues or d~liv~Lives are suitably protected as described above, the active, c~ ulli~l may be orally ~-lmini~t~red, for example, with an inert 5 diluent or with a ~imil~ble edible carrier, or it may be enclosed in hard or soft shell gelatin ç~r~lllr; or it may be coml.l~sed into tablets, or it may be incolpor~Led directly with the food of the diet. For oral therapeutic ~lministration, the active compound may be inco~ led with ~Ycirient~ and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, s~l~pPI~iol~ syrups, wafers, and the like. Such composition~ and p.~Lions should 10 contain at least 1% by weight of active compound. The percentage of the compositions and pl~dl~Liorls may, of course, be varied and may co,.v~;"iel,~y be between about S to about 80%
of the weight of the unit. The amount of active co~poLI~d in such th~l~p~uLically useful compositions in such that a suitable dosage will be obtained. Preferred composition~ or pr~Lions according to the present invention are plepared so that a oral dosage unit form 15 contains between about 0.1 ~g and 2000 mg of active colupuulld.

The tablets, troches, pills, r,~p.~lllP~ and the like may also contain the following: A binder such as gum tr~g~r~nth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a ~licinte~rating agent such as corn starch, potato starch, alginic acid and the like; a lubricant 20 such as m~gnP~i-lm ~ le; and a sweele~ agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppt;lminL, oil of w,l~Lel~leell, or cherry flavouring.
When the dosage unit form is a capsule, it may contain, in ~dtlitit~n to m~tPri~l~ of the above type, a liquid carrier. Various other m~t~.ri~l~ may be present as co~ting.~ or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or r~rSl~ may be 25 coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a swt;e~ agent, methyl and ~l~ylpal~bens as pres~lvaLivts, a dye and flavouring such as cherry or orange flavour. Of course, any m~t~ri~l used in pl~a iug any dosage unit form should be ph~rm~ceutically pure and substantially non-toxic in the amounts employed. In ~d-lition, the active compound may be incol~ol~led into s l~t~ined-release 30 pl~ Lions and form~ tinn~

CA 0224ll29 l998-06-l9 A rh~rm~r,~ltir,~lly ~ccept~hle carrier and/or diluent inclll~es any and all solvents, dispersion media, co~tinge~ antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for ph~rm~re~1tir.~1 active substances is well known in the art. Except insofar as any couvt;l~ional media or agent is incoL,lp&Lible with the 5 active ingredient, use thereof in the therapeutic comrositi~ns is co~ plated. Suppl~m~nt~ry active ingredients can also be incol~ol~ted into the compositions.

It is especially adv~nt~geQ le to form~ te p~wlL~ ,ro~; I;one in dosage unit form for ease of arlmini.ctration and uniformity of dosage. Dosage unit form as used herein refers to 10 physically discrete units suited as unitary dosages for the m~mm~ n subjects to be treated;
each unit coating a pled~;;Lr~ "~ u~LiLy of active material c~lcul~ted to produce the desired therapeutic effect in association with the required pharm~relltic~l carrier. ~he 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 th~pt;ulic effect to be 15 achieved, and (b) the 1;1ll;l;~ inherent in the art of coLupuui~ding such a active material for the tr~tment of disease in living subjects having a ~liee~ed condition in which bodily health is Llpail ed as herein disclosed in detail.

The prinr.ir~l active ingredient is compounded for convenient and effective ~tlmini~etration in 20 effective amounts with a suitable ph~rm~celltir.~lly acceptable carrier in dosage unit form as hereinbefore di.eclosed A unit dosage form can, for example, contain the princir~l active coLIlp.~ d in ~molmte ranging from 0.5 ~lg to about 2000 mg. Expressed in pl~ po~ ~ions, the active compound is generally present in from about 0.5 ,ug to about 2000 mg/ml of carrier.
In the case of compositions coating supplementary active ingredients, the dosages are 25 det~rmined by reference to the usual dose and manner of ~rlminietration of the said ingredients.

The ph~rm~celltical composition may also coL~p,ise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable 30 of mod~ ting IAP homologue eA~lession or L~P homologue activity. The vector may, for WO 97/23501 PCTlAUg6~00827 example, be a viral vector.

Another aspect of the present invention col~l~plates the use of an animal cell homologue of an baculovirus IAP to morl~ t.o. ~u~osis in ~n;m~ ,urrt;lih.g firom a degeneld~ivci disease, 5 an infectious disease, cancer or an autoimml-ne disease.

F~rt;ldbly, 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: 17or is a d~ liv~ or chemir.~l analogue thereof.
The present invention further conlelllplates antibodies to the IAP homologues and the deliva~ives of the present invention. Antibodies are useful in ~ gnostic assays for the h~m~ln~l~,c as well as ~ irj,llg the homologues or isolating the homologues from biological fluid or culture medium. Such antibodies may be monoclonal or polyclonal and may be 15 selected from naturally occ~nTin~ antibodies to an L~P homologue or may be specific~lly raised to an IAP homologue or a de~iv~live thereof. In the case of the latter, an IAP
homologue or its d~liv~Lives may first need to be associated with a carrier molecule. The antibodies and/or recombinant IAP homologue or its deliv~lives of the present invention are particularly useful as therapeutic or f~ nostic agents.
For example, an IAP homologue and its deliv~lives can be used to screen for naturally oc~rring antibodies to the homologue. These may occur, for example in some autoimmune diseases. ~lt~rn~tively, specific antibodies can be used to screen for an IAP homologue.
Techniques for such assays are well known in the art and incll-de, for example, sandwich 25 assays and ELISA. Knowledge of IAP homologue levels may be hllpollall~ 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.
Al~ll~Lively, fr~ents of antibodies may be used such as Fab fragments. Furthermore, the 30 present invention extends to recombinant and synthetic antibodies and to antibody hybrids.

A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.
The antibodies of this aspect of the present invention are particularly useful for immlmntherapy and may also be used as a rli~gnostic tool for ~ ing apoptosis or monitoring the program of a the~ ;uLic regim~n 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 l~u-iry-~g IAP homologue made by recombinant means from culture sup~ "l fluid. Techniques for the assays cont~ plated herein are 10 known in the art and in~.h~(lP; 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 f~st and second antibodies may be used in detectinn 15 assays or a first antibody may be used with a commercially available anti-imml-noglobulin antibody. An antibody as conl~...plated herein inçl~l~lP~ 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.

20 Both polyclonal and monoclonal antibodies are obtainable by i.. ""~ ion with the enzyme or protein and either type is utilizable for ;.. ~ y~. The me~ho~ of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily pre~ed by injection of a suitable laboratory animal with an effective amount of IAP
homologue, or ~ntigl~.nic parts thereof, c llecting serum from the animal, and isolating specific 25 sera by any of the known immlmr ~ orbent te~hni~l~. Although antibodies produced by this method are utilizable in virtually any type of immllnnassay~ they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immlm- ~s~y is pa~ticularly preferred because of the 30 ability to produce them in large quantities and the homogeneity of the product. The W0 97/23501 PCT/AU96/00~27 l~,~al~lion of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sen.~iti7ed against the immlmt)genic plel~al~ion can be done by t~s~.hni~x which are well known to those who are skilled in the art.

S Another aspect of the present invention co~ tes a method for detecting an LAP
homologue in a biological sample from a subject said method comp.isi~,g c~nt~ctin~ said biological sample with an antibody specific for the IAP homologue or its deliv~ es or homologues for a time and under conclitionx sllfifici~nt for an antibody-IAP homologue complex to form, and then det~cting said complex.
The presence of IAP homologue may be accomplished in a number of ways such as byWestern blotting and ELISA procedures. A wide range of immtm-~x.~y 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, inch~ both single-site and two-site or "sandwich" assays of the non-15 competitive types, as well as in the tr~(lition~l co-~lpelilive 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 v~ri~tiQns of the sandwich assay technique exist, 20 and all are int~n~lell to be ~.nf~ p~.~;xed by the present invention. Briefly, in a typical folw~ud 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 fo~m~tion of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a 25 detectable signal is then added and in~lb~te-l allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted m~pri~l 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 ~luS~ ed by compalillg with a control sample cont~ining known 30 amounts of hapten. V~ri~tion~ on the rOl w~ud assay include a ~imlllt~n~Qus assay, in which both sample and labelled antibody are added ~imlllt~neously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily al~p~. In accordance with the present invention the sample is one which might contain an IAP homologue inclllrlin~ a cell extract, tissue biopsy or possibly serum, saliva, 5 mucosal secretions, lymph, tissue fluid and l~;S~ Oly fluid. The sample is, therefore, generally a biological sample co.~pl;~ biological fluid but also extends to fermentation fluid and sup~m~t~nt fluid such as from a cell culture.

In the typical L~lW~lld sandwich assay, a first antibody having specificity for the IAP
10 homologue or ~nti~Pnic 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 celll-l~ sP; polyacryl~mide, nylon, poly.,lyl~e, polyvinyl chloride or polyplupylene. The solid ~;U~Ul l~ may be in the form of tubes, beads, discs of microplates, or any other surface suitable for con~lct;ng an immimo~ y The binding processes are well-known in the art and 15 generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in p~ ion for the test sample. An aliquot of the sample to be tested is then added to the solid phase comrlPY and ;~ b~ed for a period of time sufficient (e.g. 2~û minlltp-~) and under suitable con~lition~ (e.g. 25 ~C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is 20 washed and dried and in~lb~ted with a second antibody specific for a portion of the hapten.
The second antibody is linked to a ~ )ollel molecule which is used to indicate the binding of the second antibody to the hapten.

An ~ltP.m~five method involves immobilizing the target molecules in the biological sample and 2~ then ~osi,-g the immobilized target to specific antibody which may or may not be labelled with a l~c,llt;l molecule. Depending on the amount of target and the strength of the lepol ~el molecule signal, a bound target may be detect~hle by direct labelling with the antibody.
Alt~ ivt;ly, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The 30 complex is ~letected by the signal emitted by the lepollel molecule.

By "le~ul~ mnle~lle" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically icl~ntift~ble signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or q~ ve. The most comm- nly used lel)olLel molecules in this type of assay are either enzymes, fluorophores or S r~Ai-ml~ lide cont~ininF molecules (i.e. radioisotopes) and chemill-min~c~nt molecules.
In the case of an enzyme immlm~ ~eeay, 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 teçhniqll~.e exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, 10 beta~ ctn.ci~ e and alk~line phosph~t~ee amongst others. The substrates to be used with the specific enzymes are generally chosen for the productic-n, upon hydrolysis by the co~ )ding enzyme, of a detectable colour change. Examples of suitable enzymes include ~Ik~line phosphatase and peroxi(l~e It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all 15 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 cont~ining the applcpliate ~ul ~LI~e is then added to the complex of antibody-antigen-antibody. The ~uL,sLI~k; will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further ~ eA usually spectrophotometrically, to give an indication of the amount of~0 hapten which was present in the sample. "Reporter molecule" also extends to use of cell n or inhibition of ~ such as red blood cells on latex beads, and the like.

~ltern~tlo.ly, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illllmin~tion 25 with light of a particular wavel~n~h, the fluorochrome-labelled antibody adsorbs the light energy, ind~çing a state to excitability in the molecule, followed by ~mi~inn of the light at a characteristic colour visually ~etect~ble 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 rPm~inin~ tertiary complex is then exposed to the light 30 of the applupli~ wavelength the fluorescence observed indicates the presence of the hapten of interest. Tmmllnc)fl~ rescene and EIA techniques are both very well established in the art and are particularly pr~;rt;lled for the present metht~l However, other l~)Ul Lel molecules, such as radioisotope, rhPmilllminescent or bit lllminP.scPnt molecules, may also be employed.

5 The present invention also co..~ lates genetic assays such as involving PCR analysis to detect IAP homolgoue gene or its deliva~ives. ~ ;ve methods or methods used in conjunction include direct nucleotide sequencing or mutation sç~nnin~ such as single stranded conrnl ",~ti~n polymorphoms analysis (SSCP) as specific oli~om-~leotide hybridisation, as methods such as direct protein tluncation 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 dedllced peptide seq~lpnr~p~ of IAP proteins.
(A) Comr~ri~on of MIHA (SEQ ID NO:2), MIHB (SEQ ID NO:4), MIHC (SEQ D~
NO:6), and DIHA (SEQ ID NO:8). Amino acids shared by three or more of the proteins are hi~hlighte~ Arrows in(li~te the three BIRs. The RING finger domain is indicated by a dashed arrow.
(13) Comparison of BIRs of IAP proteins. Strongly conserved residues are highlighte~l CPI derives from Cydia pomonP.ll~ m-losi~ virus; OpIAP derives from Orgyia pseudotsugata PV; CiIAP derives from Chilo iri~PAC~Pnt virus; AIP is a c~n~lid~te gene for SMA; AcIAP derives from Autographa C~lifornic~ PV; ASV derives from IAP-like sequences from African swine fever virus.
(C) Comp~r~ n of RrNG finger motifs of IAPs and other proteins. Strongly conserved residues are highli~hte(l CRAF (TRAF3) and TRA~2 are TRAF family members;
BCRAl and RAGl are two m~mm~ n proteins with RING finger motifs; c-CBL is a cellular oncogene and SLI-l is its C. elgans homologue; cpi derives from Cydiapomonella gr~mlln~i~ virus; OpL~P derives from Orgyia pseudotsugata PV; CiIAP

W O 97/235~ PCT/AUgC/~ - '7 derives from Chilo iri~lP-~c~nt virus, AcIAP derives from Autographa californica PV;
UKMT derives from the Drosophila Un*empt gene; EctV derives from the p28 proteinof Ectromel;a Virus.

5 Figure 2 is a photographic re~l~e~ ion ~1WWi~Z3 m~mm~ n IAP homologues are expressed in a variety of tissues.
(A) An adult mouse tissue total RNA Northern blot was probed with the mMlHA cDNAcoding region at high stringency. This Northern was also probed with GAPDH as a indicator of each lane's k ~flin~
10 (B-D) A adult mouse tissue poly (A)+ RNA Northern blot (CLONTFCH) was probed with the ~MIHB CDNA coding region ( B) and the hMIHC CDNA coding region (C) at low strin~P.nt~,y, Figure 3 is a graphical repres~nt~tinn showing that MIHA and MI~IB protect against death 15 in~llced by ~ver~Al~lession of ICE, but not FADD.
(A) Tnr~ n of a~o~.Losis by L,~re-,Lion with ICE. Columns 1-6 (In black) in~lir~te the p~LcenLage of dead cells cotransfected with p32ICE-lacZ fusion plasrnid and the pl~mi~ bearing either the IAP homologues or controls. Death of cells coLLansrected with lac~ only together with the same test plasrnids is show in colurnns 7-12 (in white) and indicates the amount of cell death due to the transfection procedure itself.(B) Tnd~1ctic)n of apopL~sis by transfection with FADD. Plasmids encoding the MIH
proteins were coL~ s~ected with a lacZ vector and a construct bearing the FADD
coding region (columns 1-6). As with the ICE tA~nl ;...P.nt, ba~h~l.,ulld death was m~nit ~red in a parallel set of cultures (columns 7-12).
Figure 4 is a s~hem~tic repreS~nt~tion 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 in-lic~ted by a quto-~tion mark. Spontaneous self-association of molecules such as TRAFs and 30 death-domain bearing proteins may generate a intrin~ic activation signal that in normal W O 97~350t PCT/AU96/00827 circumstances 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 pl~,teins may act to decrease the spontaneous activation signals to various extents depending on their affinity for their target(s). According to this 5 model, OpIAP may be able to offer greater protection against FADD than the m~mm,qli~n 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 apop~osis on matter how ICE is a~liva~ed.

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

Figure 6 is a representation of the nucleotide sequence and collespol-ding amino acid 15 sequenceofMIHB.

Figure 7 is a reprP.~nt~tion of the nucleotide sequence and corresponding amino acid sequence of MIHC.

20 Figure 8 is a repr~s~nt~tion of the nucleotide sequence and coll~s~Jol-ding amino acid sequence of DIHA.

Figure 9A and 9B show protection by MIHA, MIHB and M~IC against death incluced by over t;~-ylcission of ICH-l protease.
2~
Figure 10 is a photographic represçnt~tion of a Western blot of 293T cells transfected with MIHA constructs.

WO 97/23501 PCT/ATU~6~'~l Y'7 The following single and three letter abbreviations are used for amino acid residues:

5 Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala A
Arginine Arg R
10 Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Gl~1t~mine Gln Q
(Tlllt~mic acid Glu E
15 (~lycine Gly G
~i~ti~ine His H
Isoleucine Ile Leucine Leu L
Lysine Lys K
20 Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
25 Tly~luphall Trp W
Tyrosine Tyr Y
Valine Val V
Anyresidue Xaa X

W 0 97/23501 PCT/AU96tO0827 A summary of SEQ ID NOs is as follows:

5 SEQ ID NO: DESCR~TION

Nucleotide sequence of MIHA
2 Amino acid sequence of MIHA
0 3 NucleotidesequenceofMIHB
4 Amino acid sequence of MI~B
Nucleotide sequence of MIHC
6 Amino acid sequence of MIHC
7 Nucleotide sequPnce of DIHA
8 Amino acid sequence of DIHA
9 Concensus amino acid sequence Nucleotide sequP!nce of MIHE
11 Amino acid sequence of MIHE
12 Nucleotide sequence of CIA-l 13 Amino acid sequence of (: IA- 1 14 Nucleotide sequ~n~e of CIA-2 Amino acid sequence of CIA-2 16 Nucleotide sequence of YIA-l 17 Arnino acid sequPnce of YIA-l 18 Amino acid sequence of MIHA peptide CA 02241129 1998-06-l9 WO 97/23501 PCT~AU96/00827 EXAM[PLE 1 cDNA CLONING
The GenBank database was searched using the GCG Sequence Analysis Software Package ~Madison, WI) for tr~n.~l~ted sequences resembling the OpIAP BIR alld RING finger amino 5 acid motifs. A human X chromosome genomic STS thus identified (GPnR~nk T .~4579~ was used to design PCR primers fl~nking the pul~live RING ~mger motif. This region was amplified and used to scree a human g~nnmic DNA library (Stratagene). A fragment isolated from this library was used to probe a mouse liver cDNA library (Skatagene) at low skingency yielding three murine cDNA clones that were d~i n~ted m~mm~ n IAP homologueA
10 (M~A). Tr~n.~l~tion~ ofthe human EST sequences GenBank:R19628 and GPnR~nk T96284 were found to resemble the BIR repeats of OpIAP. These sequences were used to design PC~
primers within their putative BIR ~lnm~in.~ which were used to generate probes that were used to scree a human fetal liver cDNA library (Stratagene). The hybridising cDNA clones were d~ ;n~t~-l MIEIB and MIHC, respectively.
The Drosophila genomic seq~l~.nce (G~nR~nk l~ROCCAAT) 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 20 isolated. For peptide sequence co ,p~isons (Figure 1) these residues declllced from the genomic sequence GenR~nk nROCCAAT were added.

EXAMPLI~ 2 RNA ANALYSIS
25 Radiolabelled mMIHA and GAPDH were hybridised at high stringency to a mouse tissue Northern blot bearing 5 ~g/lane of total RNA. A mouse multiple tissue Northern blot (CLONIECH) bearing 2 ,ug poly(A)+ RNA was probed with radiolabelled hMIHC at lowslrin~ency, stripped, probed with hMIHB at low stringency, stripped again and probed at high stringency to a ~-actin probe according to the m~mlf~c~lrer's instructions.

YEAST TWO-HYBR~) SYSIEM
The coding regions of M~IA, MI~ and MIHC were amplified by PCR using prirners inco-~u,~Ling an Eco~ site at the 5 ' end and a BamHI site at the 3 ' end (or a BglII site in the S case of MIHB), such that the proteins would be e~lcssed as in frame fusions with the GAL4 DNA binding dom~in The PCR products were the ligated into the pGBT9 vector (CLONTECH~. The OpIAP gene from the HindIII site 17 codons uy~LIea m of the ini~i~tin~
ATG was cloned into the p(~BT9 vector such that a in frame fusion would result. PCR-derived inserts were sequenced to check for misinco-~ol~Lions by Taq polymerase. TRAFl, 10 TRAF2 and TRAF3 c~.cission vectors have been previously described (Rothe ef al, 1994;
Hu et al, 1 994b). Vectors with the coding regions of c-jun in GBT9 and fos were used as controls for the ~etection of interacting proteins. The yeast strain ~7c was transformed with these plasmids using the lithium acetate protocol (Gietz et al, 1992).

TRANSIENT TRANS~ECTION ASSAYS
A 1.8 kb SacI-EcoRI fragment from a MIHA cDNA clone in pBluescript (Stratagene) was subcloned into pSP72 (Promega) to generate pSPM~IA, and recloned into pEF, a d~liv~Live of the pEFBOS vector (l~i~l~him~ and Nagata, 1990) as a 1.8 kb BamHI-~coRV fragment.
20 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 incl~lded at the ends of the primers for the cloning of these products into pEF.

The p32-lacZ filsion plasmid p~actMl lZ was as des~;lil)ed by Miura et al (1993). The FADD
25 ~ression construct FADD-AUl was as described by Chinnaiyan ef al (1995). The coding regions of bc1-2, crmA, p35 from AcPV, and IAP from OpPV were inserted into the pEF
vector. The tmncated OpIAP plasmid was constructed by digestion of the pEF vector coating full length IAP with ruI and SmaI, and re-li~tin~ This deleted sequences 3' of the ruI site in the OpIAP gene which encode the R~G finger dom~in WO 97/23501 PCT/A~J96~00827 EXA~LE S
CLONING OF DIHA, M~IA, MIHB and MIHC
Searches of GenBank revealed a Drosophila ~nnmic sequence (GenBank:DROCCAAT) that resembled baculoviral IAP genes. A Drosophila cDNA library was screened using PCR
5 generated probes and clones isolated which encodes this protein, d~sign~tetl herein Drosophila IAP homologue A (DIHA). The GenBank searches also revealed a number of m~mm~ n sequP.nrPA that resembled either the BIRs or the RING finger dc-m~in~ of viral IAPs. The first m~mm~ n IAP homologue idf~ntifi~d ~IA), was cloned using a PCR generated probe corresponding to a STS seguence on the X chromosome, HUMSWX595, which when 10 tr~n~1~te-l encoded a peptide resembling a baculoviral IAP RING finger motif. The il,ve~
isolated partial length human ~nnmic clones and mouse MI~IA cDNA clones coml)a~sillg the entire coding region. A further pair of closely related human IAP homologues (MlHB and MIHC) were isolated using PCR ~ el~Led probes co,lespol-ding to GenBank EST sequences Rl 9628 and T96284, which encode BIR-like motifs.
Figure lA CO~p~t::S the predicted amino acid sequences for DIHA (predicted molecular weight 55 kD), MIHA (56 kD), MIHB (70 kD~ and MIEIC (68 kD). Start codons were chose as the most 5 ' methionine with up~ alll, in frame stop codons. All four pl~ ~s bear three BIR repeats in the amino t~ormin~l half and a single RING finger domain close to the carboxy 20 tP-rminus. MIHB and MIHC are the most closely related, with 73% amino acid identity.
MIHA shares 43% identity and 62% .~imil~rity with MIHB and MIHC. DIHA has 36%
identity and 57% similarity to MIHC.

The BlR repeats of these proteins are co~-~paled to each other, the BIR repeats of AIP and the 25 viral IAPs in Figure lB. Among the m~mm~ n IAPs some of the BIRs resemble the~n~ ;ml~ BIR from another IAP more closely than t_ey resemble the rf~m~inin~ B~s in the same protein, indicating that duplication of a primordial BIR was a early event. Four of the most highly conserved residues in the B~s are three cysteines and a hi.ctitline in the pattern CX2CXI6HX6C. These may bind metal ions (Birnbaum et al, 1994).

CA 02241129 1998-06-l9 Figure 1C shows a ~ lph~ of the C3HC4 RlNG finger ~c m~in~ ofthe IAP molecules with RING f~gers from other selected proteins. The RING fingers from LAP proteins resernble each other more than they resemble the RING fingers from other proteins, such as TRAF2 (Rotheetal, 1994)andCRAF(Huetal, 1994b; Chengetal, 1995; Satoetal, l995),thatdo S not bear BIRs. The most similar RING fingers in o-BIR bearing ploLei~s are in the m~mms.li~n oncogene, c-Cbl ~3lake et al, 1991) and its C elgans homologue, Sli-l (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 e~s~nti~l for virulence (Senkevich et al, 1994). However, p28 does not have BIRs and its function is unknown.

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 ~letectecl was in the lung, with 15 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 mP.c~E~o.c of ~4.0 kb and ~5.5 kb on a mouse multiple tissue Northern blot (Figure 2B). The 20 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 ~ itiQn~l transcript of ~9.S
kb was detected in the testes but not see in other tissues.
A full length human MlHC cDNA probe hybridised at low str;n~nc,y to two messages of ~3 .0 and ~4.0 kb (Figure 2C). on 4.0 kb message was (letect~hle in the spleen, high levels were d~otected 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, 30 kidney and testes. As the hMIHB and hMIHC probes were hybridised at low stringency to W O 97n3501 PCT/AU96/00827 mouse RNA, it is possible the upper (4.0 kb) transcript is the same as that det~cted by the MIHB probe. Some of the transcripts may also represent other closely related m~mm~ n IAP homologues.

EXA~LE~ 7 PREVENTION OF ICE OR FADD ~DUCED APOPTOSIS
The m~mm~ n IAP homologues were tested for their ability to prevent apoptosis due to two stimuli,uvt~ AL3~ ionofp32ICEandov~w~ ~sionofFADD. Transfectionwithconstructs ~,Al"essi~ the Ced-3-like cysteine protease ICE have previously been show to cause apoptosis ~Miura et al, 1993). Baculoviral k4P can prevent this death, as can other anti-a~o~Losis genes such as bc1-2, crm~ and p35 (Miura et al, 1993; Xue et al, 1995). The illvenl~l~ tested MIHA, MrHB and ~HC to det~nnine whether they too could block a~o~losis caused by ICE
overt;A~ression. HeLa cells were cotransfected with a plasmid bearing a ICE-IacZ fusion construct together with pl~mit1.~ encoding the IAP homologues or controls. The cells were 15 stained with X-gal to identify those that hand been transfected, and these were ~s~ed visually for viability. As show in Figure 3A, MIHA, MIEIB and OpL~P signiflcantly reduced the arnount of death caused by ICE, whereas MIHC did not provide detect~ble protection.

~ .,l~,ed cAyl~ion of the Cd95 associated protein FADD also causes cell death (Chinnaiyan 20 et al, 1995; Boldin et al, 1995). HeLa cells were cotransfected with three plasmids: a FADD
expression construct, a ~ .cmid carrying the lacZ gene and vectors encoding the m~mm~ n L9P hr)m~ P.~ or OpL9P. 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 2). The illV~ n:~ could not detect any red~ )n in the amount of FADD indl~ced cell death by MIHA, 25 ~B or MIHC.

~NIERACTIONS WITH TRAF FAMILY MEMBERS
Two of the m~mm~ n IAP homologues, MIHB and MIHC, were isolated as part of a protein 30 complex that binds to the cytoplasmic domain ofthe TNF-R2 (p75) together with the TNF-R2 W O 97/23501 PCT/AU~/00827 associated molecules TRA~l and TRA~2 ~Rothe et al, 1994). Using the yeast two hybrid system (:Fields and Song, 1989), the three m~mm~ n IAP homologues and viral OpL9P were tested for their ability to bind TRAFl, TRAF2 and TRAF3, a related protein also known as CD40BP / CRAP-l / CAP (~Iu et al, 1994b; Cheng ef al, 1995; Sato et al, 1995) As show 5 in Table 2, yeast cotransfected with MIHB or MIHC and TRA~l 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 detect~hle in this system between any of the TRAFs tested and OpIAP or MIHA. These results show that MIHB and ~HC bind to TRAF 1 and TRA~2, but suggest tnat OpIAP and MIHA interact with other proteins.
I~XAMPLE 9 IDENTI~ICATION OF MIHE, CIA-l, CIA-2 and YIA-l A c ln~n~ sequence of baculovirus L9P repeats (BIR) was ded~lced as follows:
Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro [Xaa]m [Xaa]n Ala Xaa Ala Gly Phe [Xaa]O Asp Xaa [Xaalp 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]q [Xaa]r 20 wherein Xaa is an amino acid residue;

[Xaa]m is a series of at least 5 and preferably at least 9 amino acids;
[Xaa]n is Met or Leu;
[Xaa]O is a series of at least 3 and pl~r~ bly 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~AU~6~00827 - 37 ~

or a homologue, chemical analogue or d~ivalive thereof.

A database search using this con~ L sequence resulted in id~ntif c~tion of IAP homologes from mouse (M~), yeast (YIA-l) and C. elegans (CIA-l, CIA-2). The nucleotide and5 collespollding amino acid seqll~once from these L~P homologes is given in SEQ ID NOS: 10 to 17. I~ Lillgly, MIHE, YIA-l, CIA-l and CIA-2 do not bear RING finger ~lom~ins.

EXA~LE 10 PROTECTION BY M~IA, MIHB, and MIHC

Figures 9A and B shows the protection from MIHA, MIHB and MIHC against death in~luced by over t;~lession of the ICH-l protease.

WEST~RN BLOT

A polyclonal rabbit antibody was prepared and aff~ity purified against the MIHA peptide KDIKKTMEEKIQTSG (SEQ ID NO: 18). This antibody was used in a we~lelll blot of 293T
cells transfected infer alia MIHA. The results are shown in Figure 10.
Those skilled in the art will appreciate that the invention described herein is susceptible to v~ri~ti~ni and mo-lific~ti~ n~ other than those sper.ifir.~lly described. It is to be understood that the invention inrllld~ all such variations and modifications. The invention also incllld~,s all of the steps, features, compositions and compounds referred to or intli~ted in this 2~ specificati~ n, individually or collectively, and any and all combin~tic n~ of any two or more of said steps or features.

W O 97/23501 PCT/AU96/0~827 ~7east TWO flybrid Assays for r- ~ing Between TRAF1, TRAF2, TRAF3 and 7l~mm~ n IAP EIomologues Transformant Growth on Trp DNA binding Activation hybrid Leu- Eis- Colony hybrid medium Colour OpIAP TRAF 1 - -MIHB TRAF 1 + ++
MIHC TRAF1 + ++
cjun TRAFl OpIAP TRAF2 MIHB TRAF2 + +
MIHC TRAF2 + +
cjun TRAF2 - -OpIAP TRAF3 cjun TRA1~3 OpIAP fos MIHA fos c-IAPl fos MIHC fos cjun fos +

WO 97/23501 PCT/AU96~00827 The yeast strain ~7c was cul, ~ xr~ ...ed with constructs which express filsion 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 GALA activation dr~main Expression ~rom the his and lacZ l~)Ul~L genes ~which in~lic~tes interactions) was analysed by growt~
of double ll~sr.., l ~ ~ant.c on me~ lm lacking hi~ti~line (Colu_n 3), and blue staining of colr ni~,s, with X-gal (Column 4), c-jun andfos were used as controlgenes encoding interacting plo~eil~s in the DNA binding vector and activat,ion vector, respectively.

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W O 97/23~01 PCT/~U96~08Z7 SEQUENCE LISTING

(13 GENERAL INFORMATION:

(i) APPLICANT: AMRAD OPERATIONS PTY LTD
(US ONLY): DAVID LAURENCE VAUX

(ii) TITLE OF rNVENTION: THERAPEUTIC COMPOSITIONS

(iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DAVIES COLLISON CAVE
~B) STREET: 1 LITTLE COLLINS STREET
(C) CITY: MELBOURNE
(1:)) STATE: VICTORIA
(E) COUNTRY: AUSTRALIA
(F) ZIP: 3000 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk Q3) COMPUIER: IBM PC comIl~tihle (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT INTERNATIONAL
(B) FILING DATE: 20-DEC-1996 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: ~AU] PN 7275 ~13) FILING DATE: 22-DEC-1995 (viii) ATTORNEY/AGENT INFORMATION:
- (A) NAME: HUGHES DR, E JOHN L
(C) REFERENCE/DOCKET NUMBER: EJH/EK

CA 0224ll29 l998-06-l9 W O 97/23501 PCT/AU96/~0827 (i~) TELECOMMlJNICATION IN~Ol~MATION:
(A) TELEPHONE: +61 3 9254 2777 (B) TELEFAX: +61 3 9254 2770 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1988 base pairs (B) TYPE: nucleic acid (C) sTR~Mn~N~q~s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 212..1702 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GAATTCCTGC AGTTAGTTGT ATTGCTGTGG TAGGGGGTCT A~AGATTCTA r~T~rAr.TGA 60 T~Ar~TTTG GAA~ ACTTGGCA~A TTGAGGCAGG AAGCTAACGT TTTCCAGAAC 120 GGCCTGGGCT G~llll~AGA GCTCCGATCT r~r.Ar.AAr.~ GCACTA~AAG ATGAATATGA 180 AAAGGTGGAC AAGTCCTATT TTcr~r-~r~ G ATG ACT TTT AAC AGT TTT GAA 232 Met Thr Phe Asn Ser Phe Glu Gly Thr Arg Thr Phe Val Leu Ala ABP Thr Asn Lys Asp Glu Glu Phe GTA GAA GAG TTT AAT AGA TTA A~A 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 Ser Pro Val Ser Ala Ser Thr Leu Ala Arg Ala Gly Phe Leu Tyr Thr CA 0224ll29 l998-06-l9 Gly Glu Gly Asp Thr Val Gln CYB Phe Ser Cys His Ala Ala Ile Asp AGA TGG CAG TAT GGA GAC TCA GCT GTT GGA AGA CAC AGG AGA ATA TCC ~72 Arg Trp Gln Tyr Gly A~p Ser Ala Val Gly Arg Hi~ Arg Arg Ile Ser Pro Asn Cys Arg Phe Ile Asn Gly Phe Tyr Phe Glu Asn Gly Ala Ala CAG TCT ACA AAT CCT GGT ATC CAA AAT GGC CAG TAC A~A TCT GAA AAC 568 Gln Ser Thr Asn Pro Gly Ile Gln Asn Gly Gln Tyr Lys Ser Glu A~n cys Val Gly Asn Arg Asn Pro Phe Ala Pro A6p Arg Pro Pro Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly Gln Val Val Asp Ile Ser A~p Thr Ile Tyr Pro Arg Asn Pro Ala Met CYB Ser Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro A~p Tyr Ala His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Ala ABP Asp Gln Val Gln TGC TTT TGT TGT GGG GGA A~A CTG GAA AAT TGG GAA CCC TGT GAT CGT 856 Cys Phe Cys Cys Gly Gly Lys Leu Glu Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser Glu His Arg Arg His Phe Pro A~n CYB Phe Phe Val Leu Gly Arg Asn Val Asn Val Arg Ser Glu Ser Gly Val Ser Ser A~p Arg CA 0224ll29 l998-06-l9 Asn Phe Pro Asn Ser Thr Asn Ser Pro Arg Asn Pro Ala Met Ala Glu Tyr Glu Ala Arg Ile Val Thr Phe Gly Thr Trp Thr Ser Ser Val A~n Lys Glu Gln Leu Ala Arg Ala Gly Phe Tyr Ala Leu Gly Glu Gly Asp Lys Val LYB Cy~ Phe His CYB Gly Gly Gly Leu Thr ABP Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln His Ala LYB Trp Tyr Pro Gly Cy~ Ly~

Tyr Leu Leu ABP Glu Lys Gly Gln Glu Tyr Ile Asn Asn Ile His Leu ACC CAT TCA CTT GAG GAA TCT TTG GGA AGA ACT GCT GAA A~A ACA CCA 1288 Thr His Ser Leu Glu Glu Ser Leu Gly Arg Thr Ala Glu LYB Thr Pro TCG CTA ACT A~A AAA ATC GAT GAT ACC ATC TTC CAG AAT CCT ATG GTG 1336 Ser Leu Thr Lys Lys Ile Asp Asp Thr Ile Phe Gln Asn Pro Met Val CAA GAA GCT ATA CGA ATG GGA TTT AGC TTC AAG GAC ATT AAG A~A ACA 1384 Gln Glu Ala Ile Arg Met Gly Phe Ser Phe Lys Asp Ile LYB Lys Thr Met Glu Glu Ly~ Ile Gln Thr Ser Gly Ser Ser Tyr ~eu Ser Leu Glu GTC CTG ATT GCA GAT CTT GTG AGT GCT CAG A~A GAT AAT ACG GAG GAT 1480 Val Leu Ile Ala Asp Leu Val Ser Ala Gln Lys Asp Asn Thr Glu Asp CA 0224ll29 l998-06-l9 WO 97/23501 PCT/AU96~00X27 GAG TCA AGT CAA ACT TCA TTG CAG A~A GAC ATT AGT ACT GAA GAG CAG 1528 Glu Ser Ser Gln Thr Ser Leu Gln Ly~ A~p Ile Ser Thr Glu Glu Gln r 425 CTA AGG CGC CTA CAA GAG GAG AAG CTT TGC AA~ ATC TGT ATG GAT AGA 1576 _ Leu Arg Arg Leu Gln Glu Glu Ly~ Leu Cys Ly~ Ile Cy~ Met A~p Arg AAT ATT GCT ATC GTT TTT GTT CCT TGT GGA CAT CTG GTC ACT TGT A~A 1624 A8n Ile Ala Ile Val Phe Val Pro CYB Gly HiS Leu Val Thr CYB LYB

CAG TGT GCA GAA GCA GTT GAC A~A TGT CCC ATG TGC TAC ACC GTC ATT 1672 Gln Cy~ Ala Glu Ala Val A~p Ly~ Cy~ Pro Met Cys Tyr Thr Val Ile ACG TTC AAG CAA A~A ATT TTT ATG TCT TAGTGGGGCA CCACATGTTA 1719 Thr Phe Lys Gln ~y~ Ile Phe Met Ser ~ lCTT GCTCTAATTG AA'~ ~AT GGGAGCGAAC TTTAAGTAAT CTGCATTGCA 1779 TTCCATTAGC ATCTGCTGTT TCCA~ATGGA GACCAATGCT AACAGCACTG TTTCCGTCTA 1839 AACATTCAAT ~LclGGATCT TTCGAGTTAT CAGCTGTATC ATTTAGCCAG l~llllACTC 1899 GATTGA~ACC TTA~.~AG AAGCATTTTA TAGCTTTTCA CATGTATATT GGTAGTACAC 1959 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 496 amino acids (B) TYPE: amino acid (D) 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 A~p J

Thr Asn Lys A~p Glu Glu Phe Val Glu Glu Phe A~n Arg Leu Lys Thr ~he Ala Asn Phe Pro Ser Ser Ser Pro Val Ser Ala Ser Thr Leu Ala Arg Ala Gly Phe Leu Tyr Thr Gly Glu Gly Asp Thr Val Gln Cys Phe Ser Cy~ His Ala Ala Ile Asp Arg Trp Gln Tyr Gly Asp Ser Ala Val ~ly Arg His Arg Arg Ile Ser Pro Asn Cy8 Arg Phe Ile AE:n Gly Phe ~yr Phe Glu Asn Gly Ala Ala Gln Ser Thr Asn Pro Gly Ile Gln Asn Gly Gln Tyr Lys Ser Glu Asn Cys Val Gly Asn Arg Asn Pro Phe Ala Pro ABP Arg Pro Pro Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly Gln Val Val Asp Ile Ser A~3p Thr Ile Tyr Pro Arg Asn Pro Ala Met ~ys Ser Glu Glu Ala Arg Leu Lys Ser Phe Gln A:~n Trp Pro A~p Tyr ~la His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Ala A~p Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Glu Asn Trp Glu Pro Cya AE~p Arg Ala Trp Ser Glu Hi~ Arg Arg His Phe Pro ARrl CYB Phe Phe Val Leu Gly Arg Asn Val A~n Val Arg Ser Glu ~er Gly Val Ser Ser AE;p Arg Asn Phe Pro A~n Ser Thr A~n Ser Pro ~rg Al3n Pro Ala Met Ala Glu Tyr Glu Ala Arg Ile Val Thr Phe Gly WO 97/23501 rCT/AU96/00827 Thr Trp Thr Ser Ser Val Asn Lys Glu Gln Leu Ala Arg Ala Gly Phe Tyr Ala Leu Gly Glu Gly Asp LYR Val Lys Cy~ Phe His Cys Gly Gly Gly Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln His Ala ~y~ Trp Tyr Pro Gly Cy~ Ly~ Tyr Leu Leu ABP Glu Lys Gly Gln Glu ~yr Ile Asn Asn Ile His Leu Thr His Ser Leu Glu Glu Ser Leu Gly Arg Thr Ala Glu Lys Thr Pro Ser Leu Thr Lys Lys Ile A~p Asp Thr Ile Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg Met Gly Phe Ser Phe Lys Asp Ile Lys Lys Thr Met Glu Glu Lys Ile Gln Thr Ser Gly ~er Ser Tyr Leu Ser Leu Glu Val Leu Ile Ala Asp Leu Val Ser Ala ~ln Lys A~p Asn Thr Glu Asp Glu Ser Ser Gln Thr Ser heu Gln LYB

Asp Ile Ser Thr Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Lys Leu Cys Lys Ile Cy~ Met Asp Arg Asn Ile Ala Ile Val Phe Val Pro Cys Gly His Leu Val Thr Cys Lys Gln Cys Ala Glu Ala Val Asp Lys Cys Pro Met Cys Tyr Thr Val Ile Thr Phe Lys Gln Lys Ile Phe Met Ser CA 0224ll29 l998-06-l9 (2) INFORMATION FOR 8EQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3532 ba~e pairs (B) TYPE: nucleic acid (C) STRANnEnNRRS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 1160..3016 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GAATTCTATG GAGTGTAATT ~Ll~l~LATGA ATTATATTTT T~AA-~ATTG AAGAGTTTTC 60 AGAAAGAAGG CTAGTAGAGT TGATTACTGA TACTTTATGC TAAGCAGTAC llll-llG~-lA 120 GTACAATATT TTGTTAGGCG lll~l~ATAA CACTA~-~A~G GACAAGTTTT Alcll~l~AT 180 AAATTGATTA ATGTTTACAA CATGACTGAT A~TTATAGCT GAATAGTCCT TAAATGATGA 240 ACAGGTTATT TA~l~ AA ATGCAGTGTA AAAAGTGTGC TGTGGAAATT TTATGGCTAA 300 CTAAGTTTAT G~A~-AAAATA CCTTCAGTTG ATrAA~AATA ATAGTGGTAT ACAAAGTTAG 360 ~AA~AAAr~TC AACATGATGC TGCAGGAAAT G~AAA~AA~T ACAAATGATA TTTAA~AAA~- 420 ATAGAGTTTA CA~lllll~A ACTTTAAGCC AAATTCATTT GACATCAAGC ACTATAGCAG 480 GCACAGGTTC A~AA~cTT ~lGG~lATTG ACTTCCCCCA A~AGTTGTCA GCTGAAGTAA ~40 TTTAGCCCAC TTAAGTA~AT ACTATGATGA TAAGCTGTGT GAACTTAGCT TTTA~AT~.T 600 GTGACCATAT GAAGGTTTTA ATTACTTTTG TTTATTGGA~ TAAAATGAGA TTTTTTGGGT 660 TGTCATGTTA AAGTGCTTAT AGG~.~ A GCCTGCATAT AAlllLllAC ~Ll~GG~AT 720 AATCAGTAAT lG~lcl~l,A TTCAGGCTTC ATAGCTTGTA ACCAAATATA A~TA~P~.GC 780 ATAATTTAGG TATTCTATAG TTGCTTAGAA l~ AAT ATA~ATCTCT GT~-AAAAATC 840 CA 0224ll29 l998-06-l9 W O 97/23501 PCT~AU96~0U827 AAGGAGTTTT AATATTTTCA GAAGTGCATC CACCTTTCAG GGCTTTAAGT TAGTATTACT soo CAAGATTATG AArAA~TArC ACTTAGGTTA CCTr-~AAr-AG TTACTACAAC CC8AAA~.T 960 AA GTAGTATCTT GGTAATTCAG Ar-~TACTC ATCCTACCTG A~TATA~cT 1020 ~r~T~AATc cAGTAAAr~A~ AGTGTAGTAA ATTCTACATA AGAGTCTATC ATTGATTTCT 1080 ~ G~lA AAAATCTTAG TTCATGTGAA GAAATTTCAT GTGAATGTTT TAGCTATCAA 1140 ACAGTACTGT CACCTACTC ATG CAC A~A ACT GCC TCC CAA AGA CTT TTC CCA 1192 Met His Ly~ Thr Ala Ser Gln Arg Leu Phe Pro Gly Pro 8er Tyr Gln Asn Ile Ly~ Ser Ile Met Glu A~p Ser Thr Ile Leu Ser A~p Trp Thr Asn Ser Asn Ly~ Gln Ly~ Met Ly~ Tyr Asp Phe Ser Cys Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr GGT GTG AAT GAC AAG GTC A~A TGC TTC TGT TGT GGC CTG ATG CTG GAT 1432 Gly Val A~n Asp Lys Val Ly~ Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Leu Gly A~p Ser Pro Ile Gln Lys His Ly~ Gln Leu Tyr Pro Ser Cy8 Ser Phe Ile Gln A~n Leu Val Ser Ala Ser Leu Gly Ser Thr Ser Lys A~n Thr Ser Pro Met Arg A~n Ser Phe Ala Hi~ Ser Leu CA 0224ll29 l998-06-l9 W O 97/23~01 PCT/AU96/00827 Ser Pro Thr Leu Glu His Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser Leu Ser Pro Asn Pro Leu A~n Ser Arg Ala Val Glu Asp Ile Ser Ser Ser Arg Thr Asn Pro Tyr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe Leu Thr Tyr His Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gly A~p Arg Val Ala CYB Phe Ala Cys Gly Gly Ly~ Leu Ser Asn Trp Glu Pro Lys Asp A~p Ala Met Ser Glu His Arg Arg His Phe Pro A~n Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr Leu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr Hi~ Ala Ala Arg Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg A~n Asp A~p Val LYB Cy~ Phe Cys Cy~ Asp Gly Gly Leu Arg Cy8 Trp Glu Ser Gly Asp Asp Pro Trp Val Glu Hi~ Ala Ly~ Trp Phe Pro AGG TGT GAG TTC TTG ATA CGA ATG A~A GGC CAA GAG TTT GTT GAT GAG 2200 Arg Cy8 Glu Phe Leu Ile Arg Met LYB Gly Gln Glu Phe Val ABP Glu ATT CA~ GGT AGA TAT CCT CAT CTT CTT GAA CAG CTG TTG TCA ACT TCA 2248 Ile Gln Gly Arg Tyr Pro Hi~ Leu Leu Glu Gln Leu Leu Ser Thr Ser ABP Thr Thr Gly Glu Glu Asn Ala ABP Pro Pro Ile Ile His Phe Gly CCT GGA GA~ AGT TCT TCA GAA GAT GCT GTC ATG ATG AAT ACA CCT GTG 2344 Pro Gly Glu Ser Ser Ser Glu ABP Ala Val Met Met Asn Thr Pro Val GTT A~A TCT GCC TTG GAA ATG GGC TTT AAT AGA GAC CTG GTG A~A CAA 2392 Val Lys Ser Ala Leu Glu Met Gly Phe Asn Arg Asp Leu Val LYB Gln ACA GTT CAA AGT A~A ATC CTG ACA ACT GGA GAG AAC TAT A~A ACA GTT 2440 Thr Val Gln Ser LYB Ile Leu Thr Thr Gly Glu Asn Tyr LYB Thr Val AAT GAT ATT GTG TCA GCA CTT CTT AAT GCT GAA GAT GAA A~A AGA GAA 2488 Asn Asp Ile Val Ser Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu GAG GAG AAG GAA A~A CAA GCT GAA GAA ATG GCA TCA GAT GAT TTG TCA 2536 Glu Glu LYB Glu Lys Gln Ala Glu Glu Met Ala Ser Asp Asp Leu Ser Leu Ile Arg LYB Asn Arg Met Ala Leu Phe Gln Gln Leu Thr CYB Val CTT CCT ATC CTG GAT A~T CTT TTA AAG GCC AAT GTA ATT AAT A~A CAG 2632 Leu Pro Ile Leu Asp Asn Leu Leu Lys Ala Asn Val Ile Asn Ly~ Gln GA~ CAT GAT ATT ATT A~A CAA A~A ACA CAG ATA CCT TTA CAA GCG AGA 2680 Glu His ABP Ile Ile Lys Gln Ly~ Thr Gln Ile Pro Leu Gln Ala Arg CA 0224ll29 l998-06-l9 GA~ CTG ATT GAT ACC ATT TTG GTT AAA GGA AAT GCT GCG GCC AAC ATC 2728 Glu Leu Ile ABP Thr Ile Leu Val LYB Gly A6n Ala Ala Ala Asn Ile Phe Lys Asn CYB Leu LYB Glu Ile ABP Ser Thr Leu Tyr LYB Asn Leu Phe Val ABP LYB Asn Met Lya Tyr Ile Pro Thr Glu ABP Val Ser Gly Leu Ser Leu Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cy~

A~A GTG TGT ATG GAC AAA GAA GTT TCT GTT GTA TTT ATT CCT TGT GGT 2920 LYB Val CYB Met A~p LYR Glu Val Ser Val Val Phe Ile Pro CYB Gly CAT CTG GTA GTA TGC CAG GAA TGT GCC CCT TCT CTA AGA A~A TGC CCT 2968 His Leu Val Val CYB Gln Glu CYB Ala Pro Ser Leu Arg Ly~ CYB Pro ATT TGC AGG GGT ATA ATC AAG GGT ACT GTT CGT ACA TTT CTC TCT TAAAGA~AAA 3023 Ile CYB Arg Gly Ile Ile LYB Gly Thr Val Arg Thr Phe Leu Ser TAGTCTATAT TTTAACCTGC AT~AAArGT CTTTAA~ATA ~ ~AACA CTTGAAGCCA 3083 TCTA~AGTAA AAAGGGAATT ATGAGTTTTT CAATTAGTAA CATTCATGTT CTAGTCTGCT 3143 TTGGTACTAA TAATCTTGTT TCTr~ r-~ TGGTATCATA TATTTAATCT TAAl~l~lLl 3203 ATTTACAAGG GAAGATTTAT ~lllG~l~AA CTATATTAGT ATGTATGTGT ACCTAAGGGA 3263 GTAGTGTCAC TG~ AT GCATCATTTC AGGAGTTACT GGALLL~llG l l~L ' lCAGA 3323 ATCAGAGTTA TGGTGCCGAA L~ LLlGG TGCTTTTCAC ll~l~Llll~A A~AT~rr-~T 3443 TTTTCTCTTA TTTCTCCCCC TA~lll~lGA GAAACATCTC AATAAAGTGC TTTCCA~AAA 3503 ~ r~ TCGACGCGGC CGCGAATTC 3532 --=
CA 0224ll29 l998-06-l9 WO 97/23501 PCT/AU~ 327 (2) INFORMATION FOR 8EO ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 618 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met His Lys Thr Ala Ser Gln Arg Leu Phe Pro Gly Pro Ser Tyr Gln Asn Ile Lys Ser Ile Met Glu A~p Ser Thr Ile Leu Ser Asp Trp Thr A~n Ser Asn Lys Gln Lys Met Lys Tyr Asp Phe Ser Cys Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp hys Val Lys Cy8 Phe Cy~ Cy8 Gly Leu Met Leu Asp Asn Trp Lys Leu Gly so 95 Asp Ser Pro Ile Gln Lys His Lys Gln Leu Tyr Pro Ser Cy5 Ser Phe Ile Gln A~n Leu Val Ser Ala Ser Leu Gly Ser Thr Ser Lys Asn Thr Ser Pro Met Arg Asn Ser Phe Ala His Ser Leu Ser Pro Thr Leu Glu Hi~ Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser Leu Ser Pro Asn Pro Leu Asn Ser Arg Ala Val Glu Asp Ile Ser Ser Ser Arg Thr Asn Pro 16~ 170 175 CA 0224ll29 l998-06-l9 W O 97/23501 PCT/~U96/00827 ~yr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe 1eu Thr Tyr His Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys ABP Asp Ala Met Ser Glu His ~rg Arg His Phe Pro Asn Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr ~eu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Afip Asp Val Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly A~p ~sp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu Phe Leu ~le Arg Met Lys Gly Gln Glu Phe Val Asp Glu Ile Gln Gly Arg Tyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Thr Thr Gly Glu Glu Asn Ala Asp Pro Pro Ile Ile His Phe Gly Pro Gly Glu Ser Ser Ser Glu Asp Ala Val Met Met Asn Thr Pro Val Val Ly6 Ser Ala Leu ~lu Met Gly Phe Asn Arg Asp Leu Val Lys Gln Thr Val Gln Ser Lys ~le Leu Thr Thr Gly Glu Asn Tyr Lys Thr Val Asn Asp Ile Val Ser CA 0224ll29 l998-06-l9 WO 97/23501 PCT~AU~6/~ ~ '7 Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu Glu Glu LYB Glu Lys Gln Ala Glu Glu Met Ala Ser A~p A~p Leu Ser Leu Ile Arg Ly~ Asn Arg Met Ala Leu Phe Gln Gln Leu Thr Cys Val Leu Pro Ile Leu Asp ~sn Leu Leu Lys Ala Asn Val Ile Asn Lys Gln Glu His Asp Ile Ile ~ys Gln Lys Thr Gln Ile Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr Ile Leu Val Lys Gly Asn Ala Ala Ala A~n Ile Phe Lys Asn Cys Leu Lys Glu Ile Asp Ser Thr Leu Tyr Ly~ Asn Leu Phe Val Asp Lys Asn Met LYB Tyr Ile Pro Thr Glu A~p Val Ser Gly Leu Ser Leu Glu Glu ~ln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys Met Asp ~ys Glu Val Ser Val Val Phe Ile Pro Cys Gly His Leu Val Val Cys ~ln Glu Cys Ala Pro Ser Leu Arg Ly~ Cys Pro Ile Cys Arg Gly Ile Ile Lys Gly Thr Val Arg Thr Phe Leu Ser ~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTE~ISTICS:
(A) LENGTH: 3076 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single W O 97/23501 PCTtAU96/00827 -~8-(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
~A) NAME/KEY: CDS
(B) LOCATION: 725..2539 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GAATTCAAAA l~l~llCAGT TGTAAATCTT ACCATTATTT TACGTACCTC TAA~AATAA 60 AAGTGCTTCT AATTAA~AATA TGATGTCATT AATTATGAAA TA~l~Cll~A TAACAGAAGT 120 TTAGGTCTTG TG~~ ll TCCTGGC QC TAAATTTCAC AATTTCCAAA AAG~AAAATA 240 A~ATATTcT GAATATTTTT GCTGTGA~AC ACTTGACAGC AGAGCTTTCC ACCATGA~AA 300 GAAGCTTCAT GAGTCACACA TTACATCTTT GG~ll~ATTG AATGCCACTG AAACATTCTA 360 CTTA~T~ ATCACTCTTC TGTGAAGGGT TTTAATTTTC AACACAGCTT A~l8l~lAGC 480 ATCATGTTTA CATTGTATGT ATAAArATTA TACAAAGGTG CAAll~l~lA TTTCTTCCTT 540 A~AATGTATC AGTATAGGAT TTAGAATCTC CATGTTGAAA CTCTAAATGC ATA~.~AATAA 600 AAATAAT~AA AAATTTTTCA TTTTGGCTTT TCAGCCTAGT ATTA~AACTG ATAAAA~CAA 660 AGCCATGCAC A~AACTACCT CCCTA~A~-~ AGGCTAGTCC ~llll~llCC CCATTCATTT 720 CATT ATG AAC ATA GTA GAA AAC AGC ATA TTC TTA TCA AAT TTG ATG AAA 769 Met Asn Ile Val Glu A~n Ser Ile Phe Leu Ser Asn Leu Met Ly~

Ser Ala Asn Thr Phe Glu Leu Ly~ Tyr A~p Leu Ser Cys Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu _ 59 _ AGG AGT CTT GCT CGT GCT GGT TTC TAT TAC ACT GGT GTG AAT GAC AAG 913Arg 8er Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Ly6 GTC AAA TGC TTC TGT TGT GGC CTG ATG CTG GAT AAC TGG A~A AGA GGA 961 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp A6n Trp LYB Arg Gly GAC AGT CCT ACT GAA AAG CAT A~A AAG TTG TAT CCT AGC TGC AGA TTC 1009 Asp Ser Pro Thr Glu Ly~ His Lys Lys Leu Tyr Pro Ser Cy~ Arg Phe 80 85 go 95 Val Gln Ser Leu Asn Ser Val Asn A~n Leu Glu Ala Thr Ser Gln Pro Thr Phe Pro Ser Ser Val Thr Asn Ser Thr His Ser Leu Leu Pro Gly Thr Glu Asn Ser Gly Tyr Phe Arg Gly Ser Tyr Ser Asn Ser Pro Ser AAT CCT GTA AAC TCC AGA GCA A~T CAA GAT TTT TCT GCC TTG ATG AGA 1201 Asn Pro Val Asn Ser Arg Ala Asn Gln Asp Phe Ser Ala Leu Met Arg 145 lS0 155 Ser Ser Tyr His Cy~ Ala Met Asn Asn Glu Asn Ala Arg Leu Leu Thr Phe Gln Thr Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp Leu Ala LYB

Ala Gly Phe Tyr Tyr Ile Gly Pro Gly A~p Arg Val Ala Cys Phe Ala TGT GGT GGA A~A TTG AGC AAT TGG GAA CCG AAG GAT AAT GCT ATG TCA 1393 Cys Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys A~p Asn Ala Met Ser -GAA CAC CTG AGA CAT TTT CCC A~A TGC CCA TTT ATA GAA AAT CAG CTT 1441 Glu His Leu Arg Hi~ Phe Pro Ly~ Cys Pro Phe Ile Glu Asn Gln Leu Gln A6p Thr Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr His GCA GCC CGC TTT A~A ACA TTC TTT AAC TGG CCC TCT AGT GTT CTA GTT 1537 Ala Ala Arg Phe LYB Thr Phe Phe Asn Trp Pro Ser Ser Val Leu Val Asn Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser ABP ABP Val LYB Cy8 Phe CYB Cy8 ABP Gly Gly Leu Arg CYB Trp Glu Ser Gly Asp A~p Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cy8 GAG TAC TTG ATA AGA ATT A~A GGA CAG GAG TTC ATC CGT CAA GTT CAA 1729 Glu Tyr Leu Ile Arg Ile Lys Gly Gln Glu Phe Ile Arg Gln Val Gln Ala Ser Tyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser A~p Ser 340 3~5 350 Pro Gly Asp Glu Asn Ala Glu Ser Ser Ile Ile His Phe Glu Pro Gly Glu Asp His Ser Glu Asp Ala Ile Met Met Asn Thr Pro Val Ile Asn 370 ~75 380 GCT GCC GTG GAA ATG GGC TTT AGT AGA AGC CTG GTA A~A CAG ACA GTT 19 21 Ala Ala Val Glu Met Gly Phe Ser Arg Ser Leu Val Lys Gln Thr Val 385 390 39~
CAA AGA A~A ATC CTA GCA ACT GGA GAG AAT TAT AGA CTA GTC AAT GAT 19 69 Gln Arg LYB Ile Leu Ala Thr Gly Glu A~3n Tyr Arg Leu Val Asn ABP

CA 0224ll29 l998-06-l9 W O 97/23501 PCT/AU~

Leu Val Leu ABP Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu Arg Glu Arg Ala Thr Glu Glu Lys Glu Ser A~n Asp Leu Leu Leu Ile Arg Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr Cy8 Val Ile Pro Ile Leu Asp Ser Leu Leu Thr Ala Gly Ile Ile Asn Glu Gln Glu His GAT GTT ATT A~A CAG AAG ACA CAG ACG TCT TTA CAA GCA AGA GAA CTG 2209 Asp Val Ile Lys Gln Lys Thr Gln Thr Ser Leu Gln Ala Arg Glu Leu ATT GAT ACG ATT TTA GTA A~A GGA AAT ATT GCA GCC ACT GTA TTC AGA 2257 Ile Asp Thr Ile Leu Val Ly~ Gly Asn Ile Ala Ala Thr Val Phe Arg Asn Ser Leu Gln Glu Ala Glu Ala Val Leu Tyr Glu Hi~ Leu Phe Val Gln Gln Asp Ile Lys Tyr Ile Pro Thr Glu Asp Val Ser Asp Leu Pro GTG GAA GAA CAA TTG CGG AGA CTA CAA GAA GAA AGA ACA TGT A~A GTG 2401 Val Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys Met ABP Lys Glu Val Ser Ile Val Phe Ile Pro Cys Gly His Leu Val Val Cy8 Lys Asp Cy8 Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys , AGG AGT ACA ATC AAG GGT ACA GTT CGT ACA TTT CTT TCA T~.~A~-A~-A~ 2546 Arg Ser Thr Ile Ly~ Gly Thr Val Arg Thr Phe Leu Ser CA 0224ll29 l998-06-l9 CCA~AACATC ATCTAAACTT TAGAATTAAT TTATTAAATG TATTATAACT TTAACTTTTA 2606 TCCTAATTTG GTTTCCTTAA AATTTTTATT TATTTACAAC T~AAAAPA~A '11~L1L1~1G 2666 TAACATATTT ATATATGTAT CTAAA~TA T~AA~ATATA LLllLLAGAA ACTAAGAGAA 2726 TGATAGGCTT lL~Ll~LLAT GAACGAPAAA GAGGTAGCAC TA~AAA~ArA ATATTCAATC 2786 A~AATTTCAG CATTATTGAA ATTGTAAGTG AAGTAAAACT TAA~ATATTT GAGTTAACCT 2846 TTAAGAATTT TAAATATTTT GGCATTGTAC TAATACCTGG ll l'l'll'LL~ L ~1 L'l-l ~L lll 2906 TTTGTACAGA CAGGGCAGCA TACTGAGACC CTGCCTTTAA AAA~PA~CAG AA~AAA~AcA 2966 AAACACCAGG GACACATTTC TCTGTCTTTT TTGATCAGTG TCCTATA~PT CGAAGGTGTG 3026 CATATATGTT GAATGACATT TTAGGGACAT G~~ LA TA~AGAATTC 3076 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 604 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Asn Ile Val Glu Asn Ser Ile Phe Leu Ser Asn Leu Met Lys Ser ~la Asn Thr Phe Glu Leu Lys Tyr Asp Leu Ser Cys Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn ABP LYB Val WO 97/23501 PCT/AU96J~ ~ - 7 Ly6 Cys Phe CYB Cys Gly Leu Met Leu Asp Asn Trp Lys Arg Gly Asp ~er Pro Thr Glu Lys ~is Lys Lys Leu Tyr Pro Ser Cys Arg Phe Val ~ln Ser Leu A~n Ser Val Asn Asn Leu Glu Ala Thr Ser Gln Pro Thr Phe Pro Ser Ser Val Thr Asn Ser Thr His Ser Leu Leu Pro Gly Thr Glu Asn Ser Gly Tyr Phe Arg Gly Ser Tyr Ser A~n Ser Pro Ser Aan Pro Val A~n Ser Arg Ala Asn Gln Asp Phe Ser Ala Leu Met Arg Ser ~er Tyr His Cy~ Ala Met A~n Asn Glu Asn Ala Arg Leu Leu Thr Phe ~ln Thr Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp Leu Ala LYB Ala 180 185 l9o Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cy8 Phe Ala Cys Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asn Ala Met Ser Glu His Leu Arg His Phe Pro Lys Cys Pro Phe Ile Glu Asn Gln Leu Gln ~sp Thr Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr ~is Ala ~la Arg Phe Lys Thr Phe Phe Asn Trp Pro Ser Ser Val Leu Val Asn Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser Asp A~p Val Lys Cy~ Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly A~p Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cys Glu CA 0224ll29 l998-06-l9 ~yr Leu Ile Arg Ile Ly~ Gly Gln Glu Phe Ile Arg Gln Val Gln Ala ~er Tyr Pro His heu Leu Glu Gln Leu Leu Ser Thr Ser Asp Ser Pro Gly ABP Glu Asn Ala Glu Ser Ser Ile Ile His Phe Glu Pro Gly Glu A6p His Ser Glu A~p Ala Ile Met Met A~n Thr Pro Val Ile Asn Ala Ala Val Glu Met Gly Phe Ser Arg Ser Leu Val Lys Gln Thr Val Gln ~rg Lys Ile Leu Ala Thr Gly Glu Asn Tyr Arg Leu Val Asn Asp Leu ~al Leu Asp Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu Arg Glu Arg Ala Thr Glu Glu Lys Glu Ser A~n A~p Leu Leu Leu Ile Arg Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr Cys Val Ile Pro Ile Leu Asp Ser Leu Leu Thr Ala Gly Ile Ile Asn Glu Gln Glu His ABP

~al Ile Lys Gln Lys Thr Gln Thr Ser Leu Gln Ala Arg Glu Leu Ile ~sp Thr Ile Leu Val Lys Gly Asn Ile Ala Ala Thr Val Phe Arg Asn Ser Leu Gln Glu Ala Glu Ala Val Leu Tyr Glu His Leu Phe Val Gln Gln Asp Ile Lys Tyr Ile Pro Thr Glu Asp Val Ser Asp Leu Pro Val Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys CA 0224ll29 l998-06-l9 WO 97/23501 PCT~AU96/00827 Met Asp Lys Glu Val Ser Ile Val Phe Ile Pro Cy8 Gly His Leu Val ~al Cy8 Ly~ Asp Cys Ala Pro Ser Leu Arg Ly~ Cys Pro Ile Cy8 Arg Ser Thr Ile Lys Gly Thr Val Arg Thr Phe Leu Ser (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 1552 ba~e pair6 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 6..1502 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Thr Glu Leu Gly Met Glu Leu Glu Ser Val Arg Leu Ala Thr Phe Gly Glu Trp Pro Leu Asn Ala Pro Val Ser Ala Glu Asp Leu Val Ala Asn Gly Phe Phe Ala Thr Gly Asn Trp Leu Glu Ala Glu Cys His Phe Cy~ His Val Arg Ile A~p Arg Trp Glu Tyr Gly Asp Gln Val Ala Glu Arg His Arg Arg Ser Ser Pro Ile Cys Ser Met Val Leu Ala CA 0224ll29 l998-06-l9 CCC AAT CAC TGC GGC AAT GTT CCC AGG AGC CAG GAG AGC GAC A~C GAG 287 Pro Asn His Cy8 Gly Asn Val Pro Arg Ser Gln Glu Ser Asp Asn Glu Gly A#n Ser Val Val Asp Ser Pro Glu Ser Cys Ser Cys Pro Asp Leu Leu Leu Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Asn Pro Asn Ile Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu Asp His Val Lys Cys Val Trp Cys Asn Gly Val Ile Ala Lys Trp Glu Lys Asn A~p Asn Ala Phe Glu Glu Hls Lys Arg Phe Phe Pro Gln Cys Pro Arg Val Gln Met Gly Pro Leu Ile Glu Phe Ala Thr Gly 175 180 185 l90 Lys Asn Leu A6p Glu Leu Gly Ile G}n Pro Thr Thr Leu Pro Leu Arg Pro Lys Tyr Ala CYB Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp Pro Ile Ser Asn Ile Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly Leu Tyr Tyr Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His Cys Asn Ile Gly Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala CA 0224ll29 l998-06-l9 W O 97/23$01 PCT~AU96~00827 25~ 260 265 270 Lys Trp Ser Pro Lys Cy~ Gln Phe Val Leu Leu Asp Ly~ Gly Pro Ala Tyr Val Ser Glu Val Leu Ala Thr Thr Ala Ala A~n Ala Ser Ser Pro Thr Ala Thr Ala Pro Ala Pro Thr Leu Gln Ala Asp Val Leu Met Asp Glu Ala Pro Ala Lys Glu Ala Leu Ala Leu Gly Ile Asp Gly Gly Val Val Arg Asn Ala Ile Gln Arg Ly~ Leu Leu Ser Ser Gly Cy8 Ala Phe 8er Thr Leu Asp Glu Leu Leu Hi~ Asp Ile Phe Asp A~p Ala Gly Ala Gly Ala Ala ~eu Glu Val Arg Glu Pro Pro Glu Pro Ser Ala Pro Phe Ile Glu Pro Cys Gln Ala Thr Thr Ser Lys Ala Ala Ser Val Pro Ile CCG GTG GCC GAT TCC ATC CCA GCC A~A CCA CAG GCA GCT GAA GCA GTG 1247 Pro Val Ala Asp Ser Ile Pro Ala Ly~ Pro Gln Ala Ala Glu Ala Val GCG AAT ATA TCG A~A ATC ACA GAC GAA ATA CAA AAG ATG TCG GTG GCC 1295 Ala A~n Ile Ser Ly~ }le Thr Asp Glu Ile Gln Ly~ Met Ser Val Ala Thr Pro Asn Gly Asn Leu Ser Leu Glu Glu Glu A~n Arg Gln Leu Lys CA 0224ll29 l998-06-l9 W O 97/23501 PCTtAU~6t0C827 Asp Ala Arg Leu Cy~ Ly~ Val Cys Leu Asp Glu Glu Val Gly Val Val Phe Leu Pro Cys Gly His Leu Ala Thr Cys Asn Gln Cys Ala Pro Ser Val Ala Asn Cys Pro Met Cys Arg Ala Asp Ile Lys Gly Phe Val Arg Thr Phe Leu Ser (2) INFORMATION FOR SEQ ID NO:8:

ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 498 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Thr Glu Leu Gly Met Glu Leu Glu Ser Val Arg Leu Ala Thr Phe ~ly Glu Trp Pro Leu Asn Ala Pro Val Ser Ala Glu Asp Leu Val Ala Asn Gly Phe Phe Ala Thr Gly Asn Trp Leu Glu Ala Glu Cys His Phe Cys His Val Arg Ile Asp Arg Trp Glu Tyr Gly Asp Gln 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 His Cys Gly Asn Val Pro Arg Ser Gln Glu Ser Asp A6n Glu Gly A6n ~er Val Val Asp Ser Pro Glu Ser Cy8 Ser CYB Pro Asp Leu Leu Leu Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro AJ3n Pro Asn Ile Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu Asp His Val Lys Cys Val Trp Cys Asn Gly Val Ile Ala Lys Trp Glu ~ys Asn Asp Asn Ala Phe Glu Glu His Lys Arg Phe Phe Pro Gln CYE~

~ro Arg Val Gln Met Gly Pro Leu Ile Glu Phe Ala Thr Gly Lys Asn Leu Asp Glu Leu Gly Ile Gln Pro Thr Thr Leu Pro Leu Arg Pro Lys Tyr Ala Cys Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp Pro Ile Ser Asn Ile Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly Leu Tyr Tyr ~ln Lys Ile Gly Asp Gln Val Arg Cys Phe His CYB Asn Ile Gly Leu ~rg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala Lys Trp Ser Pro Lys Cys Gln Phe Val Leu Leu Asp Lys Gly Pro Ala Tyr Val Ser Glu Val Leu Ala Thr Thr Ala Ala Asn Ala Ser Ser Pro Thr Ala Thr Ala Pro Ala Pro Thr Leu Gln Ala Asp Val Leu Met Asp Glu Ala Pro Ala Lys Glu Ala Leu Ala Leu Gly Ile Asp Gly Gly Val Val Arg CA 0224ll29 l998-06-l9 W O 97/23~01 PCT/AU96/00827 A~n Ala Ile Gln Arg Lys Leu Leu Ser Ser Gly Cys Ala Phe Ser Thr Leu A~p Glu Leu Leu HiE~ Ai3p Ile Phe Asp Asp Ala Gly Ala Gly Ala Ala Leu Glu Val Arg Glu Pro Pro Glu Pro Ser Ala Pro Phe Ile Glu Pro Cy~ Gln Ala Thr Thr Ser Lys Ala Ala Ser Val Pro Ile Pro Val Ala Asp Ser Ile Pro Ala Lys Pro Gln Ala Ala Glu Ala Val Ala A~n 405 41~ 415 ~le Ser LYB Ile Thr AE:p Glu Ile Gln Lys Met Ser Val Ala Thr Pro 420 425 43~
Asn Gly A~n Leu Ser Leu Glu Glu Glu Asn Arg Gln Leu Lys Asp Ala Arg Leu Cy~ Lys Val Cyfl Leu Asp Glu Glu Val Gly Val Val Phe Leu Pro Cy~ Gly His Leu Ala Thr Cys Asn Gln Cys Ala Pro Ser Val Ala ~sn Cys Pro Met Cy~ Arg Ala Asp Ile Lys Gly Phe Val Arg Thr Phe ~eu Ser WO 97~23~01 PCT/~U96~00827 (2) INFORMATION FOR SEQ ID NO:9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids (B) TYPE: amino acid (D) 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 Xaa,3 Xaa3 Ala Xaa Ala Gly Phe XaaO Asp Xaa Xaap Xaa Cy~ Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa Xaa Hi~ Xaa Xaa Xaa Xaa Pro Xaa Cyg Xaa Xaaq Xaar (2) INFORMATION FOR SEQ ID NO:l0:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 500 base pair3 (B) TYPE: nucleic acid (C) sTR~n~nN~q~q: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 48..467 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:
TGA~TCCGCG TTTGAGTCGT CTTGGCGGAG GTTGTGGTGA CGCCATC ATG GGA GCT 56 Met Gly Ala CCG GCG CTG CCC CAG ATC TGG CAG CTG TAC CTC AAG AAC TAC CGC ATC l04 Pro Ala Leu Pro Gln Ile Trp Gln Leu Tyr Leu Lys Asn Tyr Arg Ile 5 l0 15 Ala Thr Phe Lys Asn Trp Pro Phe Leu Glu Asp Cy8 Ala Cys Thr Pro CA 0224ll29 l998-06-l9 Glu Arg Met Ala Glu Ala Gly Phe Ile His Cy~ Pro Thr Glu A~n Glu Pro A~p Leu Ala Gln Cy~ Phe Phe Cy~ Phe Ly~ Glu Leu Glu Gly Trp Glu Pro Asp Asp Asn Pro Ile Glu Glu His Arg LYB His Ser Pro Gly CYG Ala Phe Leu Thr Val Lys Ly~ Gln Met Glu Glu Leu Thr Val Ser Glu Phe Leu LYB Leu A~p Arg Gln Arg Ala Lys A~n Lys Ile Ala Lys GAG ACC AAC AAC A~G CAA A~A GAG TTT GAA GAG ACT GCA AAG ACT ACC 440 Glu Thr Asn Asn Lys Gln Ly~ Glu Phe Glu Glu Thr Ala Lys Thr Thr Arg Gln Ser Ile Glu Gln Leu Ala Ala *

(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 140 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Gly Ala Pro Ala Leu Pro Gln Ile Trp Gln Leu Tyr Leu Lys Asn Tyr Arg Ile Ala Thr Phe Lys A~n Trp Pro Phe Leu Glu Asp Cy~ Ala WO 97/23501 PCTJAU96J0~827 Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Ly~ Glu Leu Glu Gly Trp Glu Pro Asp Asp Asn Pro Ile Glu Glu His Arg Lys His ~er Pro Gly Cys Ala Phe Leu Thr Val Lys Lys Gln Met Glu Glu Leu so gs ~hr Val Ser Glu Phe Leu Lys Leu Asp Arg Gln Arg Ala Lys Asn LYB

Ile Ala Lys Glu Thr Asn Asn Lys Gln Lys G1U Phe Glu Glu Thr Ala Ly~ Thr Thr Arg Gln Ser Ile G1U Gln Leu Ala Ala *

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1075 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix~ FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 18..941 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Met Pro Tyr Thr Phe Glu Asn Ser Glu Ala Leu CA 0224ll29 l998-06-l9 Leu LYB Asn Leu Lys Asp Ala Ala Pro Tyr Ile Ser Ala Ala Glu Arg Phe Ala Ser Phe Lys Gly Phe Val Tyr Asp Lys Arg Ile Asn Ile Ala Cy~ Thr Ser Glu Lys Leu Ala Arg Ala Gly Phe Tyr Ser Thr Ala Ser CCA GAA TTC CCC GCT TCA GCC A~A TGC CCG TTT TGT ATG CTG GAA ATC 242 Pro Glu Phe Pro Ala Ser Ala Ly~ Cy~ Pro Phe Cys Met Leu Glu Ile AAT TTC GAA CAG TGC GAC GAT CCA TGG GAG A~A CAC A~A TCA GGA AGC 290 Asn Phe Glu Gln Cys Asp Asp Pro Trp Glu LYB His Lys Ser Gly Ser Pro Hi6 Cys Glu Phe Val Met Ile Gly Glu Ile Glu Glu Ser Glu Leu Ser Phe Arg Ile Ile Ser Asn Leu Ala Ile Arg His Ala Thr Val Arg Leu Tyr Glu Glu Leu Leu Gly Ile Val Ala Thr Leu Glu Asn Gly Asp ATT GCT AAT GAA AAC CCG ATT ACC CGC GCT GAC GCC ACC AGG A~A TTG 482 Ile Ala Asn Glu Asn Pro Ile Thr Arg Ala Asp Ala Thr Arg Lys Leu Ile Ser Phe Arg Ser Ser Ser Lys Leu Leu Thr Phe A~p His Arg Leu GCC ACA TTC CAA AAT TTC ATT TTC GAC A~A AAA AGG AAT GTA AAA TGC 578 Ala Thr Phe Gln A~n Phe Ile Phe Asp Lys Ly~ Arg A~n Val Lys Cys ACT TCA AAG AAG CTC GCC AAA GCT GGA TGG TTT TCT ATT GCC AAC A~A 626 Thr Ser Lys Ly~ Leu Ala Lys Ala Gly Trp Phe Ser Ile Ala Asn Lys WO 97/23501 PC~r/AU96~00827 AAA GAT A~A ACT TCT GCA AAA TGT CCA TTC TGC CTT GTT GAG CTG GAC 674 Ly~ Asp Lys Thr Ser Ala Lys cys Pro Phe Cys Leu Val Glu Leu Asp Phe A~p Glu Ser Asp A~p Pro Trp Glu Glu HiR Gln Ly~ Phe Ser Ala TCC TGC GAT TTT ATC AAA CTA GGC AAG CTT GAT GAG A~A AAA TGG ACT 770 Ser CY6 Asp Phe Ile LYB Leu Gly Lys Leu A~p Glu Ly~ LYR Trp Thr GAA AAT GAA GCA TTA ATG CTG GGA GCA AGA ATC ACG ATT ATG CAA A~A 818 Glu Asn Glu Ala Leu Met Leu Gly Ala Arg Ile Thr Ile Met Gln Lys TAC GAA AAA GGA AGT TGG TTG ATT GAC GA~ CTC GAG A~A GAA AAT AGA 866 Tyr Glu Lys Gly Ser Trp Leu Ile Asp Glu Leu Glu Lys Glu Asn Arg Ile A~p Glu Ile Ile Lys Ile Arg Lys Ile Met Ile Lys Pro Asn His GTT CTC A~A CGA CGT CGA TGC AGT ATT TAA TTTATATTTC GA~ll L ~lAT 944 Val Leu Lys Arg Arg Arg Cy~ Ser Ile *

CTGTAAACTT TTGCCTCCCT CCCCGACCTG TTTAACATCC GTAATTCATT ~C~AATGT 1004 TCTAATTCGA ATGAATATGT TTTCCTAA~A A~A~ A~ AAAAACTCGA G 1055 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 308 amino acidR
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Pro Tyr Thr Phe Glu Asn Ser Glu Ala Leu Leu Lys Asn Leu Ly~

~sp Ala Ala Pro Tyr Ile Ser Ala Ala Glu Arg Phe Ala Ser Phe Lys ~ly Phe Val Tyr ABP LYB Arg Ile Asn Ile Ala Cys Thr Ser Glu Lys Leu Ala Arg Ala Gly Phe Tyr Ser Thr Ala Ser Pro Glu Phe Pro Ala Ser Ala LYB CYB Pro Phe CYB Met Leu Glu Ile Asn Phe Glu Gln CYB
~sp ABP Pro Trp Glu Lys His LYB Ser Gly Ser Pro His Cys Glu Phe ~al Met Ile Gly Glu Ile Glu Glu Ser Glu Leu Ser Phe Arg Ile Ile Ser A~n Leu Ala Ile Arg His Ala Thr Val Arg Leu Tyr Glu Glu Leu Leu Gly Ile Val Ala Thr Leu Glu Asn Gly ABP Ile Ala Asn G1U Asn Pro Ile Thr Arg Ala ABP Ala Thr Arg LYB Leu Ile Ser Phe Arg Ser ~er Ser Lys Leu Leu Thr Phe Asp Hi~ Arg Leu Ala Thr Phe Gln Asn ~he Ile Phe Asp Ly~ Ly~ Arg Asn Val Lys CYB Thr Ser Ly~ Ly~ Leu Ala Ly~ Ala Gly Trp Phe Ser Ile Ala A~n LYB LYB A~p Ly~ Thr Ser Ala Ly~ CYB Pro Phe CYB Leu Val Glu Leu ABP Phe ABP Glu Ser ABP

ABP Pro Trp Glu Glu His Gln Lys Phe Ser Ala Ser CYB A~p Phe Ile LYB Leu Gly LYB Leu Aap Glu Lys Ly~ Trp Thr Glu Asn Glu Ala Leu CA 0224ll29 l998-06-l9 WO 97/235(11 PC7:1/AU96~00827 Met Leu Gly Ala Arg Ile Thr Ile Met Gln Ly~ Tyr Glu LYB Gly Ser Trp Leu Ile ABP Glu Leu Glu Ly~ Glu Asn Arg Ile Asp Glu Ile Ile Ly~ Ile Arg Ly~ Ile Met Ile Ly~ Pro A~n Hi~ Val Leu Ly~ Arg Arg Arg Cy8 Ser Ile *

(2) INPORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTX: 512 baee pair~
~B) TYPE: nucleic acid (C) STRANDEDNESS: 6ingle (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix) PEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..123 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 168..512 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATG GCA CCC GGG ACC AAA A~A AAG TCG GAT ATG GCA AAA TTC ACA TTC 48 Met Ala Pro Gly Thr Ly6 Ly~ Lys Ser A6p Met Ala Lyc Phe Thr Phe Tyr Ly~ A6p Arg Leu Met Thr Phe Ly~ Asn Phe Glu Tyr A~p Arg Acp CCG GAT GCA A~A TGC ACG TCT CAA GCG GTTTGTCCAT TTTCGTGTTT 143 Pro A~p Ala Ly~ Cy~ Thr Ser Gln Ala CA 0224ll29 l998-06-l9 W O 97/2350l PCT/AU96/0~827 TA~ATA~AAT TATTCTTGTT TTAG GTT GCT CAA GCC GGA TTT TAC TGC ACC 194 Val Ala Gln Ala Gly Phe Tyr Cys Thr GGT CCT CAG TCT GGC A~A TGT GCA TTT TGC AAC AAG GAA CTT GAT TTT 242 Gly Pro Gln Ser Gly Ly~ Cys Ala Phe Cys Asn hys Glu heu Asp Phe ABP Pro Glu Asp Asp Pro Trp Tyr Glu His Thr Lys Arg Asp Glu Pro Cy~ Glu Phe Val Arg Ile Gly Ly~ Leu Asp Asp Ser Glu Leu Thr Ile Asn A~p Thr Val Arg Leu Ser Gln Thr Ala Met Ile Met Thr Ly~ Leu Phe Glu His Glu Met Met Ile Asn Asn heu Ser Asn His Ser Ser Ser GAT GCT CTC TTC GAT CAG CTG A~A A~A GTA CCG AAC ACA GCA TCG ACA 482 Asp Ala heu Phe Asp Gln Leu hys Lys Val Pro Asn Thr Ala Ser Thr ACA A~A TCT AAC AGC CGC CGC GGC AAA TA 512 Thr Lys Ser Asn Ser Arg Arg Gly Ly~

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids (B) TYPE: amino acid (D) TOPOhOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

CA 0224ll29 l998-06-l9 WO 97~3501 PC~AU96~00827 Met Ala Pro Gly Thr Lys Lys Lys Ser Acp Met Ala Lys Phe Thr Phe ~yr Lys Asp Arg Leu Met Thr Phe Lys Asn Phe Glu Tyr Asp Arg Asp Pro A~p Ala Lys Cys Thr Ser Gln Ala Val Ala Gln Ala Gly Phe Tyr CYB Thr Gly Pro Gln Ser Gly Lys Cys Ala Phe Cys Asn LYB Glu Leu ABP Phe A~p Pro Glu Asp Asp Pro Trp Tyr Glu ~is Thr Ly~ Arg ABP

~lu Pro Cy~ Glu Phe Val Arg Ile Gly Lys Leu Asp Asp Ser Glu Leu ~hr Ile Asn Asp Thr Val Arg Leu Ser Gln Thr Ala Met Ile Met Thr Lys Leu Phe Glu His Glu Met Met Ile Asn Asn Leu Ser Asn His Ser Ser Ser A6p Ala Leu Phe Asp Gln Leu Ly~ Ly~ Val Pro Asn Thr Ala Ser Thr Thr Lys Ser Asn Ser Arg Arg Gly Lys (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2866 base pairs tB) TYPE: nucleic acid (C) sT~Nn~n~ : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..2863 CA 0224ll29 l998-06-l9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Met Asp Gly Gln Ile Asp Lys Met Glu Ly~ Arg Tyr Ser Met Thr Lys Leu Glu Asn Arg Leu Arg Thr Phe Gln Asp Gly Val Ala Leu Glu Lys Lys Ly~ Leu Lys Trp Ser Phe Lys Val Ile Pro Tyr Gln Ala Met Ala Lys Leu Gly Phe Tyr Phe Asp Pro Val Ile ABP Pro Lys Thr Ser LYB

Leu Lys Lys Asp Ser Val Arg Cys Cys Tyr Cys His Arg Gln Thr Tyr A~n Val Arg Asp Cys Arg Ser Lys Arg Lys Asp Val Leu Glu Thr Leu Ser Asn Ile Met Arg Gln His Leu Thr Val Thr Asp Asn Lys Gln Val Cys Leu Leu Ile Tyr Leu Arg Asn Ly~ Leu Leu Thr Asp Tyr Ser Phe His Met Gly Val Ser Aep Trp Lys Asn Asp Lys Tyr Phe Ser Asn Pro A~p Asp Glu A~n Val Ile Asn Leu Arg Lys Phe Thr Phe Gln Asp Asn TGG CCT CAC AGT GGT TCT CAA A~T GAA CAT CCG CTG GGT ATA GAA AAG 528 Trp Pro His Ser Gly Ser Gln Asn Glu His Pro Leu Gly Ile Glu Lys ATG GTG A~T GCA GGA CTC ATG CGA TAC GAC TCC AGC ATA GAA GGC TTA 576 Met Val Asn Ala Gly Leu Met Arg Tyr Asp 8er Ser Ile Glu Gly Leu Gly Asp Pro Ser Met Asp Lys Thr Leu Met Asn Asp Thr Cy~ Tyr CYB

Ile Tyr Cy8 Lys Gln Leu Leu Gln Gly Trp Ser Ile Asn Asp ABP Pro Met Ser Arg His Tyr Lys Val Ser Gln Asn Gly Asn cys Tyr Phe Phe CAG ACA CGT AAT CGA TTT GAG AGA ATA A~A AAT GAC AAT GAC AGT ATC 768 Gln Thr Arg Asn Arg Phe Glu Arg Ile Lys Asn A~p Asn Asp Ser Ile ACG AAA A~T TGC GAA GTC TCT CCA ACT TTA GGC GAA AAT GGG A~A AGA 816 Thr Lys Asn Cys Glu Val Ser Pro Thr Leu Gly Glu Asn Gly Ly~ Arg Glu Val Ile Asn Thr Lys Thr Ala Ser Gln Arg Gln Cys Pro Leu Phe Glu Ser Pro Pro Ser Ser Thr Gly Pro Gln Leu Asp Asp Tyr Asn Glu Lys Thr Asp Ile Ser Val Ile Gln His Asn Ile Ser Val Leu Asp Gly GCA CAA GGC GAA AAT GTA A~A CGT AAT AGT GTG GAA GAA AAA GAA CAA 1008 Ala Gln Gly Glu Asn Val Ly6 Arg Asn Ser Val Glu Glu Lys Glu Gln Ile Asn Met Glu Asn Gly Ser Thr Thr Leu Glu Glu Gly Asn Ile Asn CGT GAT GTT TTA GCA GAT AAG A~A GAA GTT ATT TCG ACA C Q ACT GCA 1104 Arg Asp Val Leu Ala Asp Lys Lys Glu Val Ile Ser Thr Pro Thr Ala CA 0224ll29 l998-06-l9 LYB Glu Ile Ly~ Arg Pro A~n Val Gln Leu Thr Gln Ser Ser Ser Pro Ile Ly~ Ly~ Ly~ Arg Lys Phe Lys Arg Ile Ser Pro Arg Ly~ Ile Phe Asp Glu Glu ABP Ser Glu His Ser Leu Asn A~n A~n Ser Ala Asn Gly GAC AAC A~A GAT AAG GAT TTG GTT ATA GAT TTT ACA AGC CAT ATC ATA 1296 ABP A~n LYB ABP Lys Asp Leu Val Ile Asp Phe Thr Ser His Ile Ile Lys Asn Arg Asp Val Gly Arg Lys Asn Ala Ile Leu Asp Asp Ser Thr Asp Glu Phe Ser Phe Ser A~n Gln Gly His A~n Thr Phe Asp Ile Pro Ile Pro Thr Ser Ser Hi~ Leu Leu Lys Gly Ile Asp Ser ABP ARn Asp AAT GTT ATA CGA GAA GAT GAT ACT GGC ATA AAT ACA GAC ACA A~A GGA 1488 Asn Val Ile Arg Glu A~p Asp Thr Gly Ile Asn Thr Asp Thr Ly~ Gly Ala Ser Ser Ly~ His Glu Ly6 Phe Ser Val Asn Ser Glu Glu A~p Leu Asn Phe Ser Glu Val Lys Leu Thr Gly Arg Asp Ser Ser Thr Asn Ile Leu Ile Arg Thr Gln Ile Val Asp Gln A~n Leu Gly Asp Ile Asp Arg CA 0224ll29 l998-06-l9 W O 97/23501 PCT/AU96~08Z7 Asp Lys Val Pro A~n Gly Gly 8er Pro Glu Val Pro Lys Thr His Glu Leu Ile Arg A~p Asn Ser Glu Lys Arg Glu Ala Gln Asn Gly Glu Phe CGA CAT CAA A~A GAT TCA ACT GTA CGG CAA TCT CCA GAT ATA TTA CAT 17 7 6Arg His Gln Lys Asp Ser Thr Val Arg Gln Ser Pro ABP Ile Leu His TCT AAT A~A AGT GGT GAT AAT TCC AGT AAT ATT ACC GCA ATA CCC A~A 1824 Ser Asn LYB Ser Gly Asp Asn Ser Ser Asn Ile Thr Ala Ile Pro Lys GAG GAA CAA AGG AGA GGC AAT AGC A~A ACA TCC AGC ATT CCC GCT GAT 1872 Glu Glu Gln Arg Arg Gly Asn Ser Lys Thr Ser Ser Ile Pro Ala ABP

ATT CAT CCT A~A CCA AGG A~A AAC TTG CAA GAA CCA AGA AGC CTA TCA 1920 Ile His Pro Lys Pro Arg Lys Asn Leu Gln Glu Pro Arg Ser Leu Ser ATA AGT GGA AAA GTT GTT CCA ACA GAA AGA A~A TTA GAT AAC ATC AAT 1968 Ile Ser Gly Lys Val Val Pro Thr Glu Arg Lys Leu Asp Asn Ile Asn Ile Asp Leu Asn Phe Ser Ala Ser Asp Phe Ser Pro Ser Ser Gln Ser GAG CAA TCA TCA A~A AGC TCA AGC GTT ATT TCA ACG CCG GTA GCG AGC 2064 Glu Gln Ser Ser Ly~ Ser Ser Ser Val Ile Ser Thr Pro Val Ala Ser CCT A~A ATT AAT CTC ACA CGC AGT TTG CAT GCA GTC A~A GAG CTT TCT 2112 Pro Lys Ile Asn Leu Thr Arg Ser Leu His Ala Val Lys Glu Leu Ser GGC CTC A~A A~A GAA ACT GAC GAT GGT AAG TAT TTT ACT AAT AAG CAG 2160 Gly Leu Lys LYB Glu Thr Asp Asp Gly Lys Tyr Phe Thr Asn Lys Gln GAA ACT ATA A~A ATA CTG GAA GAT GTG TCT GTG AAG AAT GAG ACC CCA 2208 Glu Thr Ile Lys Ile Leu Glu Asp Val Ser Val Lys Asn Glu Thr Pro A~n Asn Glu Met Leu Leu Phe Glu Thr Gly Thr Pro Ile Ala Ser Gln GAA AAC AAG TCA AGA AI~A TTA TTT GAT GAA GAG TTT TCT GGG AAG GAA 2304 Glu Asn LYE Ser Arg Lys Leu Phe A~p Glu Glu Phe Ser Gly Lys Glu Leu A~p Ile Pro Ile A6p Ser Ser Thr Val Glu Ile Lys Lys Val Ile Ly~ Pro Glu Phe Glu Pro Val Pro Ser Val Ala Arg Asn Leu Val Ser Gly Thr 8er Ser Tyr Pro Arg Asn Ser Arg Leu Glu Glu Gln Arg Lys Glu Thr Ser Thr Ser Leu Ala A~p A~n Ser Lys Ly~ Gly Ser Ser Phe AAT GAG GGC AAC AAC GAA AAA GAA CCG AAT GCA GCA GAA TGG TTT A~A 2544 Asn Glu Gly Asn Asn Glu Lys Glu Pro Asn Ala Ala Glu Trp Phe Lys Ile A~p Glu Asn Arg His Leu Val Lys A~n Tyr Phe His Asp Leu Leu 8s0 855 860 AAA TAT ATA AAT AAT AAT GAT GCT ACG CTA GCT AAT GAC A?~A GAC GGT 2640 Lys Tyr Ile Asn Asn A~n Asp Ala Thr Leu Ala Asn Asp Lys Asp Gly A~p Leu Ala Phe Leu Ile Lys Gln Met Pro Ala Glu Glu Leu Asp Met Thr Phe Asn Asn Trp Val Asn Leu Lys Val Gln Ser Ile Lys Arg Glu CA 0224ll29 l998-06-l9 WO 97~35~1 PCT~AU96~00827 TTT ATA GAT GAC TGT GAT AAG ~AA TTG GAC ATA TTG AGA AGG GAT TAC 2784 Phe Ile Asp Asp Cys ABP LYB Ly~ Leu ABP Ile Leu Arg Arg Asp Tyr TAT ACT GCC ACA AAT TTT ATT GAA ACT TTG GAA GAT GAC AAT CA~ TTG 2832 Tyr Thr Ala Thr Asn Phe Ile Glu Thr Leu Glu Asp ABP Asn Gln Leu Ile A~p Ile Ala Lys Lys Met Gly Ile Leu *

(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 954 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Met Asp Gly Gln Ile Asp LYB Met Glu Lys Arg Tyr Ser Met Thr Lys Leu Glu Asn Arg Leu Arg Thr Phe Gln Asp Gly Val Ala Leu Glu Lys Lys Lys Leu LYR Trp Ser Phe Lys Val Ile Pro Tyr Gln Ala Met Ala Lys Leu Gly Phe Tyr Phe Asp Pro Val Ile Asp Pro Lys Thr Ser Lys Leu Lys Lys Asp Ser Val Arg Cys Cys Tyr Cys His Arg Gln Thr Tyr Asn Val Arg Asp Cys Arg Ser Lys Arg LYB Asp Val Leu Glu Thr Leu r85 90 95 Ser Asn Ile Met Arg Gln His Leu Thr Val Thr Asp Asn Lys Gln Val Cys Leu Leu Ile Tyr Leu Arg Asn Lys Leu Leu Thr Asp Tyr Ser Phe W O 97/23501 PCTtAU96/00827 Hi~3 Met Gly Val Ser AE~p Trp Lya AE~n AE3p Ly~ Tyr Phe Ser Asn Pro Asp A~p Glu A~n Val Ile A~n Leu Arg Ly~ Phe Thr Phe Gln A~p Aeln ~rp Pro His Ser Gly Ser Gln Asn Glu His Pro Leu Gly Ile Glu Ly~

~et Val A~n Ala Gly Leu Met Arg Tyr Asp Ser Ser Ile Glu Gly Leu Gly Asp Pro Ser Met ABP Lys Thr Leu Met AEIn Asp Thr CYFI Tyr CYB

Ile Tyr CYF: Lys Gln Leu Leu Gln Gly Trp Ser Ile Asn A~3p Asp Pro Met Ser Arg His Tyr Lys Val Ser Gln A~ln Gly Asn Cy~ Tyr Phe Phe ~ln Thr Arg A~n Arg Phe Glu Arg Ile Lys AEln Al3p Asn A~p Ser Ile ~hr Lys A~n CYE~ Glu Val Ser Pro Thr Leu Gly Glu Asn Gly Lys Arg Glu Val Ile Asn Thr Lys Thr Ala Ser Gln Arg Gln Cys Pro Leu Phe Glu Ser Pro Pro Ser Ser Thr Gly Pro Gln Leu Asp Asp Tyr A~n Glu Lyrl Thr Asp Ile Ser Val Ile Gln His Al3n Ile Ser Val Leu A~p Gly ~la Gln Gly Glu Asn Val Ly~ Arg A6n Ser Val Glu Glu Lys Glu Gln ~le Al3n Met Glu Asn Gly Ser Thr Thr Leu Glu Glu Gly Asn Ile Asn 340 3~5 350 ~rg Asp Val Leu Ala Al3p Ly~ Lys Glu Val Ile Ser Thr Pro Thr Ala CA 0224ll29 l998-06-l9 WO 97/23501 PCTJAU96~00827 LYB Glu Ile Lys Arg Pro Asn Val Gln Leu Thr Gln Ser Ser Ser Pro Ile LYB Lys Lys Arg Lys Phe Lys Arg Ile Ser Pro Arg Lys Ile Phe ABP Glu Glu ABP Ser Glu Hia Ser Leu Asn Asn Asn Ser Ala Asn Gly Asp Asn Lys A6p Lys ABP Leu Val Ile ABP Phe Thr Ser His Ile Ile Lys Asn Arg ABP Val Gly Arg Lys Asn Ala Ile Leu ABP Asp Ser Thr ABP Glu Phe Ser Phe Ser Asn Gln Gly His Asn Thr Phe Asp Ile Pro Ile Pro Thr Ser Ser His Leu Leu LYB Gly Ile Asp Ser Asp Asn Asp Asn Val Ile Arg Glu ABP ABP Thr Gly Ile Asn Thr Asp Thr LYB Gly Ala Ser Ser Lys His Glu Lys Phe Ser Val Asn Ser Glu Glu ABP Leu Asn Phe Ser Glu Val Lys Leu Thr Gly Arg ABP Ser Ser Thr Asn Ile Leu Ile Arg Thr Gln Ile Val Asp Gln Asn Leu Gly Asp Ile A~p Arg Asp Lys Val Pro Asn Gly Gly Ser Pro Glu Val Pro Lys Thr His Glu Leu Ile Arg Asp Asn Ser Glu Lys Arg Glu Ala Gln Asn Gly Glu Phe Arg His Gln Lys Asp Ser Thr Val Arg Gln Ser Pro ABP Ile Leu His Ser Asn LYB Ser Gly Asp Asn Ser Ser Asn Ile Thr Ala Ile Pro Lys _ 595 600 605 Glu Glu Gln Arg Arg Gly ABn Ser LYB Thr Ser Ser Ile Pro Ala Asp W O 97/23501 rCT/AU96/00827 Ile Hi~ Pro Ly~ Pro Arg Lys A~n Leu Gln Glu Pro Arg Ser Leu Ser ~le Ser Gly Lys Val Val Pro Thr Glu Arg Lya Leu Asp Asn Ile Aan ~le Asp Leu Asn Phe Ser Ala Ser Asp Phe Ser Pro Ser Ser Gln Ser Glu Gln Ser Ser Lya Ser Ser Ser Val Ile Ser Thr Pro Val Ala Ser Pro Lys Ile Asn Leu Thr Arg Ser Leu His Ala Val Lys Glu Leu Ser Gly Leu LYG Lys Glu Thr Asp Asp Gly Lys Tyr Phe Thr Asn Ly~ Gln ~lu Thr Ile Lys Ile Leu Glu A~p Val Ser Val Lys Asn Glu Thr Pro ~~n Asn Glu Met Leu Leu Phe Glu Thr Gly Thr Pro Ile Ala Ser Gln Glu Asn Lys Ser Arg Lys Leu Phe ABP Glu Glu Phe Ser Gly Lys Glu Leu Asp Ile Pro Ile Asp Ser Ser Thr Val Glu Ile Lys Lys Val Ile Lys Pro Glu Phe Glu Pro Val Pro Ser Val Ala Arg Asn Leu Val Ser ~ly Thr Ser Ser Tyr Pro Arg Asn Ser Arg Leu Glu Glu Gln Arg Lys ~lu Thr Ser Thr Ser Leu Ala A~p Asn Ser Lys Ly~ Gly Ser Ser Phe A~n Glu Gly Asn Asn Glu Ly~ Glu Pro Asn Ala Ala Glu Trp Phe Ly~

Ile Asp Glu Asn Arg Hi~ Leu Val Lys Asn Tyr Phe His Asp Leu Leu CA 0224ll29 l998-06-l9 WO 97~3501 PCT/AU96/00827 Lys Tyr Ile Asn Asn Asn Asp Ala Thr Leu Ala Asn Asp Lys A~p Gly Asp Leu Ala Phe Leu Ile Lys Gln Met Pro Ala Glu Glu Leu Asp Met .

Thr Phe Asn Asn Trp Val Aen Leu Lya Val Gln Ser Ile Lys Arg Glu Phe Ile Asp Asp Cya ABP Lys Ly~ Leu Asp Ile Leu Arg Arg ABP Tyr Tyr Thr Ala Thr Asn Phe Ile Glu Thr Leu Glu Asp Asp Asn Gln Leu Ile Asp Ile Ala Lys Lys Met Gly Ile Leu (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein Lys ABP Ile Lys Lys Thr Met Glu Glu LYB Ile Gln Thr Ser Gly

Claims (43)

CLAIMS:

1. An isolated proteinaceous molecule or derivative of chemical analogue thereofcapable 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 - 1.beta. converting enzyme (ICE) protease or death domain bearing protein FADD.

4. An isolated proteinaceous molecule according to claim 1 wherein the homologue is from an animal cell.

5. An isolated proteinaceous molecule according to claim 4 wherein the cell is from a mammal, nematode, yeast or insect.

6. An isolated proteinaceious molecule according to claim 5 having the amino acid sequence:

Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa Xaa Trp Pro [Xaa]m [Xaa]n 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]q [Xaa]r wherein Xaa is an amino acid residue;

[Xaa]m is a series of at least 5 and preferably at least 9 amino acids;
[Xaa]n 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;

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.

10. An isolated proteinaceous molecule according to claim 6 having an amino acidsequence 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 acidsequence 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 acidsequence 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 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 acidsequence substantially as set forth in SEQ ID NO: 17 or having at least 40% similarly thereto.

15. 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.

20. 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:15.

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.

25. 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.

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.

30. 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.

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.

35. 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 acidsequence 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 comprising 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 comprising an amino acid sequenre 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 acidsequence substantially as set forth in SEQ ID NO: 8 or has at least about 40% similarity thereto.

40. Use according to claim 35 wherein the cell homologue comprising 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 acidsequence substantially as set forth in SEQ ID NO: 13 or has at least about 40% similarity thereto.

42. Use according to claim 35 wherein the cell homologue comprises an amino acidsequence substantially as set forth in SEQ ID NO: 15 or has at least about 40% similarity thereto.

43. Use according to claim 35 wherein the cell homologue comprises an amino acidsequence substantially as set forth in SEQ ID NO: 17 or has at least about 40% similarity thereto.