Note: Descriptions are shown in the official language in which they were submitted.
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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
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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~).
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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
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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
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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
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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
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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
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--8-
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
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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
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- 10-
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.
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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-
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-12-
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.
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TABLE 1
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
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- 14-
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
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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
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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~
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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.
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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
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WO 97/23501 PCT~AU96~00827
- 19-
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~
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-20-
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
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-21-
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.
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-22-
A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.
The antibodies of this aspect of the present invention are particularly useful for
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
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- 23 -
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
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-24-
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.
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-25-
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
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- 26 -
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
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-27-
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
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-28-
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.
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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
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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
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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.
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EXAMPLE 3
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).
EXAMPLE 4
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
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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).
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- 34 -
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.
EXAMPLE 6
EXPRESSION OF MAMMALIAN HOMOLOGUES
The message for murine MIHA is about 7.5 kb, and is expressed in most mouse tissues with
the except of skeletal and cardiac muscle. The highest level ~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
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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.
EXAMPLE 8
~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
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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;
.,
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- 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.
EXAMPLE 11
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.
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TA7BLE 2
~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 - -
MIHA TRAF 1
MIHB TRAF 1 + ++
MIHC TRAF1 + ++
cjun TRAFl
OpIAP TRAF2
MIHA TRAF2
MIHB TRAF2 + +
MIHC TRAF2 + +
cjun TRAF2 - -
OpIAP TRAF3
MIHA TRAF 3
MIHB TRAF3
MIHC TRAP3
cjun TRA1~3
OpIAP fos
MIHA fos
c-IAPl fos
MIHC fos
cjun fos +
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- 39 -
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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-40-
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Rabizadeh, S, T ~count DJ, Friesen, PD and Bredesen, DE (1993) J. Neurochem. 61: 2318-
2321.
Rothe, M, Wog, SC, ~T~n7:~l, WJ and Goeddel, DV (1994) Cell 78: 681-692.
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Sato, T, Irie, S and Reed, JC (1995) Febs ~etters 358: 113-118.
Senkevich,TG,Koonin,EVandBuller,RM(1994) Virology 198: 118-128.
Stager, BZ, Leder, P, Lee, TH, Kim, E and Seed, B (1995) Cell 81: 513-523.
Thornberry, A, Bull, HG, Calaycay, JR, Ch~rm~n, KT, Howard, AD, Kostura, MJ, Miller,
DK, Molineau~, SM, Weidner, JR, Aunins, J, Flli~ton, KO, Ayala, JM, Casano, FJ, Chi, J,
Dig, GJ, Egger, LA, Gaffney, EP, Limjuco, G, Paylha, OC, Rayu, SM, Rolando, AM, Salley,
JP, Yanin, TT, Lee, TD, Shively, JE, MacCross, JE, Mumford, RNA, Schmidt, JA and Tocci,
MJ(1992) Nafure 356: 768-774.
Vaux, DL (1993) Proc. nafl. Aca~ Sci. US~ 90: 786-789.
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Vaux, DL, ~e~ r, G and Strasser, A (1994) Cell 76: 777-779.
Vaux, DL, WP;~m~n, IL and Kim, SK (1992) Science 258: 1955-1957.
Walker, PC, T~l~ni~n, RV, Brady, KD, Dang, LC, J, B, Ferenz, CR, Franklin, S, Ghayur, T,
~kett, MC, Hamill, LD, Herzog, L, E~ nin, M, Houy, W, Mankovich, JA, McGuiness,
L, Orlewicz, E, Paskind, M, Pratt, C, Reis, P, Sllmm~ni~ A, Terra~ova, M, Welch, JP, Xiong,
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Wilson, KP, Black J, Thomson, JA, Kim, EE, (~riffith, JP, Navia, MA, Murcko, MA,Chambers, SP, Aldape, RNA, Raybuck, SA and Livingston, DJ (1994) Nature 370: 270-275.
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Yoon, CH, Lee, JH, Jongeward, GD, and Sternberg, PW (1995) Science 2~9: 1102-1105.
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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
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(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
1 5
GGA ACT AGA ACT TTT GTA CTT GCA GAC ACC AAT AAG GAT GAA GAA TTT 280
Gly Thr Arg Thr Phe Val Leu Ala ABP Thr Asn Lys Asp Glu Glu Phe
10 15 20
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
25 30 35
AGT CCT GTT TCA GCA TCA ACA TTG GCG CGA GCT GGG TTT CTT TAT ACC 376
Ser Pro Val Ser Ala Ser Thr Leu Ala Arg Ala Gly Phe Leu Tyr Thr
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GGT GAA GGA GAC ACC GTG CAA TGT TTC AGT TGT CAT GCG GCA ATA GAT 424
Gly Glu Gly Asp Thr Val Gln CYB Phe Ser Cys His Ala Ala Ile Asp
60 65 70
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
75 80 85
CCA AAT TGC AGA TTT ATC AAT GGT TTT TAT TTC GAA AAT GGT GCT GCA 520
Pro Asn Cys Arg Phe Ile Asn Gly Phe Tyr Phe Glu Asn Gly Ala Ala
90 95 100
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
105 110 115
TGT GTG GGA AAT AGA AAT CCT TTT GCC CCT GAC AGG CCA CCT GAG ACT 616
cys Val Gly Asn Arg Asn Pro Phe Ala Pro A6p Arg Pro Pro Glu Thr
120 125 130 135
CAT GCT GAT TAT CTC TTG AGA ACT GGA CAG GTT GTA GAT ATT TCA GAC 664
His Ala Asp Tyr Leu Leu Arg Thr Gly Gln Val Val Asp Ile Ser A~p
140 145 150
ACC ATA TAC CCG AGG AAC CCT GCC ATG TGT AGT GAA GAA GCC AGA TTG 712
Thr Ile Tyr Pro Arg Asn Pro Ala Met CYB Ser Glu Glu Ala Arg Leu
155 160 165
AAG TCA TTT CAG AAC TGG CCG GAC TAT GCT CAT TTA ACC CCC AGA GAG 760
Lys Ser Phe Gln Asn Trp Pro A~p Tyr Ala His Leu Thr Pro Arg Glu
170 - 175 180
TTA GCT AGT GCT GGC CTC TAC TAC ACA GGG GCT GAT GAT CAA GTG CAA 808
Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Ala ABP Asp Gln Val Gln
185 190 195
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
200 205 210 215
GCC TGG TCA GAA CAC AGG AGA CAC TTT CCC AAT TGC TTT TTT GTT TTG 904
Ala Trp Ser Glu His Arg Arg His Phe Pro A~n CYB Phe Phe Val Leu
220 225 230
GGC CGG AAC GTT AAT GTT CGA AGT GAA TCT GGT GTG AGT TCT GAT AGG 952
Gly Arg Asn Val Asn Val Arg Ser Glu Ser Gly Val Ser Ser A~p Arg
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235 240 245
AAT TTC CCA AAT TCA ACA AAC TCT CCA AGA AAT CCA GCC ATG GCA GAA 1000
Asn Phe Pro Asn Ser Thr Asn Ser Pro Arg Asn Pro Ala Met Ala Glu
250 255 260
TAT GAA GCA CGG ATC GTT ACT TTT GGA ACA TGG ACA TCC TCA GTT AAC 1048
Tyr Glu Ala Arg Ile Val Thr Phe Gly Thr Trp Thr Ser Ser Val A~n
265 270 275
AAG GAG CAG CTT GCA AGA GCT GGA TTT TAT GCT TTA GGT GAA GGC GAT 1096
Lys Glu Gln Leu Ala Arg Ala Gly Phe Tyr Ala Leu Gly Glu Gly Asp
280 285 290 295
AAA GTG AAG TGC TTT CAC TGT GGA GGA GGG CTC ACG GAT TGG AAG CCA 1144
Lys Val LYB Cy~ Phe His CYB Gly Gly Gly Leu Thr ABP Trp Lys Pro
300 305 310
AGT GAA GAC CCT TGG GAA CAG CAT GCG AAG TGG TAC CCA GGG TGC AAA 1192
Ser Glu Asp Pro Trp Glu Gln His Ala LYB Trp Tyr Pro Gly Cy~ Ly~
315 320 325
TAC CTA TTG GAT GAG AAG GGG CAA GAA TAT ATA AAT AAT ATT CAT TTA 1240
Tyr Leu Leu ABP Glu Lys Gly Gln Glu Tyr Ile Asn Asn Ile His Leu
330 335 340
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
345 350 355
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
360 365 370 375
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
380 385 390
ATG GAA GAA AAA ATC CAA ACA TCC GGG AGC AGC TAT CTA TCA CTT GAG 1432
Met Glu Glu Ly~ Ile Gln Thr Ser Gly Ser Ser Tyr ~eu Ser Leu Glu
395 400 405
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
410 415 420
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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
440 445 450 455
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
460 465 470
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
475 480 485
ACG TTC AAG CAA A~A ATT TTT ATG TCT TAGTGGGGCA CCACATGTTA 1719
Thr Phe Lys Gln ~y~ Ile Phe Met Ser
490 495
~ 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
TGACTTGATT TCTATATGTA AGTGAATTC 1988
(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
1 5 10 15
J
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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
100 105 110
Gly Gln Tyr Lys Ser Glu Asn Cys Val Gly Asn Arg Asn Pro Phe Ala
115 120 125
Pro ABP Arg Pro Pro Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly
130 135 140
Gln Val Val Asp Ile Ser A~3p Thr Ile Tyr Pro Arg Asn Pro Ala Met
145 150 155 160
~ys Ser Glu Glu Ala Arg Leu Lys Ser Phe Gln A:~n Trp Pro A~p Tyr
165 170 175
~la His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr
180 185 190
Gly Ala A~p Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Glu
195 200 205
Asn Trp Glu Pro Cya AE~p Arg Ala Trp Ser Glu Hi~ Arg Arg His Phe
210 215 220
Pro ARrl CYB Phe Phe Val Leu Gly Arg Asn Val A~n Val Arg Ser Glu
225 230 235 240
~er Gly Val Ser Ser AE;p Arg Asn Phe Pro A~n Ser Thr A~n Ser Pro
245 250 255
~rg Al3n Pro Ala Met Ala Glu Tyr Glu Ala Arg Ile Val Thr Phe Gly
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260 265 270
Thr Trp Thr Ser Ser Val Asn Lys Glu Gln Leu Ala Arg Ala Gly Phe
275 280 285
Tyr Ala Leu Gly Glu Gly Asp LYR Val Lys Cy~ Phe His Cys Gly Gly
290 295 300
Gly Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln His Ala
305 310 315 320
~y~ Trp Tyr Pro Gly Cy~ Ly~ Tyr Leu Leu ABP Glu Lys Gly Gln Glu
325 330 335
~yr Ile Asn Asn Ile His Leu Thr His Ser Leu Glu Glu Ser Leu Gly
340 345 350
Arg Thr Ala Glu Lys Thr Pro Ser Leu Thr Lys Lys Ile A~p Asp Thr
355 360 365
Ile Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg Met Gly Phe Ser
370 375 380
Phe Lys Asp Ile Lys Lys Thr Met Glu Glu Lys Ile Gln Thr Ser Gly
385 390 395 400
~er Ser Tyr Leu Ser Leu Glu Val Leu Ile Ala Asp Leu Val Ser Ala
405 410 415
~ln Lys A~p Asn Thr Glu Asp Glu Ser Ser Gln Thr Ser heu Gln LYB
420 425 430
Asp Ile Ser Thr Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Lys Leu
435 440 445
Cys Lys Ile Cy~ Met Asp Arg Asn Ile Ala Ile Val Phe Val Pro Cys
450 455 460
Gly His Leu Val Thr Cys Lys Gln Cys Ala Glu Ala Val Asp Lys Cys
465 470 475 480
Pro Met Cys Tyr Thr Val Ile Thr Phe Lys Gln Lys Ile Phe Met Ser
485 490 495
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(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
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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
1 5 10
GGT CCC TCG TAT CAA AAC ATT AAG AGT ATA ATG GAA GAT AGC ACG ATC 1240
Gly Pro 8er Tyr Gln Asn Ile Ly~ Ser Ile Met Glu A~p Ser Thr Ile
15 20 25
TTG TCA GAT TGG ACA AAC AGC AAC AAA CAA AAA ATG AAG TAT GAC TTT 1288
Leu Ser A~p Trp Thr Asn Ser Asn Ly~ Gln Ly~ Met Ly~ Tyr Asp Phe
30 35 40
TCC TGT GAA CTC TAC AGA ATG TCT ACA TAT TCA ACT TTC CCC GCC GGG 1336
Ser Cys Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly
45 50 55
GTG CCT GTC TCA GAA AGG AGT CTT GCT CGT GCT GGT TTT TAT TAT ACT 1384
Val Pro Val Ser Glu Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr
60 65 70 75
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
80 85 90
AAC TGG AAA CTA GGA GAC AGT CCT ATT CAA AAG CAT AAA CAG CTA TAT 1480
Asn Trp Lys Leu Gly A~p Ser Pro Ile Gln Lys His Ly~ Gln Leu Tyr
95 100 105
CCT AGC TGT AGC TTT ATT CAG AAT CTG GTT TCA GCT AGT CTG GGA TCC 1528
Pro Ser Cy8 Ser Phe Ile Gln A~n Leu Val Ser Ala Ser Leu Gly Ser
110 115 120
ACC TCT AAG AAT ACG TCT CCA ATG AGA AAC AGT TTT GCA CAT TCA TTA 1576
Thr Ser Lys A~n Thr Ser Pro Met Arg A~n Ser Phe Ala Hi~ Ser Leu
125 130 135
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TCT CCC ACC TTG GAA CAT AGT AGC TTG TTC AGT GGT TCT TAC TCC AGC 1624
Ser Pro Thr Leu Glu His Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser
140 145 150 155
CTT TCT CCA AAC CCT CTT AAT TCT AGA GCA GTT GAA GAC ATC TCT TCA 1672
Leu Ser Pro Asn Pro Leu A~n Ser Arg Ala Val Glu Asp Ile Ser Ser
160 165 170
TCG AGG ACT AAC CCC TAC AGT TAT GCA ATG AGT ACT GAA GAA GCC AGA 1720
Ser Arg Thr Asn Pro Tyr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg
175 180 185
TTT CTT ACC TAC CAT ATG TGG CCA TTA ACT TTT TTG TCA CCA TCA GAA 1768
Phe Leu Thr Tyr His Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu
190 195 200
TTG GCA AGA GCT GGT TTT TAT TAT ATA GGA CCT GGA GAT AGG GTA GCC 1816
Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gly A~p Arg Val Ala
205 210 215
TGC TTT GCC TGT GGT GGG AAG CTC AGT AAC TGG GAA CCA AAG GAT GAT 1864
CYB Phe Ala Cys Gly Gly Ly~ Leu Ser Asn Trp Glu Pro Lys Asp A~p
220 225 230 235
GCT ATG TCA GAA CAC CGG AGG CAT TTT CCC AAC TGT CCA TTT TTG GAA 1912
Ala Met Ser Glu His Arg Arg His Phe Pro A~n Cys Pro Phe Leu Glu
240 245 250
AAT TCT CTA GAA ACT CTG AGG TTT AGC ATT TCA AAT CTG AGC ATG CAG 1960
Asn Ser Leu Glu Thr Leu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln
255 260 265
ACA CAT GCA GCT CGA ATG AGA ACA TTT ATG TAC TGG CCA TCT AGT GTT 2008
Thr Hi~ Ala Ala Arg Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val
270 275 280
CCA GTT CAG CCT GAG CAG CTT GCA AGT GCT GGT TTT TAT TAT GTG GGT 2056
Pro Val Gln Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly
285 290 295
CGC AAT GAT GAT GTC AAA TGC TTT TGT TGT GAT GGT GGC TTG AGG TGT 2104
Arg A~n Asp A~p Val LYB Cy~ Phe Cys Cy~ Asp Gly Gly Leu Arg Cy8
300 305 310 315
TGG GAA TCT GGA GAT GAT CCA TGG GTA GAA CAT GCC AAG TGG TTT CCA 2152
Trp Glu Ser Gly Asp Asp Pro Trp Val Glu Hi~ Ala Ly~ Trp Phe Pro
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320 325 330
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
335 340 345
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
350 355 360
GAT ACC ACT GGA GAA GAA AAT GCT GAC CCA CCA ATT ATT CAT TTT GGA 2296
ABP Thr Thr Gly Glu Glu Asn Ala ABP Pro Pro Ile Ile His Phe Gly
365 370 375
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
380 385 390 395
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
400 405 410
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
415 420 425
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
430 435 440
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
445 450 455
TTA ATT CGG AAG AAC AGA ATG GCT CTC TTT CAA CAA TTG ACA TGT GTG 2584
Leu Ile Arg LYB Asn Arg Met Ala Leu Phe Gln Gln Leu Thr CYB Val
460 465 470 475
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
480 485 490
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
495 500 505
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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
510 515 520
TTC AAA AAC TGT CTA AAA GAA ATT GAC TCT ACA TTG TAT AAG AAC TTA 2776
Phe Lys Asn CYB Leu LYB Glu Ile ABP Ser Thr Leu Tyr LYB Asn Leu
525 530 535
TTT GTG GAT AAG AAT ATG AAG TAT ATT CCA ACA GAA GAT GTT TCA GGT 2824
Phe Val ABP LYB Asn Met Lya Tyr Ile Pro Thr Glu ABP Val Ser Gly
540 545 550 555
CTG TCA CTG GAA GAA CAA TTG AGG AGG TTG CAA GAA GAA CGA ACT TGT 2872
Leu Ser Leu Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cy~
560 565 570
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
575 580 585
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
590 595 600
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
605 610 615
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
AAGCTTTGAA TACTAAATTA TAGTGTAGAA AAGAACTGGA AACCAGGAAC TCTGGAGTTC 3383
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
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- 55 -
(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
1 5 10 15
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
100 105 110
Ile Gln A~n Leu Val Ser Ala Ser Leu Gly Ser Thr Ser Lys Asn Thr
115 120 125
Ser Pro Met Arg Asn Ser Phe Ala His Ser Leu Ser Pro Thr Leu Glu
130 135 140
Hi~ Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser Leu Ser Pro Asn Pro
145 150 155 160
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
180 185 190
Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala Arg Ala Gly
195 200 205
Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys Gly
210 215 220
Gly Lys Leu Ser Asn Trp Glu Pro Lys ABP Asp Ala Met Ser Glu His
225 230 235 240
~rg Arg His Phe Pro Asn Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr
245 250 255
~eu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg
260 265 270
Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu
275 280 285
Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Afip Asp Val
290 295 300
Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly A~p
305 310 315 320
~sp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu Phe Leu
325 330 335
~le Arg Met Lys Gly Gln Glu Phe Val Asp Glu Ile Gln Gly Arg Tyr
340 345 350
Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Thr Thr Gly Glu
355 360 365
Glu Asn Ala Asp Pro Pro Ile Ile His Phe Gly Pro Gly Glu Ser Ser
370 375 380
Ser Glu Asp Ala Val Met Met Asn Thr Pro Val Val Ly6 Ser Ala Leu
385 390 395 400
~lu Met Gly Phe Asn Arg Asp Leu Val Lys Gln Thr Val Gln Ser Lys
405 410 415
~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
420 425 430
Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu Glu Glu LYB Glu Lys
435 440 445
Gln Ala Glu Glu Met Ala Ser A~p A~p Leu Ser Leu Ile Arg Ly~ Asn
450 455 460
Arg Met Ala Leu Phe Gln Gln Leu Thr Cys Val Leu Pro Ile Leu Asp
465 470 475 480
~sn Leu Leu Lys Ala Asn Val Ile Asn Lys Gln Glu His Asp Ile Ile
485 490 495
~ys Gln Lys Thr Gln Ile Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr
500 505 510
Ile Leu Val Lys Gly Asn Ala Ala Ala A~n Ile Phe Lys Asn Cys Leu
515 520 525
Lys Glu Ile Asp Ser Thr Leu Tyr Ly~ Asn Leu Phe Val Asp Lys Asn
530 535 540
Met LYB Tyr Ile Pro Thr Glu A~p Val Ser Gly Leu Ser Leu Glu Glu
545 550 555 560
~ln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys Met Asp
565 570 575
~ys Glu Val Ser Val Val Phe Ile Pro Cys Gly His Leu Val Val Cys
580 585 590
~ln Glu Cys Ala Pro Ser Leu Arg Ly~ Cys Pro Ile Cys Arg Gly Ile
595 600 605
Ile Lys Gly Thr Val Arg Thr Phe Leu Ser
610 615
~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTE~ISTICS:
(A) LENGTH: 3076 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02241129 1998-06-19
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
TTTAAAATAG CCATCTTAGA ATCAGTGAAA TATGGTAATG TATTATTTTC CTCCTTTGAG 180
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
GTAGCCTGGA GAAGTTGACC TACCTGTGGA GATGCCTGCC ATTAAATGGC ATCCTGATGG 420
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~
1 5 10 15
AGC GCC AAC ACG TTT GAA CTG AAA TAC GAC TTG TCA TGT GAA CTG TAC 817
Ser Ala Asn Thr Phe Glu Leu Ly~ Tyr A~p Leu Ser Cys Glu Leu Tyr
20 25 30
CGA ATG TCT ACG TAT TCC ACT TTT CCT GCT GGG GTT CCT GTC TCA GAA 865
Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu
CA 02241129 1998-06-19
W O 97/23501 PCT/AU96/00827
_ 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
50 55 60
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
65 70 75
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
GTT CAG AGT CTA AAT TCC GTT AAC AAC TTG GAA GCT ACC TCT CAG CCT 1057
Val Gln Ser Leu Asn Ser Val Asn A~n Leu Glu Ala Thr Ser Gln Pro
100 105 110
ACT TTT CCT TCT TCA GTA ACA AAT TCC ACA CAC TCA TTA CTT CCG GGT 1105
Thr Phe Pro Ser Ser Val Thr Asn Ser Thr His Ser Leu Leu Pro Gly
115 120 125
ACA GAA AAC AGT GGA TAT TTC CGT GGC TCT TAT TCA AAC TCT CCA TCA 1153
Thr Glu Asn Ser Gly Tyr Phe Arg Gly Ser Tyr Ser Asn Ser Pro Ser
130 135 140
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
AGT TCC TAC CAC TGT GCA ATG AAT AAC GAA AAT GCC AGA TTA CTT ACT 1249
Ser Ser Tyr His Cy~ Ala Met Asn Asn Glu Asn Ala Arg Leu Leu Thr
160 165 170 175
TTT CAG ACA TGG CCA TTG ACT TTT CTG TCG CCA ACA GAT CTG GCA AAA 1297
Phe Gln Thr Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp Leu Ala LYB
180 185 190
GCA GGC TTT TAC TAC ATA GGA CCT GGA GAC AGA GTG GCT TGC TTT GCC 1345
Ala Gly Phe Tyr Tyr Ile Gly Pro Gly A~p Arg Val Ala Cys Phe Ala
195 200 205
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
210 215 220
-
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
CA 02241129 1998-06-19
W O 97/23501 PCT/AU96/00827
-60-
225 230 235
CAA GAC ACT TCA AGA TAC ACA GTT TCT AAT CTG AGC ATG CAG ACA CAT 1489
Gln A6p Thr Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr His
240 245 250 255
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
260 265 270
AAT CCT GAG CAG CTT GCA AGT GCG GGT TTT TAT TAT GTG GGT AAC AGT 1585
Asn Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser
275 280 285
GAT GAT GTC AAA TGC TTT TGC TGT GAT GGT GGA CTC AGG TGT TGG GAA 1633
ABP ABP Val LYB Cy8 Phe CYB Cy8 ABP Gly Gly Leu Arg CYB Trp Glu
290 295 300
TCT GGA GAT GAT CCA TGG GTT QA CAT GCC AAG TGG TTT CCA AGG TGT 1681
Ser Gly Asp A~p Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cy8
305 310 315
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
320 325 330 335
GCC AGT TAC CCT CAT CTA CTT GAA CAG CTG CTA TCC AQ TCA GAC AGC 1777
Ala Ser Tyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser A~p Ser
340 3~5 350
CCA GGA GAT GAA AAT GCA GAG TCA TCA ATT ATC CAT TTT GAA CCT GGA 1825
Pro Gly Asp Glu Asn Ala Glu Ser Ser Ile Ile His Phe Glu Pro Gly
355 360 365
GAA GAC CAT TCA GAA GAT GCA ATC ATG ATG AAT ACT CCT GTG ATT AAT 1873
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
400 405 410 415
CA 0224ll29 l998-06-l9
W O 97/23501 PCT/AU~
-61-
CTT GTG TTA GAC TTA CTC AAT GCA GAA GAT GAA ATA AGG GAA GAG GAG 2017
Leu Val Leu ABP Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu
420 425 430
AGA GAA AGA GCA ACT GAG GAA AAA GAA TCA AAT GAT TTA TTA TTA ATC 2065
Arg Glu Arg Ala Thr Glu Glu Lys Glu Ser A~n Asp Leu Leu Leu Ile
435 440 445
CGG AAG AAT AGA ATG GCA CTT TTT CAA CAT TTG ACT TGT GTA ATT CCA 2113
Arg Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr Cy8 Val Ile Pro
450 455 460
ATC CTG GAT AGT CTA CTA ACT GCC GGA ATT ATT AAT GAA CAA GAA CAT 2161
Ile Leu Asp Ser Leu Leu Thr Ala Gly Ile Ile Asn Glu Gln Glu His
465 470 475
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
480 485 490 495
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
500 505 510
AAC TCT CTG CAA GAA GCT GAA GCT GTG TTA TAT GAG CAT TTA TTT GTG 2305
Asn Ser Leu Gln Glu Ala Glu Ala Val Leu Tyr Glu Hi~ Leu Phe Val
515 520 525
CAA CAG GAC ATA AAA TAT ATT CCC ACA GAA GAT GTT TCA GAT CTA CCA 2353
Gln Gln Asp Ile Lys Tyr Ile Pro Thr Glu Asp Val Ser Asp Leu Pro
530 535 540
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
545 550 555
TGT ATG GAC AAA GAA GTG TCC ATA GTG TTT ATT CCT TGT GGT CAT CTA 2449
Cys Met ABP Lys Glu Val Ser Ile Val Phe Ile Pro Cys Gly His Leu
560 565 570 575
GTA GTA TGC AAA GAT TGT GCT CCT TCT TTA AGA AAG TGT CCT ATT TGT 2497
Val Val Cy8 Lys Asp Cy8 Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys
580 585 590
,
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
W O 97/23501 PCT/AU96/00827
-62-
595 600 605
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
1 5 10 15
~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
CA 02241129 1998-06-19
WO 97/23501 PCT/AU96J~ ~ - 7
- 63 -
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
100 105 110
Phe Pro Ser Ser Val Thr Asn Ser Thr His Ser Leu Leu Pro Gly Thr
115 120 125
Glu Asn Ser Gly Tyr Phe Arg Gly Ser Tyr Ser A~n Ser Pro Ser Aan
130 135 140
Pro Val A~n Ser Arg Ala Asn Gln Asp Phe Ser Ala Leu Met Arg Ser
145 150 155 160
~er Tyr His Cy~ Ala Met A~n Asn Glu Asn Ala Arg Leu Leu Thr Phe
165 170 175
~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
195 200 205
Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asn Ala Met Ser Glu
210 215 220
His Leu Arg His Phe Pro Lys Cys Pro Phe Ile Glu Asn Gln Leu Gln
225 230 235 240
~sp Thr Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr ~is Ala
245 250 255
~la Arg Phe Lys Thr Phe Phe Asn Trp Pro Ser Ser Val Leu Val Asn
260 265 270
Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser Asp
275 280 285
A~p Val Lys Cy~ Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser
290 295 300
Gly A~p Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cys Glu
CA 0224ll29 l998-06-l9
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-64-
305 310 315 320
~yr Leu Ile Arg Ile Ly~ Gly Gln Glu Phe Ile Arg Gln Val Gln Ala
325 330 335
~er Tyr Pro His heu Leu Glu Gln Leu Leu Ser Thr Ser Asp Ser Pro
340 345 350
Gly ABP Glu Asn Ala Glu Ser Ser Ile Ile His Phe Glu Pro Gly Glu
355 360 365
A6p His Ser Glu A~p Ala Ile Met Met A~n Thr Pro Val Ile Asn Ala
370 375 380
Ala Val Glu Met Gly Phe Ser Arg Ser Leu Val Lys Gln Thr Val Gln
385 390 395 400
~rg Lys Ile Leu Ala Thr Gly Glu Asn Tyr Arg Leu Val Asn Asp Leu
405 410 415
~al Leu Asp Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu Arg
420 425 430
Glu Arg Ala Thr Glu Glu Lys Glu Ser A~n A~p Leu Leu Leu Ile Arg
435 440 445
Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr Cys Val Ile Pro Ile
450 455 460
Leu Asp Ser Leu Leu Thr Ala Gly Ile Ile Asn Glu Gln Glu His ABP
465 470 475 480
~al Ile Lys Gln Lys Thr Gln Thr Ser Leu Gln Ala Arg Glu Leu Ile
485 490 495
~sp Thr Ile Leu Val Lys Gly Asn Ile Ala Ala Thr Val Phe Arg Asn
500 505 510
Ser Leu Gln Glu Ala Glu Ala Val Leu Tyr Glu His Leu Phe Val Gln
515 520 525
Gln Asp Ile Lys Tyr Ile Pro Thr Glu Asp Val Ser Asp Leu Pro Val
530 535 540
Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys
545 550 555 560
CA 0224ll29 l998-06-l9
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- 65 -
Met Asp Lys Glu Val Ser Ile Val Phe Ile Pro Cy8 Gly His Leu Val
565 570 575
~al Cy8 Ly~ Asp Cys Ala Pro Ser Leu Arg Ly~ Cys Pro Ile Cy8 Arg
580 585 590
Ser Thr Ile Lys Gly Thr Val Arg Thr Phe Leu Ser
595 600
(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:
CCACG ATG ACG GAG CTG GGC ATG GAG CTG GAG AGC GTT CGC CTG GCG 47
Met Thr Glu Leu Gly Met Glu Leu Glu Ser Val Arg Leu Ala
1 5 10
ACA TTT GGG GAA TGG CCC CTG AAT GCC CCA GTT TCC GCG GAG GAT CTG 95
Thr Phe Gly Glu Trp Pro Leu Asn Ala Pro Val Ser Ala Glu Asp Leu
15 20 25 30
GTC GCC AAT GGT TTC TTT GCC ACG GGA AAC TGG CTG GAG GCC GAG TGC 143
Val Ala Asn Gly Phe Phe Ala Thr Gly Asn Trp Leu Glu Ala Glu Cys
35 40 45
CAT TTC TGC CAC GTG CGC ATC GAC CGC TGG GAA TAC GGC GAT CAA GTG 191
His Phe Cy~ His Val Arg Ile A~p Arg Trp Glu Tyr Gly Asp Gln Val
50 55 60
GCG GAG CGC CAT CGC CGC TCC TCG CCC ATC TGC TCC ATG GTT CTG GCT 239
Ala Glu Arg His Arg Arg Ser Ser Pro Ile Cys Ser Met Val Leu Ala
CA 0224ll29 l998-06-l9
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-66-
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
80 85 90
GGA AAC AGC GTA GTG GAC AGC CCG GAG TCC TGC TCT TGT CCC GAT CTC 335
Gly A#n Ser Val Val Asp Ser Pro Glu Ser Cys Ser Cys Pro Asp Leu
95 100 105 110
TTG TTG GAG GCC AAT CGA TTG GTA ACT TTC AAG GAC TGG CCG AAT CCC 383
Leu Leu Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Asn Pro
115 120 125
AAC ATC ACG CCG CAG GCT CTG GCA AAG GCA GGT TTC TAC TAC CTG AAC 431
Asn Ile Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn
130 135 140
CGT CTG GAT CAC GTG AAG TGT GTT TGG TGC AAC GGA GTG ATT GCC AAG 479
Arg Leu Asp His Val Lys Cys Val Trp Cys Asn Gly Val Ile Ala Lys
145 150 155
TGG GAG AAG AAC GAC AAT GCC TTT GAA GAG CAC AAG CGC TTT TTT CCC 527
Trp Glu Lys Asn A~p Asn Ala Phe Glu Glu Hls Lys Arg Phe Phe Pro
160 165 170
CAA TGT CCT CGT GTG CAA ATG GGC CCC CTT ATA GAG TTT GCC ACC GGG 575
Gln Cys Pro Arg Val Gln Met Gly Pro Leu Ile Glu Phe Ala Thr Gly
175 180 185 l90
AAG AAC CTG GAT GAG CTG GGC ATC CAG CCC ACC ACT CTG CCA CTG CGT 623
Lys Asn Leu A6p Glu Leu Gly Ile G}n Pro Thr Thr Leu Pro Leu Arg
195 200 205
CCC AAG TAC GCC TGC GTG GAC GCC AGA TTG AGA ACC TTC ACC GAT TGG 671
Pro Lys Tyr Ala CYB Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp
210 215 220
CCC ATT TCC AAT ATT CAG CCC GCT TCG GCT TTG GCG CAG GCT GGC CTA 719
Pro Ile Ser Asn Ile Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly Leu
225 230 235
TAT TAT CAG AAA ATA GGC GAC CAG GTG CGC TGT TTC CAC TGC AAC ATT 767
Tyr Tyr Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His Cys Asn Ile
240 245 250
GGA TTG CGC TCC TGG CAG AAG GAG GAC GAG CCA TGG TTC GAA CAC GCC 815
Gly Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala
CA 0224ll29 l998-06-l9
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25~ 260 265 270
AAG TGG TCG CCA AAG TGT CAG TTC GTT TTG CTG GAC AAG GGT CCA GCC 863
Lys Trp Ser Pro Lys Cy~ Gln Phe Val Leu Leu Asp Ly~ Gly Pro Ala
275 280 285
TAT GTA AGC GAA GTG CTC GCG ACA ACA GCT GCC AAC GCC AGT TCC CCG 911
Tyr Val Ser Glu Val Leu Ala Thr Thr Ala Ala A~n Ala Ser Ser Pro
290 295 300
ACA GCA ACT GCT CCG GCT CCA ACG CTC CAA GCA GAC GTA CTG ATG GAT 959
Thr Ala Thr Ala Pro Ala Pro Thr Leu Gln Ala Asp Val Leu Met Asp
305 310 315
GAA GCT CCG GCC AAG GAG GCC CTA GCC TTG GGT ATC GAT GGA GGA GTT 1007
Glu Ala Pro Ala Lys Glu Ala Leu Ala Leu Gly Ile Asp Gly Gly Val
320 325 330
GTG CGC AAT GCC ATC CAG CGC AAG CTC TTG AGT TCC GGC TGC GCT TTC 1055
Val Arg Asn Ala Ile Gln Arg Ly~ Leu Leu Ser Ser Gly Cy8 Ala Phe
335 340 345 350
TCT ACG TTG GAC GAA CTA TTG CAT GAC ATC TTT GAC GAT GCC GGC GCA 1103
8er Thr Leu Asp Glu Leu Leu Hi~ Asp Ile Phe Asp A~p Ala Gly Ala
355 360 365
GGA GCT GCA CTG GAG GTG CGC GAG CCG CCG GAG CCA AGT GCT CCT TTT 1151
Gly Ala Ala ~eu Glu Val Arg Glu Pro Pro Glu Pro Ser Ala Pro Phe
370 375 380
ATT GAA CCA TGT CAG GCC ACC ACC AGC AAG GCG GCA TCT GTG CCA ATA 1199
Ile Glu Pro Cys Gln Ala Thr Thr Ser Lys Ala Ala Ser Val Pro Ile
385 390 395
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
400 405 410
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
415 420 425 430
ACG CCG AAC GGA AAC CTG TCG CTG GAG GAG GAA AAC CGC CAG CTG AAG 1343
Thr Pro Asn Gly Asn Leu Ser Leu Glu Glu Glu A~n Arg Gln Leu Lys
435 440 445
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GAT GCA CGC TTA TGC AAG GTA TGC TTG GAC GAG GAG GTT GGC GTA GTG 1391
Asp Ala Arg Leu Cy~ Ly~ Val Cys Leu Asp Glu Glu Val Gly Val Val
450 455 460
TTC CTG CCC TGC GGC CAC TTG GCC ACC TGC AAT CAG TGC GCC CCC AGT 1439
Phe Leu Pro Cys Gly His Leu Ala Thr Cys Asn Gln Cys Ala Pro Ser
465 470 475
GTA GCC AAT TGT CCC ATG TGC CGC GCA GAC ATC AAG GGA TTC GTG CGC 1487
Val Ala Asn Cys Pro Met Cys Arg Ala Asp Ile Lys Gly Phe Val Arg
480 485 490
ACG TTC CTT TCG TGAAGTGCAT ATCGACATCA TTTAGAGTTC ACCAGCTAGT 1539
Thr Phe Leu Ser
495
TAGCAGTTAA GCA 1552
(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
1 5 10 15
~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
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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
100 105 110
Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro AJ3n Pro Asn Ile
115 120 125
Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu
130 135 140
Asp His Val Lys Cys Val Trp Cys Asn Gly Val Ile Ala Lys Trp Glu
145 150 155 160
~ys Asn Asp Asn Ala Phe Glu Glu His Lys Arg Phe Phe Pro Gln CYE~
165 170 175
~ro Arg Val Gln Met Gly Pro Leu Ile Glu Phe Ala Thr Gly Lys Asn
180 185 190
Leu Asp Glu Leu Gly Ile Gln Pro Thr Thr Leu Pro Leu Arg Pro Lys
195 200 205
Tyr Ala Cys Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp Pro Ile
210 215 220
Ser Asn Ile Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly Leu Tyr Tyr
225 230 235 240
~ln Lys Ile Gly Asp Gln Val Arg Cys Phe His CYB Asn Ile Gly Leu
245 250 255
~rg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala Lys Trp
260 265 270
Ser Pro Lys Cys Gln Phe Val Leu Leu Asp Lys Gly Pro Ala Tyr Val
275 280 285
Ser Glu Val Leu Ala Thr Thr Ala Ala Asn Ala Ser Ser Pro Thr Ala
290 295 300
Thr Ala Pro Ala Pro Thr Leu Gln Ala Asp Val Leu Met Asp Glu Ala
305 310 315 320
Pro Ala Lys Glu Ala Leu Ala Leu Gly Ile Asp Gly Gly Val Val Arg
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325 330 335
A~n Ala Ile Gln Arg Lys Leu Leu Ser Ser Gly Cys Ala Phe Ser Thr
340 345 350
Leu A~p Glu Leu Leu HiE~ Ai3p Ile Phe Asp Asp Ala Gly Ala Gly Ala
355 360 365
Ala Leu Glu Val Arg Glu Pro Pro Glu Pro Ser Ala Pro Phe Ile Glu
370 375 380
Pro Cy~ Gln Ala Thr Thr Ser Lys Ala Ala Ser Val Pro Ile Pro Val
385 390 395 400
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
435 440 445
Arg Leu Cy~ Lys Val Cyfl Leu Asp Glu Glu Val Gly Val Val Phe Leu
450 455 460
Pro Cy~ Gly His Leu Ala Thr Cys Asn Gln Cys Ala Pro Ser Val Ala
465 470 475 480
~sn Cys Pro Met Cy~ Arg Ala Asp Ile Lys Gly Phe Val Arg Thr Phe
485 490 495
~eu Ser
CA 02241129 1998-06-19
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(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
GCC ACC TTC AAG AAC TGG CCC TTC CTG GAG GAC TGC GCC TGC ACC CCA 152
Ala Thr Phe Lys Asn Trp Pro Phe Leu Glu Asp Cy8 Ala Cys Thr Pro
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GAG CGA ATG GCG GAG GCT GGC TTC ATC CAC TGC CCT ACC GAG AAC GAG 200
Glu Arg Met Ala Glu Ala Gly Phe Ile His Cy~ Pro Thr Glu A~n Glu
40 45 50
CCT GAT TTG GCC CAG TGT TTT TTC TGC TTT AAG GAA TTG GAA GGC TGG 248
Pro A~p Leu Ala Gln Cy~ Phe Phe Cy~ Phe Ly~ Glu Leu Glu Gly Trp
55 60 65
GAA CCC GAT GAC AAC CCG ATA GAG GAG CAT AGA AAG CAC TCC CCT GGC 296
Glu Pro Asp Asp Asn Pro Ile Glu Glu His Arg LYB His Ser Pro Gly
70 75 80
TGC GCC TTC CTC ACT GTC AAG AAG CAG ATG GAA GAA CTA ACC GTC AGT 344
CYG Ala Phe Leu Thr Val Lys Ly~ Gln Met Glu Glu Leu Thr Val Ser
85 90 95
GAA TTC TTG AAA CTG GAC AGA CAG AGA GCC AAG AAC AAA ATT GCA AAG 392
Glu Phe Leu LYB Leu A~p Arg Gln Arg Ala Lys A~n Lys Ile Ala Lys
100 105 110 115
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
120 125 130
CGT CAG TCA ATT GAG CAG CTG GCT GCC TAATGCTGAG CGTTTGCTGA G 488
Arg Gln Ser Ile Glu Gln Leu Ala Ala *
135 140
ATA ACT TGG ACG 500
(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
1 5 10 15
Tyr Arg Ile Ala Thr Phe Lys A~n Trp Pro Phe Leu Glu Asp Cy~ Ala
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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
100 105 110
Ile Ala Lys Glu Thr Asn Asn Lys Gln Lys G1U Phe Glu Glu Thr Ala
115 120 125
Ly~ Thr Thr Arg Gln Ser Ile G1U Gln Leu Ala Ala *
130 135 140
(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:
GAATTCGGCA CGAGAGA ATG CCA TAT ACA TTC GAG AAC TCA GAA GCT CTG 50
Met Pro Tyr Thr Phe Glu Asn Ser Glu Ala Leu
1 5 10
CTC AAA AAC TTG AAA GAC GCG GCT CCG TAC ATA TCA GCT GCC GAA CGC 98
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Leu LYB Asn Leu Lys Asp Ala Ala Pro Tyr Ile Ser Ala Ala Glu Arg
TTT GCA TCT TTC AAA GGA TTC GTT TAT GAT AAG AGA ATT AAC ATC GCC 146
Phe Ala Ser Phe Lys Gly Phe Val Tyr Asp Lys Arg Ile Asn Ile Ala
30 35 40
TGT ACT TCC GAA AAG CTT GCA CGT GCA GGA TTC TAT TCG ACA GCC TCA 194
Cy~ Thr Ser Glu Lys Leu Ala Arg Ala Gly Phe Tyr Ser Thr Ala Ser
45 50 55
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
60 65 70 75
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
80 85 90
CCA CAC TGT GAA TTT GTC ATG ATT GGA GAA ATC GAA GAA AGT GAA CTT 338
Pro Hi6 Cys Glu Phe Val Met Ile Gly Glu Ile Glu Glu Ser Glu Leu
95 100 105
TCT TTC CGA ATC ATA TCC AAT CTT GCG ATA CGA CAC GCA ACA GTG AGA 386
Ser Phe Arg Ile Ile Ser Asn Leu Ala Ile Arg His Ala Thr Val Arg
110 115 120
CTT TAC GAA GAG CTA CTT GGA ATC GTA GCG ACA TTG GAA AAT GGA GAC 434
Leu Tyr Glu Glu Leu Leu Gly Ile Val Ala Thr Leu Glu Asn Gly Asp
125 130 135
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
140 145 150 155
ATT TCA TTC CGA TCT TCT AGC AAG CTT CTC ACT TTT GAT CAT CGA CTT 530
Ile Ser Phe Arg Ser Ser Ser Lys Leu Leu Thr Phe A~p His Arg Leu
160 165 170
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
175 180 185
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
190 195 200
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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
205 210 215
TTT GAC GAA TCA GAT GAT CCG TGG GAA GAG CAT CAG AAG TTC AGT GCA 722
Phe A~p Glu Ser Asp A~p Pro Trp Glu Glu HiR Gln Ly~ Phe Ser Ala
220 225 230 235
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
240 245 250
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
255 260 265
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
270 275 280
ATT GAT GAA ATA ATC AAG ATT CGA AAA ATT ATG ATC AAA CCA AAT CAT 914
Ile A~p Glu Ile Ile Lys Ile Arg Lys Ile Met Ile Lys Pro Asn His
285 290 295
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 *
300 305
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~
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1 5 10 15
~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
100 105 110
Ser A~n Leu Ala Ile Arg His Ala Thr Val Arg Leu Tyr Glu Glu Leu
115 120 125
Leu Gly Ile Val Ala Thr Leu Glu Asn Gly ABP Ile Ala Asn G1U Asn
130 135 140
Pro Ile Thr Arg Ala ABP Ala Thr Arg LYB Leu Ile Ser Phe Arg Ser
145 150 155 160
~er Ser Lys Leu Leu Thr Phe Asp Hi~ Arg Leu Ala Thr Phe Gln Asn
165 170 175
~he Ile Phe Asp Ly~ Ly~ Arg Asn Val Lys CYB Thr Ser Ly~ Ly~ Leu
180 185 190
Ala Ly~ Ala Gly Trp Phe Ser Ile Ala A~n LYB LYB A~p Ly~ Thr Ser
195 200 205
Ala Ly~ CYB Pro Phe CYB Leu Val Glu Leu ABP Phe ABP Glu Ser ABP
210 215 220
ABP Pro Trp Glu Glu His Gln Lys Phe Ser Ala Ser CYB A~p Phe Ile
225 230 235 240
LYB Leu Gly LYB Leu Aap Glu Lys Ly~ Trp Thr Glu Asn Glu Ala Leu
245 250 255
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Met Leu Gly Ala Arg Ile Thr Ile Met Gln Ly~ Tyr Glu LYB Gly Ser
260 265 270
Trp Leu Ile ABP Glu Leu Glu Ly~ Glu Asn Arg Ile Asp Glu Ile Ile
275 280 285
Ly~ Ile Arg Ly~ Ile Met Ile Ly~ Pro A~n Hi~ Val Leu Ly~ Arg Arg
290 295 300
Arg Cy8 Ser Ile *
305
(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
1 5 10 15
TAC AAA GAT CGC TTG ATG ACA TTC AAA AAT TTC GAA TAT GAT AGA GAC 96
Tyr Ly~ A6p Arg Leu Met Thr Phe Ly~ Asn Phe Glu Tyr A~p Arg Acp
20 25 30
CCG GAT GCA A~A TGC ACG TCT CAA GCG GTTTGTCCAT TTTCGTGTTT 143
Pro A~p Ala Ly~ Cy~ Thr Ser Gln Ala
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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
10 15 20 25
GAC CCA GAA GAC GAT CCG TGG TAC GAG CAC ACG AAA CGT GAT GAA CCG 290
ABP Pro Glu Asp Asp Pro Trp Tyr Glu His Thr Lys Arg Asp Glu Pro
30 35 40
TGC GAG TTT GTA CGG ATT GGA AAG CTC GAT GAC TCG GAA TTA ACT ATT 338
Cy~ Glu Phe Val Arg Ile Gly Ly~ Leu Asp Asp Ser Glu Leu Thr Ile
45 50 55
AAC GAT ACC GTT CGT CTC TCA CAA ACC GCC ATG ATT ATG ACT AAA CTC 386
Asn A~p Thr Val Arg Leu Ser Gln Thr Ala Met Ile Met Thr Ly~ Leu
60 65 70
TTT GAG CAT GAG ATG ATG ATA AAT AAT TTG TCT AAT CAT TCT TCT TCT 434
Phe Glu His Glu Met Met Ile Asn Asn heu Ser Asn His Ser Ser Ser
75 80 85
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
90 95 100 105
ACA A~A TCT AAC AGC CGC CGC GGC AAA TA 512
Thr Lys Ser Asn Ser Arg Arg Gly Ly~
110 115
(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
-79-
Met Ala Pro Gly Thr Lys Lys Lys Ser Acp Met Ala Lys Phe Thr Phe
1 5 10 15
~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
100 105 110
Lys Leu Phe Glu His Glu Met Met Ile Asn Asn Leu Ser Asn His Ser
115 120 125
Ser Ser A6p Ala Leu Phe Asp Gln Leu Ly~ Ly~ Val Pro Asn Thr Ala
130 135 140
Ser Thr Thr Lys Ser Asn Ser Arg Arg Gly Lys
145 150 155
(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
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ATG GAT GGT CAA ATA GAT AAA ATG GAG AAG CGA TAT AGC ATG ACC AAA 48
Met Asp Gly Gln Ile Asp Lys Met Glu Ly~ Arg Tyr Ser Met Thr Lys
1 5 10 15
CTG GAG AAT AGG TTA AGG ACT TTC CAA GAT GGT GTT GCA CTT GAA AAA 96
Leu Glu Asn Arg Leu Arg Thr Phe Gln Asp Gly Val Ala Leu Glu Lys
20 25 30
AAA AAG TTA AAG TGG AGT TTT AAA GTT ATT CCG TAT CAG GCA ATG GCC 144
Lys Ly~ Leu Lys Trp Ser Phe Lys Val Ile Pro Tyr Gln Ala Met Ala
35 40 45
AAG CTT GGA TTT TAC TTT GAT CCA GTG ATC GAT CCT AAG ACA TCT AAA 192
Lys Leu Gly Phe Tyr Phe Asp Pro Val Ile ABP Pro Lys Thr Ser LYB
50 55 60
CTG AAA AAA GAC TCT GTA AGA TGT TGC TAT TGT CAT CGT CAA ACA TAC 240
Leu Lys Lys Asp Ser Val Arg Cys Cys Tyr Cys His Arg Gln Thr Tyr
65 70 75 80
AAT GTG AGA GAC TGC AGA TCC AAA AGA AAA GAT GTT CTT GAG ACT CTA 288
A~n Val Arg Asp Cys Arg Ser Lys Arg Lys Asp Val Leu Glu Thr Leu
85 90 95
AGC AAT ATA ATG AGG CAA CAT CTG ACT GTT ACT GAT AAC AAA CAA GTT 336
Ser Asn Ile Met Arg Gln His Leu Thr Val Thr Asp Asn Lys Gln Val
100 105 110
TGC CTC CTT ATT TAC TTG CGC AAT AAG CTA TTG ACG GAC TAT AGC TTC 384
Cys Leu Leu Ile Tyr Leu Arg Asn Ly~ Leu Leu Thr Asp Tyr Ser Phe
115 120 125
CAT ATG GGG GTT TCG GAC TGG AAA AAT GAC AAG TAC TTC AGC AAT CCA 432
His Met Gly Val Ser Aep Trp Lys Asn Asp Lys Tyr Phe Ser Asn Pro
130 135 140
GAT GAT GAA AAC GTG ATA AAC CTA AGA AAA TTT ACT TTT CAA GAT AAC 480
A~p Asp Glu A~n Val Ile Asn Leu Arg Lys Phe Thr Phe Gln Asp Asn
145 150 155 160
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
165 170 175
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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
180 185 190
GGC GAT CCA AGC ATG GAT AAA ACA CTT ATG AAC GAT ACC TGT TAT TGT 624
Gly Asp Pro Ser Met Asp Lys Thr Leu Met Asn Asp Thr Cy~ Tyr CYB
195 200 205
ATT TAT TGC AAA CAG TTG TTA CAA GGT TGG TCA ATA AAT GAC GAT CCG 672
Ile Tyr Cy8 Lys Gln Leu Leu Gln Gly Trp Ser Ile Asn Asp ABP Pro
210 215 220
ATG AGC CGA CAT TAT AAG GTT TCT CAA AAT GGG AAC TGT TAT TTC TTT 720
Met Ser Arg His Tyr Lys Val Ser Gln Asn Gly Asn cys Tyr Phe Phe
225 230 235 240
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
245 250 255
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
260 265 270
GAA GTG ATC AAC ACG AAA ACC GCT TCT CAA AGG CAG TGT CCT TTA TTT 864
Glu Val Ile Asn Thr Lys Thr Ala Ser Gln Arg Gln Cys Pro Leu Phe
275 280 285
GAG TCA CCT CCA TCT AGT ACT GGT CCT CAA CTT GAC GAT TAT AAT GAG 912
Glu Ser Pro Pro Ser Ser Thr Gly Pro Gln Leu Asp Asp Tyr Asn Glu
290 295 300
AAG ACA GAT ATA TCT GTT ATT CAA CAT AAT ATA AGT GTG CTT GAT GGA 960
Lys Thr Asp Ile Ser Val Ile Gln His Asn Ile Ser Val Leu Asp Gly
305 310 315 320
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
325 330 335
ATC AAC ATG GAA AAT GGA AGC ACT ACA TTA GAA GAG GGC AAT ATA AAT 1056
Ile Asn Met Glu Asn Gly Ser Thr Thr Leu Glu Glu Gly Asn Ile Asn
340 345 350
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
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355 360 365
AAA GAA ATC AAA CGT CCA AAT GTC CAG CTA ACC CAA TCA TCG TCA CCT 1152
LYB Glu Ile Ly~ Arg Pro A~n Val Gln Leu Thr Gln Ser Ser Ser Pro
370 375 380
ATA AAA AAG AAA AGA AAG TTT AAG AGA ATA TCG CCT AGA AAA ATC TTT 1200
Ile Ly~ Ly~ Ly~ Arg Lys Phe Lys Arg Ile Ser Pro Arg Ly~ Ile Phe
385 390 395 400
GAT GAG GAA GAT AGC GAA CAC TCC CTT AAT AAT AAT TCT GCC AAC GGA 1248
Asp Glu Glu ABP Ser Glu His Ser Leu Asn A~n A~n Ser Ala Asn Gly
405 410 415
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
420 425 430
AAA AAT AGA GAT GTA GGG AGA AAG AAT GCT ATT TTA GAT GAC AGC ACA 1344
Lys Asn Arg Asp Val Gly Arg Lys Asn Ala Ile Leu Asp Asp Ser Thr
435 440 445
GAT GAA TTC AGT TTC AGT AAC CAA GGA CAT AAC ACA TTT GAC ATA CCT 1392
Asp Glu Phe Ser Phe Ser A~n Gln Gly His A~n Thr Phe Asp Ile Pro
450 455 460
ATA CCA ACC TCA TCA CAT TTG CTC AAA GGT ATA GAT TCA GAT AAT GAC 1440
Ile Pro Thr Ser Ser Hi~ Leu Leu Lys Gly Ile Asp Ser ABP ARn Asp
465 470 475 480
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
485 490 495
GCG TCT TCT AAA CAC GAA AAA TTT TCT GTT AAT TCT GAA GAA GAT TTA 1536
Ala Ser Ser Ly~ His Glu Ly6 Phe Ser Val Asn Ser Glu Glu A~p Leu
500 505 510
AAT TTC AGT GAA GTG AAA CTC ACA GGT AGA GAC AGT AGT ACT AAT ATT 1584
Asn Phe Ser Glu Val Lys Leu Thr Gly Arg Asp Ser Ser Thr Asn Ile
515 520 525
CTT ATA AGA ACG CAA ATT GTA GAT CAG AAT TTG GGT GAT ATC GAC AGA 1632
Leu Ile Arg Thr Gln Ile Val Asp Gln A~n Leu Gly Asp Ile Asp Arg
530 535 540
CA 0224ll29 l998-06-l9
W O 97/23501 PCT/AU96~08Z7
-83-
GAT AAG GTC CCC AAT GGT GGA TCC CCA GAA GTT CCA AAG ACA CAT GAG 1680
Asp Lys Val Pro A~n Gly Gly 8er Pro Glu Val Pro Lys Thr His Glu
545 550 555 560
TTA ATT AGA GAT AAC TCT GAA AAG AGG GAA GCA CAA AAC GGG GAA TTT 1728
Leu Ile Arg A~p Asn Ser Glu Lys Arg Glu Ala Gln Asn Gly Glu Phe
565 570 575
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
580 585 590
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
595 600 605
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
610 615 620
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
625 630 635 640
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
645 650 655
ATA GAT CTA AAT TTT TCG GCG TCC GAT TTT TCA CCA TCA TCA CAA TCT 2016
Ile Asp Leu Asn Phe Ser Ala Ser Asp Phe Ser Pro Ser Ser Gln Ser
660 665 670
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
675 680 685
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
690 695 700
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
705 710 715 720
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
CA 02241129 1998-06-19
W O 97/23501 PCT/AU96/00827
- 84 -
725 730 735
AAT AAT GAA ATG TTA CTT TTT GAG ACT GGA ACC CCG ATC GCA TCT CAA 2256
A~n Asn Glu Met Leu Leu Phe Glu Thr Gly Thr Pro Ile Ala Ser Gln
740 745 750
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
755 760 765
TTG GAT ATA CCT ATA GAT TCA TCA ACT GTA GAA ATA AAA AAG GTT ATT 2352
Leu A~p Ile Pro Ile A6p Ser Ser Thr Val Glu Ile Lys Lys Val Ile
770 775 780
AAA CCC GAA TTT GAG CCA GTC CCA TCA GTT GCA AGG AAT TTG GTG TCT 2400
Ly~ Pro Glu Phe Glu Pro Val Pro Ser Val Ala Arg Asn Leu Val Ser
785 790 795 800
GGA ACC TCT TCA TAT CCA CGG AAT TCA CGT TTA GAA GAA CAA AGA AAG 2448
Gly Thr 8er Ser Tyr Pro Arg Asn Ser Arg Leu Glu Glu Gln Arg Lys
805 810 815
GAA ACA AGC ACA AGT TTG GCT GAT AAT TCT AAG AAA GGA AGT TCA TTT 2496
Glu Thr Ser Thr Ser Leu Ala A~p A~n Ser Lys Ly~ Gly Ser Ser Phe
820 825 830
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
835 840 845
ATT GAT GAA AAT AGA CAC CTT GTA AAA AAT TAC TTC CAT GAT TTG TTA 2592
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
865 870 875 880
GAT CTG GCT TTT TTG ATT AAG CAA ATG CCT GCA GAA GAG TTG GAC ATG 2688
A~p Leu Ala Phe Leu Ile Lys Gln Met Pro Ala Glu Glu Leu Asp Met
885 890 895
ACA TTC AAC AAT TGG GTG AAC CTA AAA GTG CAA TCT ATT AAG CGC GAA 2736
Thr Phe Asn Asn Trp Val Asn Leu Lys Val Gln Ser Ile Lys Arg Glu
900 905 910
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
915 920 925
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
930 935 940
ATC GAT ATT GCT AAG AAA ATG GGC ATT TTA TAG 2865
Ile A~p Ile Ala Lys Lys Met Gly Ile Leu *
945 950 955
(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
1 5 10 15
Leu Glu Asn Arg Leu Arg Thr Phe Gln Asp Gly Val Ala Leu Glu Lys
20 25 30
Lys Lys Leu LYR Trp Ser Phe Lys Val Ile Pro Tyr Gln Ala Met Ala
35 40 45
Lys Leu Gly Phe Tyr Phe Asp Pro Val Ile Asp Pro Lys Thr Ser Lys
50 55 60
Leu Lys Lys Asp Ser Val Arg Cys Cys Tyr Cys His Arg Gln Thr Tyr
65 70 75 80
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
-100 105 110
Cys Leu Leu Ile Tyr Leu Arg Asn Lys Leu Leu Thr Asp Tyr Ser Phe
CA 02241129 1998-06-19
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-86-
115 120 125
Hi~3 Met Gly Val Ser AE~p Trp Lya AE~n AE3p Ly~ Tyr Phe Ser Asn Pro
130 135 140
Asp A~p Glu A~n Val Ile A~n Leu Arg Ly~ Phe Thr Phe Gln A~p Aeln
145 150 155 160
~rp Pro His Ser Gly Ser Gln Asn Glu His Pro Leu Gly Ile Glu Ly~
165 170 175
~et Val A~n Ala Gly Leu Met Arg Tyr Asp Ser Ser Ile Glu Gly Leu
180 185 190
Gly Asp Pro Ser Met ABP Lys Thr Leu Met AEIn Asp Thr CYFI Tyr CYB
195 200 205
Ile Tyr CYF: Lys Gln Leu Leu Gln Gly Trp Ser Ile Asn A~3p Asp Pro
210 215 220
Met Ser Arg His Tyr Lys Val Ser Gln A~ln Gly Asn Cy~ Tyr Phe Phe
225 230 235 240
~ln Thr Arg A~n Arg Phe Glu Arg Ile Lys AEln Al3p Asn A~p Ser Ile
245 250 255
~hr Lys A~n CYE~ Glu Val Ser Pro Thr Leu Gly Glu Asn Gly Lys Arg
260 265 270
Glu Val Ile Asn Thr Lys Thr Ala Ser Gln Arg Gln Cys Pro Leu Phe
275 280 285
Glu Ser Pro Pro Ser Ser Thr Gly Pro Gln Leu Asp Asp Tyr A~n Glu
290 295 300
Lyrl Thr Asp Ile Ser Val Ile Gln His Al3n Ile Ser Val Leu A~p Gly
305 310 315 320
~la Gln Gly Glu Asn Val Ly~ Arg A6n Ser Val Glu Glu Lys Glu Gln
325 330 335
~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
355 360 365
CA 0224ll29 l998-06-l9
WO 97/23501 PCTJAU96~00827
-87-
LYB Glu Ile Lys Arg Pro Asn Val Gln Leu Thr Gln Ser Ser Ser Pro
370 375 380
Ile LYB Lys Lys Arg Lys Phe Lys Arg Ile Ser Pro Arg Lys Ile Phe
385 390 395 400
ABP Glu Glu ABP Ser Glu Hia Ser Leu Asn Asn Asn Ser Ala Asn Gly
40S 410 415
Asp Asn Lys A6p Lys ABP Leu Val Ile ABP Phe Thr Ser His Ile Ile
420 425 430
Lys Asn Arg ABP Val Gly Arg Lys Asn Ala Ile Leu ABP Asp Ser Thr
435 440 445
ABP Glu Phe Ser Phe Ser Asn Gln Gly His Asn Thr Phe Asp Ile Pro
450 455 460
Ile Pro Thr Ser Ser His Leu Leu LYB Gly Ile Asp Ser Asp Asn Asp
465 470 475 480
Asn Val Ile Arg Glu ABP ABP Thr Gly Ile Asn Thr Asp Thr LYB Gly
485 490 495
Ala Ser Ser Lys His Glu Lys Phe Ser Val Asn Ser Glu Glu ABP Leu
500 505 510
Asn Phe Ser Glu Val Lys Leu Thr Gly Arg ABP Ser Ser Thr Asn Ile
515 520 525
Leu Ile Arg Thr Gln Ile Val Asp Gln Asn Leu Gly Asp Ile A~p Arg
530 535 540
Asp Lys Val Pro Asn Gly Gly Ser Pro Glu Val Pro Lys Thr His Glu
545 550 555 560
Leu Ile Arg Asp Asn Ser Glu Lys Arg Glu Ala Gln Asn Gly Glu Phe
565 570 575
Arg His Gln Lys Asp Ser Thr Val Arg Gln Ser Pro ABP Ile Leu His
580 585 590
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
CA 02241129 1998-06-19
W O 97/23501 rCT/AU96/00827
610 615 620
Ile Hi~ Pro Ly~ Pro Arg Lys A~n Leu Gln Glu Pro Arg Ser Leu Ser
625 630 635 640
~le Ser Gly Lys Val Val Pro Thr Glu Arg Lya Leu Asp Asn Ile Aan
645 650 655
~le Asp Leu Asn Phe Ser Ala Ser Asp Phe Ser Pro Ser Ser Gln Ser
660 665 670
Glu Gln Ser Ser Lya Ser Ser Ser Val Ile Ser Thr Pro Val Ala Ser
675 680 685
Pro Lys Ile Asn Leu Thr Arg Ser Leu His Ala Val Lys Glu Leu Ser
690 695 700
Gly Leu LYG Lys Glu Thr Asp Asp Gly Lys Tyr Phe Thr Asn Ly~ Gln
705 710 715 720
~lu Thr Ile Lys Ile Leu Glu A~p Val Ser Val Lys Asn Glu Thr Pro
725 730 735
~~n Asn Glu Met Leu Leu Phe Glu Thr Gly Thr Pro Ile Ala Ser Gln
740 745 750
Glu Asn Lys Ser Arg Lys Leu Phe ABP Glu Glu Phe Ser Gly Lys Glu
755 760 765
Leu Asp Ile Pro Ile Asp Ser Ser Thr Val Glu Ile Lys Lys Val Ile
770 775 780
Lys Pro Glu Phe Glu Pro Val Pro Ser Val Ala Arg Asn Leu Val Ser
785 790 795 800
~ly Thr Ser Ser Tyr Pro Arg Asn Ser Arg Leu Glu Glu Gln Arg Lys
805 810 815
~lu Thr Ser Thr Ser Leu Ala A~p Asn Ser Lys Ly~ Gly Ser Ser Phe
820 825 830
A~n Glu Gly Asn Asn Glu Ly~ Glu Pro Asn Ala Ala Glu Trp Phe Ly~
835 840 845
Ile Asp Glu Asn Arg Hi~ Leu Val Lys Asn Tyr Phe His Asp Leu Leu
850 855 860
CA 0224ll29 l998-06-l9
WO 97~3501 PCT/AU96/00827
-89-
Lys Tyr Ile Asn Asn Asn Asp Ala Thr Leu Ala Asn Asp Lys A~p Gly
865 870 875 880
Asp Leu Ala Phe Leu Ile Lys Gln Met Pro Ala Glu Glu Leu Asp Met
885 890 895
.
Thr Phe Asn Asn Trp Val Aen Leu Lya Val Gln Ser Ile Lys Arg Glu
900 905 910
Phe Ile Asp Asp Cya ABP Lys Ly~ Leu Asp Ile Leu Arg Arg ABP Tyr
915 920 925
Tyr Thr Ala Thr Asn Phe Ile Glu Thr Leu Glu Asp Asp Asn Gln Leu
930 935 940
Ile Asp Ile Ala Lys Lys Met Gly Ile Leu
945 950
(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
