Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL TNF RECEPTOR
DEATH DOMAIN LIGAND PROTEINS
AND INHIBITORS OF LIGAND B~NDING
This application is a continulation-in-part of application Ser. No.
08/494,440, filed June 19, 1995, which was a corltin~1~tion-in-part of application Ser.
No. 08/327,514, filed October 19, 1994.
BACKGROUND OF THE INVENTION
The present invention relates to the field of anti-infl~m~tory
substances and other s~hst~nces which act by inhibiting binding to the intracellular
domain of a tumor necrosis factor receptor (hereinafter "TNF-R"), such as, for
example, the P55 type (or TNF-Rl) TNF receptor. More particularly, the present
invention is directed to novel ligands which bind to the TNF-R intracellular domain
and to inhibition or modulation of signal transduction by this receptor.
Tumor necrosis factor (herein "TNF") is a cytokine which produces
a wide range of cellular activities. TNF causes an infl~rnm~tQry response. which can
be beneficial, such as in mounting an immune response to a pathogen, or when
overexpressed can lead to other detrimental effects of infl~mm~tion.
The cellular effects of TNF are initiated by the binding of TNF to its
receptors (TNF-Rs) on the surface of target cells. The isolation of polynucleotides
encoding TNF-Rs and variant forms of such receptors has been described in
European patent publication Nos. EP 308.378, EP 393.438, EP 433,900, EP 526,90~
and EP 568,925; in PCT patentpublication Nos. WO91/03553 and WO93/19777: and
by Schall et al., Cell 61:361-370 (1990) (disclosing the P5~ type TNF receptor)
Processes for purification of TNF-Rs have also been disclosed in U.S. Patent No
5,296,592.
Native TNF-Rs are characterized bv distinct extracellular.
transmembrane and intracellular domains. The primary purpose of the extracellular
domain is to present a binding site for TNF on the ou~side of the cell. When TNPis bound to the billdin~ site~ a "signal" is 1ranslllitted to the inside of the cell throu~h
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the transmembrane and intracellular domains, indicating that binding has occurred.
Tr~n~mi~.sion or "tr~n.~ ction" of the signal to the inside of the cell occurs by a
change in conformation of the transmembrane and/or intracellular domains of the
receptor. This signal is "received" by the binding of proteins and other molecules
5 to the intracellular domain of the receptor, resulting in the effects seen upon TNF
stim~ tion Two distinct TNF l~ece~tols of ~5~ kd (~TMF-Rl ") and ~75 kd ("TNF-
R2") have been identified. Numerous studies with anti-TNF receptor antibodies have
demonstrated that TNF-RI is the receptor which signals the majority of the
pleiotropic activities of TNF. Recently, the domain required for cign~ling
l0 cvtotoxicity and other TNF-mediated responses has been mapped to the ~80 amino
acid near the C-terminus of TNF-RI. This domain is therefore termed the "death
domain" (hereinafter referred to as "TNF-R death domain" and "TNF-RI-DD") (see~
Tartaglia et al.. Cell 74:845-853 (1993)).
While TNF binding by TNF-Rs results in beneficial cellular effects.
it is oRen desirable to prevent or deter TNF binding from cal-cin~ other detrimental
cellular effects. Although substantial effort has been expended investigating
inhibition of TNF binding to the extracellular domain of TNF-Rs. e~min~tion of
binding of proteins and other molecules to the intracellular domain of TNF-Rs has
received much less attention.
However~ ligands which bind to the TNF-R intracellular domain have
vet to be identified. It would be desirable to identify and isolate such ligands to
examine their effects upon TNF-R signal transduction and their use as therapeutic
agents for treatment of TNF-induced conditions. Furtherrnore, identification of such
ligands would provide a means for screening for inhibitors of TNF-R/intracellular
ligand binding, which will also be useful as anti-inflarnrnatorv agents.
SUMMARY OF THE INVENTIOi~T
Applicants have for the first time identified novel TNF-RI-DD ligand
proteins and have isolated pol ~nucleotides encoding such lioands. Applicants have
also identified a known protein which ma~ also bind to the death domain of TNF-R.
In one embodiment. the present invention provides a composition
comprisinlJ an i~olated pol! nuclcotide encoding a protein havin~ ~F-R 1 -Dl) li~and
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protein activity. In preferred embodiments, the polynucleotide is selected from the
group concicting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:l from nucleotide 2 to nucleotide 1231;
(b) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:l;
(c) a polynucleotide encoding an TNF-R1-DD ligand protein
comprising the amino acid sequence of SEQ ID NO:2;
(d) a polynucleotide encoding an TNF-Rl-DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:2;
(e) - a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:3 from nucleotide 2 to nucleotide 415;
(f) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:3;
(g) a polynucleotide encoding an TNF-RI-DD ligand protein
comprising the amino acid sequence of SEQ ID NO:4;
(h) a polynucleotide encoding an TNF-R1-DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:9 from nucleotide 2 to nucleotide 931;
(j) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:9;
(k) a polynucleotide encoding an TNF-R l -DD ligand protein
comprising the amino acid sequence of SEQ ID NO:lO;
(l) a polynucleotide encodinv an TNF-RI-DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:lO;
(m) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO: I l from nucleotide 2 to nucleotide l 822;
(n) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO: l l:
(o) a polynucleotide encoding an TNF-RI-DD ligand protein
comprisine the amino acid seq~lence of SEQ ID ~ 12:
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(p) a polynucleotide encoding an TNF-RI-DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:12;
(q) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:13 from nucleotide 3 to nucleotide 2846;
(r) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:13, which encodes a protein having TNF-Rl-DD
ligand protein activity;
(s) a polynucleotide encoding an TNF-Rl-DD ligand protein
comprising the amino acid sequence of SEQ ID NO:14;
l O (t) a polynucleotide encoding an TNF-Rl -DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:14 and
having TNF-RI-DD ligand protein activity; and
(u) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(t).
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell. includin~
bacterial, yeast, insect and m~mm~ n cells, transforrned with such polynucleotide
compositions.
Processes are also provided for producing an TNF-RI-DD ligand protein.
which comprises:
(a) growing a culture of the host cell transforrned with such
polynucleotide compositions in a suitable culture medium: and
(b) purifying the TNF-RI-DD ligand protein from the culture.
The ligand protein produced according to such methods is also provided by the
present invention.
Compositions comprisin~ a protein having TNF-RI -DD ligand protein activit~
are also disclosed. In preferred embodiments the protein comprises an amino acidsequence selected from the group consistin~ of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) fragments of the amino acid sequence of SEQ ID NO:2;
(c) the amino acid sequence of SEQ ID NO:4:
(d) I`ragments ol the a~ lo acid sequence of SEQ ID NO:4:
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(e) the amino acid sequence of SEQ ID N0:6;
(f) fr~m-ont~ of the amino acid sequence of SEQ ID N0:6;
(g) the amino acid sequence of SEQ ID N0:10;
(h) fr~ ent.c of the arnino acid sequence of SEQ ID N0:10;
(i) the amino acid sequence of SEQ ID N0:12;
(j) fi .~ of the amino acid sequence of SEQ ID N0:12;
(k) the amino acid sequence of SEQ ID N0:14; and
(I) fragments of the amino acid sequence of SEQ ID N0:14;
the protein being subst~nt~ y free from other m~mm~ n proteins. Such
compositions may further comprise a pharmaceutically acceptable carrier.
Compositions comprising an antibody which specificall~ reacts with
such TNF-RI-DD ligand protein are also provided by the present invention.
Methods are also provided for identifying an inhibitor of rNF-R death
domain binding which comprise:
(a) combining an TNF-R death domain protein with an TNF-RI-
DD ligand protein said combination forming a first binding mixture;
(b) ~,.e~su,i~,g the amount of binding between the rNF-R death
domain protein and the lNF-Rl-DD ligand protein in the first binding
mixture;
(c) combining a compound with the TNF-R death domain protein
and an TNF-RI-DD ligand protein to form a second binding mixture;
(d) measuring the amount of binding in the second binding
mixture: and
(e) comparing the amount of binding in the first binding mixture
with the amount of binding in the second binding mixture;
wherein the compound is capable of inhibitine TNF-R death domain binding when
a decrease in the amount of binding of the second binding mi:cture occurs. In certain
preferred embodiments the TNF-RI-DD ligand protein used in such method
comprises an amino acid sequence selected from the group consistin~ ot`:
(a) the amino acid sequence of SEQ ID N0:2:
(b) fragments of the amino acid sequence of SEQ ID N0:2;
(c) the amino acid sequence of SEQ lO N():~:
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(d) fragments of the amino acid sequence of SEQ ID NO:4;
(e) the a~nino acid sequence of SEQ ID NO:6;
(f) fr~m.ont~ of the amino acid sequence of SEQ ID NO:6;
(g) the amino acid sequence of SEQ ID NO:8;
(h) fr~gm~nt.c of the arnino acid sequence of SEQ ID NO:8
(i) the arnino acid sequence of SEQ ID NO:10;
(j) fragments of the amino acid sequence of SEQ ID NO:10;
(k) the arnino acid sequence of SEQ ID NO:12;
(l) fragments of the arnino acid sequence of SEQ ID NO:12;
(m) the amino acid sequence of SEQ ID NO:14; and
(n) fragments of the amino acid sequence of SEQ ID NO: 14.
Compositions comprising inhibitors identified according to such method are also
provided. Such compositions may include pharmaceutically acceptable carriers.
Methods are also provided for preventing or ameliorating an
15 infl~mm~tory condition which comprises a~mini~tering a therapeutically effective
amount of a composition comprising a protein having TNF-Rl-DD ligand protein
activity and a pharmaceutically acceptable carrier.
Other embodiments provide methods of inhibiting TNF-R death
domain binding comprising a~mini.~tering a therapeutically effective amount of a20 composition comprising a protein having TNF-RI-DD ligand protein activity and a
pharrnaceutically acceptable carrier.
Methods are also provided for preventing or amelioratinv an
infl~mm~tory condition which comprises administering to a m~mm~ n subject a
therapeutically effective amount of a composition comprising a pharmaceutlcally
25 acceptable carrier and a protein selected from the group consisting of insulin-like
gro~vth factor binding protein-5 ("IGFBP-5"), and fragments thereof having TNF-RI -
DD ligand protein activity. Such proteins may also be a~lmini.~tered for inhibiting
TNF-R death domain binding.
Methods of preventing or amelioratin~ an inflammatory condition or
30 of inhibiting TNF-R death domain bindin~ are provided~ ~ hich comprise
a-lministçring to a mammalian subject a therapeuticallv effective amount of inhibitors
of TNF-R death domain hinding. are also provided.
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Methods of identifying an inhibitor of TNF-R death domain binding
are also provided by the present invention which comprise:
(a) transforrning a cell with a first polynucleotide encoding an
TNF-R death domain protein, a second polynucleotide encoding an TNF-R1-
S DD ligand protein, and at least one reporter gene, wherein the c~ ession of
the reporter gene is regulated by the binding of the TNF-R1-DD ligand
protein encoded by the second polynucleotide to the TNF-R death domain
protein encodecl by the first polynucleotide;
(b) growing the cell in the presence of and in the absence of a
compound; and
(c) comparing the degree of ~lession of the reporter gene in the
presence of and in the absence of the compound:
wherein the compound is capable of inhibiting TNF-R death domain binding when
a decrease in the degree of e~ Jsion of the reporter gene occurs. . In preferredl 5 embodiments, the cell is a yeast cell and the second polynucleotide is selected from
the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:1 from nucleotide 2 to nucleotide 1231;
(b) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:l, which encodes a protein having T~F-R1-DD
ligand protein activity;
(c) a polynucleotide encoding an TNF-R I -DD ligand protein
comprising the amino acid sequence of SEQ ID NO:2;
(d) a polynucleotide encoding an TNF-R1-DD ligand protein
2j comprising a fragment of the amino acid sequence of SEQ ID NO:2 and
having TNF-R1-DD ligand protein activity;
(e) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:3 from nucleotide 2 to nucleotide 415;
(f) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:3. which encodes a protein ha~in Tl~'F-RI-DD
Iigand protein activity;
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(g) a polynucleotide encoding an TNF-R1-DD ligand protein
comprising the arnino acid sequence of SEQ ID NO:4;
(h) a polynucleotide encoding an TNF-R1-DD ligand protein
comprising a fragment of the arnino acid sequence of SEQ ID-NO:4 and
having TNF-R1-DD ligand protein activity;
(i) a polynucleotide c~ g the nucleotide se~ ce of SEQ
ID NO:S from nucleotide 2 to nucleotide 559;
(j) a polynucleotide colnyl;sillg a fragment of the nucleotide
sequence of SEQ ID NO:5, which encodes a protein having TNF-RI-DD
ligand protein activity;
(k) a polynucleotide encoding an TNF-R I -DD ligand protein
comprising the amino acid sequence of SEQ ID NO:6;
(l) a polynucleotide encoding an TNF-RI-DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:6 and
having TNF-RI-DD ligand protein activity;
(m) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:7 from nucleotide 57 to nucleotide 87S;
(n) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:7, which encodes a protein having TNF-RI-DD
ligand protein activity;
(o) a polynucleotide encoding an TNF-RI-DD ligand protein
comprisin~ the amino acid sequence of SEQ ID NO:8:
(p) a polynucleotide encoding an TNF-R I -DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:8 and
having TNF-RI-DD ligand protein activity;
(q) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:9 from nucleotide 2 to nucleotide 931;
(r) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:9;
(s) a polynucleotide encoding an TNF-R1-DD ligand protein
comprising the amino acid sequence of SEQ ID NO:lO:
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(t) a polynucleotide encoding an TNF-Rl-DD ligand protein
comprising a fragment of the arnino acid sequence of SEQ ID NO:10;
(u) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO: l l from nucleotide 2 to nucleotide 1822;
(v) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:11;
(w) a polynucleotide encoding an TNF-Rl-DD ligand protein
comprising the arnino acid sequence of SEQ ID NO:12;
(x) a polynucleotide encoding an TNF-Rl-DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO:l':
(y) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:13 from nucleotide 3 to nucleotide 2846;
(z) a polynucleotide comprising a fragment of the nucleotide
sequence of SEQ ID NO:13, which encodes a protein having TNF-Rl-DD
ligand protein activity;
(aa) a polynucleotide encoding an TNF-R1-DD ligand protein
comprising the amino acid sequence of SEQ ID NO:14:
(bb) a polynucleotide encoding an TNF-R1-DD ligand protein
comprising a fragment of the amino acid sequence of SEQ ID NO: 1 l and
having TNF-Rl-DD ligand protein activity: and
(cc) a polynucleotide capable of hybridizing under stringent
conditions to anv one of the polynucleotides specified in (a)-(bb). which
encodes a protein having TNF-R1-DD ligand protein acti~ity.
BRIEF DESCRIPTION OF THE FIGURES
Figs. I and 2 depict autoradiographs demonstrating the e~;pression of
TNF-RI-DD ligand proteins of the present invention.
Fig. 3 depicts an autoradiograph demonstrating the expression of
clones ITU. 15TU and 27TU.
Fig. 4 demonstrates the binding of ITU and 27TU to TNF-RI-DD.
MBP. MBP-lTU or MBP-27TU (3~g) was incubated with sglutathione beads
containil1g 3~1g of either (`JST or GST-TNF-Rl -Dr) in 1 00ul of bindins~ buffer 1().2%
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Triton, 20 mM Tris pH 7.5, 140 mM NaCl, 0.1 mM EDTA, 10 mM DTT and 5%
glycerol). The reaction ws performed at 4C for 2 hours and centrifuged to remove
unbound fraction (Unbound). The beads were then washed with 500~11 binding
buffer four times and re~,u~ ded into SDS-sarnple buffer (Bound). These samples
were analyzed by Western blot using anti-MBP antibody (New Fn~l~nd Biolab).
Fig. 5 demonstrates the ability of 1 5TU and 27TU to activate the JNK
pathway. COS cells were colltl~n~Çected with HA-tagged JNKl and clones l~tu or
27TU. Cells were left untreated or treated for 15 min with 50 ng/ml TNF, and HA-JNK1 was immunoprecipitated with anti-HA antibody. JNK activit~ was measured
in an in vitro kinase assay using GST-cjun (amino acids 1-79) as substrate. and
reactions were electrophoresed on SDS-PAGE.
Fig. 6 is an autoradiograph of an SDS-PAGE gel of conditioned media
from COS cells transfected with clone 3TW.
Fig. 7 is an autoradiograph which demonstrates that.an antisense
IS oligonucleotide derived from the sequence of clone 3TW inhibits TNF-induced
cPLA, phosphorylation.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have for the first time identified and isolated
novel polynucleotides which encode proteins which bind to the TNF-R death domain.
As used herein "TNF-R" includes all receptors for tumor necrosis factor. The P~5tvpe TNF-R is the preferred receptor for practicing the present invention.
The sequence of a polynucleotide encoding one such protein is set
forth in SEQ ID NO:I from nucleotides 2 to 1231. This polynucleotide has been
identified as "clone 2DD" The amino acid sequence of the TNF-R1-DD li_and
protein encoded by clone 2DD is set forth in SEQ ID NO:2. It is belie~ ed that clone
'DD is a partial cDNA clone of a longer full length coding sequence. However. asdemonstrated herein the protein encoded by clone 2DD does bind the death domain
of TNF-R (i.e.~ has "TNF-RI-DD ligand protein activity" as defined herein). Clone
~DD was deposited with the American T~pe Culture Collection on October 13. 1994
and ~iven the accession number ATCC 69706.
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Thc prutein erY~oded by clone 2DD is 410 amino acids in length. Nn
identical nr closely related sequence~ were found using BLASTN/BLASIX or
FASTA searchcs. Theref~rc, clone 2DD ë.r~?des a no~el prote~n.
The ~qUcncc of a polynuclcotide encoding one 6uch proteill is ~et
forth in SQ Il~ NO:3 from nucleotides 2 to 415. l'his polymlclcotide has been
ider-tifi-~ a~ "clone 3DD". I~e amino acid sequence ~ f the TNF-R1-DL) ligand
protein ent~ d by clone 3DD is ~set forth in SEQ ID N0:4. It is beli~ved that
clone 31~D is a partial cDNA clone of a longcr full length codin~ ce.
However, as de~nonstrated hercin the protein eneo-lrd by clone 3DD does bind thedeath domain of TNF-R (i.c., has PTNF-I~1-DD ligand pMtein aCti~ fi de~
herein~. Clone 3DD was deposit~ h the ~4m~ri~an Typc Culalre Collec~o~ on
October 13, 19g4 and given the ~-c~ss~on numbe~ ATCC 69705.
The plOte~n ~nr~d by clonc 3DD is 138 amino acid~. No j~1~ntir~1
or closcly ~elated 6e~ P~ were found using BLAS~BI~STX or FAST~
se~rcl~s. Tl~c.~ol~, clon~ 3DD enrode~ a novel pro~ein. -
A full-leng~ clone eorresponding to clone 3DD was also isolated ~
ide~fed as "~lon~ 31~". '~e nllcleot;~ n~e of clonc 3TW is reportcd as
SEQ ID NO:13. Nucleolides 3 to ~846 of SEQ ID NO:13 encodc a TNF-R1~
li~and protein. the amino a~:id seq~rre of which is reported a~ SFQ ID NO:14.
Amino acids ~11 to 948 of SEQ ID NO:14 cnJ-~yu~ to amino ~ci~ls 1 tu 138 of
SEQ ID NO:4 (clone 3DD~. Cl~ne 3TW was deposited with tlle Al,lelic~L~ Typc
Cult~u~ Collection on Septembc~ 2~, lg~5 and giYen the a~c~ n n~er ATCC
69904.
The seqn~n~ vf a pnlyr-~rlcoti~le en~odinp ano~er such protein is set
for~ in SEQ ID NO:S ~om nucJeotidcs 2 ts~ 559. Tl is poly~u~;leotide has ~een
ntifi~d as "clouc 20DD.r The amino aci~l sequence of ~ T~ R1-DD ligand
protcin enco~led hy clone 20DD is ~et ford~ in SEQ ID ~0:6. Tl is bclieved tl~t
clone 20DD is a partial cl~NA clone of a longer ti~ll lcngth coding scqu~ n~r.
HowcYer, ~ demonstrated herein ~e ~rotein e~ode~ by clone 20DD does bir~l the
death domain vf TN~:-R (i.e., ha~ "TNF-Rl-DD ligand protein acdvityr as defined
herein) Clone 20DD was dcpoailc~ with the American Type ~ulture Collection on
October 13, 1994 and given th~ accession numb~r ATCC 69704.
A~l.t,.LJ~ T
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The protein encoded by clone 20DD is identical to arnino acids 87 to
272 of insulin-like growth factor binding protein-5 ("IGFBP-5"), a sequence for
which was disclosed in J. Biol. Chem. 266: 10646-10653 (1991) by Shim~c~ki et al.,
which is incoll-olaled herein by reference. The polynucleotide and ~ino acid
5 sequences of IGFBP-5 are set forth in SEQ ID NO:7 and SEQ ID NO:8,
respectively. Based upon the sequence identity between clone 20DD and IGFBP-5,
IGFBP-5 and certain fr~gm~rlt.~ thereof will exhibit TNF-R1-DD ligand binding
activity (as defined herein).
The sequence of a polynucleotide encoding another such protein is set
forth in SEQ ID NO:9 from nucleotides 2 to 931. This polynucleotide has been
identified as "clone ITU" The amino acid sequence of the TNF-R1-DD ligand
protein encoded by clone ITU is set forth in SEQ ID NO:10. It is believed that
clone ITU is a partial cDNA clone of a longer full length coding sequence.
However, as demonstrated herein the protein encoded by clone lTU does bind the
death domain of TNF-R (i.e., has "TNF-R1-DD ligand protein activity" as defined
herein). Clone lTU was deposited with the American Type Culture Collection on
June 7, 1995 and given the accession number ATCC 69848.
The protein encoded by clone ITU is 310 amino acids in length. No
identical or closely related sequences were found using BLASTN/BLASTX or
FASTA searches. Therefore, clone ITU encodes a novel protein.
The sequence of a polynucleotide encoding another such protein is set
forth in SEQ ID NO: 11 from nucleotides 2 to 1822. This polynucleotide has been
identified as "clone 27TU" The amino acid sequence of the TNF-RI-DD !igand
protein encoded bv clone 27TU is set forth in SEQ ID NO:12. It is believed that
clone 27TU is a partial cDNA clone of a longer full length coding sequence.
However~ as demonstrated herein the protein encoded by clone 27TU does bind the
death domain of TNF-R (i.e., has "TNF-RI-DD ligand protein activity" as defined
herein). Clone 27TU was deposited with the American Type Culture Collection on
June 7, 1995 and given the accession number ATCC 69846.
The protein encoded by clone 27TU is 607 amino acids in length. No
identical or closely related sequences were found using BLASTN/BLASTX or
FASTA searches Therefore. clone 27TIJ encodes a novel protein. 27TU mav be
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a longer version of clone 2DD. 2DD encodes the same amino acid sequence (SEQ
ID NO:2) as amino acids 198-607 encoded by 27TU (SEQ ID NO:12). The
nucleotide sequences of 2DD and 27TU are also identical within this region of
identity.
An additional "clone 15TU" was isolated which encoded a portion of
the 27TU sequence (a~oxi~llately amino acids 289-607 of SEQ ID NO: 12). Clone
15TU was deposited with the American Type Culture Collection on June 7, 1995 andgiven the accession number ATCC 69847. 15TU comprises the same nucleotide
sequence as 27TU over this region of amino acids.
Polynucleotides hybridizing to the polynucleotides of the present
invention under stringent conditions and highly stringent conditions are also part of
the present invention. As used herein. "highly stringent conditions" include, for
example~ 0.2xSSC at 65C; and "stringent conditions" include~ for example, 4xSSCat 65C or 50% forrnamide and 4xSSC at 42C.
For the purposes of the present application, "TNF-RI-DD ligand
protein" includes proteins which exhibit TNF-RI-DD ligand protein activity. For the
purposes of the present application, a protein is defined as having "TNF-RI-DD
ligand protein activity" when it binds to a protein derived from the TI~F-R death
domain. Activity can be measured by using any assay which will detect binding toan TNF-R death domain protein. Examples of such assays include ~ithout limitation
the interaction trap assays and assays in which TNF-R death domain protein whiclis affixed to a surface in a manner conducive to observing binding. including without
limitation those described in Exarnples I and 3. As used herein an "TNF-R death
domain protein" includes the entire death domain or fragments thereof.
Fragments of the TNF-Rl-DD ligand protein which are capable of
interacting with the TNF-R death domain or which are capable of inhibitill J TNF-R
death domain binding (i.e.~ exhibit TNF-RI-DD ligand protein activity) are also
encompassed by the present invention. Fragments of the TNF-RI-DD ligand protein
may be in linear form or they may be cyclized usin~ knowll methods. f`or example.
as described in H.U. Saragovi. et al., Bio/Technology 1 O, 773-778 (1992) and in R.S.
McDowell~ et al.. J. Amer. Chem. Soc. 114. 9245-9253 (1992). both of wllicll areincorporated herein h~ reference. Such fragments ma~ he fuscd to carrier nlolecules
CA 02202912 1997-04-16
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such as immunoglobulins for many purposes, including increasing the valency of
TNF-RI-DD ligand protein binding sites. For exarnple, fr~ment~ of the TNF-Rl-
DD ligand protein may be fused through "linker" sequences to the Fc portion of an
irnmunoglobulin. For a bivalent form of the TNF-Rl-DD ligand protein, such a
S fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin
isotypes may also be used to ge,lcl~le such fusions. For example, an TNF-RI-DD
ligand protein - IgM fusion would generate a decavalent forrn of the TNF-R1-DD
ligand protein of the invention.
The isolated polynucleotide of the invention may be operably linked
10 to an ex~,ession control sequence such as the pMT2 or pED expression vectors
disclosed in ~ fm~n et al.. Nucleic Acids Res. 19, 4485-4490 (1991), in order toproduce the TNF-Rl-DD ligand protein recombinantly. Many suitable expression
control sequences are known in the art. General methods of expressing recombinant
proteins are also known and are exemplified in R. ~C~ufm~n~ Methods in Enzymology
185, 537-566 (1990). As defined herein "operably linked" means that the isolatedpolynucleotide of the invention and the expression control sequence are situatedwithin a vector or cell in such a way that the TNF-RI-DD ligand protein is ~xl~lessed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for
expression of the TNF-RI-DD ligand protein. Host cells include. for example.
monkey CO~ cells Chinese Hamster Ovary (CHO) cells. human kidnev 293 cells.
human epidermal A431 cells. human Colo205 cells. 3T3 cells. CV-I cells.,other
transformed primate cell lines, normal diploid cells, cell strains derived from in vitro
culture of primary tissue, primary explants, HeLa cells. mouse L cells, BHK, HL-60
U937. HaK or Jurkat cells.
The TNF-RI-DD ligand protein may also be produced by operably
linking the isolated polynucleotide of the invention to suitable control sequences in
one or more insect expression vectors, and employing an insect expression system.
Materials and methods for baculovirus/insect cell expression svstems are
commercially available in kit form from. L'.,L,~., lnvitrogen. San Die~o. California.
h!S.~ (thc MaxBac(~ kit). and such methods are ~ell ~ 11 ill thc 'llt. a~i dcscribcd
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in Summers and Smith, Texas A~ricultural E~ hl,~nt Station Bulletin No. 1555
(1987), incol~oraled herein by reference.
Alternatively, it may be possible to produce the TNF-RI-DD ligand
protein in lower eukaryotes such as yeast or in prok~yot~s such as bacteria.
5 Potentially suitable yeast strains include Saccharomyces cerevisiae,
Schi70sa~charomyces pombe, Kluyveromyces strains, Candida, or any yeast strain
capable of ~ les~ g heterologous proteins. Potentially suitable bacterial strains
include ~scherichia coli, Bacillus subtilis, Salmonella typhim?lrium, or any bacterial
strain capable of e~pl~ea~ g heterologous proteins. If the TNF-Rl-DD ligand protein
10 is made in yeast or bacteria, it may be necessary to modify the protein produced
therein. for example- by phosphorylation or glycosylation of the appropliate sites, in
order to obtain the functional TNF-Rl-DD ligand protein. Such covalent ~tt~t~hmçntc
may be accomplished using known chemical or enzymatic methods.
The TNF-RI-DD ligand protein of the invention m. ay also be
15 expressed as a product of transgenic ~nim~lc e.g., as a component of the milk of
transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ
cells containing a nucleotide sequence encoding the TNF-Rl-DD ligand protein.
The TNF-RI-DD ligand protein of the invention may be prepared by
culturing transformed host cells under culture conditions suitable to express the
20 recombinant protein. The resulting expressed protein may then be purified from such
culture (i.e.. from culture medium or cell extracts) using known purification
processes. such as gel filtration and ion exchange chromatography The purification
of the TNF-RI-DD ligand protein may also include an affinity column cont~inin~ the
TNF-R death domain or other TNF-R death domain protein; one or more column
25 steps over such affinity resins as concanavalin A-agarose. heparin-toyopearl~ or
Cibacrom blue 3GA Sepharose~); one or more steps involving hydrophobic
interaction chromatography using such resins as phenyl ether, butyl ether. or propyl
ether; or immunoaffinity chromatography.
Alternatively~ the TNF-RI-DD ligand protein of the invention may
30 also be expressed in a form which will facilitate purification. For example. it may
be expressed as a fusion protein, such as those of maltose bindin~ protein (MBP) or
lutathiolle-~-transferase (CJST). Kits for expression and purification of such fusion
CA 02202912 1997-04-16
WO 9611273S Pcr/uS95112724
proteins are commercially available from New Fngl~n~1 BioLab (Beverly, MA) and
Pharmacia (Pisca~w~y, NJ), le~l,ecLi~ely. The TNF-R ligand protein can also be
tagged with an epitope and subsequently purified by using a specific antibody
directed to such epitope. One such epitope ("Flag") is co~ ially available from
5 Kodak (New Haven, CT).
Finally, one or more reverse-phase high pc,r~ lallce liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g.,
silica gel having pendant methyl or other aliphatic groups, can be employed to
further purify the TNF-RI-DD ligand protein. Some or all of the foregoing
10 purification steps, in various combinations. can also be employed to provide a
subst~nti~lly homogeneous isolated recombinant protein. The TNF-Rl-DD ligand
protein thus purified is substantially free of other m~mm~ n proteins and is defined
in accordance with the present invention as an "isolated TNF-Rl-DD ligand protein."
TNF-Rl-DD ligand proteins may also be produced by known
15 conventional chemical synthesis. Methods for constructing the proteins of the present
invention by synthetic means are known to those skilled in the art. The
synthetically-constructed protein sequences, by virtue of sharing primary, secondary
or tertiary structural and/or conformational characteristics with TNF-Rl-DD ligand
proteins may possess biological properties in common therewith, including TNF-RI-
20 DD ligand protein activity. Thus, they may be employed as biologically active orimmunological substitutes for natural. purified TNF-Rl-DD ligand proteins in
screening of therapeutic compounds and in immunological processes for the
development of antibodies.
The TNF-RI-DD ligand proteins provided herein also include proteins
25 characterized by amino acid sequences similar to those of purified TNF-Rl-DD
ligand proteins but into which modification are naturally provided or deliberately
engineered. For example, modifications in the peptide or DNA sequences can be
made by those skilled in the art using known techniques. Modifications of interest
in the TNF-RI-DD ligand protein sequences may include the replacement~ insertion30 or deletion of a selected amino acid residue in the coding sequence. For example.
one or more of the cysteine residues may be deleted or replaced with another amino
acid to alter the conformation of the molecule. Mutagenic techniques for such
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replacement, insertion or deletion are well known to those skilled in the art (see, e.g.,
U.S. Patent No. 4,518,584).
Other fragments and derivatives of the sequences of TNF-RI-DD
ligand proteins which would be expected to retain TNF-R1-DD ligand protein
S activity in whole or in part and may thus be usefi~l for scr~enillg or other
immunological m~thoclologies may also be easily made by those skilled in the artgiven the disclosures herein. Such modifications are believed to be encomp~cce~l by
the present invention.
TNF-RI-DD ligand protein of the invention may also be used to
10 screen for agents which are capable of inhibiting or blocking binding of an TNF-R1-
DD ligand protein to the death domain of TNF-R, and thus may act as inhibitors of
l'NF-R death domain binding and/or TNF activity. Binding assays using a desired
binding protein. immobilized or not, are well known in the art and may be used for
this purpose using the TNF-RI-DD ligand protein of the invention. Examples 1 and15 3 describe examples of such assays. Appropriate s~lee~ g assays may be cell-based
or cell-free. Altematively, purified protein based screening assays may be used to
identify such agents. For exarnple, TNF-RI-DD ligand protein may be immobilized
in purified form on a carrier and binding to purified TNF-R death domain may be
measured in the presence and in the absence of potential inhibiting agents. A
20 suitable binding assay may alternatively employ purified TNF-R death domain
immobilized on a carrier. with a soluble form of a TNF-RI-DD ligand protein of the
invention. Any TNF-RI-DD li~and protein may be used in the screenin assays
described above.
In such a screening assay, a first binding mixture is formed bv
25 combining TNF-R death domain protein and TNF-RI-DD ligand protein. and the
amount of binding in the first binding mixture (Bo) is measured. A second bindin~
mixture is also formed by combining TNF-R death domain protein, TNF-RI-DD
ligand protein. and the compound or agent to be screened. and the amount of binding
in the second binding mixture (B) is measured. The amounts of binding in the first
30 and second binding mixtures are compared. for example. by performing a B/Bo
calculation. A compound or agent is considered to be capable of inhibitin~ TNF-Rdeath domain hinding if a decrease in bindin in the second hindin~ nli~;ture
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compared to the first binding mixture is observed. The formulation and optimization
of binding mixtures is within the level of skill in the art. Such binding mixtures may
also contain buffers and salts necess~ry to enhance or to optimize binding, and
additional control assays may be included in the sclee~ g assay of the rnvention.
Alternatively, applo~liale sclcening assays may be cell based. For
example, the binding or interaction betw~een an TNF-R ligand protein and the TNF-R
death domain can be measured in yeast as described below in Exarnples l and 3.
Compounds found to reduce, preferably by at least about 10%. more
preferably greater than about 50% or more, the binding activity of TNF-Rl-DD
ligand protein to TNF-R death domain may thus be identified and then secondaril~screened in other binding assays, including in vivo assays. By these means
compounds having inhibitory activity for TNF-R death domain binding which may
be suitable as anti-infl~mm~tory agents may be identified.
Isolated TNF-Rl-DD ligand protein may be useful in treating,
preventing or ameliorating infl~mrn~tory conditions and other conditions, such as
cachexia, autoimmllne disease, graft versus host reaction~ osteoporosis, colitis.
myelogenous lellk~mi~ diabetes, wasting, and atherosclerosis. Isolated TNF-Rl-DDligand protein may be used itself as an inhibitor of TNF-R death domain binding or
to design inhibitors of TNF-R death domain binding. Inhibitors of binding of TNF-
Rl-DD ligand protein to the TNF-R death domain ("TNF-R intracellular binding
inhibitors") are also useful for treating such conditions.
The present invention encompasses both pharmaceutical compositions
and therapeutic methods of treatment or use which emplov isolated TNF-Rl-DD
ligand protein andlor binding inhibitors of TNF-R intracellular binding.
Isolated TNF-Rl-DD ligand protein or binding inhibitors (from
whatever source derived, including without limitation from recombinant and non-
recombinant cell lines) may be used in a pharmaceutical composition when combined
with a pharmaceutically acceptable carrier. Such a composition may also contain (in
addition to TNF-RI-DD ligand protein or binding inhibitor and a carrier) diluents.
fillers. salts. buffers, stabilizers. solubilizers, and other materials well known in the
art. The term "pharmaceuticallv acceptable" means a non-toxic material that doesnot interfere ~vith the et`fectiveness of the biological activitv of the active
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ingredient(s). The characteristics of the carrier will depend on the route of
a~1mini~tration. The ph~ eeutical corll~o~iLion of the invention may also contain
cytokines, Iymphokines, or other hematopoietic factors such as M-CSF, GM-CSF,
TNF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, G-CSF, Meg-CSF; stem cell
5 factor, and erythropoietin. The ph~.n~ce~lic~l composition may further containother anti-infl~mm~tory agents. Such additional factors and/or agents may be
included in the l ha,...~rei-tical composition to produce a synergistic effect with
isolated TNF-Rl-DD ligand protein or binding inhibitor, or to minimi7~ side effects
caused by the isolated TNF-RI-DD ligand protein or binding inhibitor. Conversely,
10 isolated TNF-R I -DD ligand protein or binding inhibitor may be included in
formulations of the particular cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-infl~mm~tory agent to minimi7~ side
effects of the cytokine, Iymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic factor, or anti-infl~mm~tQry agent.
The pharmaceutical composition of the invention may be in the form
of a liposome in which isolated TNF-Rl-DD ligand protein or binding inhibitor iscombined, in addition to other pharm~eutically acceptable carriers, with amphipathic
agents such as lipids which exist in aggregated form as micelles, insoluble
monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids
20 for liposomal formulation include, without limitation, monoglycerides, diglycerides~
sulfatides, Iysolecithin, phospholipids, saponin. bile acids, and the like. Preparation
of such liposomal formulations is within the level of skill in the art~ as disclosed. for
example. in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No.
4,837,028: and U.S. Patent No. 4.737,323. all of which are incorporated heréin by
'5 reference.
As used herein. the term "therapeutically effective amount" means the
total amount of each active component of the pharmaceutical composition or method
that is sufficient to show a meaningful patient benefit, i.e.. treatment. healing,
prevention or amelioration of an infl~mm~torv response or condition. or an increase
30 in rate of treatment. healing. prevention or amelioration of such conditions. When
applied to an individual active ingredient. administered alone. the term refers to that
inoredient alone. When applied to a combination. the term refers to combined
13
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arnounts of the active ingredients that result in the therapeutic effect, whether
aAmini.etered in combination, serially or simultaneously.
In practicing the method of ~rcidL~ llt or use of the present invention,
a therapeutically effective arnount of isolated TNF-RI-DD ligand protein or binding
5 inhibitor is af~mini~tered to a m~mm~l having a condition to be treated. Isolated
TNF-Rl-DD ligand protein or binding inhibitor may be ?~lminictered in accordancewith the method of the invention either alone or in combination with other therapies
such as ~c~ cnts employing cytokines, lymphokines or other ht",~o~oietic factors.
When co-~lmini.~tered with one or more cytokines, Iymphokines or other
10 hematopoietic factors~ isolated TNF-Rl-DD ligand protein or binding inhibitor may
be a~lmini~tered either simultaneously with the cytokine(s), Iymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If
a~mini~tered sequentially, the ~ n(line physician will decide on the appropriatesequence of ~f~mini.~tering isolated TNF-Rl-DD ligand protein or binding inhibitor
15 in combination with cytokine(s), Iymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors.
Atlmini~tration of isolated TNF-Rl-DD ligand protein or binding
inhibitor used in the pharmaceutical composition or to practice the method of the
present invention can be carried out in a variety of conventional ways, such as oral
20 ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection.
Intravenous ~mini.~tration to the patient is preferred.
When a therapeutically effective amount of isolated TNF-RI-DD
ligand protein or binding inhibitor is adminietered orally, isolated TNF-RI-DD ligand
protein or binding inhibitor will be in the form of a tablet. capsule, powder, soiution
25 or elixir. When ~lministered in tablet form~ the pharmaceutical composition of the
invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
The tablet, capsule, and powder contain from about 5 to 95% isolated TNF-Rl-DD
ligand protein or binding inhibitor, and preferably from about 25 to 90% isolated
TNF-RI-DD ligand protein or binding inhibitor. When administered in liquid form.30 a liquid carrier such as water. petroleum. oils of animal or plant origin such as
peanut oil. mineral oil. soybean oil. or sesame oil. or svnthetic oils may be added.
The liquid t`orm of the pharmaceuticai composition mav t`urther contain phvsiolo~ical
2G
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saline solution~ dextrose or other saccharide solution, or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol. When ~rlminictered in li~uid form,
the pharmaceutical composition contains from about 0.5 to 90% by weight of isolated
TNF-Rl-DD ligand protein or binding inhibitor, and preferably from about 1 to 50%
isolated TNF-R1-DD ligand protein or binding inhibitor.
When a therareuti~lly effective arnount of isolated TNF-Rl-DD
ligand protein or binding inhibitor is a~minict~red by intravenous, cul~leous orsubcutaneous injection, isolated TNF-RI-DD ligand protein or binding inhibitor will
be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The
prepaI~tion of such parenterally acceptable protein solutions, having due regard to
pH. isotonicity, stability, and the like, is within the skill in the art. A preferred
pharmaceutical composition for intravenous, cutaneous. or subcutaneous injectionshould contain. in addition to isolated TNF-RI-DD ligand protein or binding
inhibitor, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's
Injection, or other vehicle as known in the art. The pharm~ce~-tical composition of
the present invention may also contain stabiliærs, preservatives, buffers, antioxidants,
or other additives known to those of skill in the art.
The amount of isolated TNF-RI-DD ligand protein or binding
inhibitor in the pharmaceutical composition of the present invention will dependupon the nature and severity of the condition being treated, and on the nature of
prior treatments which the patient has undergone. Ultimately, the attending physician
will decide the amount of isolated TNF-RI-DD ligand protein or binding inhibitorwith which to treat each individual patient. Initially, the attending physician will
a~3minister low doses of isolated TNF-RI-DD ligand protein or binding inhibitor and
observe the patient`s response. Larger doses of isolated TNF-RI-DD ligand protein
or bindin~ inhibitor may be ~rnini.ctered until the optimal therapeutic effect is
obtained for the patient, and at that point the dosage is not increased further. It is
contemplated that the various pharmaceutical compositions used to practice the
method of the present invention should contain about 0.1 ~ to about 100 mg of
isolated TN~-RI-DD ligand protein or bindin~ inhibitor per kg body weight.
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The duration of intravenous therapy using the pharmaceutical
composition of the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncratic response of each
individual patient. It is cont~ lated that the duration of each application of the
5 isolated TNF-Rl-DD ligand protein or binding inhibitor will be in the range of 12
to 24 hours of con~ nuS in~avenous ~mini~tration. Ultimately the ~ onrline
physician will decide on the al)propl,ate duration of intravenous therapy using the
pharmaceutical composition of the present invention.
Isolated TNF-RI-DD ligand protein of the invention may also be used
10 to immunize animals to obtain polyclonal and monoclonal antibodies which
specificallv react with the TNF-RI-DD ligand protein and which may inhibit TNF-Rdeath domain binding. Such antibodies may be obtained using either the entire TNF-
R1-DD ligand protein or fragments of TNF-R1-DD ligand protein as an immunogen.
The peptide immunogens additionally may contain a cysteine residue at.the carboxyl
15 terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
Methods for synthesizing such peptides are known in the art, for example, as in R.P.
Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 ( 1963); J.L. Krsterl~n.~ky, et al., FEBS
Lett. 211, 10 (1987).
Monoclonal antibodies binding to TNF-R1-DD ligand protein or to
~0 complex carbohydrate moieties characteristic of the TNF-R1 -DD ligand glycoprotein
may be useful diagnostic agents for the immunodetection of TNF-R ligand protein.Neutralizing monoclonal antibodies binding to TNF-R1-DD ligand
protein or to complex carbohydrates characteristic of TNF-R1-DD ligand
glycoprotein may also be useful therapeutics for both infl~mm~tory conditions and
~5 also in the treatment of some forms of cancer where abnormal expression of TNF-
R1-DD ligand protein is involved. These neutralizing monoclonal antibodies are
capable of blocking the signaling function of the TNF-R1-DD ligand protein. By
blocking the binding of TNF-R1-DD ligand protein, certain biological responses to
TNF are either abolished or markedl~ reduced. In the case of cancerous cells or
30 leukemic cells. neutralizing monoclonal antibodies aSgainst TNF-RI-DD ligand
protein may be useful in detectin and preventing the metastatic spread of the
cancerous cells. ~hicll ma!~ he mediated h! the TNF-Rl-DD ligand r)rotein.
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Due to the similarity of their sequences to the insulin growth factor
binding protein ("IGFBP-5") and fragments thereof which bind to the TNF-R death
domain are proteins having TNF-RI-DD ligand protein activity as defined herein.
As a result, they are also useful in pharrnaceutical compositions, for treating
5 infl~mm~tory conditions and for inhibiting TNF-R death domain binding as described
above for TNF-R1-DD ligand proteins generally.
EXAMPLE 1
CLONING OF TNF-R DEATH DOMAIN LIGAND
PROTEIN ENCODING POLYNUCLEOTIDE
A yeast genetic selection method, the "interaction trap ' [G~uris et al,
Cell 75:791-803, 1993, which is incorporated herein by reference]? was used to
screen WI38 cell cDNA libraries (preparation, see below) for proteins that interact
I ~ with the death domain of the P55 type I TNF receptor (TNF-R I -DD). A
polynucleotide encoding arnino acids 326 to 413 of the P55 type TNF receptor, TNF-
R1-DD, was obtained via the polymerase chain reaction (PCR) using a graRing
method. This TNF-RI-DD DNA was then cloned into pEG202 by BamHI alld Sall
sites, g~ "d~ing the bait plasmid, pEG202-TNF-R~-DD. This plasmid contains the
20 HIS3 selectable marker, and expression of the bait, the LexA-TNF-RI-DD fusionprotein, is from the strong constitutive ADH I promoter. To create the reporter strain
carrying the bait protein, yeast strain EGY48, cont~inin~ the reporter sequence
LexAop-Leu2 in place of the chromosomal LEU2. was transformed with
pEG202-TNF-RI -DD and pSH 18-34 (Ura+), which carries another reporter sequence..5 LexAop-lacZ. For screening cDNAs encoding proteins that interact: with
TNF-RI-DD. the expression vector pJG4-5 (TRPI)~ containing the WI38 cell cDNA
library (see below for the cDNA library construction), was transformcd into the
above strain (EGY48/pEG202-TNF-R I -DD/pSH 18-34) according to the method
described by Gietz et al., Nucleic Acids Res., 20.1425 (1992).
cDNA Librarv Construction:
WI38 cell cDNA library: Double stranded cDNA was prepared rrom
3u~ of WI38 mRNA using re~gellts provided b! Ihe Superscript Choice Syste
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WO 96/12735 PCr/US95/12724
(Gibco/BRL, Gaithersberg, MD) with the following substitutions: the first strandsynthesis was primed using an oligo dT/XhoI primer/linker, and the dNTP mix was
substituted with a mix cont~ining methyl dCTP (Stratagene, LaJolla, CA). The cDNA
was mo~lified at both ends by addition of an EcoR~/NotI/SalI adapter-linker and
S subsequently digested with XhoI. This produced cDNA molecules po.csessin~ an
EcoRI/NotI/SalI overhang at the 5' end of the gene and an XhoI overhang at the 3'
end. These fragments were then ligated into the yeast expression/fusion vector
pJG4-5 (Gy~uris et al., Cell, 75, 791-803, 1993), which contains at its amino
terminus, the influenza virus HAI epitope tag, the B42 acidic transcription activation
domain, and the SV40 nuclear localization signal. all under the control of the
galactose-dependent GAL I promoter. The resulting plasmids ~ ere then electroporated
into DHIOB cells (Gibco/BRL). A total of 7.1 x l06 colonies were plated on LB
plates cont~ining 1OO ug/ml of ampicillin. These E coli were scraped. pooled anda large scale plasmid prep was performed using the Wizard Maxi Prep kit (Promega.
Madison, WI), yielding 3.2mg of supercoiled plasmid DNA.
WI38 Cell cDNA Screenin~ Results:
I x l06 transformants were obtained on glucose Ura-His~Trp~ plates.
These transformants were pooled and resuspended in a solution of 65% glycerol.
IOmM Tris-HCI (pH 7.5), 10 mM MgCI~ and stored at -80C in ImL aliquots. For
screening purposes. aliquots of these were diluted 10-fold into Ura-His-Trp~ CM
dropout gal/raff medium (containinSJ 2% alactose 1% rafi'inose) which induces the
expresssion of the library encoded proteins and incubated at 30C for 4 hours. 12
x 106 colony forming units (CFUs) were then plated on standard IOcm galactose
X-Gal Ura-His~Trp-Leu~ plates at a densitv of 2 :; 10~ CFU!plate. After three days at
30C about 1,000 colonies were formed (Leu' ) and of those. si:;ty-four colonies were
LacZ~. In order to test if the Leu'/LacZ~ phenotype was due to the library-encoded
protein, the galactose dependencv of the phenotvpe was tested. Expression of thelibrary-encoded proteins was turned off by ,~rowth on glucose Ura~His~Trp~ master
plates and then retested for galactose-dependency on olucose Ura~His~Trp-Leu-.
galactose Ura~His~Trp'Leu~ ~lucose X-Gal Ura'His~'l`rp-. and galactose X-Gal
Ura'His-Trp~ plates. Of these 3~ colollics showcd s~alactose-dependent S~rowth on
CA 02202912 1997-04-16
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Leu~ plates and galactose-dependent blue color on X-Gal-cont~inin~ medium (LacZ~phenotype). Total yeast DNA was ~ ,aled from these colonies according to the
method described previously (Hoffman and Winston, 1987). In order to analyze thecDNA sequences, PCR re~tiorlC were pelro~ ed using the above yeast-DNA as a
5 template and oligo primers specific for the vector pJG4-5, fl~nkin~ the cDNA
insertion point. PCR products were purified (Qiagen PCR purification kit), subjected
to restriction digest with the enzyme HaeIII, run on 1.8% agarose gels, and the
restriction paKerns compared. Similar and identical restriction pal~llls were grouped
and ~e~leselll~Lives of each group were sequenced and compared to Genbank and
10 other (l~t~baces to identify any sequence homologies.
One clone of unique sequence ("2DD") and three clones with identical
sequence ("3DD") were isolated and showed no signficant sequence homologies
compared to Genbank and other databases. Additionally. four other clones ("20DD")
with identical sequence to a portion of hurnan insulin-like growth factor binding
protein-5 (Shunichi Shim~c~ki et al., ~. Biol. Chem. 266: 10646- 10653 (1991)) were
isolated. The clones "2DD," "3DD" and "20DD" were chosen for further analysis.
Library vector pJG4-5 cont~inin~ these clones sequences were rescued from yeast by
transforming the total yeast DNAs into the E. coli strain KC8 and selecting for
growth on Trp-ampicillin plates. These putative TNFRI interacting proteins were
20 then tested further for specificity of interaction with the TNF-Rl-DD by the
reintroduction of JG4-5 clone into EGY48 derivatives containing a panel of different
baits. including bicoid. the cytoplasmic domain of the IL-I receptor and TNF-RI-DD. The above clones were found to interact only ~ ith the TNF-RI-DD. The
interaction between these clones and TNF-RI-DD was thus jud_ed to be spécific.
U937 cDNA Screening Results:
A U937 cDNA library was also constructed and screened as described
above. 1.020 Leu+ colonies were found and of those, 326 colonies were also LacZ+.
62 colonies of these Leu+/LacZ+ colonies showed a galactose-dependent phenotype..
30 One of these clones. ITU. encodes a novel sequence. Interestingly, two clones.
15TU and 27TU~ encode related or identical sequences. e~cept that 27TU contains
CA 02202912 1997-04-16
W O96/12735 PCTrUS95/12724
about 864 additional nucleotides (or about 288 amino acids) at the 5' end. 1 5/27TU
also encode a novel sequence.
EXAMPLE 2
EXPRESSION OF THE TNF-Rl-DD ligand PROTErN
cDNAs ~nro~in~ TNF-R intracellular ligand l)r~teills were released
from the plG4-5 vector with the ap~)lolJIiate restriction enzymes. For exarnple, EcoRI
and XhoI or NotI and XhoI were used to release cDNA from clone 2DD and clone
10 20DD. Where the restriction sites were also present in the internal sequence of the
cDNA, PCR was performed to obtain the cDNA. For example, the cDNA fragment
encoding "clone 3~D" was obtained through PCR due to the presence of an internalXhol site. These cDNAs were then cloned into various expression vectors. These
included pGEX (Pharrnacia) or pMAL (New Fngl~n~l Biolabs) for expression as a
15 GST (Glutathione-S-transferase) or MBP (maltose binding protein) fusion protein in
E. coli, a pED-based vector for m~mm~ n expression, and pVL or pBlueBacHis
(In~itrogen) for baculovirus/insect expression. For the immunodetection of TNF-Rintracellular ligand expression in m~mm~ n cells, an epitope sequence? Flag," was
inserted into the translational start site of the pED vector, generating the pED-Flag
'0 vector. cDNAs were then inserted into the pED-Flag vector. Thus, the expression of
cDNA from pED-Flag yields a protein with an amino terminal Met, followed by the
"Flag` sequence. Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. Standard DEAE-Dextran or
lipofectamine methods were used to transfect COS or CHO dukx cells.
Immunodetection of Flag-tagged proteins was achieved using the M2 antibody
(Kodak). Moreover. an immunoaffinity column using the M2 antibody. followed by
elution with the Flag" peptide. can be used for the rapid purification of the
flag-tagged protein. Similarly, affinity purification of GST-, MBP- or His-tagged
fusion proteins can be performed using glutathione~ amylose or nickel columns.
Detailed purification protocols are provided by the manufacturers. For manv fusion
30 proteins, the TNF-R intracellular ligand can be released bv the action of thrombin,
factor Xa~ or enterokinase cleavage. In the case where highly purified material is
required. standard purification procedures. such as ion-e~;change. hydrophobic. and
26
CA 02202912 1997-04-16
WO g6112735 PCT/USg5/12724
gel filtration chromatography will be applied in addition to the affinity purification
step.
Figs. 1 and 2 depict autoradiographs demonstrating the expression of
TNF-Rl-DD ligand proteins in yeast and m~mm~ n cells. Fig. 1 shows the results
5 of expression of isloated clones of the present invention in yeast. EGY48 was
transformed with pJG4-5 co~ clone 2DD, 3DD or 20DD. Cells were then
grown overnight in the galactose/raffinose mediurn. Cell Iysates were prepared and
subject to 4-20% SDS gel electrophoresis, followed by Western blot analysis using
anti-HA antibody (12CA5. Boehringer Mannheim, Tn~ n~l~olis, IN). Fig. 2 shows
10 the results of expression of Flag-2DD and Flag-20DD in COS cells. COS cells were
transfected with either pED-Flag (Vector control), Flag-2DD or Flag-20DD plasmidby the lipofectamine method. Thirty llg of each cell lysate were prepared and
subjected to 4-20% SDS gel electrophoresis, followed by Western blot analysis using
M2 antibody (Kodak). The bands in the Flag-2DD and Flag-20DD lanes indicate
15 significant expression of the re~e-;live TNF-RI-DD ligand proteins.
EXAMPLE 3
ASSAYS OF TNF-R DEATH DOMAIN BINDING
Two different methods were used to assay for TNF-R1-DD ligand
protein activity. The first assay measures binding in the yeast strain in "interaction
trap.`' the system used here to screen for TNF-Rl-DD interacting proteins. In this
system. the expression of reporter genes from both LexAop-Leu' and LexAop-LacZ
relies on the interaction between the bait protein, in this case TNF-RlDD. and the
prey. the TNF-R intracellular ligand. Thus, one can measure the strength of the
interaction by the level of Leu2 or LacZ expression. The most simple method is to
measure the activity of the LacZ encoded protein, ~-galactosidase. This activity can
be judged by the degree of blueness on the X-Gal containing medium or filter. For
the quantitative measurement of ~-galactosidase activitv. standard assavs can befound in "Methods in Yeast Genetics" Cold Spring Harbor, New York. 1990 (bv
Rose. M.D., Winston~ F.. and Hieter, P.).
The second assay for measurin~ binding is a cell-free svslem. An
exalllple of a typical assay is described below. Purified G~T-TNF-RI-DD fusion
- - -
CA 02202912 1997-04-16
W O96112735 PCTrUS95/12724
protein (2 ug) was mixed with amylose resins bound with a GST-TNF-Rl-DD
intracellular ligand for 2 hour at 4C. The mixture was then centrifuged to se~
bound (rçm~in~d with the beads) and unbound (rern~ined in the supernatant)
GST-TNF-Rl-DD. After extensive washing, the bound GST-TNF-Rl-DD-was eluted
5 with maltose and detçcted by Westem blot analysis using a GS~ antibody. The
TNF-R1-DD or the intracellular ligand can also be irn~nobiliæd on other solid
aLIpl)oll~, such as on plates or fluorobeads. The binding can then be measured using
ELISA or SPA (scintillation ploxil"ily assay).
EXAMPLE 4
CHARACTERIZATION OF TNF-R
DEATH DOMAIN LIGAND PROTEIN
MapPin~ the interaction site in TNF-R1
Many of the key amino acids for TNF-R sign~ling have been
determined by site-directed mutagenesis (Tataglia et a/., Cell 74:845-853 (1993).
These amino acids are conserved between TNF-R and the Fas antigen, which is
required for me~ tin~ cytotoxicity and other cellular responses. In order to test if
20 the TNF-R intracellular proteins interact with these residues. the following mutations
were constructed: F345A (substitution of phe at amino acid 345 to Ala)~ R347A,
L351A, F345A/R347A/L351A, E369A, W378A and I408A. The ability of the
mutant protein to interact with the intracellular ligand in the "interaction trap'` system
was tested.
Effcct on the TNF-mediated response
The effect of the TNF-R intracellular ligands on the TNF-mediated
response can be evaluated in cells overexpressing the ligands. A number of TNF-
mediated responses~ including transient or prolonged responses, can be measured. For
30 example, TNF-induced kinase activity toward either MBP (myelin basic protein) or
the N-terminus (amino acids 1-79) of cjun can be measured in COS cells or CHO
cells either transiently or stably overexpressing clone 2DD~ 3DD or clone 20DD.
The significance of these ligand proteins in TNF-mediated cytoto:~icity alld other
ccllulal rcspollses call be measured in L9'9 or ~J937 overe~;pressin~ cclls.
28
CA 02202912 1997-04-16
W O 96/12735 PCTAUS95/12724
Alternatively, other functional assays~ such as the induction of gene expression or
PGE2 production after prolonged incubation with TNF, can also be used to measurethe TNF mecli~tçd response. Conversely, the significance of the TNF-RI-DD ligandproteins in lNF si~n~lin~ can be established by lowering or elimin~tin~ the
5 expression of the ligands. These experiments can be performed using ~nti~n~e
s~;on or ~Sg~lliC mice.
Enzymatic or functional assavs
The signal transduction events initiated by TNF binding to its receptor
10 are still largely unknown. However, one major result of TNF bindin~ is the
stimulation of cellular serine/threonine kinase activity. In addition, TNF has been
shown to stimulate the activity of PC-PLC~ PLA" and sphingomyelinase. Therefore.some of the TNF-RI -DD ligand proteins may possess intrinsic enzymatic activity that
is responsible for these activities. Therefore, enzymatic assays can be performed to
15 test this possibility, particularly with those clones that encode proteins with sequence
homology to known enzymes. In addition to enzymatic activity, based on the
sequence homology to proteins with known function, other functional assays can
also be measured.
EXAMPLE S
ISOLATION OF FULL LENGTI~ CLONES
In many cases. cDNAs obtained from the interaction trap method each
encode only a portion of the full length protein. For example~ based on identity and
sequence and the lack of the initiating methionine codon. clones 2DD. 3DD and
20DD appal~ntly do not encode full lengtll proteins. Therefore, it is desirable to
isolate full length clones. The cDNAs obtained from the screening, SUCIl as clone
2DD, are used as probes, and the cDNA libraries described herein. or alternatively
phage cDNA libraries, are screened to obtain full length clones in accordance with
known methods (see for example, "Molecular Cloning, ~ Laborator~ Manual"~ b~
Sambrook et al., 1989 Cold Spring Harbor).
CA 022029I2 I997-04-I6
WO 9611273S PCrlUS95112724
EXAMPLE 6
ANTIBODIES SPECIFIC FOR TNF-R
INTRACELLULAR LIGAND PROTEIN
Antibodies specific for TNF-R intracellular ligand proteins can be
produced using purified recombinant protein, as described in Example 2. as antigen.
Both polyclonal and monoclonal antibodies will be produced using standard
techniques, such as those described in '~Antibodies, a Laboratory Manual" by Ed
Harlow and David Lane (1988)~ Cold Spring Harbor Laborat~
EXAMPLE 7
CHARACTERIZATION OF
CLONES lTU AND 15/27TU
Specificitv of Interaction
The specificity of clones ITU, I STU and 27TU was tested usin~ a panel of
baits. The ability of these clones to bind the TNF-R death domain ~:as compared
to their binding to the intracellular domain of the second TNF-R(TNF-R P751c), the
entire intracellular domain of TNF-R (TNF-R P551c), the death domain of the fas
antigen (which shares 28% identity with TNF-R-DD) (FasDD), the Drosophila
transcription factor bicoid. and a region of the IL-I receptor known to be critical for
sign~llin~ (IL-IR4~752,). As shown in Table 1, none of these clones interacted with
TNF-R P751C or FasDD, and only lTU interacted with bicoid In contrast. both ITU
and 1 5TU bound the cytoplasmic domain of the p55 TNF-R. as well as residues 477-
527 of the IL-IR. 27TU interacted relati~ely weakly with these sequences.
Table I
clone TNF-RDD TNF-R TNF-R FasDD bicoid IL-IR
p75,~ P551c (177-527)
ITU + ++ - ++ + - + + + + +
30 15TU +++ + + ++ - - + +
27TU +++ - +
Interaction ~ith Amino Acids Critical for Si~nallin~
CA 02202912 1997-04-16
WO g6/12735 Pcr/usssll2724
The ability of each clone to interact with four single-site mutations
in the TNF-R death domain (each known to abolish .~i~n~llin~) was measured. As
shown in Table 2, each of the clones interacted less strongly with the death ~om~in
mllt~nt.c than with the wild type death dQrn~in~ sugg~;n~ that these clones may bind
5 - critical residues in vivo.
Table 2
clone TNF-RDD F345A L351A W378A 1408A
ITU + ++ +
15TU + + + + + + + ++
27TU ++ + + + + ++
Expression of lTU, 15TU and 27TU
1~ Fig. 3 depicts an autoradiograph demonstrating the expression of
clones lTU, 15TU and 27TU in yeast (A) and COS cells (B).
In (A): EGY48 was transformed with pJG4-5 cont~inin~ clones lTU,
15TU or 27TU, Cells were then grown overnight in galactose/raffinose mto~ m.
Cell Iysates were prepared and subjected to 4-20% SDS gel electrophoresis,
20 followed by Western blot analysis using anti-HA antibody (12CA5, Boehringer
Mannheim).
In (B): COS cells were transfected with pED-Flag cont~ining clones
lTU, l5TU and 27TU. Cell Iysates were prepared and analyzed by Western blot
using anti-Flag antibody (M2, Kodak).
Specific Bindin~ of lTU and 27TU to TNF-Rl-DD
The interaction of lTU and 27TU with TNF-Rl-DD was tested using
purified bacterially expressed fusion proteins. As shown in Fig. 4, MBP fusion
proteins containing lTU or 27TU bound only to TNF-R1-DD expressed as a GST
~0 fusion protein. but not to GST protein alone. In the control experiment, MBP
protein did not bind either GST or GST/TNF-RI-DD. These r~sults indicate that
CA 02202912 1997-04-16
W O 96/12735 PCTnUSg5/12724
lTU and 27TU bound specifically to the TNF-R1 death domain in vitro, confiIming
the data obtained in the interaction trap.
15TU and 27TU Activation of JNK Activity
S The jun N-termin~l kinase (JNK) is normally activated within 15 min
of TNF treatment in COS cells. 15TU and 27TU were cotlallsrected with an
epitope tagged version of JNK, HA-JNK, in duplicate. After TNF ~l~A~ nl, JNK
was i.,..~ op~cci~ ted with anti-HA antibody and JNK activity was measured in
immllnoprecipitation kinase assays, using GST-cjun (amino acids 1-79) as
10 substrate). Reactions were electrophoresed on SDS-PAGE. As shown in Fig. 5,
transfection of 15TIJ and 27TU, but not vector alone, into COS cells activated JNK
even in the absence of TNF, suggesting that these clones are involved in signal
transduction of TNF and the pathway leading to JNK activation in vivo.
EXAMPLE 8
ISOLATION, EXPRESSION AND ASSAY
OF CLONE 3TW
Clone 3TW was isolated from the WI38 cDNA library using clone 3DD as
a porbe. Clone 3TW was expressed. Fig. 6 is an autoradiograph which
demonstrates expression of 3TW (indicated by arrow).
An antisense oligonucleotide was derived from the sequence of clone 3TW.
The antisense oligonucleotide was assayed to determine its ability to inhibit TNF-
induced cPLA. phosphorylation. Fig. 7 depicts the results of that experiment.
Activity of the anitsense oligonucleotide (3TWAS) was compared with the full-
length clone (3TWFL), Flag-3TW full length (3TWFLflag) and pED-flag vector
(pEDflag). The antisense oligonucleotide inhibited phosphorylation.
CA 02202912 1997-04-16
WO 96/12735 PCI/US95/12724
~EYU~N~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Lin, Lih-Ling
Chen, Jennifer H.
Schievella, Andrea
Graham, James
(ii) TITLE OF lNv~N~lON: NOVEL TNF R~LOK DEATH DOMAIN LIGAND
PROTEINS AND INHIBITORS OF LIGAND BINDING
(iii) NUMBER OF ~:yu~N~S: 14
(iv) CORRESPON~N~ ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Brown, Scott A,
(B) REGISTRATION NUMBER: 32,724
(C) REFERENCE/DOCKET NUMBER: GI5232B
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8224
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2158 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..1231
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
C AGC AAT GCA GGT GAT GGA CCA GGT GGC GAG GGC AGT GTT CAC ^TG --
Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly Ser Val His 'eu
33
CA 02202912 1997-04-16
WO 96/12735 PCTrUS95/12724
1 5 lo 15
GCA AGC TCT CGG GGC ACT TTG TCT GAT AGT GAA ATT GAG ACC AAC TCT 94
Ala Ser Ser Arg Gly Thr Leu Ser Asp Ser Glu Ile Glu Thr Asn Ser
20 25 30
GCC ACA AGC ACC ATC TTT GGT AAA GCC CAC AGC TTG AAG CCA AGC ATA 142
Ala Thr Ser Thr Ile Phe Gly Lys Ala His Ser Leu Lys Pro Ser Ile
35 40 45
AAG GAG AAG CTG GCA GGC AGC CCC ATT CGT ACT TCT GAA GAT GTG AGC 19 0
Lys Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser Glu Asp Val Ser
50 55 60
CAG CGA GTC TAT CTC TAT GAG GGA CTC CTA GGC AAA GAG CGT TCT ACT 23 B
Gln Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys Glu Arg Ser Thr
65 70 75
TTA TGG GAC CAA ATG CAA TTC TGG GAA GAT GCC TTC TTA GAT GCT GTG 286
Leu Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val
80 85 go 95
ATG TTG GAG AGA GAA GGG ATG GGT ATG GAC CAG GGT CCC CAG GAA ATG 334
Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met
loo 105 llo
ATC GAC AGG TAC CTG TCC CTT GGA GAA CAT GAC CGG AAG CGC CTG GAA 382
Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu
115 120 125
GAT GAT GAA GAT CGC TTG CTG GCC ACA CTT CTG CAC AAC CTC ATC TCC 430
Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser
130 135 140
TAC ATG CTG CTG ATG AAG GTA AAT AAG AAT GAC ATC CGC AAG AAG GTG 478
Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val
145 150 155
AGG CGC CTA ATG GGA AAG TCG CAC ATT GGG CTT GTG TAC AGC CAG CAA 526
Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln
160 165 170 175
ATC AAT GAG GTG CTT GAT CAG CTG GCG AAC CTG AAT GGA CGC GAT CTC 5 7 4
Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu
180 185 lgo
TCT ATC TGG TCC AGT GGC AGC CGG CAC ATG AAG AAG CAG ACA TTT GTG 622
Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val
195 200 205
GTA CAT GCA GGG ACA GAT ACA AAC GGA GAT ATC TTT TTC ATG GAG GTG 6 7 0
Val His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe Phe Met Glu Val
210 215 220
TGC GAT G..C TGT GTG GTG TTG CGT AGT AAC ATC GGA ACA GTG TAT GAG 718
Cys Asp ASD Cys Val Val Leu Arg Ser Asn I le Gly Thr Val Tyr Glu
225 230 235
CGC TGG TGG TAC GAG AAG CTC ATC AAC ATG ACC TAC TGT CCC AAG ACG 7 6 6
Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lvs Thr
240 245 250 2s5
AAG GTG TTG TGC TTG TGG CGT AGA AAT GGC TCT GAG ACC CAG CTC AAC 814
Lys Val Lu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn
260 265 270
34
CA 02202912 1997-04-16
W O 96112735 P~ S/12724
AAG TTC TAT ACT AAA AAG TGT CGG GAG CTG TAC TAC TGT GTG AAG GAC 862
Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr Cys Val Lys Asp
275 280 285
AGC ATG GAG CGC GCT GCC GCC CGA CAG CAA AGC ATC AAA CCC GGA CCT 910
Ser Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile Lys Pro Gly Pro
290 295 300
GAA TTG GGT GGC GAG TTC CCT GTG CAG GAC CTG AAG ACT GGT GAG GGT 958
Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly
305 310 315
GGC CTG CTG CAG GTG ACC CTG GAA GGG ATC AAC CTC AAA TTC ATG CAC 1006
Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His
320 325 330 335
AAT CAG GTT TTC ATA GAG CTG AAT CAC ATT AAA AAG TGC AAT ACA GTT 1054
Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val
340 345 350
CGA GGC GTC TTT GTC CTG GAG GAA TTT GTT CCT GAA ATT AAA GAA GTG 1102
Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val
355 360 365
GTG AGC CAC AAG TAC AAG ACA CCA ATG GCC CAC GAA ATC TGC TAC TCC 1150
Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser
370 375 380
GTA TTA TGT CTC TTC TCG TAC GTG GCT GCA GTT CAT AGC AGT GAG GAA 1198
Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu
385 390 395
GAT CTC AGA ACC CCG CCC CGG CCT GTC TCT AGC TGATGGAGAG GGGCTACGCA 1251
Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser
400 405 410
GCTGCCCCAG CCCAGGGCAC GCCCCTGGCC CCTTGCTGTT CCCAAGTGCA CGATGCTGCT 1311
GTGACTGAGG AGTGGATGAT G-lC~l~l~l CCTCTGCAAG CCCCCTGCTG TGGCTTGGGT 1371
GGGTACCGGT TAl~l~lCCC TCTGAGTGTG TCTTGAGCGT GTCCACCTTC TCCCTCTCCA 1431
CTCCCAGAAG ACCAAACTGC CTTCCCCTCA GGGCTCAAGA ATGTGTACAG TCTGTGGGGC 1491
CGGTGTGAAC CCACTATTTT GTGTCCTTGA GACATTTGTG TTGTGGTTCC TTGTCCTTGT 1551
CCCTGGCGTT AACTGTCCAC TGCAAGAGTC TGGCTCTCCC l~ ~lGA CCCGGCATGA 1611
CTGGGCGCCT GGAGCAGTTT CA~ ~lGA GGAGTGAGGG AACCCTGGGG CTCACCCTCT 1671
CAGAGGAAGG GCACAGAGAG GAAGGGAAGA ATTGGGGGGC AGCCGGAGTG AGTGGCAGCC 1731
TCCCTGCTTC CTTCTGCATT CCCAAGCCGG CAGCTACTGC CCAGGGCCCG CAGTGTTGGC 1791
TGCTGCCTGC CACAGCCTCT GTGACTGCAG TGGAGCGGCG AATTCCCTGT GGCCTGCCAC 1851
GCCTTCGGCA TCAGAGGATG GAGTGGTCGA GGCTAGTGGA GTCCCAGGGA CCGCTGGCTG 1911
CTCTGCCTGA GCATCAGGGA GGGGGCAGGA AAGACCAAGC TGGGTTTGCA CAT~ lG 1971
CAGGCTGTCT CTCCAGGCAC GGGGTGTCAG GAGGGAGAGA CAGCCTGGGT ATGGGCAAGA 2031
AATGACTGTA AATATTTCAG CCCCACATTA TTTATAGAAA ATGTACAGTT GTGTGAATGT 2091
GAAATAAATG TCCTCACCTC CcAAAAAAAA AAAAAAAPAA AAAAAAA.~AA AAAAAAAAAA 2151
AAAAAAA 2158
CA 022029l2 l997-04-l6
96/1273S PCTnUS95/12724
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 410 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) S:~u~N~ DESCRIPTION: SEQ ID NO:2:
Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly Ser Val His Leu Ala
1 5 10 15
er Ser Arg Gly Thr Leu Ser Asp Ser Glu Ile Glu Thr Asn Ser Ala
Thr Ser Thr Ile Phe Gly Lys Ala His Ser Leu Lys Pro Ser Ile Lys
Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser Glu Asp Val Ser Gln
Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys Glu Arg Ser Thr Leu
rp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val Met
eu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met Ile
100 105 110
Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu Asp
115 120 125
Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser Tyr
130 135 140
Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val Arg
145 150 155 160
rg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln Ile
165 170 175
sn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu Se-
180 185 190
Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val Val
195 200 205
His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe Phe Met Glu Val Cys
210 215 220
Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly Thr Val Tyr Glu Arg
225 230 235 240
rp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lys Thr Lys
245 250 255
al Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn Lys
260 265 270
he Tyr Thr Lys Lvs Cys Arg Glu Leu Tyr Tyr Cys Val Lvs Asp Ser
~75 280 285
- CA 022029l2 1997-04-16
W O 96/12735 PCTn~S95/12724
Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile Lys Pro Gly Pro Glu
290 295 300
Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly Gly
305 310 315 320
Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His Asn
325 330 335
Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val Arg
340 345 350
Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val Val
355 360 365
Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser Val
370 375 380
Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu Asp
385 390 395 400
Leu Arg Thr Pro Pro Arg Pro Val Ser Ser
405 410
(2) INFO~MATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
tD) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..415
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
G GAG GTG CAG GAC CTC TTC GAA GCC CAG GGC AAT GAC CGA CTG AAG 46Glu Val Gln Asp Leu Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys
1 5 10 15
CTG CTG GTG CTG TAC AGT GGA GAG GAT GAT GAG CTG CTA CAG CGG GCA 94Leu Leu Val Leu Tyr Ser Gly Glu Asp Asp Glu Leu Leu Gln Arg Ala
20 25 30
GCT GCC GGG GGC TTG GCC ATG CTT ACC TCC ATG CGG CCC ACG CTC TGC 142
Ala Ala Gly Gly Leu Ala Met Leu Thr Ser Met Arg Pro Thr Leu Cys
35 40 45
AGC CGC ATT CCC CAA GTG ACC ACA CAC TGG CTG GAG ATC CTG CAG GCC 190
Ser Arg Ile Pro Gln Val Thr Thr His Trp Leu Glu Ile Leu Gln Ala
50 55 60
CTG CTT CTG AGC TCC AAC CAG GAG CTG CAG CAC CGG GGT GCT GTG GTG 238
Leu Leu Leu Ser Ser Asn Gln Glu Leu Gln His Arg Gly Ala Val Val
65 70 75
GTG CTG AAC ATG GTG GAG GCC TCG AGG GAG ATT GCC AGC ACC CTG ATG 286
Val Leu Asn Met Val Glu Ala Ser Arg Glu Ile Ala Ser Thr Leu Met
80 85 90 95
37
CA 02202912 1997-04-16
WO 96/12735 PCT/US95/12724
GAG AGT GAG ATG ATG GAG ATC TTG TCA GTG CTA GCT AAG GGT GAC CAC 334
Glu Ser Glu Met Met Glu Ile Leu Ser Val Leu Ala Lys Gly Asp His
100 105 110
AGC CCT GTC ACA AGG GCT GCT GCA GCC TGC CTG GAC AAA GCA GTG GAA 382
Ser Pro Val Thr Arg Ala Ala Ala Ala Cys Leu Asp Lys Ala Val Glu
115 120 125
TAT GGG CTT ATC CAA CCC AAC CAA GAT GGA GAG TGAGGGGGTT GTCCCTGGGC 435
Tyr Gly Leu Ile Gln Pro Asn Gln Asp Gly Glu
130 13S
CCAAGGCTCA TGCACACGCT ACCTATTGTG GCACGGAGAG TAAGGACGGA AGCAGCTTTG 495
GCTGGTGGTG GCTGGCATGC CCAATACTCT TGCCCATCCT CGCTTGCTGC CCTAGGATGT 555
C~ ~llCT GAGTCAGCGG CCACGTTCAG TCACACAGCC CTGCTTGGCC AGCACTGCCT 615
GCAGCCTCAC TCAGAGGGGC C~ L-lG TACTACTGTA GTCAGCTGGG AATGGGGAAG 675
GTGCATCCCA ACACAGCCTG TGGATCCTGG GGCATTTGGA AGGGCGCACA CATCAGCAGC 735
CTCACCAGCT GTGAGCCTGC TATCAGGCCT GCCCCTCCAA TAAAAGTGTG TAGAACTCCA 795
AAAAA~A~A AAAAAAAAAA ~U~AAAAA A 826
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xl ) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Glu Val Gln Asp Leu Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu
1 5 10 15
Leu Val Leu Tyr Ser Gly Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala
Ala Gly Gly Leu Ala Met Leu Thr Ser Met Arg Pro Thr Leu Cvs Ser
Arg Ile Pro Gln Val Thr Thr His Trp Leu Glu Ile Leu Gln Ala Leu
Leu Leu Ser Ser Asn Gln Glu Leu Gln His Arg Gly Ala Val val Val
Leu Asn Met Val Glu Ala Ser Arg Glu Ile Ala Ser Thr Leu Met Glu
Ser Glu Met Met Glu Ile Leu Ser Val Leu Ala Lys Gly Asp Hls Ser
100 105 110
Pro Val Thr Arg Ala Ala Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr
1'5 120 125
Gly Leu Ile Gln Pro Asn Gln Asp Gly Glu
130 135
(2~ 'NFOR"~TION FOR SEQ ID NO:5:
38
CA 02202912 1997-04-16
WO 96/12735 PCT/US95112724
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 722 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
~B) LOCATION: 2..559
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
G GAG AAG CCG CTG CAC GCC CTG CTG CAC GGC CGC GGG GTT TGC CTC 46
Glu Lys Pro Leu His Ala Leu Leu His Gly Arg Gly Val Cys Leu
1 5 10 15
AAC GAA AAG AGC TAC CGC GAG CAA GTC AAG ATC GAG AGA GAC TCC CGT 94
Asn Glu Lys Ser Tyr Arg Glu Gln Val Lys Ile Glu Arg Asp Ser Arg
20 25 30
GAG CAC GAG GAG CCC ACC ACC TCT GAG ATG GCC GAG GAG ACC TAC TCC 142
Glu His Glu Glu Pro Thr Thr Ser Glu Met Ala Glu Glu Thr Tyr Ser
35 40 45
CCC AAG ATC TTC CGG CCC AAA CAC ACC CGC ATC TCC GAG CTG AAG GCT 190
Pro Lys Ile Phe Arg Pro Lys His Thr Arg Ile Ser Glu Leu Lys Ala
50 - 55 60
GAA GCA GTG AAG AAG GAC CGC AGA AAG AAG CTG ACC CAG TCC AAG TTT 238
Glu Ala Val Lys Lys Asp Arg Arg Lys Lys Leu Thr Gln Ser Lys Phe
65 70 75
GTC GGG GGA GCC GAG AAC ACT GCC CAC CCC CGG ATC ATC TCT GAA CCT 286
Val Gly Gly Ala Glu Asn Thr Ala His Pro Arg Ile Ile Ser Glu Pro
80 85 90 95
GAG ATG AGA CAG GAG TCT GAG CAG GGC CCC TGC CGC AGA CAC ATG GAG 334
Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg Arg His Met Glu
100 105 110
GCT TCC CTG CAG GAG CTC AAA GCC AGC CCA CGC ATG GTG CCC CGT GCT 382
Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala
115 120 125
GTG TAC CTG CCC AAT TGT GAC CGC AAA GGA TTC TAC AAG AGA AAG CAG 430
Val Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe Tyr Lys Arg Lys Gln
130 135 140
TGC AAA CCT TCC CGT GGC CGC AAG CGT GGC ATC TGC TGG TGC GTG GAC 478
Cys Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys Trp Cys Val Asp
145 150 155
AAG TAC GGG ATG AAG CTG CCA GGC ATG GAG TAC GTT GAC GGG GAC TTT 526
Lvs Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe
160 165 170 175
CAG TGC CAC ACC TTC GAC AGC AGC AAC GTT GAG TGATGCGTCC CCCCCCAACC 579
Gln Cvs His Thr Phe Asp Ser Ser Asn Val Glu
180 185
TTTCCCTCAC CCCCTTCCAC CCCCAGCCCC GACTCCAGCC AGCGCCTCCC ,CCACCCCAG ~39
3q
CA 02202912 1997-04-16
96/12735 PCTrUS95/12724
GACGCCACTC ATTTCATCTC ATTTAAGGGA AAAATATATA TCTATCTATT TGAGGAAAAA 699
AAAAAAAAAA AAAAAAAAAA AAA 722
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 186 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Glu Lys Pro Leu His Ala Leu Leu His Gly Arg Gly Val Cys Leu Asn
1 5 10 15
lu Lys Ser Tyr Arg Glu Gln Val Lys Ile Glu Arg Asp Ser Arg Glu
His Glu Glu Pro Thr Thr Ser Glu Met Ala Glu Glu Thr Tyr Ser Pro
Lys Ile Phe Arg Pro Lys His Thr Arg Ile Ser Glu Leu Lys Ala Glu
Ala Val Lys Lys Asp Arg Arg Lys Lys Leu Thr Gln Ser Lys Phe Val
ly Gly Ala Glu Asn Thr Ala His Pro Arg Ile Ile Ser Glu Pro Glu
et Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg Arg His Met Glu Ala
100 105 110
Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala Val
115 120 125
Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe Tyr Lys Arg Lys Gln Cys
130 135 140
Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys Trp Cys Val Asp Lys
145 150 155 160
Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe Gln
165 170 175
ys His Thr Phe Asp Ser Ser Asn Val Glu
180 185
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1023 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
( ~:) FEATURE:
(A) NAME/KEY: CDS
CA 022029l2 l997-04-l6
96/12735 PCTAUS95/12724
(B) LOCATION: 57..875
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCCTGCACTC TCG~ C~l GCCCCACCCC GAGGTAAAGG GGGCGACTAA GAGAAG 56
ATG GTG TTG CTC ACC GCG GTC CTC CTG CTG CTG GCC GCC TAT GCG GGG 104
Met Val Leu Leu Thr Ala Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly
l 5 10 15
CCG GCC CAG AGC CTG GGC TCC TTC GTG CAC TGC GAG CCC TGC GAC GAG 152
Pro Ala Gln Ser Leu Gly Ser Phe Val His Cys Glu Pro Cys Asp Glu
20 25 30
AAA GCC CTC TCC ATG TGC CCC CCC AGC CCC CTG GGC TGC GAG CTG GTC 200
Lys Ala Leu Ser Met Cys Pro Pro Ser Pro Leu Gly Cys Glu Leu Val
35 40 45
AAG GAG CCG GGC TGC GGC TGC TGC ATG ACC TGC GCC CTG GCC GAG GGG 248
Lys Glu Pro Gly Cys Glv Cys Cys Met Thr Cys Ala Leu Ala Glu Gly
50 55 60
CAG TCG TGC GGC GTC TAC ACC GAG CGC TGC GCC CAG GGG CTG CGC TGC 296
Gln Ser Cys Gly Val Tvr Thr Glu Arg Cys Ala Gln Gly Leu Arg Cys
65 70 75 80
CTC CCC CGG CAG GAC GAG GAG AAG CCG CTG CAC GCC CTG CTG CAC GGC 344
Leu Pro Arg Gln Asp Glu Glu Lys Pro Leu His Ala Leu Leu His Gly
85 90 95
CGC GGG GTT TGC CTC AAC GAA AAG AGC TAC CGC GAG CAA GTC AAG ATC 392
Arg Gly Val Cys Leu Asn Glu Lys Ser Tyr Arg Glu Gln Val Lys Ile
100 105 110
GAG AGA GAC TCC CGT GAG CAC GAG GAG CCC ACC ACC TCT GAG ATG GCC 440
Glu Arg Asp Ser Arg Glu His Glu Glu Pro Thr Thr Ser Glu Met Ala
115 120 125
GAG GAG ACC TAC TCC CCC AAG ATC TTC CGG CCC AAA CAC ACC CGC ATC 488
Glu Glu Thr Tyr Ser Pro Lys Ile Phe Arg Pro Lys His Thr Arg Ile
130 135 140
TCC GAG CTG AAG GCT GAA GCA GTG AAG AAG GAC CGC AGA AAG AAG CTG 536
Ser Glu Leu Lys Ala Glu Ala Val Lys Lys Asp Arg Arg Lys Lvs Leu
1~5 150 155 160
ACC CAG TCC AAG TTT GTC GGG GGA GCC GAG AAC ACT GCC CAC CCC CGG .584
Thr Gln Ser Lys Phe Val Gly Gly Ala Glu Asn Thr Ala His Pro Arg
165 170 175
ATC ATC TCT GCA CCT GAG ATG AGA CAG GAG TCT GAG CAG GGC CCC TGC 632
Ile Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys
180 185 190
CGC AGA CAC ATG GAG GCT TCC CTG CAG GAG CTC AAA GCC AGC CCA CGC 680
Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg
195 200 205
ATG GTG CCC CGT GCT GTG TAC CTG CCC AAT TGT GAC CGC AAA GGA TTC 728
Met Val Pro Arg Ala Val Tyr Leu Pro Asn Cys Asp Arg Lvs Gly Phe
210 215 220
TAC AAG AGA AAG CAG TGC AAA CCT TCC CGT GGC CGC AAG CGT GGC ATC 776
Tyr Lvs Ar~ Lys Gln Cvs Lvs Pro Ser Arg Glv Arg Lys Arg Gly Ile
~-c ~3G ~3c ~1v
4I
-
CA 022029l2 l997-04-l6
WO 96/12735 PCTfUS95/12724
TGC TGG TGC GTG GAC AAG TAC GGG ATG AAG CTG CCA GGC ATG GAG TAC 824
Cys Trp Cys Val Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr
245 250 255
GTT GAC GGG GAC TTT CAG TGC CAC ACC TTC GAC AGC AGC AAC GTT GAG 872
Val Asp Gly Asp Phe Gln Cys His Thr Phe Asp Ser Ser Asn Val Glu
260 265 270
TGATGCGTCC CCCCCCAACC TTTCCCTCAC CCCCTCCCAC CCCCAGCCCC GACTCCAGCC 932
AGCGCCTCCC TCCACCCCAG GACGCCACTC ATTTCATCTC ATTTAAGGGA AAAATATATA 992
TCTATCTATT TGAAAAAAAA AAAAAAAACC C 1023
(2) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 272 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Val Leu Leu Thr Ala Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly
1 5 10 15
Pro Ala Gln Ser Leu Gly Ser Phe Val His Cys Glu Pro Cys Asp Glu
Lys Ala Leu Ser Met Cys Pro Pro Ser Pro Leu Gly Cys Glu Leu Val
Lys Glu Pro Gly Cys Gly Cys Cys Met Thr Cys Ala Leu Ala Glu Gly
Gln Ser Cys Gly Val Tyr Thr Glu Arg Cys Ala Gln Gly Leu Arg Cys
Leu Pro Arg Gln Asp Glu Glu Lys Pro Leu His Ala Leu Leu His Gly
Ara Gly Val Cys Leu Asn Glu Lys Ser Tyr Arg Glu Gln Val Lys Ile
100 105 110
Glu Arg Asp Ser Arg Glu His Glu Glu Pro Thr Thr Ser Glu Met Ala
115 120 125
Glu Glu Thr Tyr Ser Pro Lys Ile Phe Arg Pro Lys His Thr Arg Ile
130 135 140
Ser Glu Leu Lys Ala Glu Ala Val Lys Lys Asp Arg Arg Lys Lys Leu
145 150 155 160
Thr Gln Ser Lys Phe Val Gly Gly Ala Glu Asn Thr Ala His Pro Arg
165 170 175
Ile Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys
180 185 190
Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg
195 200 205
r:e~ ~al 2~-o Ara Ala Val T~r L~u Prc Asn C-y5 Asp Arg Lys G'y Phe
~l0 215 220
q _
CA 02202912 1997-04-16
WO 96/12735 PCTIUS95/12724
Tyr Lys Arg Lys Gln Cys Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile
225 230 235 240
Cys Trp Cys Val Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr
245 250 255
al Asp Gly Asp Phe Gln Cys His Thr Phe Asp Ser Ser Asn Val Glu
260 265 270
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1694 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..931
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
C TCT CTC AAG GCC AAC ATC CCT GAG GTG GAA GCT GTC CTC AAC ACC 46
Ser Leu Lys Ala Asn Ile Pro Glu Val Glu Ala Val Leu Asn Thr
1 5 10 15
GAC AGG AGT TTG GTG TGT GAT GGG AAG AGG GGC TTA TTA ACT CGT CTG 94
Asp Arg Ser Leu Val Cys Asp Gly Lys Arg Gly Leu Leu Thr Arg Leu
20 25 30
CTG CAG GTC ATG AAG AAG GAG CCA GCA GAG TCG TCT TTC AGG TTT TGG 142
Leu Gln Val Met Lys Lys Glu Pro Ala Glu Ser Ser Phe Arg Phe Trp
35 40 45
CAA GCT CGG GCT GTG GAG AGT TTC CTC CGA GGG ACC ACC TCC TAT GCA 190
Gln Ala Arg Ala Val Glu Ser Phe Leu Arg Gly Thr Thr Ser Tyr Ala
50 55 60
GAC CAG ATG TTC CTG CTG AAG CGA GGC CTC TTG GAG CAC ATC CTT TAC 238
Asp Gln Met Phe Leu Leu Lys Arg Gly Leu Leu Glu His Ile Leu Tyr
65 70 75
TGC ATT GTG GAC AGC GAG TGT AAG TCA AGG GAT GTG CTC CAG AGT TAC 286
Cys Ile Val Asp Ser Glu Cys Lys Ser Arg Asp Val Leu Gln Ser Tyr
80 85 90 95
TTT GAC CTC CTG GGG GAG CTG ATG AAG TTC AAC GTT GAT GCA TTC AAG 334
Phe Asp Leu Leu Gly Glu Leu Met Lys Phe Asn Val Asp Ala Phe Lys
100 105 110
AGA TTC AAT AAA TAT ATC AAC ACC GAT GCA AAG TTC CAG GTA TTC CTG 38,
Arg Phe Asn Lys Tyr Ile Asn Thr Asp Ala Lys Phe Gln Val Phe Leu
115 120 125
~3
CA 02202912 1997-04-16
96/12735 PCTnUS95/12724
AAG CAG ATC AAC AGC TCC CTG GTG GAC TCC AAC ATG CTG GTG CGC TGT 430
Lys Gln Ile Asn Ser Ser Leu Val Asp Ser Asn Met Leu Val Arg Cys
130 135 140
GTC ACT CTG TCC CTG GAC CGA TTT GAA AAC CAG GTG GAT ATG AAA GTT 478
Val Thr Leu Ser Leu Asp Arg Phe Glu Asn Gln Val Asp Met Lys Val
145 150 155
GCC GAG GTA CTG TCT GAA TGC CGC CTG CTC GCC TAC ATA TCC CAG GTG 526
Ala Glu Val Leu Ser Glu Cys Arg Leu Leu Ala Tyr Ile Ser Gln Val
160 165 170 175
CCC ACG CAG ATG TCC TTC CTC TTC CGC CTC ATC AAC ATC ATC CAC GTG 574
Pro Thr Gln Met Ser Phe Leu Phe Arg Leu Ile Asn Ile Ile His Val
180 185 190
CAG ACG CTG ACC CAG GAG AAC GTC AGC TGC CTC AAC ACC AGC CTG GTG 622
Gln Thr Leu Thr Gln Glu Asn Val Ser Cys Leu Asn Thr Ser Leu Val
195 200 205
ATC CTG ATG CTG GCC CGA CGG AAA GAG CGG CTG CCC CTG TAC CTG CGG 670
Ile Leu Met Leu Ala Arg Arg Lys Glu Arg Leu Pro Leu Tyr Leu Arg
210 215 220
CTG CTG CAG CGG ATG GAG CAC AGC AAG AAG TAC CCC GGC TTC CTG CTC 718
Leu Leu Gln Arg Met Glu His Ser Lys Lys Tyr Pro Gly Phe Leu Leu
225 230 235
AAC AAC TTC CAC AAC CTG CTG CGC TTC TGG CAG CAG CAC TAC CTG CAC 766
Asn Asn Phe His Asn Leu Leu Arg Phe Trp Gln Gln His Tyr Leu His
240 245 2S0 255
AAG GAC AAG GAC AGC ACC TGC CTA GAG AAC AGC TCC TGC ATC AGC TTC 814
Lys Asp Lys Asp Ser Thr Cys Leu Glu Asn Ser Ser Cys Ile Ser Phe
260 265 270
TCA TAC TGG AAG GAG ACA GTG TCC ATC CTG TTG AAC CCG GAC CGG CAG 862
Ser Tyr Trp Lys Glu Thr Val Ser Ile Leu Leu Asn Pro Asp Arg Gln
275 280 285
TCA CCC TCT GCT CTC GTT AGC TAC ATT GAG GAG CCC TAC ATG GAC ATA 910
Ser Pro Ser Ala Leu Val Ser Tyr Ile Glu Glu Pro Tyr Met Asp Ile
290 295 300
GAC AGG GAC TTC ACT GAG GAG TGACCTTGGG CCAGGCCTCG GGAGGCTGCT 96'
Asp Arg Asp Phe Thr Glu Glu
305 310
GGGCCAGTG- GGGTGAGCGT GGGTACGATG CCACACGCCC TGCCCTGTTC CCGTTCCTCC 1021
CTGCTGCTCT CTGCCTGCCC CAGGTCTTTG GGTACAGGCT TGGTGGGAGG GAAGTCCTAG 1081
AAGCCCTTGG TCCCCCTGGG TCTGAGGGCC CTAGGTCATG GAGAGCCTCA GTCCCCATAA 1141
TGAGGACAGG GTACCATGCC CACCTTTCCT TCAGAACCCT GGGGCCCAGG GCCACCCAGA 1201
GGTAAGAGGA CATTTAGCAT TAG~r~l~lG TGAGCTCCTG CCG~~ ll GGCTGTCAGT 1261
CAGTCCCAGA GTGGGGAGGA AGATATGGGT GACCCCCACC CCCCATCTGT GAGCCAAGCC 1321
TCCCTTGTCC CTGGCCTTTG GACCCAGGCA AAGGCTTCTG AGCCCTGGGC AGGGGTGGTG 1381
GGTACCAGAG AATGCTGCCT TCCCCCAAGC CTGCCCCTCT GCCTCATTTT CCTGTAGCTC 1441
CTCTGGTTCT GTTTGCTCAT TGGCCGCTGT GTTCATCCAA GGGGGTTCTC CCAGAAGTG.`. 1501
GGGGCCTTTC CCTCCATCCC TTGGGGCACG GGGCAGCTGT GCCTGCCCTG CCTCTGCCTG 1561
~4
CA 02202912 1997-04-16
96112735 PCTnUS95/12724
AGGCAGCCGC TCCTGCCTGA GCCTGGACAT GGGGCCCTTC ~ ~llGC CAATTTATTA 1621
ACAGCAAATA AACCAATTAA ATGGAGACTA TTAAATAACT TTATTTTAAA AATGAAAAAA 1681
AAAAA~AAAA AAA 1694
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 310 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ser Leu Lys Ala Asn Ile Pro Glu Val Glu Ala Val Leu Asn Th- Asp
1 5 10 :_
rg Ser Leu Val Cys Asp Gly Lvs Arg Gly Leu Leu Thr Arg Leu Leu
Gln Val Met Lys Lys Glu Pro Ala Glu Ser Ser Phe Arg Phe Trp Gln
Ala Arg Ala Val Glu Ser Phe Leu Arg Gly Thr Thr Ser Tyr Ala Asp
Gln Met Phe Leu Leu Lys Arg Gly Leu Leu Glu His Ile Leu Tyr Cys
le Val Asp Ser Glu Cys Lys Ser Arg Asp Val Leu Gln Ser Tyr Phe
sp Leu Leu Gly Glu Leu Met Lys Phe Asn Val Asp Ala Phe Lys Arg
100 105 110
Phe Asn Lys Tyr Ile Asn Thr Asp Ala Lys Phe Gln Val Phe Leu Lys
115 120 125
Gln Ile Asn Ser Ser Leu Val Asp Ser Asn Met Leu Val Arg Cys Val
130 135 140
Thr Leu Ser Leu Asp Arg Phe Glu Asn Gln Val Asp Met Lys Va_ Ala
145 150 155 160
lu Val Leu Ser Glu Cys Arg Leu Leu Ala Tyr Ile Ser Gln Val Pro
165 170 17~
hr Gln Met Ser Phe Leu Phe Arg Leu Ile Asn Ile Ile Hls Val Gln
180 185 190
Thr Leu Thr Gln Glu Asn Val Ser Cys Leu Asn Thr Ser Leu Va~ Ile
195 200 205
Leu Met Leu Ala Arg Arg Lys Glu Arg Leu Pro Leu Tyr _eu Arg Leu
210 215 220
Leu Gln Arg Met Glu His Ser Lys Lys Tyr Pro Gly Phe Leu Leu Asn
225 230 ~35 240
sn Phe His Asn Leu Leu Arg Phe Trp Gln 51n His Tyr :eu His Lys
245 250 25~
sp L,s Asp Ser Thr Cys Leu Glu Asn Ser Ser Cys Ile -er Phe Ser
CA 02202912 1997-04-16
WO 96/12735 PCTrUS9Stl2724
260 265 270
Tyr Trp Lys Glu Thr Val Ser Ile Leu Leu Asn Pro Asp Arg Gln Ser
27s 280 28s
Pro Ser Ala Leu Val Ser Tyr Ile Glu Glu Pro Tyr Met Asp Ile Asp
290 295 300
Arg Asp Phe Thr Glu Glu
305 310
(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273s base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2. .1822
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
G GAG ATC AGT CGG AAG GTG TAC AAG GGA ATG TTA GAC CTC CTC AAG 46
Glu Ile Ser Arg Lys Val Tyr Lys Gly Met Leu Asp Leu Leu Lys
5 10 15
TGT ACA GTC CTC AGC TTG GAG CAG TCC TAT GCC CAC GCG GGT CTG GGT 9 4
Cys Thr Val Leu Ser Leu Glu Gln Ser Tyr Ala His Ala Gly Leu Gly
20 25 30
GGC ATG GCC AGC ATC TTT GGG CTT TTG GAG ATT GCC CAG ACC CAC TAC 142
Gly Met Ala Ser Ile Phe Gly Leu Leu Glu Ile Ala Gln Thr His Tyr
35 40 45
TAT AGT AAA GAA CCA GAC AAG CGG AAG AGA AGT CCA ACA GAA AGT GTA 190
Tyr Ser Lys Glu Pro Asp Lys Arg Lys Arg Ser Pro Thr Glu Ser Val
50 55 60
AAT ACC CCA GTT GGC AAG GAT CCT GGC CTA GCT GGG CGG GGG GAC CCA 238
Asn Thr Pro Val Gly Lys Asp Pro Gly Leu Ala Gly Arg Gly Asp Pro
65 70 75
AAG GCT ATG GCA CAA CTG AGA GTT CCA CAA CTG GGA CCT CGG GCA CCA 286
Lys Ala Met Ala Gln Leu Arg Val Pro Gln Leu Gly Pro Arg Ala Pro
80 85 90 95
AGT GCC ACA GGA AAG GGT CCT AAG GAA CTG GAC ACC AGA AGT TTA AAG 334
Ser Ala Thr Gly Lys Gly Pro Lys Glu Leu Asp Thr Arg Ser Leu Lys
oo 105 llo
~.~A GAA AAT TTT ATA GCA TCT ATT GGG CCT GAA GTA ATC AAA CCT GTC 382
Glu Glu Asn Phe Ile Ala Ser Ile Gly Pro Glu Val Ile Lys Pro Val
115 120 125
-TT GAC C-T GGT GAG ACA GAG GAG AAA AAG TCC CAG ATC AGC GCA GAC 430
~he ASD L-~ Glv Glu Thr Glu Glu Lys Lys Ser Gln Ile Ser Ala Asp
1'? 135 140
~C~
CA 022029l2 l997-04-l6
PCTrUS95/12724
W 096112735
AGT GGT GTG AGC CTG ACG TCT AGT TCC CAG AGG ACT GAT CAA GAC TCT 478
Ser Gly Val Ser Leu Thr Ser Ser Ser Gln Arg Thr Asp Gln Asp Ser
145 150 155
GTC ATC GGC GTG AGT CCA GCT GTT ATG ATC CGC AGC TCA AGT CAG GAT 526
Val Ile Gly Val Ser Pro Ala Val Met Ile Arg Ser Ser Ser Gln Asp
160 165 170 175
TCT GAA GTT AGC ACC GTG GTG AGT AAT AGC TCT GGA GAG ACC CTT GGA 574
Ser Glu Val Ser Thr Val Val Ser Asn Ser Ser Gly Glu Thr Leu Gly
180 185 190
GCT GAC AGT GAC TTG AGC AGC AAT GCA GGT GAT GGA CCA GGT GGC GAG 622
Ala Asp Ser Asp Leu Ser Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu
195 200 205
GGC AGT GTT CAC CTG GCA AGC TCT CGG GGC ACT TTG TCT GAT AGT GAA 670
Gly Ser Val His Leu Ala Ser Ser Arg Gly Thr Leu Ser Asp Ser Glu
210 215 220
ATT GAG ACC AAC TCT GCC ACA AGC ACC ATC TTT GGT AAA GCC CAC AGC 718
Ile Glu Thr Asn Ser Ala Thr Ser Thr Ile Phe Gly Lys Ala His Ser
225 230 235
TTG AAG CCA AGC ATA AAG GAG AAG CTG GCA GGC AGC CCC ATT CGT ACT 766
Leu Lys Pro Ser Ile Lys Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr
240 245 250 255
TCT GAA GAT GTG AGC CAG CGA GTC TAT CTC TAT GAG GGA CTC CTA GGC 814
Ser Glu Asp Val Ser Gln Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly
260 265 270
AAA GAG CGT TCT ACT TTA TGG GAC CAA ATG CAA TTC TGG GAA GAT GCC 862
Lys Glu Arg Ser Thr Leu Trp Asp Gln Met Gln Phe Trp Glu Asp Ala
275 280 285
TTC TTA GAT GCT GTG ATG TTG GAG AGA GAA GGG ATG GGT ATG GAC CAG 910
Phe Leu Asp Ala Val Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln
290 295 300
GGT CCC CAG GAA ATG ATC GAC AGG TAC CTG TCC CTT GGA GAA CAT GAC 958
Gly Pro Gln Glu Met Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp
305 310 315
CGG AAG CGC CTG GAA GAT GAT GAA GAT CGC TTG CTG GCC ACA CTT CTG 1006
Arg Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu
320 325 330 335
CAC AAC CTC ATC TCC TAC ATG CTG CTG ATG AAG GTA AAT AAG AAT GAC 1054
His Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp
340 345 350
ATC CGC AAG AAG GTG AGG CGC CTA ATG GGA AAG TCG CAC ATT GGG CTT 1102
Ile Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu
355 360 365
GTG TAC AGC CAG CAA ATC AAT GAG GTG CTT GAT CAG CTG GCG AAC CTG 1150
Val Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu
370 375 380
AAT GGA CGC GAT CTC TCT ATC TGG TCC AGT GGC AGC CGG CAC ATG AAG 1198
Asn Gly Arg Asp Leu Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys
385 390 39s
AAG CAG ACA TTT GTG GTA CAT GCA GGG ACA GAT ACA AAC GGA GAT ATC 1246
.s Gln T!-r Phe Val Val His Ala Glv Thr Asp Thr Asn Gly Asp Ile
o 405 410 415
47
CA 02202912 1997-04-16
WO 96/12735 PCrrUS9S/12724
TTT TTC ATG GAG GTG TGC GAT GAC TGT GTG GTG TTG CGT AGT AAC ATC 1294
Phe Phe Met Glu Val Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile
420 425 430
GGA ACA GTG TAT GAG CGC TGG TGG TAC GAG AAG CTC ATC AAC ATG ACC 1342
Gly Thr Val Tyr Glu Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr
435 440 445
TAC TGT CCC AAG ACG AAG GTG TTG TGC TTG TGG CGT AGA AAT GGC TCT 1390
Tyr Cys Pro Lys Thr Lys Val Leu Cys Leu Trp Arg Arg Asn Gly Ser
450 455 460
GAG ACC CAG CTC AAC AAG TTC TAT ACT AAA AAG TGT CGG GAG CTG TAC 1438
Glu Thr Gln Leu Asn Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr
465 470 475
TAC TGT GTG AAG GAC AGC ATG GAG CGC GCT GCC GCC CGA CAG CAA AGC 1486
Tyr Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala Arg Gln Gln Ser
4ao 485 490 495
ATC AAA CCC GGA CCT GAA TTG GGT GGC GAG TTC CCT GTG CAG GAC CTG 1534
Ile Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu
500 505 510
AAG ACT GGT GAG GGT GGC CTG CTG CAG GTG ACC CTG GAA GGG ATC AAC 1582
Lys Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn
515 520 525
CTC AAA TTC ATG CAC AAT CAG GTT TTC ATA GAG CTG AAT CAC ATT AAA 1630
Leu Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys
530 535 540
AAG TGC AAT ACA GTT CGA GGC GTC TTT GTC CTG GAG GAA TTT GTT CCT 1678
Lys Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro
545 550 555
GAA ATT AAA GAA GTG GTG AGC CAC AAG TAC AAG ACA CCA ATG GCC CAC 1726
Glu Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His
560 565 570 575
GAA ATC TGC TAC TCC GTA TTA TGT CTC TTC TCG TAC GTG GCT GCA GTT 1774
Glu Ile Cys Tyr Ser Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val
580 585 590
CAT AGC AGT GAG GAA GAT CTC AGA ACC CCG CCC CGG CCT GTC TCT AGC 1822
His Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser
595 600 605
TGATGGAGAG GGGCTACGCA GCTGCCCCAG CCCAGGGCAC GCCCCTGGCC CCTTGCTGTT 1882
CCCAAGTGCA CGATGCTGCT GTGACTGAGG AGTGGATGAT GCTCG~ CCTCTGCAAG 1942
CCCCCTGCTG TGGCTTGGTT GGTTACCGGT TATGTGTCCC TCTGAGTGTG TCTTGAGCGT 2002
GTCCACCTTC TCCCTCTCCA CTCCCAGAAG ACCAAACTGC CTTCCCCTCA GGGCTCAAGA 2062
ATGTGTACAG TCTGTGGGGC CGGTGTGAAC CCACTATTTT GTGTCCTTGA GACATTTGTG 2122
TTGTGGTTCC TTGTCCTTGT CCCTGGCGTT ATAACTGTCC ACTGCAAGAG TCTGGCTCTC 2182
C~ lGT GACCCGGCAT GACTGGGCGC CTGGAGCAGT TTCACTCTGT GAGGAGTGAG 2242
GGAACCCTGG GGCTCACCCT CTCAGAGGAA GGGCACAGAG AGGAAGGGAA GAATTGGGGG 2302
GCAGCCGGAG TGAGTGGCAG CCTCCCTGCT TCCTTCTGCA TTCCCAAGCC 5GCAGCTACT 2362
SCCCAGGGCC CGCAGT5TTG GCTGCTGCCT GCCACAGCCT CTGTGACTGC AGTGGAGCGG 2422
~8
CA 02202912 1997-04-16
W O96112735 PCTnUS95/12724
CGAATTCCCT GTGGCCTGCC ACGCCTTCGG CATCAGAGGA TGGAGTGGTC GAGGCTAGTG 2482
GAGTCCCAGG GACCGCTGGC TGCTCTGCCT GAGCATCAGG GAGGGGGCAG GAAAGACCAA 2542
GCTGGGTTTG CACATCTGTC TGCAGGCTGT CTCTCCAGGC ACGGGGTGTC AGGAGGGAGA 2602
GACAGCCTGG GTATGGGCAA GAAATGACTG TAAATATTTC AGCCCCACAT TATTTATAGA 2662
AAATGTACAG ll~l~lGAAT GTGAAATAAA l~lc~-l~AAc TCCCAAAAAA AAAAAAAAAA 2722
AAAAAAAAAA AAA 2735
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 607 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Glu Ile Ser Arg Lys Val Tyr Lvs Gly Met Leu Asp Leu Leu Lys Cys
1 5 10 15
Thr Val Leu Ser Leu Glu Gln Ser Tyr Ala His Ala Gly Leu Gly Gly
Met Ala Ser Ile Phe Gly Leu Leu Glu Ile Ala Gln Thr His Tyr Tyr
Ser Lys Glu Pro Asp Lys Arg Lys Arg Ser Pro Thr Glu Ser Val Asn
Thr Pro Val Gly Lys Asp Pro Gly Leu Ala Gly Arg Gly Asp Pro Lys
Ala Met Ala Gln Leu Arg Val Pro Gln Leu Gly Pro Arg Ala Pro Ser
Ala Thr Gly Lys Gly Pro Lys Glu Leu Asp Thr Arg Ser Leu Lvs Glu
100 105 110
Glu Asn Phe Ile Ala Ser Ile Gly Pro Glu Val Ile Lys Pro Val Phe
115 120 125
Asp Leu Gly Glu Thr Glu Glu Lys Lys Ser Gln Ile Ser Ala Asp Ser
130 135 140
Gly Val Ser Leu Thr Ser Ser Ser Gln Arg Thr Asp Gln Asp Ser Val
145 150 155 160
Ile Gly Val Ser Pro Ala Val Met Ile Arg Ser Ser Ser Gln Asp Ser
165 170 175
Glu Val Ser Thr Val Val Ser Asn Ser Ser Gly Glu Thr Leu Gly Ala
180 185 190
Asp Ser Asp Leu Ser Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly
195 200 205
Ser Val His Leu Ala Ser Ser Arg Glv Thr Leu Ser Asp Ser Glu Ile
210 215 220
Glu Thr Asn Ser Ala Thr ser Thr iie Phe Glv Lys Ala ~ms Ser Leu
4~
CA 02202912 1997-04-16
WO 96112735 PCTrUS95/12724
225 230 235 240
Lys Pro Ser Ile Lys Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser
245 250 2s5
Glu Asp Val Ser Gln Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys
260 265 270
Glu Arg Ser Thr Leu Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe
275 280 285
Leu Asp Ala Val Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly
290 295 300
Pro Gln Glu Met Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg
305 310 315 320
Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His
325 330 335
Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile
340 345 350
Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val
355 360 365
Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn
370 375 380
Gly Arg Asp Leu Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys
385 390 395 400
Gln Thr Phe Val Val His Ala Gly Thr Asp Thr Asn Gly ASD Ile Phe
405 410 415
Phe Met Glu Val Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly
420 425 430
Thr Val Tyr Glu Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr
435 440 445
Cys Pro Lys Thr Lys Val Leu Cys Leu Trp Arg Arg Asn Glv Ser Glu
450 455 460
Thr Gln Leu Asn Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr
465 470 475 480
Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala Ara Gln Gln Ser Ile
485 490 495
Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys
500 505 510
Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu
515 520 525
Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys
530 535 540
Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu
545 550 555 560
Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu
565 570 575
' e Cys T~. r Ser Val Leu C~. s Leu P~e Ser T .-r ~'a' Ala Ala ~ a_ Y._~
580 s&s sgo
CA 02202912 1997-04-16
WO 96/12735 PCI/US95/12724
Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser
595 600 605
(2) INFORMATION FOR SEQ ID NO:13:
(i) S~Qu~:N~ CHARACTERISTICS:
(A) LENGTH: 3225 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~:SS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..2846
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
CC CAG ACT CGC CCC GCC CCA GAG ACT GCG CCT GCG CGG GCA CGA GAC 47
Gln Thr Arg Pro Ala Pro Glu Thr Ala Pro Ala Arg Ala Arg Asp
1 5 10 15
ACC CTC TCC GCG ATG ACT GCC AGC TCA GTG GAG CAG CTG CGG AAG GAG 95
Thr Leu Ser Ala Met Thr Ala Ser Ser Val Glu Gln Leu Arg Lys Glu
20 25 30
GGC AAT GAG CTG TTC AAA TGT GGA GAC TAC GGG GGC GCC CTG GCG GCC 143
Gly Asn Glu Leu Phe Lys Cys Gly Asp Tyr Gly Gly Ala Leu Ala Ala
35 40 45
TAC ACT CAG GCC CTG GGT CTG GAC GCG ACG CCC CAG GAC CAG GCC GTT 191
Tyr Thr Gln Ala Leu Gly Leu Asp Ala Thr Pro Gln Asp Gln Ala Val
50 55 60
CTG CAC CGG AAC CGG GCC GCC TGC CAC CTC AAG CTG GAA GAT TAC GAC 239
Leu His Arg Asn Arg Ala Ala Cys His Leu Lys Leu Glu Asp Tyr Asp
65 70 75
AAA GCA GAA ACA GAG GCA TCC AAA GCC ATT GAA AAG GAT GGT GGG GAT 287
Lys Ala Glu Thr Glu Ala Ser Lys Ala Ile Glu Lys Asp Gly Gly Asp
80 85 90 95
GTC AAA GCA CTC TAC CGG CGG AGC CAA GCC CTA GAG AAG CTG GGC CGC 335
Val Lys Ala Leu Tyr Arg Arg Ser Gln Ala Leu Glu Lys Leu Gly Arg
100 105 110
CTG GAC CAG GCT GTC CTT GAC CTG CAG AGA TGT GTG AGC TTG GAG CCC 383
Leu Asp Gln Ala Val Leu Asp Leu Gln Arg Cys Val Ser Leu Glu Pro
115 120 125
AAG AAC AAA GTT TTC CAG GAG GCC TTG CGG AAC ATC GGG GGC CAG ATT 431
Lys Asn Lys Val Phe Gln Glu Ala Leu Arg Asn Ile Gly Gly Gln Ile
130 135 140
CAG GAG AAG GTG CGA TAC ATG TCC TCG ACG GAT GCC AAA GTG GAA CAG ~75
Gln Glu Lys Val Arg Tyr Met Ser Ser Thr Asp Ala Lys Val Glu Gln
145 150 155
CA 022029l2 l997-04-l6
WO 96112735 PCIIUS95/12724
ATG TTT CAG ATA CTG TTG GAC CCA GAA GAG AAG GGC ACT GAG AAA AAG 527
Met Phe Gln Ile Leu Leu Asp Pro Glu Glu Lys Gly Thr Glu Lys Lys
160 165 170 175
CAA AAG GCT TCT CAG AAC CTG GTG GTG CTG GCC AGG GAG GAT GCT GGA 575
Gln Lys Ala Ser Gln Asn Leu Val Val Leu Ala Arg Glu Asp Ala Gly
180 185 l90
GCG GAG AAG ATC TTC CGG AGT AAT GGG GTT CAG CTC TTG CAA CGT TTA 623
Ala-Glu Lys Ile Phe Arg Ser Asn Gly Val Gln Leu Leu Gln Arg Leu
195 200 205
CTG GAC ATG GGA GAG ACT GAC CTC ATG CTG GCG GCT CTG CGT ACG CTG 671
Leu Asp Met Gly Glu Thr Asp Leu Met Leu Ala Ala Leu Arg Thr Leu
210 215 220
GTT GGC ATT TGC TCT GAG CAT CAG TCA CGG ACA GTG GCA ACC CTG AGC 719
Val Gly Ile Cys Ser Glu His Gln Ser Arg Thr Val Ala Thr Leu Ser
225 230 235
ATA CTG GGA ACT CGG CGA GTA GTC TCC ATC CTG GGC GTG GAA AGC CAG 767
Ile Leu Gly Thr Arg Arg Val Val Ser Ile Leu Gly Val Glu Ser Gln
240 245 250 255
GCT GTG TCC CTG GCT GCC TGC CAC CTG CTG CAG GTT ATG TTT GAT GCC 815
Ala Val Ser Leu Ala Ala Cys His Leu Leu Gln Val Met Phe Asp Ala
260 265 270
CTC AAG GAA GGT GTC AAA AAA GGC TTC CGA GGC AAA GAA GGT GCC ATC 863
Leu Lys Glu Gly Val Lys Lys Gly Phe Arg Gly Lys Glu Gly Ala Ile
275 280 285
ATT GTG GAT CCT GCC CGG GAG CTG AAG GTC CTC ATC AGT AAC CTC TTA 911
Ile Val Asp Pro Ala Arg Glu Leu Lys Val Leu Ile Ser Asn Leu Leu
290 295 300
GAT CTG CTG ACA GAG GTG GGG GTC TCT GGC CAA GGC CGA GAC AAT GCC 959
Asp Leu Leu Thr Glu Val Gly Val Ser Gly Gln Gly Arg Asp Asn Ala
305 310 315
CTG ACC CTC CTG ATT AAA GCG GTG CCC CGG AAG TCT CTC AAG GAC CCC 1007
Leu Thr Leu Leu Ile Lys Ala Val Pro Arg Lys Ser Leu Lys Asp Pro
320 325 330 335
AAC AAC AGC CTC ACC CTC TGG GTC ATC GAC CAA GGT CTG AAA AAG ATT 1055
Asn Asn Ser Leu Thr Leu Trp Val Ile Asp Gln Gly Leu Lys Lys Ile
340 345 350
TTG GAA GTG GGG GGC TCT CTA CAG GAC CCT CCT GGG GAG CTC GCA GTG 1103
Leu Glu Val Gly Gly Ser Leu Gln Asp Pro Pro Gly Glu Leu Ala Val
355 360 365
ACC GCA AAC AGC CGC ATG AGC GCC TCT ATT CTC CTC AGC AAG CTC TTT 1151
Thr Ala Asn Ser Arg Met Ser Ala Ser Ile Leu Leu Ser Lys Leu Phe
370 375 380
GAT GAC CTC AAG TGT GAT GCG GAG AGG GAG AAT TTC CAC AGA CTT TGT ll99
Asp Asp Leu Lys Cys Asp Ala Glu Arg Glu Asn Phe His Arg Leu Cys
385 390 395
GAA AAC TAC ATC AAG AGC TGG TTT GAG GGC CAA GGG CTG GCC GGG AAG 1247
Glu Asn Tyr Ile Lys Ser Trp Phe Glu Gly Gln Gly Leu Ala Gly Lvs
400 405 410 ~15
CTA CGG GCC ATC CAG ACG GTG TCC TGC CTC CTG CAG GGC CCA TGT GAC 1295
Leu Ara Ala Ile Gln Thr Val Ser Cys Leu Leu Gln Gly Pro C~.s ASD
420 425 ~30
S ~
CA 022029l2 l997-04-l6
WO 96/12735 PCT/US95112724
GCT GGC AAC CGG GCC TTG GAG CTG AGC GGT GTC ATG GAG AGT GTG ATT 1343
Ala Gly Asn Arg Ala Leu Glu Leu Ser Gly Val Met Glu Ser Val Ile
435 440 445
GCT CTG TGT GCC TCT GAG CAG GAG GAG GAG CAG CTG GTG GCC GTG GAG 1391
Ala Leu Cys Ala Ser Glu Gln Glu Glu Glu Gln Leu Val Ala Val Glu
- 450 455 460
GCT CTG ATC CAT GCA GCC GGC AAG GCT AAG CGG GCC TCA TTC ATC ACT 1439
Ala Leu Ile His Ala Ala Gly Lys Ala Lys Arg Ala Ser Phe Ile Thr
465 470 475
GCC AAT GGT GTC TCG CTG CTG AAG GAC CTA TAT AAG TGC AGC GAG AAG 1487
Ala Asn Gly Val Ser Leu Leu Lys Asp Leu Tyr Lys Cys Ser Glu Lys
480 485 490 495
GAC AGC ATC CGC ATC CGG GCG CTA GTG GGA CTC TGT AAG CTC GGT TCG 1535
Asp Ser Ile Arg Ile Arg Ala Leu Val Gly Leu Cys Lys Leu Gly Ser
500 505 510
GCT GGA GGG ACT GAC TTC AGC ATG AAG CAG TTT GCT GAA GGC TCC ACT 1583
Ala Gly Gly Thr Asp Phe Ser Met Lys Gln Phe Ala Glu Gly Ser Thr
515 520 525
CTC AAA CTG GCT AAG CAG TGT CGA AAG TGG CTG TGC AAT GAC CAG ATC 1631
Leu Lys Leu Ala Lys Gln Cys Arg Lys Trp Leu Cys Asn Asp Gln Ile
530 535 540
GAC GCA GGC ACT CGG CGC TGG GCA GTG GAG GGC CTG GCT TAC CTG ACC 1679
Asp Ala Gly Thr Arg Arg Trp Ala Val Glu Gly Leu Ala Tyr Leu Thr
545 550 555
TTT GAT GCC GAC GTG AAG GAA GAG TTT GTG GAG GAT GCG GCT GCT CTG 1727
Phe Asp Ala Asp Val Lys Glu Glu Phe Val Glu Asp Ala Ala Ala Leu
560 565 570 575
AAA GCT CTG TTC CAG CTC AGC AGG TTG GAG GAG AGG TCA GTG CTC TTT 1775
Lys Ala Leu Phe Gln Leu Ser Arg Leu Glu Glu Arg Ser Val Leu Phe
580 585 590
GCG GTG GCC TCA GCG CTG GTG AAC TGC ACC AAC AGC TAT GAC TAC GAG 1823
Ala Val Ala Ser Ala Leu Val Asn Cys Thr Asn Ser Tyr Asp Tyr Glu
595 600 605
GAG CCC GAC CCC AAG ATG GTG GAG CTG GCC AAG TAT GCC AAG CAG CAT 1871
Glu Pro Asp Pro Lys Met Val Glu Leu Ala Lys Tyr Ala Lys Gln His
610 615 620
GTG CCC GAG CAG CAC CCC AAG GAC AAG CCA AGC TTC GTG CGG GCT CGG l919
Val Pro Glu Gln His Pro Lys Asp Lys Pro Ser Phe Val Arg Ala Arg
625 630 635
GTG AAG AAG CTG CTG GCA GCG GGT GTG GTG TCG GCC ATG GTG TGC ATG 1967
Val Lys Lys Leu Leu Ala Ala Gly Val Val Ser Ala Met Val Cys Met
640 645 650 655
GTG AAG ACG GAG AGC CCT GTG CTG ACC AGT TCC TGC AGA GAG CTG CTC 2015
Val Lys Thr Glu Ser Pro Val Leu Thr Ser Ser Cys Arg Glu Leu Leu
660 665 670
TCC AGG GTC TTC TTG GCT TTA GTG GAA GAG GTA GAG GAC CGA GGC ACT 2063
Ser Arg Val Phe Leu Ala Leu Val Glu Glu Val Glu Asp Arg Gly Thr
675 680 6B5
GTG GTT GCC CAG GGA GGC GGC AGG GCG CTG ATC CCG CTG GCC CTG GAA 211
Val ~'al Ala Gln Gly Gly Gly Arg Ala Leu le Pro 'eu ~.la Leu Glu
690 695 700
. 53
CA 02202912 1997-04-16
WO 96/12735 PCT/US95/12724
GGC ACG GAC GTG GGG CAG ACA AAG GCA GCC CAG GCC CTT GCC AAG CTC 2159
Gly Thr Asp Val Gly Gln Thr Lys Ala Ala Gln Ala Leu Ala Lys Leu
705 710 715
ACC ATC ACC TCC AAC CCG GAG ATG ACC TTC CCT GGC GAG CGG ATC TAT 2207
Thr Ile Thr Ser Asn Pro Glu Met Thr Phe Pro Gly Glu Arg Ile Tyr
720 725 730 735
GAG GTG GTC CGG CCC CTC GTC TCC CTG TTG CAC CTC AAC TGC TCA GGC 2255
Glu Val Val Arg Pro Leu Val Ser Leu Leu His Leu Asn Cys Ser Gly
740 745 750
CTG CAG AAC TTC GAG GCG CTC ATG GCC CTA ACA AAC CTG GCT GGG ATC 2303
Leu Gln Asn Phe Glu Ala Leu Met Ala Leu Thr Asn Leu Ala Gly Ile
755 760 765
AGC GAG AGG CTC CGG CAG AAG ATC CTG AAG GAG AAG GCT GTG CCC ATC- 2351
Ser Glu Arg Leu Arg Gln Lys Ile Leu Lys Glu Lys Ala Val Pro Met
770 775 780
ATA GAA GGC TAC ATG TTT GAG GAG CAT GAG ATG ATC CGC CGG GCA GC- 2399
Ile Glu Gly Tyr Met Phe Glu Glu His Glu Met Ile Arg Arg Ala Ala
785 790 795
ACG GAG TGC ATG TGT AAC TTG GCC ATG AGC AAG GAG GTG CAG GAC CTC 2447
Thr Glu Cys Met Cys Asn Leu Ala Met Ser Lys Glu Val Gln Asp Leu
800 805 810 815
TTC GAA GCC CAG GGC AAT GAC CGA CTG AAG CTG CTG GTG CTG TAC AGT 2495
Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu Leu Val Leu Tyr Ser
820 825 830
GGA GAG GAT GAT GAG CTG CTA CAG CGG GCA GCT GCC GGG GGC TTG GCC 2543
Gly Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala Ala Gly Gly Leu Ala
835 840 845
ATG CTT ACC TCC ATG CGG CCC ACG CTC TGC AGC CGC ATT CCC CAA G-^- 2591
Met Leu Thr Ser Met Arg Pro Thr Leu Cys Ser Arg Ile Pro Gln Val
850 855 860
ACC ACA CAC TGG CTG GAG ATC CTG CAG GCC CTG CTT CTG AGC TCC A~.C 2639
Thr Thr His Trp Leu Glu Ile Leu Gln Ala Leu Leu Leu Ser Ser Asn
865 870 875
CAG GAG CTG CAG CAC CGG GGT GCT GTG GTG GTG CTG AAC ATG GTG G-- 2687
Gln Glu Leu Gln Hls Arg Gly Ala Val Val Val Leu Asn Met Val Giu
880 885 890 8~-
GCC TCG AGG GAG ATT GCC AGC ACC CTG ATG GAG AGT GAG r TG ATG G,^- 2735
Ala Ser Arg Glu Ile Ala Ser Thr Leu Met Glu Ser Glu Met Met G'_
900 905 910
ATC TTG TCA GTG CTA GCT AAG GGT GAC CAC AGC CCT GTC ACA AGG GC- 2783
Ile Leu Ser Val Leu Ala Lys Gly Asp His Ser Pro Val Thr Arg Ala
915 920 925
GCT GCA GCC TGC CTG GAC AAA GCA GTG GAA TAT GGG CTT ATC CAA CC~ 2831
Ala Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr Gly Leu Ile Gln Prc
930 935 940
AAC CAA GAT GGA GAG TGAGGGGGTT GTCCCTGGGC CCAAGGCTCA TGCACACGC- 2886
Asn Gln Asp Gly Glu
945
ACCTATTGTG GCACGGAGAG TAAGGACGGA AGCAGCTTTG GCTGGTGGT- GCTGGC,- C '946
CCAATAC-CT TGCCCATCCT CGCTTGCTGC CCTAGGAlGT CCTCTGTTC- GAGTC.'.~ 3006
_ ~
CA 02202912 1997-04-16
W O g6/12735 PCTrUS95112724
CCACGTTCAG TCACACAGCC CTGCTTGGCC AGCACTGCCT GCAGCCTCAC TCAGAGGGGC 3066
C~lllrl~lG TACTACTGTA GTCAGCTGGG AATGGGGAAG GTGCATCCCA ACACAGCCTG 3126
TGGATCCTGG GGCATTTGGA AGGGCGCACA CATCAGCAGC CTCACCAGCT GTGAGCCTGC 3186
TATCAGGCCT GCCCCTCCAA TAAAAGTGTG TAGAACTCC 3225
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 948 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Gln Thr Arg Pro Ala Pro Glu Thr Ala Pro Ala Arg Ala Arg Asp Thr
1 5 10 15
Leu Ser Ala Met Thr Ala Ser Ser Val Glu Gln Leu Arg Lys Glu Gly
Asn Glu Leu Phe Lys Cys Gly Asp Tyr Gly Gly Ala Leu Ala Ala Tyr
Thr Gln Ala Leu Gly Leu Asp Ala Thr Pro Gln Asp Gln Ala Val Leu
His Arg Asn Arg Ala Ala Cys His Leu Lys Leu Glu Asp Tyr Asp Lys
Ala Glu Thr Glu Ala Ser Lys Ala Ile Glu Lys Asp Gly Gly Asp Val
Lys Ala Leu Tyr Arg Arg Ser Gln Ala Leu Glu Lys Leu Gly Arg Leu
100 105 110
Asp Gln Ala Val Leu Asp Leu Gln Arg Cys Val Ser Leu Glu Pro Lys
115 120 125
Asn Lys Val Phe Gln Glu Ala Leu Arg Asn Ile Gly Gly Gln Ile Gln
130 135 140
Glu Lys Val Arg Tyr Met Ser Ser Thr Asp Ala Lys Val Glu Gln Met
145 150 155 160
Phe Gln Ile Leu Leu Asp Pro Glu Glu Lys Gly Thr Glu Lys Lys Gln
165 170 175
Lys Ala Ser Gln Asn Leu Val Val Leu Ala Arg Glu Asp Ala Gly Ala
180 185 190
Glu Lys Ile Phe Arg Ser Asn Gly Val Gln Leu Leu Gln Arg Leu Leu
195 200 205
Asp Met Gly Glu Thr Asp Leu Met Leu Ala Ala Leu Arg Thr Leu Val
210 215 220
Gly Ile Cys Ser Glu His Gln Ser Arg Thr Val Ala Thr Leu Ser Ile
225 230 235 240
L-u Glv Thr Arg Arg Val Val S~r Il- L~u C-ly ~al Glu Ser Gln Ala
245 ~50 255
_ ;~
CA 02202912 1997-04-16
WO 96/12735 PCT/US95/12724
Val Ser Leu Ala Ala Cys His Leu Leu Gln Val Met Phe Asp Ala Leu
260 265 270
Lys Glu Gly Val Lys Lys Gly Phe Arg Gly Lys Glu Gly Ala Ile Ile
275 280 285
Val Asp Pro Ala Arg Glu Leu Lys Val Leu Ile Ser Asn Leu Leu Asp
290 295 300
Leu Leu Thr Glu Val Gly Val Ser Gly Gln Gly Arg Asp Asn Ala Leu
305 310 315 320
Thr Leu Leu Ile Lys Ala Val Pro Arg Lys Ser Leu Lys Asp Pro Asn
325 330 335
Asn Ser Leu Thr Leu Trp Val Ile Asp Gln Gly Leu Lys Lys Ile Leu
340 345 350
Glu Val Gly Gly Ser Leu Gln Asp Pro Pro Gly Glu Leu Ala Val Thr
355 360 365
Ala Asn Ser Arg Met Ser Ala Ser Ile Leu Leu Ser Lys Leu Phe Asp
370 375 380
Asp Leu Lys Cys Asp Ala Glu Arg Glu Asn Phe His Arg Leu Cys Glu
385 390 395 400
Asn Tyr Ile Lys Ser Trp Phe Glu Gly Gln Gly Leu Ala Gly Lys Leu
405 410 415
Arg Ala Ile Gln Thr Val Ser Cys Leu Leu Gln Gly Pro Cys Asp Ala
420 425 430
Gly Asn Arg Ala Leu Glu Leu Ser Gly Val Met Glu Ser Val Ile Ala
435 440 445
Leu Cys Ala Ser Glu Gln Glu Glu Glu Gln Leu Val Ala Val Glu Ala
450 455 460
Leu Ile His Ala Ala Gly Lys Ala Lys Arg Ala Ser Phe Ile Thr Ala
465 470 475 480
Asn Gly Val Ser Leu Leu Lys Asp Leu Tyr Lys Cys Ser Glu Lys Asp
485 ~90 495
Ser Ile Arg Ile Arg Ala Leu Val Gly Leu Cys Lys Leu Gly Ser Ala
500 505 510
Gly Gly Thr Asp Phe Ser Met Lys Gln Phe Ala Glu Gly Ser Thr Leu
515 520 525
Lys Leu Ala Lys Gln Cys Arg Lys Trp Leu Cys Asn Asp Gln Ile Asp
530 535 540
Ala Gly Thr Arg Arg Trp Ala Val Glu Gly Leu Ala Tyr Leu Thr Phe
545 550 555 560
Asp Ala Asp Val Lys Glu Glu Phe Val Glu Asp Ala Ala Ala Leu Lys
565 570 575
Ala Leu Phe Gln Leu Ser Arg Leu Glu Glu Arg Ser Val Leu Phe Ala
580 585 590
Val Ala Ser Ala Leu Val Asn Cys Thr Asn Ser T-. r Asp Tyr Glu Glu
595 600 605
?ro Asp 2ro Lys Met Val Glu Leu Alâ Lys Tyr .~_a L~s Gln Hls Val
_
CA 02202912 1997-04-16
W O96/12735 PCTnUS95/12724
610 615 620
Pro Glu Gln His Pro Lys Asp Lys Pro Ser Phe Val Arg Ala Arg Val
625 630 635 640
Lys Lys Leu Leu Ala Ala Gly Val Val Ser Ala Met Val Cys Met Val
645 650 655
Lys Thr Glu Ser Pro Val Leu Thr Ser Ser Cys Arg Glu Leu Leu Ser
. 660 665 670
Arg Val Phe Leu Ala Leu Val Glu Glu Val Glu Asp Arg Gly Thr Val
675 680 685
Val Ala Gln Gly Gly Gly Arg Ala Leu Ile Pro Leu Ala Leu Glu Gly
690 695 700
Thr Asp Val Gly Gln Thr Lys Ala Ala Gln Ala Leu Ala Lys Leu Thr
705 710 715 720
Ile Thr Ser Asn Pro Glu Met Thr Phe Pro Gly Glu Arg Ile Tvr Glu
725 730 735
Val Val Arg Pro Leu Val Ser Leu Leu Hls Leu Asn Cys Ser Gly Leu
740 745 750
Gln Asn Phe Glu Ala Leu Met Ala Leu Thr Asn Leu Ala Gly Ile Ser
755 760 765
Glu Arg Leu Arg Gln Lys Ile Leu Lys Glu Lys Ala Val Pro Met Ile
770 775 780
Glu Glv Tyr Met Phe Glu Glu His Glu Met Ile Arg Arg Ala Ala Thr
785 790 795 800
Glu Cys Met Cys Asn Leu Ala Met Ser Lys Glu Val Gln Asp Leu Phe
805 810 815
Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu Leu Val Leu Tyr Ser Gly
820 825 830
Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala Ala Gly Gly Leu Ala Met
835 840 845
Leu Thr Ser Met Arg Pro Thr Leu Cys Ser Arg Ile Pro Gln Val Thr
850 855 860
Thr H1s Trp Leu Glu Ile Leu Gln Ala Leu Leu Leu Ser Ser Asn Gln
865 870 875 880
Glu Leu Gln His Arg Gly Ala Val Val Val Leu Asn Met Val Glu Ala
885 890 895
Ser Arg Glu Ile Ala Ser Thr Leu Met Glu Ser Glu Met Met Glu Ile
900 905 910
Leu Ser Val Leu Ala Lys Gly Asp His Ser Pro Val Thr Arg Ala Ala
915 920 925
Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr Gly Leu Ile Gln Pro Asn
930 935 940
Gln Asp Gly Glu
945
