CA2202912A1 - Novel tnf receptor death domain ligand proteins and inhibitors of ligand binding - Google Patents

Abstract

Novel TNF receptor death domain ("TNF-R1-DD") ligand proteins are disclosed. Polynucleotides encoding the TNF-R1-DD ligand protein are also disclosed, along with vectors, host cells, and methods of making the TNF-R1-DD ligand protein. Pharmaceutical compositions containing the TNF-R1-DD ligand protein, methods of treating inflammatory conditions, and methods of inhibiting TNF-R death domain binding are also disclosed. Methods of identifying inhibitors of TNF-R death domain binding and inhibitors identified by such methods are also disclosed.

Description

W O 96/12735 PCTAUSg5112724 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 Wo 96/12735 PCTrUS95/12724 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 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:

WO 96/12735 PCr/US9S/12724 (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:

W O96/12735 1~ 3~12724 (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():~:

W O96/12735 PCTÇUS9S/12724 (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.

W O 96/12735 PCTrUS95112724 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;

W O 96/12735 P~ 35/12724 (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:

WO 96/12735 PCI~/US95/12724 (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%

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.

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

W O96/12735 PCTnUS95/12724 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 WO 96/1273S PCrlUS9S/12724 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 WO 96112735 PCr/US95/12724 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 W O 96/12735 PCT~US95112724 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 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 W O 96112735 P ~ AUS95/12724 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 Wo 96/12735 Pcr/uS9S/12724 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 W 096/12735 ~ 2724 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 Wo 96/12735 PCrnJS9S112724 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 W O g6/1273S P ~ rUS95/12724 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.

W O96/1273S PCTrUS95/12724 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.

W O 96112735 PCTnUS95/12724 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.

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 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 Wo 96/1273S PCrlUS95/12724 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 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.

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 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.

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 - - -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).

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.

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).

WO 9611273S PCrlUS95112724 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~

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~

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 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.

ISOLATION, EXPRESSION AND ASSAY

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.

~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 WO 96/12735 PCTrUS95/12724 1 5 lo 15 Ala Ser 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 Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val 80 85 go 95 Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met loo 105 llo Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu 180 185 lgo Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val Val His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe Phe Met Glu Val 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 Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lvs Thr 240 245 250 2s5 Lys Val Lu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn W O 96112735 P~ S/12724 Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser GTGACTGAGG AGTGGATGAT G-lC~l~l~l CCTCTGCAAG CCCCCTGCTG TGGCTTGGGT 1371 GGGTACCGGT TAl~l~lCCC TCTGAGTGTG TCTTGAGCGT GTCCACCTTC TCCCTCTCCA 1431 CCCTGGCGTT AACTGTCCAC TGCAAGAGTC TGGCTCTCCC l~ ~lGA CCCGGCATGA 1611 CTGGGCGCCT GGAGCAGTTT CA~ ~lGA GGAGTGAGGG AACCCTGGGG CTCACCCTCT 1671 CTCTGCCTGA GCATCAGGGA GGGGGCAGGA AAGACCAAGC TGGGTTTGCA CAT~ lG 1971 GAAATAAATG TCCTCACCTC CcAAAAAAAA AAAAAAAPAA AAAAAAA.~AA AAAAAAAAAA 2151 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 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 Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val Arg rg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln Ile sn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu Se-Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val Val His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe Phe Met Glu Val Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly Thr Val Tyr Glu Arg rp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lys Thr Lys al Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn Lys 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 Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser (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 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 Ala Ala Gly Gly Leu Ala Met Leu Thr Ser Met Arg Pro Thr Leu Cys 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 His Ser Pro Val Thr Arg Ala Ala Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr Gly Leu Ile Gln Pro Asn Gln Asp Gly Glu C~ ~llCT GAGTCAGCGG CCACGTTCAG TCACACAGCC CTGCTTGGCC AGCACTGCCT 615 GCAGCCTCAC TCAGAGGGGC C~ L-lG TACTACTGTA GTCAGCTGGG AATGGGGAAG 675 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 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 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 (2~ 'NFOR"~TION FOR SEQ ID NO:5:

(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:

Glu Lys Pro Leu His Ala Leu Leu His Gly Arg Gly Val Cys Leu Asn Glu 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 Gly Gly Ala Glu Asn Thr Ala His Pro Arg Ile Ile Ser Glu Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala Val Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe Tyr Lys Arg Lys Gln Cys Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys Trp Cys Val Asp Lvs Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe Gln Cvs His Thr Phe Asp Ser Ser Asn Val Glu TTTCCCTCAC CCCCTTCCAC CCCCAGCCCC GACTCCAGCC AGCGCCTCCC ,CCACCCCAG ~39 3q 96/12735 PCTrUS95/12724 (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 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 Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala Val Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe Tyr Lys Arg Lys Gln Cys Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys Trp Cys Val Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe Gln ys His Thr Phe Asp Ser Ser Asn Val Glu (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 (B) LOCATION: 57..875 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCCTGCACTC TCG~ C~l GCCCCACCCC GAGGTAAAGG GGGCGACTAA GAGAAG 56 Met Val Leu Leu Thr Ala Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly l 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 Glv Cys Cys Met Thr Cys Ala Leu Ala Glu Gly Gln Ser Cys Gly Val Tvr 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 Arg Gly Val Cys Leu Asn Glu 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 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 Ile Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala Val Tyr Leu Pro Asn Cys Asp Arg Lvs Gly Phe Tyr Lvs Ar~ Lys Gln Cvs Lvs Pro Ser Arg Glv Arg Lys Arg Gly Ile ~-c ~3G ~3c ~1v -CA 022029l2 l997-04-l6 WO 96/12735 PCTfUS95/12724 Cys Trp Cys Val Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe Gln Cys His Thr Phe Asp Ser Ser Asn Val Glu (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 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 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 Gly Gly Ala Glu Asn Thr Ala His Pro Arg Ile Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg 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 _ Tyr Lys Arg Lys Gln Cys Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys Trp Cys Val Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr al Asp Gly Asp Phe Gln Cys His Thr Phe Asp Ser Ser Asn Val Glu (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:

Ser Leu Lys Ala Asn Ile Pro Glu Val Glu Ala Val Leu Asn Thr Asp Arg Ser Leu Val Cys Asp Gly Lys 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 Ile Val Asp Ser Glu Cys Lys Ser Arg Asp Val Leu Gln Ser Tyr Phe Asp Leu Leu Gly Glu Leu Met Lys Phe Asn Val Asp Ala Phe Lys 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 ~3 96/12735 PCTnUS95/12724 Lys Gln Ile Asn Ser Ser Leu Val Asp Ser Asn Met Leu Val Arg Cys Val Thr Leu Ser Leu Asp Arg Phe Glu Asn Gln Val Asp Met Lys Val Ala Glu Val Leu Ser Glu Cys Arg Leu Leu Ala Tyr Ile Ser Gln Val Pro Thr Gln Met Ser Phe Leu Phe Arg Leu Ile Asn Ile Ile His Val Gln Thr Leu Thr Gln Glu Asn Val Ser Cys Leu Asn Thr Ser Leu Val Ile Leu Met Leu Ala Arg Arg Lys Glu Arg Leu Pro Leu Tyr Leu Arg Leu Leu Gln Arg Met Glu His Ser Lys Lys Tyr Pro Gly Phe Leu Leu Asn Asn Phe His Asn Leu Leu Arg Phe Trp Gln Gln His Tyr Leu His Lys Asp Lys Asp Ser Thr Cys Leu Glu Asn Ser Ser Cys Ile Ser Phe Ser Tyr Trp Lys Glu Thr Val Ser Ile Leu Leu Asn Pro Asp Arg Gln Ser Pro Ser Ala Leu Val Ser Tyr Ile Glu Glu Pro Tyr Met Asp Ile GAC AGG GAC TTC ACT GAG GAG TGACCTTGGG CCAGGCCTCG GGAGGCTGCT 96' Asp Arg Asp Phe Thr Glu Glu GGTAAGAGGA CATTTAGCAT TAG~r~l~lG TGAGCTCCTG CCG~~ ll GGCTGTCAGT 1261 CTCTGGTTCT GTTTGCTCAT TGGCCGCTGT GTTCATCCAA GGGGGTTCTC CCAGAAGTG.`. 1501 ~4 96112735 PCTnUS95/12724 AGGCAGCCGC TCCTGCCTGA GCCTGGACAT GGGGCCCTTC ~ ~llGC CAATTTATTA 1621 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 Phe Asn Lys Tyr Ile Asn Thr Asp Ala Lys Phe Gln Val Phe Leu Lys Gln Ile Asn Ser Ser Leu Val Asp Ser Asn Met Leu Val Arg Cys Val Thr Leu Ser Leu Asp Arg Phe Glu Asn Gln Val Asp Met Lys Va_ Ala 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 Thr Leu Thr Gln Glu Asn Val Ser Cys Leu Asn Thr Ser Leu Va~ Ile Leu Met Leu Ala Arg Arg Lys Glu Arg Leu Pro Leu Tyr _eu Arg Leu 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 WO 96/12735 PCTrUS9Stl2724 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 Arg Asp Phe Thr Glu Glu (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:

Glu Ile Ser Arg Lys Val Tyr Lys Gly Met Leu Asp Leu Leu Lys Cys 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 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 ~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 Ser Gly Val Ser Leu Thr Ser Ser Ser Gln Arg Thr Asp Gln Asp Ser Val Ile Gly Val Ser Pro Ala Val Met Ile Arg Ser Ser Ser Gln Asp Ser Glu Val Ser Thr Val Val Ser Asn Ser Ser Gly Glu Thr Leu Gly Ala Asp Ser Asp Leu Ser Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly Ser Val His Leu Ala Ser 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 Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys 385 390 39s .s Gln T!-r Phe Val Val His Ala Glv Thr Asp Thr Asn Gly Asp Ile o 405 410 415 WO 96/12735 PCrrUS9S/12724 Phe Phe Met Glu Val Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly Thr Val Tyr Glu Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lys Thr Lys Val Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala Arg Gln Gln Ser 4ao 485 490 495 Ile Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser CCCAAGTGCA CGATGCTGCT GTGACTGAGG AGTGGATGAT GCTCG~ CCTCTGCAAG 1942 C~ lGT GACCCGGCAT GACTGGGCGC CTGGAGCAGT TTCACTCTGT GAGGAGTGAG 2242 ~8 W O96112735 PCTnUS95/12724 AAATGTACAG ll~l~lGAAT GTGAAATAAA l~lc~-l~AAc TCCCAAAAAA AAAAAAAAAA 2722 (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 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 Glu Asn Phe Ile Ala Ser Ile Gly Pro Glu Val Ile Lys Pro Val Phe Asp Leu Gly Glu Thr Glu Glu Lys Lys Ser Gln Ile Ser Ala Asp Ser Gly Val Ser Leu Thr Ser Ser Ser Gln Arg Thr Asp Gln Asp Ser Val Ile Gly Val Ser Pro Ala Val Met Ile Arg Ser Ser Ser Gln Asp Ser Glu Val Ser Thr Val Val Ser Asn Ser Ser Gly Glu Thr Leu Gly Ala Asp Ser Asp Leu Ser Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly Ser Val His Leu Ala Ser Ser Arg Glv Thr Leu Ser Asp Ser Glu Ile Glu Thr Asn Ser Ala Thr ser Thr iie Phe Glv Lys Ala ~ms Ser Leu 4~

WO 96112735 PCTrUS95/12724 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 Glu Arg Ser Thr Leu Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val Val His Ala Gly Thr Asp Thr Asn Gly ASD Ile Phe Phe Met Glu Val Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly Thr Val Tyr Glu Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lys Thr Lys Val Leu Cys Leu Trp Arg Arg Asn Glv Ser Glu Thr Gln Leu Asn Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala Ara Gln Gln Ser Ile Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu ' e Cys T~. r Ser Val Leu C~. s Leu P~e Ser T .-r ~'a' Ala Ala ~ a_ Y._~
580 s&s sgo Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser (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:

Gln Thr Arg Pro Ala Pro Glu Thr Ala Pro Ala Arg Ala Arg Asp Thr 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 Asp Gln Ala Val Leu Asp Leu Gln Arg Cys Val Ser Leu Glu Pro Lys Asn Lys Val Phe Gln Glu Ala Leu Arg Asn Ile Gly Gly Gln Ile 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 CA 022029l2 l997-04-l6 Met Phe Gln Ile Leu Leu Asp Pro Glu Glu Lys Gly Thr Glu Lys Lys Gln Lys Ala Ser Gln Asn Leu Val Val Leu Ala Arg Glu Asp Ala Gly 180 185 l90 Ala-Glu Lys Ile Phe Arg Ser Asn Gly Val Gln Leu Leu Gln Arg Leu Leu Asp Met Gly Glu Thr Asp Leu Met Leu Ala Ala Leu Arg Thr Leu Val Gly Ile Cys Ser Glu His Gln Ser Arg Thr Val Ala Thr Leu Ser Ile Leu Gly Thr Arg Arg Val Val Ser Ile Leu Gly Val Glu Ser Gln Ala Val Ser Leu Ala Ala Cys His Leu Leu Gln Val Met Phe Asp Ala Leu Lys Glu Gly Val Lys Lys Gly Phe Arg Gly Lys Glu Gly Ala Ile Ile Val Asp Pro Ala Arg Glu Leu Lys Val Leu Ile Ser Asn Leu Leu Asp Leu Leu Thr Glu Val Gly Val Ser Gly Gln Gly Arg Asp Asn Ala Leu Thr Leu Leu Ile Lys Ala Val Pro Arg Lys Ser Leu Lys Asp Pro Asn Asn Ser Leu Thr Leu Trp Val Ile Asp Gln Gly Leu Lys Lys Ile Leu Glu Val Gly Gly Ser Leu Gln Asp Pro Pro Gly Glu Leu Ala Val Thr Ala Asn Ser Arg Met Ser Ala Ser Ile Leu Leu Ser Lys Leu Phe 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 Glu Asn Tyr Ile Lys Ser Trp Phe Glu Gly Gln Gly Leu Ala Gly Lvs 400 405 410 ~15 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 Ala Gly Asn Arg Ala Leu Glu Leu Ser Gly Val Met Glu Ser Val Ile Ala Leu Cys Ala Ser Glu Gln Glu Glu Glu Gln Leu Val Ala Val Glu Ala Leu Ile His Ala Ala Gly Lys Ala Lys Arg Ala Ser Phe Ile Thr Ala Asn Gly Val Ser Leu Leu Lys Asp Leu Tyr Lys Cys Ser Glu Lys Asp Ser Ile Arg Ile Arg Ala Leu Val Gly Leu Cys Lys Leu Gly Ser Ala Gly Gly Thr Asp Phe Ser Met Lys Gln Phe Ala Glu Gly Ser Thr Leu Lys Leu Ala Lys Gln Cys Arg Lys Trp Leu Cys Asn Asp Gln Ile Asp Ala Gly Thr Arg Arg Trp Ala Val Glu Gly Leu Ala Tyr Leu Thr Phe Asp Ala Asp Val Lys Glu Glu Phe Val Glu Asp Ala Ala Ala Leu Lys Ala Leu Phe Gln Leu Ser Arg Leu Glu Glu Arg Ser Val Leu Phe Ala Val Ala Ser Ala Leu Val Asn Cys Thr Asn Ser Tyr Asp Tyr Glu Glu Pro Asp Pro Lys Met Val Glu Leu Ala Lys Tyr Ala Lys Gln His 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 Val Lys Lys Leu Leu Ala Ala Gly Val Val Ser Ala Met Val Cys Met Val Lys Thr Glu Ser Pro Val Leu Thr Ser Ser Cys Arg Glu Leu Leu Ser Arg Val Phe Leu Ala Leu Val Glu Glu Val Glu Asp Arg Gly Thr Val ~'al Ala Gln Gly Gly Gly Arg Ala Leu le Pro 'eu ~.la Leu Glu . 53 Gly Thr Asp Val Gly Gln Thr Lys Ala Ala Gln Ala Leu Ala Lys Leu Thr Ile Thr Ser Asn Pro Glu Met Thr Phe Pro Gly Glu Arg Ile Tyr Glu Val Val Arg Pro Leu Val Ser Leu Leu His Leu Asn Cys Ser Gly Leu Gln Asn Phe Glu Ala Leu Met Ala Leu Thr Asn Leu Ala Gly Ile Ser Glu Arg Leu Arg Gln Lys Ile Leu Lys Glu Lys Ala Val Pro Met Ile Glu Gly Tyr Met Phe Glu Glu His Glu Met Ile Arg Arg Ala Ala Thr Glu Cys Met Cys Asn Leu Ala Met Ser Lys Glu Val Gln Asp Leu Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu Leu Val Leu Tyr Ser Gly Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala Ala Gly Gly Leu Ala 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 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 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'_ Ile Leu Ser Val Leu Ala Lys Gly Asp His Ser Pro Val Thr Arg Ala 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 Asn Gln Asp Gly Glu ACCTATTGTG GCACGGAGAG TAAGGACGGA AGCAGCTTTG GCTGGTGGT- GCTGGC,- C '946 CCAATAC-CT TGCCCATCCT CGCTTGCTGC CCTAGGAlGT CCTCTGTTC- GAGTC.'.~ 3006 _ ~

W O g6/12735 PCTrUS95112724 C~lllrl~lG TACTACTGTA GTCAGCTGGG AATGGGGAAG GTGCATCCCA ACACAGCCTG 3126 (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 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 Asp Gln Ala Val Leu Asp Leu Gln Arg Cys Val Ser Leu Glu Pro Lys Asn Lys Val Phe Gln Glu Ala Leu Arg Asn Ile Gly Gly Gln Ile Gln Glu Lys Val Arg Tyr Met Ser Ser Thr Asp Ala Lys Val Glu Gln Met Phe Gln Ile Leu Leu Asp Pro Glu Glu Lys Gly Thr Glu Lys Lys Gln Lys Ala Ser Gln Asn Leu Val Val Leu Ala Arg Glu Asp Ala Gly Ala Glu Lys Ile Phe Arg Ser Asn Gly Val Gln Leu Leu Gln Arg Leu Leu Asp Met Gly Glu Thr Asp Leu Met Leu Ala Ala Leu Arg Thr Leu Val Gly Ile Cys Ser Glu His Gln Ser Arg Thr Val Ala Thr Leu Ser Ile L-u Glv Thr Arg Arg Val Val S~r Il- L~u C-ly ~al Glu Ser Gln Ala 245 ~50 255 _ ;~

Val Ser Leu Ala Ala Cys His Leu Leu Gln Val Met Phe Asp Ala Leu Lys Glu Gly Val Lys Lys Gly Phe Arg Gly Lys Glu Gly Ala Ile Ile Val Asp Pro Ala Arg Glu Leu Lys Val Leu Ile Ser Asn Leu Leu Asp Leu Leu Thr Glu Val Gly Val Ser Gly Gln Gly Arg Asp Asn Ala Leu Thr Leu Leu Ile Lys Ala Val Pro Arg Lys Ser Leu Lys Asp Pro Asn Asn Ser Leu Thr Leu Trp Val Ile Asp Gln Gly Leu Lys Lys Ile Leu Glu Val Gly Gly Ser Leu Gln Asp Pro Pro Gly Glu Leu Ala Val Thr Ala Asn Ser Arg Met Ser Ala Ser Ile Leu Leu Ser Lys Leu Phe Asp Asp Leu Lys Cys Asp Ala Glu Arg Glu Asn Phe His Arg Leu Cys Glu Asn Tyr Ile Lys Ser Trp Phe Glu Gly Gln Gly Leu Ala Gly Lys Leu Arg Ala Ile Gln Thr Val Ser Cys Leu Leu Gln Gly Pro Cys Asp Ala Gly Asn Arg Ala Leu Glu Leu Ser Gly Val Met Glu Ser Val Ile Ala Leu Cys Ala Ser Glu Gln Glu Glu Glu Gln Leu Val Ala Val Glu Ala Leu Ile His Ala Ala Gly Lys Ala Lys Arg Ala Ser Phe Ile Thr Ala 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 Gly Gly Thr Asp Phe Ser Met Lys Gln Phe Ala Glu Gly Ser Thr Leu Lys Leu Ala Lys Gln Cys Arg Lys Trp Leu Cys Asn Asp Gln Ile Asp Ala Gly Thr Arg Arg Trp Ala Val Glu Gly Leu Ala Tyr Leu Thr Phe Asp Ala Asp Val Lys Glu Glu Phe Val Glu Asp Ala Ala Ala Leu Lys Ala Leu Phe Gln Leu Ser Arg Leu Glu Glu Arg Ser Val Leu Phe Ala Val Ala Ser Ala Leu Val Asn Cys Thr Asn Ser T-. r Asp Tyr Glu Glu ?ro Asp 2ro Lys Met Val Glu Leu Alâ Lys Tyr .~_a L~s Gln Hls Val _ W O96/12735 PCTnUS95/12724 Pro Glu Gln His Pro Lys Asp Lys Pro Ser Phe Val Arg Ala Arg Val Lys Lys Leu Leu Ala Ala Gly Val Val Ser Ala Met Val Cys Met Val 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 Val Ala Gln Gly Gly Gly Arg Ala Leu Ile Pro Leu Ala Leu Glu Gly Thr Asp Val Gly Gln Thr Lys Ala Ala Gln Ala Leu Ala Lys Leu Thr Ile Thr Ser Asn Pro Glu Met Thr Phe Pro Gly Glu Arg Ile Tvr Glu Val Val Arg Pro Leu Val Ser Leu Leu Hls Leu Asn Cys Ser Gly Leu Gln Asn Phe Glu Ala Leu Met Ala Leu Thr Asn Leu Ala Gly Ile Ser Glu Arg Leu Arg Gln Lys Ile Leu Lys Glu Lys Ala Val Pro Met Ile Glu Glv Tyr Met Phe Glu Glu His Glu Met Ile Arg Arg Ala Ala Thr Glu Cys Met Cys Asn Leu Ala Met Ser Lys Glu Val Gln Asp Leu Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu 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 Cys Ser Arg Ile Pro Gln Val Thr Thr H1s 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 His Ser Pro Val Thr Arg Ala Ala Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr Gly Leu Ile Gln Pro Asn Gln Asp Gly Glu

Claims (35)

What is claimed is:

1. A composition comprising an isolated polynucleotide encoding a protein having TNF-R1-DD ligand protein activity.

2. The composition of claim 1 wherein said 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:1:
(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-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2: and (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

3. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:3 from nucleotide 2 to nucleotide 415:
(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3;
(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:4:
(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4: and (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

4. A composition of claim 1 wherein said polynucleotide is operably linked to an expression control sequence.

5. A host cell transformed with a composition of claim 4.

6. The host cell of claim 5, wherein said cell is a mammalian cell.

7. A process for producing an TNF-R1-DD ligand protein, which comprises:
(a) growing a culture of the host cell of claim 5 in a suitable culture medium; and (b) purifying the TNF-R1-DD ligand protein from the culture.

8. A composition comprising a protein having TNF-R1-DD ligand protein activity.

9. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2: and (b) fragments of the amino acid sequence of SEQ ID NO:2;
said protein being substantially free from other mammalian proteins.

10. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4: and (b) fragments of the amino acid sequence of SEQ ID NO:4:
said protein being substantially free from other mammalian proteins.

11. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6: and (b) fragments of the amino acid sequence of SEQ ID NO:6:

said protein being substantially free from other mammalian proteins.

12. The composition of claim 8, further comprising a pharmaceutically acceptable carrier.

13. A composition comprising an antibody which specifically reacts with the TNF-R1-DD ligand protein of claim 8.

14. A method of identifying an inhibitor of TNF-R death domain binding which comprises:
(a) combining an TNF-R death domain protein with a composition of claim 8. said combination forming a first binding mixture;
(b) measuring the amount of binding between the TNF-R death domain protein and the TNF-R1-DD ligand protein in the first binding mixture;
(c) combining a compound with the TNF-R death domain protein and an TNF-R1-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 inhibiting TNF-R death domain binding when a decrease in the amount of binding of the second binding mixture occurs.

15. The method of claim 14 wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting 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) fragments of the amino acid sequence of SEQ ID NO:4:
(e) the amino acid sequence of SEQ ID NO:6;
(f) fragments of the amino acid sequence of SEQ ID NO:6:
(g) the amino acid sequence of SEQ ID NO:8; and (h) fragments of the amino acid sequence of SEQ ID NO:8.

16. A method of preventing or ameliorating an inflammatory condition which comprises administering a therapeutically effective amount of a composition of claim 12.

17. TNF-R1-DD ligand protein produced according to the method of claim 7.

18. A method of inhibiting TNF-R death domain binding comprising administering a therapeutically effective amount of a composition of claim 12.

19. A method of preventing or ameliorating an inflammatory condition which comprises administering to a mammalian subject a therapeutically effectiveamount of a composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity.

20. A method of inhibiting TNF-R death domain binding comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD
ligand protein activity.

21. A composition comprising an inhibitor identified according to the method of claim 14.

22. The composition of claim 21 further comprising a pharmaceutically acceptable carrier.

23. A method of preventing or ameliorating an inflammatory condition comprising administering to a mammalian subject a therapeutically effective amount of the composition of claim 22.

24. A method of inhibiting TNF-R death domain binding comprising administering to a mammalian subject a therapeutically effective amount of the composition of claim 22.

25. A composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity.

26. A method of identifying an inhibitor of TNF-R death domain binding which comprises:
(a) transforming a cell with a first polynucleotide encoding an TNF-R death domain protein, a second polynucleotide encoding an TNF-R1-DD ligand protein, and at least one reporter gene, wherein the expression 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 encoded 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 expression 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 expression of the reporter gene occurs.

27. The method of claim 26 wherein 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:1, which encodes a protein having TNF-R1-DD
ligand protein activity;
(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-R1-DD ligand protein 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 having TNF-R1-DD
ligand protein activity;
(g) a polynucleotide encoding an TNF-R1-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 and having TNF-R1-DD ligand protein activity;
(i) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:5 from nucleotide to nucleotide 559:
(i) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:5 which encodes a protein having TNF-R1-DD
ligand protein activity;
(j) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:6;
(l) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 and having TNF-R1-DD ligand protein activity:
(m) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:7 from nucleotide 57 to nucleotide 875:

(n) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:7, which encodes a protein having TNF-R1-DD
ligand protein activity;
(o) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:8;
(p) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 and having TNF-R1-DD ligand protein activity; and (q) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(p). which encodes a protein having TNF-R1-DD ligand protein activity.

28. The method of claim 26 wherein the cell is a yeast cell.

29. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:9 from nucleotide 2 to nucleotide 931;
(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9;
(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:10:
(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:10: and (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

30. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:11 from nucleotide 2 to nucleotide 1822:

(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11;
(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:12;
(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:12; and (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

31. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10: and (b) fragments of the amino acid sequence of SEQ ID NO: 10:
said protein being substantially free from other mammalian proteins.

32. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:12: and (b) fragments of the amino acid sequence of SEQ ID NO:12:
said protein being substantially free from other mammalian proteins.

33. The method of claim 14 wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10:
(b) fragments of the amino acid sequence of SEQ ID NO:10:
(c) the amino acid sequence of SEQ ID NO:12: and (d) fragments of the amino acid sequence of SEQ ID NO:12.

34. The method of claim 26 wherein the second polynucleotide is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:9 from nucleotide 2 to nucleotide 931:

(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9, which encodes a protein having TNF-R1-DD
ligand protein activity;
(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:10;
(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 and having TNF-R1-DD ligand protein activity;
(e) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:11 from nucleotide 2 to nucleotide 1822:
(f) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11. which encodes a protein having TNF-R1-DD
ligand protein activity;
(g) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO: 12: and (h) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 and having TNF-R1-DD ligand protein activity; and (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h). which encodes a protein having TNF-R1-DD ligand protein activity.

35. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ
ID NO:13 from nucleotide 3 to nucleotide 2846;
(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:13;
(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:14:
(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:14: and