AU2003203679B2 - Human toll homologues - Google Patents

Description

AUSTRALIA

Patents Act 1590 COXPLUT SPECIFICATION ST)AAD PAT= GEN1ENTECH, TNC.

Invention Title:- HUMAN TOLL MONOLOGUES The following statement is a full description of this invention, including the best method of performring it known to m[e /us The present invanfion relates gonsrslly to the Ldentifi alien an isolation of novel DN s designated herein a D)NA4D021, DNA42663 and DNA47381, an o h tecomnant productian of nel human Tall bojuolo gm (designated as PRO2$5 POMLS and PROSIs, respectively) encoded by sai DNAs.

Mentbrane-bmund proteis and receptors oum play an important role in the formtion, differentiation and mafintenac f wmultiaeliular organism The fmt, of Manty individual cells, prolifeation, migration, differentiation, or interaction with oUte cell., is typicay governed by informatio received Cram other cells and/or the immediat, environment.

Th is inftrmution is often transmtted by mceted polypeptides (for inance, mitogenic factor, survival factor, cytatoxic factors, differentiation factors, neuropeptides, andl hormones) which are, in tun, received and interpreted by divars. cell reeeptors or membranea-bound pTroten.

Such membrane-bound proteins and cell receptors iMude% hut are not Utnited to, cytokine receptors, receptor kinases, receptor ploephatases. reeptors involved in veil-cell interactions, and cellular adhesin acleculga like selectin an interins. For mnsance, transduction of signwa thtat regulate coll growth mist differentiation i-s reglated in part by phmaphorylation of various cellula proteins. Proten tyroelne kinniei, enzymes that catalyze thatt process, can also &a ag grwth factor receptor.. Examples include fibroblast growth factor receptor and nerve growth factor receptor.

Membrane-bound pruteins and recetcr molecules have various industrial applnt'cons, including as phnrmacenlical and diagnostic agents. Receptor izmuociadhesina, for instance, can be employed no therautic agents to black receptor-Jigund Interatonh The membrane-bound protein. can also be employed for screing of potenfli peptide or small molecule inhibitor of the relevant resptor/lignd interaction.

Wafrts are being undertaen- by both industry and academia to identify neaw, naive receptor protaria Many efforts a"e focusd on the screening of mammalian recombinan DNA librarims to idlentiy the coding sequenes far novel receptor proteins.

so The cloning of the Toll gene of Drosophila, a maternal effect gene that plays a central role in "h establishmen of the etnlrcic dora-venbral pattern, ha. been reported by Hasaimt et fall U. 269-479 (19W6), 'The Droaapkilcz Toll gene encode.n interal msxubrane protein with ain ertracytolasmie domain of B0B amino acids and a cytoplasmic doin of U69 mnim acids. The ertracyteplasmlo domain has a potentia membrane. spanning segment, and contains multiple copies of a leucine-rich segment, a structural moti found in mainy tranmembrane pratelna. The Toll protein control. dorsal-ventral patterning in Drosophila embryos and activates the triascription factor Dorsal upon binding to its ligand Spitle:. (Odrinto and Anderson CA L ~617-688(199%).) "naut Dreoph&o. the Toll/Banal H:\cintac\Kcep\speci\2003203679.doc 17/11/06

NO

0 signaling pathway participates in the anti-fungal immune response. (Lenaitre et al., Cell 86, 973-983 (1996).

O A human homologue of the Drosophila Toll protein has been described by Medzhitov et al., Nature 388, 394-397 (1997). This human Toll, just as Drosophila Toll, is a type I transmembrane protein, with an extracellular domain consisting of 21 tandemly repeated leucine-rich motifs (leucine-rich region LRR), separated by a non-LRR region, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin-l (IL-I) receptor. A constitutively active mutant of the human Toll transfected into human cell lines was shown to be able to induce the activation of NF-KB and the expression of NF-KB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as r" 10 the expression of the constimulatory molecule B7.1, which is required for the activation of native T cells.

O It has been suggested that Toll functions in vertebrates as a non-clonal receptor of the immune system, CI which can induce signals for activating both an innate and an adaptive immune response in vertebrates.

The human Toll gene reported by Medzhitov et al., supra was most strongly expressed in spleen and peripheral blood leukocytes (PBL), and the authors suggested that its expression in other tissues may be due to the presence of macrophages and dendritic cells, in which it could act as an early-warning system for infection. The public GenBank database contains the following Toll sequences: Toll I (DNAX# HSU88540-1, which is identical with the random sequenced full-length cDNA #HUMRSC786-1); Toll2 (DNAX# HSU88878-1); Toll3 (DNAX# HSU88879-1); and Toll4 (DNAX# HSU88880-1, which is identical with the DNA sequence reported by Medzhitov et al., supra). A partial Toll sequence (Toll5) is available from GenBank under DNAX# HSU88881-1.

Further human homologues of the Drosophila Toll protein, designated as Toll-like receptors (huTLRsl-5) were recently cloned and shown to mirror the topographic structure of the Drosophila counterpart (Rock et al., Proc. Natl. Acad. Sci. USA 95, 588-593 [1998]). Overexpression of a constitutively active mutant of one human TLR (Toll-protein homologue Medzhitov et al, supra; TLR4 Rock et al., supra) leads to the activation of NF-KB and induction of the inflammatory cytokines and constimulatory molecules. Medzhitov et al., supra.

All references, including any patents or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY OF THE INVENTION Applicants have identified three novel cDNA clones that encode novel human Toll polypeptides, designated in the present application as PRO285 (encoded by DNA40021).

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O In one embodiment, the invention provides an isolated nucleic acid molecule comprising a DNA encoding a polypeptide having at least about 95% sequence identity to a DNA O molecule encoding a PRO285 polypeptide having amino acid residues 27 to 839 of Fig. 1 (SEQ ID NO:1) or the complement of the DNA molecule of(a). The complementary DNA molecule preferably remains stably bound to such encoding nucleic acid sequence under at least moderate, and optionally, under high stringency conditions.

In a further embodiment, the isolated nucleic acid molecule comprises a polynucleotide Sthat has at least about 95% sequence identity with a polynucleotide encoding a polypeptide comprising the sequence of amino acids I to 839 of Fig. I (SEQ ID NO: I).

In a specific embodiment, the invention provides an isolated nucleic acid molecule Scomprising DNA encoding native or variant PRO285 polypeptides, with or without the N-terminal signal CK1 sequence, and with or without the transmembrane regions of the respective full-length sequences. In one aspect, the isolated nucleic acid comprises DNA encoding a mature, full-length native PR0285 polypeptide having amino acid residues 1 to 1049 of Fig. 1 (SEQ ID NO: or is complementary to such encoding nucleic acid sequence. In another aspect, the invention concerns an isolated nucleic acid molecule that comprises DNA encoding a native PRO285 polypeptide without an N-terminal signal sequence, or is complementary to such encoding nucleic acid sequence. In yet another embodiment, the invention concerns nucleic acid encoding transmembrane-domain deleted or inactivated forms of the fulllength native PRO285 protein.

In another aspect, the invention concerns an isolated nucleic acid molecule encoding a PRO285 polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about residues 85 and about 3283 inclusive, of Figure 2 (SEQ ID NO: Preferably, hybridization occurs under stringent hybridization and wash conditions.

In another aspect, the invention concerns an isolated nucleic acid molecule comprising DNA encoding a polypeptide scoring at least about 95% positives when compared with the amino acid sequence of residues 1 to 1049, inclusive of Figure 1 (SEQ ID NO: or the complement of a DNA of(a).

In another embodiment, the invention the isolated nucleic acid molecule comprises the clone (DNA 40021-1154) deposited on October 17, 1997, under ATCC number 209389.

In yet another embodiment, the invention provides a vector comprising DNA encoding PR0285 polypeptide, or its variants. Thus the vector may comprise any of the isolated nucleic acid molecules hereinabove defined.

A host cell comprising such a vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast.

A process for producing PRO285 polypeptide is further provided and comprises culturing host cells under conditions suitable for expression of PRO285, and recovering PRO285 from the cell culture.

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O

O In another embodiment, the invention provides isolated PR0285 polypeptide. In particular, the invention provides isolated native sequence PR0285 polypeptide, which in one 0 embodiment, include the amino acid sequences comprising residues 1 to 1049 and 1 to 1041 of Figure 1 (SEQ ID NO:1). The invention also provides for variants of the PR0285 polypeptide which are encoded by any of the isolated nucleic acid molecules hereinabove defined. Specific variants include, but are not limited to, deletion (truncated) variants of the full-length native sequence PR0285 polypeptide which lack the respective N-terminal signal sequences and/or have their respective transmembrane and/or 0 cytoplasmic domains deleted or inactivated.

In a further aspect, the invention concerns an isolated PRO285 polypeptide, comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 85% positives, Smore preferably at least about 90% positives, most preferably at least about 95% positives when Cl compared with the amino acid sequence of amino acid residues I to 1049, inclusive of Figure 1 (SEQ ID NO:1).

In a still further aspect, the invention provides a polypeptide produced by (I) hybridizing a test DNA molecule under stringent conditions with a DNA molecule encoding a PRO285 polypeptide having the sequence of amino acid residues from about I to about 1049, inclusive of Figure 1 (SEQ ID NO: or the complement of a DNA molecule of(a), and if the test DNA molecule has at least about a 95% sequence identity to or (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.

In another embodiment, the invention provides chimeric molecules comprising PRO285 polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a PRO285 polypeptide fused to an epitope tag sequence or a Fc region of an immunoglobulin. An example of such a chimeric molecule comprises a PRO285 polypeptide (including its signal peptide and/or transmembrane-domain and, optionally, intracellular domain, deleted variants), fused to an epitope tag sequence or a Fc region of an immunoglobulin. In a preferred embodiment, the fusion contains the extracellular domain of PR0285 fused to an immunoglobulin constant region, comprising at least the CH2 and CH3 domains.

In another embodiment, the invention provides an antibody which specifically binds to PRO285 polypeptide. Optionally, the antibody is a monoclonal antibody. The invention specifically includes antibodies with dual specificities, bispecific antibodies binding more than one Toll polypeptide.

In yet another embodiment, the invention concerns agonists and antagonists of the native PRO285 polypeptide. In a particular embodiment, the agonist or antagonist is an anti-PR0285.

In a further embodiment, the invention concerns screening assays to identify agonists or antagonists of the native PRO285 polypeptide.

In a still further embodiment, the invention concerns a composition comprising a -4- H:\cintae\Kee\speci\2003203679.doc 17/11/06

IO

O PRO285 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier.

O The invention further concerns a composition comprising an antibody specifically binding a PRO285 polypeptide, in combination with a pharmaceutically acceptable carrier.

The invention also concerns a method of treating septic shock comprising administering to a patient an effective amount of an antagonist of a PRO285 polypeptide. In a specific embodiment, the antagonist is a blocking antibody specifically binding a native PRO285 polypeptide.

\BRIEF DESCRIPTION OF THE DRAWINGS Figure I shows the derived amino acid sequence of a native sequence human Toll 10 protein, designated PRO285 (SEQ ID NO: 1).

Figure 2 shows the nucleotide sequence of a native sequence human Toll protein cDNA NC1 designated DNA40021 (SEQ ID NO: which encodes PRO285.

Figure 3 shows the derived amino acid sequence of a native sequence human Toll protein, designated PRO286 (SEQ ID NO: 3).

Figure 4 shows the nucleotide sequence of a native sequence human Toll protein cDNA designated DNA42663 (SEQ ID NO: which encodes PRO 286.

Figure 5 shows the expression pattern of human Toll receptor 2 (huTLR2) (Rock et al,.

supra). a. Northern analysis of human multiple immune tissues probed with a TLR2 probe. PBL, peripheral blood leukocytes. b. Enriched expression of TLR2 in macrophages, and transcriptional upregulation of TLR2 in response to LPS. Quantitative RT-PCR was used to determined the relative expression levels of TLR2 in PBL, T cells, macrophages and LPS-stimulated macrophages

(MD+LPS).

Figure 6 TLR2 mediates LPS-induced signaling, a. 293 cells stably expressing TLR2 acquire LPS responsiveness. Either a population of stable clones expressing gD.TLR2 (293-TLR2 pop I) or a single clone of cells expressing gD.TLR2 (293-TLR2 clone 1) or control cells (293-MSCV) that were stably transfected with the expression vector alone were transiently transfected with pGL3.ELAM.tk and then stimulated with 1 pg/ml of 055:B5 enhancer for 6 h with or without LBP in serum-free medium. Activation of the ELAM enhancer was measured as described in the Examples.

Results were obtained from two independent experiments. No stimulation was observed using the control reporter plasmid that lacked the ELAM enhancer (data not sown). Expression of the reporter plasmid was equivalent in untreated cells or cells treated with LBP alone (data not shown), b. Western blot showing expression of epitope-tagged TLR2 in 293 cells, c. Time course of TLR2-dependent LPSinduced activation and translocation of NF-KB. Nuclear extracts were prepared from cells treated with 055:B5 LPS (10 pg/ml) and H:\cintae\Jcecp'spcci\2003203679.doc 17/11106 THIS PAGE IS INTENTIONALLY BLANK -6- F1154R2 LAP frithe indateddtmes (top), or ci~pth pet6tjw'ith I iM cyvluhcmdn (CSX) for lb the stimulate wit I Ksg/mLPS for Th in tha presenc ofWBP in menum-free medium (bottom). d.

Kifect of mCDI4 aMP N-uS activation by TI-Rt. Veetor control (l9-100V) or Sfl-1LR2 popI cells were transeoted with the reporter plmi&, and a CD14 expressin vactor (+mCDU4) or vwctor contro (-mCD)14), raspsmtvely. Aftar 24h, culls~e M were r iUated with 128 for Oh in the presence of LBP' in Anrucn-free medium. The data prasented axe representative from three independent experiments.

Figure 7 DomainfunctonocJThR2 idugnalnga iLustratmicn fvarkousTLR2 eon slncu TLR2-WTP the ul-lengh sitcpe -tagge u f TLR2 2 TLRI-AIbd -6.2 reresnt a trucation of 13 or 141 amino acids at the carboxyl terminus, respectively. CD4-TLH2 1 a human 0D4-TIJR2 chimera replacing the erteellular domain of TLR2 with amino acids 1-205 of human 004. ECD, extraceluAir domain; TM1, fransnembrene region; ICD, intracellular domain, KC-terminsl rwidmp cGriticl for I-ItR and TEAS signal traneuceon Residue wtumbers are sh-own to thezrightofnech protein. Arrow indicted the pcritin of the TLUS2-M truncation efdueeasentiml for ilrl 9si nlig(HeuynscJ-M ~n&M26D2609 119923; Crouton et aL, J.Bid.flham. 27T 16514-1L6517 [1995])1 1, identical amino ed; conservative changes. c- TLR-RS variatst fifl to induce NE-KB in respause to LPS and LBP.

2V8 oulls were iranioiny tranafeeted with pOL-3,ELAM tk and expressin vwktos encodingf full- 1.ngth ThLt2 or TLR2 variants mua indicated. The cell were also tmanseaed with a CD14 expreasion plasmid (-ImCDI4) or with a control plasmnid (-mnCI)14 Equal expression of each protein is confirmed by Western blot using either anti-gD or 01)4 iantibody (boftm). The lncikfraee aseay wa. performed as; described in the Examples. Data wer obtained frmm dupliente experiments.

Nupre 9 High potocyof Ecolt K(12 LI'S (LC25) "n its binding to n=.S a.

Done-responise curve of varous LAPS preparatiout. b. Specifc Intractio of [ft -U'S (L.D2 with the extracefular domain of TLB2. Specific binding was obuerwd to ThRI-Pe, but not to either e alone, or fusian proteina ontuizing the extracelnlar domins of Roo, Am[, HerZ, or Hart. Binding to TLR2-Fc was spooioally competed wit L0D246 LP'S, but "o with detoxidfied Miume 9 TLRU is required for the [YB-Induced IL-S eqnnuion. 298-MSCV vectmor ontrol and 292-TLRZ cells transiently eragm'CDl4 wn stimulated with LBI' alone or together with the indicated typo of LI'S at onceutratjons of lji/ml in merw-free medium for 6bh Equal amount4 of pol-(A) [NA. wer, used for Northern analysts.

Figure ID NucleotidesequenceenoodinghuTujRs(SEQ I lO:ii).

N Figure 11 Amino acid sequence of huTLJlS (SEQ EID NO,-i2).

Figure. lIARB ao tbe derived amcino acid aueme of a naive sequence human Toll protein, designated PROM5 (SEQ mD No. 18). In the FgUre, amino adda I though hnn a putali signal sequmenceaino acids 20 through 575 are the putative extruaelular.

domain, with amino acids 2g) through 54 having the charactertics of leucine rink repeals, H:\cintae\Keep\speci0032 03679 doc 17/11/06

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0amino acids 576 through 595 are a putative transmembrane domain, whereas amino acids 596 through 811 form an intracellular domain.

O Figures 13A-B (SEQ ID NO: 14) show the nucleotide sequence of a native sequence Z human Toll protein cDNA designated DNA47361, which encodes the mature, full-length Toll protein, PRO358. As the sequence shown contains some extraneous sequences, the ATG start codon is underlined, and the TAA stop codon is boxed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions In the claims of this application and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or cl variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The terms "PRO285 polypeptide", "PRO286 polypeptide", "PRO285" and "PR0286",when used herein, encompass the native sequence PRO285 and PR0286 Toll proteins and variants (which are further defined herein). The PRO285 and PRO286 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods, or by any combination of these and similar techniques.

A "native sequence PRO285" or "native sequence PRO286" comprises a polypeptide having the same amino acid sequence as PRO285 or PRO286 derived from nature. Such native sequence Toll polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.

The terms "native sequence PRO285" and "native sequence PRO286" specifically encompass naturallyoccurring truncated or secreted forms of the PRO285 and PRO286 polypeptides disclosed herein an extracellular domain sequence), naturally-occurring variant forms alternatively spliced forms) and naturally-occurring allelic variants of the PR0285 and PRO286 polypeptides. In one embodiment of the invention, the native sequence PR0285 is a mature or full-length native sequence PR0285 polypeptide comprising amino acids I to 1049 of Fig. I (SEQ ID NO: while native sequence PRO286 is a mature or full-length native sequence PR0286 polypeptide comprising amino acids I to 1041 of Fig. 3 (SEQ ID NO:3). In a further embodiment, the native sequence PRO285 comprises amino acids 27-1049, or 27- 836 of Fig. 1 (SEQ ID NO: or amino acids 27-1041, or 27-825 of Fig. 3 (SEQ ID NO:3).

The terms "PRO285 variant" and "PRO286 variant" mean an active PRO285 or PRO286 polypeptide as defined below having at least about 80% amino acid sequence identity with PRO285 having the deduced amino acid sequence shown in Fig. I (SEQ ID NO: I) for a full-length native sequence PRO285, or at least about 80% amino acid sequence identity with PR0286 having the deduced amino acid sequence shown in Fig. 3 (SEQ ID NO:3) for a full-length native sequence PR0286. Such variants include, for instance, PRO285 and PRO286 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the sequences of Figs. I and 3 (SEQ ID NO: I and respectively. Ordinarily, a PRO285 or PRO286 variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the amino acid sequence of Fig. I or Fig.

3 (SEQ P1154B2 ]D NK~st-and P~~vdniS Ashow.n high degree of sequence identity with the exfraeellulr domain of a native sequete PROB or PR0286 polypep6da na aspecIal esabodinent the FROM8 and FR0266 variants of the present invention retain at least a Cterminal portiwn of the intracelular domain of the corresponding native praeuins, and amt preferably they retain moat of the Intracellular and the extracellular domains. However.

dlependig on their Intended use, sfuch varits =my have various amino acid alterations, eg., suhetboa, detetions and/or insertio within these regio.

The terms PJIUS6S polyppide, TPR03S aPROSBS Toll homologue~ and grmmatical variants thereoC as used herein, enompass the native seqee PR0358 Toll protein and variants (which are fthffer idefined herein). The PMS polyaeptide may be Isolated from a variety of sourcee euch as from hu1mank tssue types or froze another source, or prepared by rusombiinant or synthetic metkpe, or by any combination of thes and simiar techniquesg.

A 'native sequence PR35W 4 omprises a polypeptide having the ame amino acid sequenema w 0858 derived from nature. Such native sequence Toll polypeptdes can be isolated from nature or can be produced by reoozbinant or sjynthetic mea. The term l"n~tiv sequence PROW5" specifically once mpmssa na~turally-occurrin truncated Or secreted forms of the PROMS8 polypeptide disclosed heei an extracellular domain sequence), naturally.

oenrduig variant forms alternatively spliced forms.) and naturally-occuring aulic vaants, In one embodiment of the invention, the native sequence P80358 is a mature or fulllength nafrve sequence P30358 polypeptide comprising amino acds 20 to 811 of Fig. 12K-B (SEQ M1 NO: 13), with or without the N-terminal sipnl sequence (amnino acidso I to 19), and with or without ths N-tnmicul inethionine In anotber embodiment the native sequence PR0868 ia the soluble fcnn of the fulli-length PROWS, retaining thes extracellular domain of the M-legt protin (amino acdsa 29 to 5Th.), with or widihout the N-trmina~l cfigal .equence, and with ar without tho N-terninal imethionine.- The term "P308S8 variants means an active PR0355 polypeptide ex defined below having at le&a about B0%, preferably at least about 85%, more preferably at learnt about most prenbly at least about 95% amino acid sequence identity with PROMS having the So dedwred amiin acid sequenc shown in N. 12A-E (SEQ ID) Such variate include, for instance P3018 polypep tides wherein onm or more amnin acid tesidues arm added, or dilated, ttheN--or O-tarmIm. Offli thaquenes of Fig. IRA-D (SEQ ID NO:13). Variants .pecifinlly include trsnsmembrane-domain deleted and inactivated variants of native sequence which may also have p&at or whole of their intracellular domain deleted. Prefnted variants gre 235 thos which show a hig degree of sapience ideantity with the extracellular domain of the native sequence PR0358 polypeptidek. In a special emnbodimeint, the PRO M5 variants of the present invention retain at leat 0 -terminal pbrt of the inttracelular domain of a correspndn native proteun. and mabst preferably they retin root of the intracellular and the extraceilniar I I IPl5R2 domains. Horweve, deopending on tkeirintupdod urns uch variant may have various amino aci alterations, eg.. substitutions, deletions andior insertions within thee regios.

T

fteeet am~n acid sequence identiy with rwepw to the PROIS5, PR023 and PROM maquenose identfie herein io defined as the percentage of atoms acid reuiduea in a acndidat. sequence that ame identical with the amino acid residues in the P10285, PR0286, or PROM5 sequence, after uligming the sqecsand iniroducin ga&M if neceasazy, to achieve the mazcimum percent sequence identity, and not considering any conservative sub etitutions a& part of the squence identity. Alignmejt for purpowis of deternmig percent amino acid HeqUence identzity can be achieved in various ways that are withi th, skil in the art, for instance, using publi*l available computer software such as BLAOT, ALIGN or Megalign (DNASTAR) boftware. Those skilled in the eatcan dctermnina aippropriatte parameters for measurng alignment, including any algorithms needed to ac-heve maimal alignmnt ever the full length of the equences being compared. The AIGN software is preferredt to determine amnino acid sequence identity.

a sapecifi aspeat )'percet amino acid sequence idniy with respect to the PROM&5 l'R0266 and PROSS8 sequences identifiedl herein is deftnsl as the pwntge of amino aid residues in a candidate equence that are identical with the atninn acid reslidues in the PiIO28, PRO2M and FROM sequence, after aligning the sequence an~d introdueing gps.

if necessary, to achieve the maximium percent aequance identity, and not considering any conservaltive Substitution as part of the sequence identity. The %6 identiy valuev used herein are generated by WtJ-ILAST-2 which wasg obtained fr-om (Alteciwi et al., MkQUALaIn Ezwznlaag 2M.f: 460-480 (299Q), http:ffblast.wuslkdulblnt/READNMhtm]. WU-BIfAST-2 uses several search parameters, most of which "re set to the default values. The adjustable paramneters are met with the following values: overlap HIVn 1, overlap fraction 0.125, word z d bmhold ME 11. Me [ESP S and 1181 82 parameters we dynamic values and are stablished by the r~oram itself depending upon the cczupoition of the, particlar sequence and oonmetion of the particlar da-tabase againt which the sequence of interest is being searched; however, the voinea may be adjusted to incremasensitivity. A %4 amino acid sequence identity value is determined by thie numr uf malriiing identicl resdues dlivided by thea totAl number of residues Of the "1onger" sequence in the aligned rcgion. The longer" sequence Is the one having the most aotual residues in the aligned region (gaps introduced by WU.-Blaat-.2 to mmaiize the alignimnt cere are ignored).

Th. term "positives', in he contert of sequence comparison perfarmed ms descibe above, includes reidues in the sequenes compared that are not identical Wut have similarproperties as aresfult of conservAtive sbstitutions). The%5 value of positives is determined by the fraction of residues scoing a Positive value in the BLOSUM 62 matrix divided by the total number of rewiduus in the longer sequence, as defined .bov, *Percent 6) nucleic adid sequence idtty with respet to the DNA&=2I, DNA42663an IPNA4T8OI sequence identified herein is defined asi the percentage of d11 11 nucleoddes in a candldatae asimx that are identicl wit the nuiwaota in the DNA4OO2i, DNA42668 and D*JA473O1 eequencs% after 5hgnwg the svquwn and intrduin gap, if nacfnearyp to achieve the maximum percent mequenee ldentity. Alipgna for purposee of determining perent nucleic acdd sequence identity can be achieed in variou ways that are 54 within the skil in the art, for insta&nce, using publicly available computer software sfuch am BLAST, AUON or Megalipi (DNASTAR) .niftwarv. Thcme sfkilld in the mrt can determine appropriate parameters for nmauring ahignmmnt~ including any algorithms needed to achieve tninvl alignment ovor Lbs AUi length of the sequences being eompssw& The AUG14 software is prefered to determine nucleic acid sequence identity.

1n S3pecifically, 'percent nucleic acid sequence identlV with respet to the coding sequence of thePRO2M, PR0286 and FROM polypptides identified herein in defined as the percentage of nuclooidn residues in a candlidate Eeqitene that are identcal wite nuckotid. resiue in it. FROM,8 PRO2S and PR10358 coding sequence. The identity value.

used herein were generated by the BLASTN modul, of WLJ-BLAST-2 net to the default paameters, with overlap span and overlap fraction set to land 0. 125, reepectively, "holated," when used to describe the various polypeptides disclosed herein, means polypeptde that has been identified and speparated and/or recovered from a component of ita natural environmet. Contamnant cmponents of ita oatural environment are materia that would typically interfere with diagnostic or theampeutic usa for the polypeptide, and nml hrlude enzyznes hornmes, and othe ra~wm or non-piuteiziau solutes. 1n preferred embodfiments, the polypeptido wil he purified to a degre sufficient to obtain at lenst residue. of N-terminal or internal amino aci meuence by use -of a spinning cup iequenato, or to homogeneity by 5DS-PAGE under non-reducing or reducing conditions using Co:omauuie bins or, preferabWy, silver stui Isolated polypeptde includes polypeptide in asitu within recombinant cella, since at leasft one component of the PROM~, PR286 or P11085 natural euvwnoomct wil not be present Ordinarily, however, isoated polypptide will be prepared by at least one purifiationa step.

A 9alatid" DNA400ZI DNA4ZGG or DNA47BO1 nucleaic acid moleule s aa nucleic acid molecule that is identified and separated from ait leuat one contaminant nucleic acid S0 molecule with which it is ordinaril associated in the natural source of the DNA4002I, DNA4268B or DNA47861 nirleic cid. An isoated DNXA4OO2i, DNA4 2SS or DNA47SGI nucleic acid molecule is other than in the form or setting in which it is forund in natur. Msoated DNA400I, DNA4288 amd DNA473S1 nucleic avid mauciueg therfore are d~stigUished from the DNA40021, DNA4IEGS or DNA478OI nucle acoid molecule no it ezisfta in natura vlls.

39 lHowover. an isolated DNAEOO2Z, DNA42888 or DNA47381 nucleic acid molecule includes DNA400Sl, DNA42683 and DNA47361 nuclein acd moleule contained ina cello tat orarily express DN4A4002 1. DNAt266 -or DNA4736I where, for ezampip, the nucleic acid molecule is ina chromosomal location different from that of natural cell.

P11I54B2 -TcIl receptar2', hTM r~d are' mind interchangeably, and refer to .a human Toll reptor desigate as "HuTLEI" by Rock A a.L, Proc. Nd. As&- 8d, USA 2L.

58B-59(t9M8. The nualotdide ind minea acid zequen of huTLE2 are shown in Figures (SQID NO: 11) and I1I (SEQ U)D NO: 12), respectively The term vectoron iN Und to dsfinea vsctor in which a nuclei acid encoding a Toll homologue protein herein ig operably linked to control sequences capable of sffectw its exparession is a suitable host ceilt Vector. ordinarily carry a replication uite (although this is not necessary where chromosomal integratio will occur). Rx~simvector.

aiso inilude mnare squencea whic are capable of providing phenotypic selection in transformed Aein. For example, E. ccli is typically transornmed using pBH322, a plamid derived from an A tali spaee (Bolivar. vi ed, Gene 1- 95 [1977]). pBR322 containa genes for anmpiillin and tetracycln resistance and thus provide easy ma for identifyin transformed cella, whether fur parpcesaof cloning or nprsniont. Expresion vectors also optimalfly will contain aequenm which arm us"M for the control of tranocription and trnfationa, proem and Shine-Palgarno sequences (for prokaryotes) or promoters and enhancer. (for mam malian cells). The promoters may be, but need wAn be, Inducible; even powerful CoDnStitUtiVe promoters such aB the CMV promoter fair marnmalinn hosts hove been found to produce the Tilt without host cell toxicity, While it isi conceivable that expression veo~rs needl not contain any expression control, replicatie sequences or selection gees, their absence ay hamper the 2D idendiffit of hybrid trinfonnamt. and the acievement of high level hybrid inugobulin erpr-3,ion.

The term 1 conirl .equences" referse to DNA sequences necesary for the expression of an operably linked coding sequence in a particular host organism. The control sequence that are suitable for prokaryotes, for example, include a promoter, Optionally amn 26 operator sequence, and a rllxnie binding aite Eukuzyoti cells are known to ulili promoters, polyadenylation signals, and enhancers.

Nuclei acid-in holietably linked' when it is placed into a ftinetional relat~onxhi with antothr nuclic acid sequence. For example, DNA for ak presequence or secretory leader ia operably linkd to DNA fo~r a polYPeptde if it Is Oxrmdas a preprotuin that parinipates in thu swsreio of the potpeptkid; a promoter or enhancer is operably linked to a coding sequence if it affects the troeiptioa of the aee; or aL ribosM.e bindin sits Is operably linked to a coding sequence if it is pohtiamhid soaus to faidltate translation. Clennily, 'operabl linke" means that the DNA sEquences being linked are contguo, a~nd, in the case of a saereto leader, contiguu and in reading phase- However, enhancers do not have to be ccmtgi~mus. Unkng issccwnuplishd by lgationat cnvenient retrictio~nsites. Ifsuch Hit..do twt exist the synthetic oligunucleotide adaptors -or linker, ane ued in acoangce with conventionalI Practice.

The tfrm "anibdy is used in the broadest sense and .peefinsily coven inlea m"i-PROW. moti-PROMB and anti-NOSES monoelousl a"tbodies (including .goaiut ante gonint, and neutralizing antibodi &Aid anS-Pfl2t, anti-PROMB and and-fl038 antibody conmoiion, with polymopiopspeiiity. The term %nocloonal antibody au used herein'refers to an antibody obtulned fm a population of subetni6y homogfeneou@ snkibocheu, Le-, 6ha individual iatibodies comprisin the Popukatin ane identica ezoept for pomibi. natually-occurring mutations that may be presen ina minor amounts, The ktrm 'nagonist" is -oe in the broadest sense, and icludes any molecule that partially or fuilly blocks, prevents, inhibits, or naeutralsiz a biological activity ofta native TOi receptor disclosed herein. In a .imnilar manner, the term "agonise is used in the broadest ense and incudes any nolecule that mimicsm, or enhances a biological actvity of a native Toill receptor disclosed herein. Suitable agonist at antagoais molecules specifically include agoist or antagonist andibndie. or antibody fragments, fragments or amino adid sequence varint. of native Toll receptor polypeptides, peptides, small organia molecules, etc, 'Active" or "activity' for the purposes heroin refers to form(s) of PRO2BS, P110286 and PR0358 which retain the biologic and/or immunologic activitie of native or naturally-oomrring P110285, PXO2M6 and PROM8i, respectively. A preferred 'activity- is the ability to induce the activation of NF-rB and/or the exprakin of NF-xlJ-oontoiled genes for the inflammnatory cytokines IL-I, HL-G and tLrS, Mnother preferr~ed "~activity' is the ability to acetiviate an innate and/or adaptive immune response in vertq'brates, A further preferred

T

"ativity is the ability to sense the praenee of tanner-ed molecular structurea Present on microbes, and specifically the ability to meodiate lipopolyssocharide (Li'S) signaling. The ame -Wctvity'. definition applies toe agonists agonist antibodies) ofT PR0OM, P10286 and P30358 polypeptides. Aim noted above, the 4activity an anitagonist (xIluding agqonist antibodies) of a PROM8, P110286 or PROM35 polypeptiae is defined as thie abily to ounteracl, e4;. partially or fulfly block, prevent, inhibit or neutralie a&y of the above-identified activities 26 of.a PROM8, PRO286 or P30S58 polypeptide.

'8tingezcy' of hybridization ractionsis readily dbeen iusby one of ordinary AM~ in the art, and generally is an empirical calculation dependent upon probe length, wsshing temperature, and salt connmaion. In general, longer probes "equire higher temperature. fox proper annealing 1 while shorter probes need lower temperatures. Hybridization generally depend on the ability of denatuued DNA to reenneal when cumplementary srand s ar present in an environent below their mielting temperatue. The highe the degree of desired homolgy between the probe and hybridizable sequence, the higher the relative temperature whichca be used. As a result, it follows that higher relative temp~eraue would tend to make the rotatio aonditin MMO strnn~t whie lower teMperature les ao. For additoal details and explanation of stringency of hybridization reactions, see Auzubal et fk 'Mga1% 4 Strinvent ccndidontr or Idgi, atrimgency conditions", as defined herein, may be identfiemd by thoe that amplo low ioni strenth and high trperattn for washing. for sample G.0151 sodfum ohloridu/0.O15 M1 bsodm Sir~a 1% sodium dodsoy sMt. at ISO- F1154R2 employ durin hybrijation a denatuing agent such as formamide, foyr enmple, (vNv) formamide with 0.1% hoin serum alhumla/O.l% FicoWD.% sudium phosphate buffer at pH 0.5 wit 750 mM sodum ch~oide, 76.14 sodium citrate at 42Q(8) employ 50% fornamids 5 xB 88(0.75 LINaCi, 0.075MhI nin cirate) 1 S 0M sodium phosphate (pH 0.1% sodium pyro~hoephate, 5 x D.Dnhard's solution, sonicated sainmn sperm DNA (50 pg/mI), 51)6, and 10% dextranmzalfte at 42C, with washues at 42t0C in D,2 x SEC (slodium ehkride/mxllum citte) and 50% Lormamide at WC. followed by a highstringency wash coitinge of 0.11 xSAG containing EDTA at 5500.

"Moderately stringet OcndiMon May be identifed. as described by Sambrock ef tjn&a==ffMnul- New Yorle Cold Spring Hlarbor Froms, IM8, and include the use of washing uolution and hybridization onditions temaperatur, Ionic strength and %SDS) lam sftinget that thos. described above. An eample of moderately stringent onditions is overnigh incubation at 37' C ins solution compriuinr 20% for roaside, 6 x NO0 (150 mM NaC, 15 mM trisodiunz citrate), 50 nM sodium ;hosphatn (~pH B x Denhsrdeu solution 1 dexta sulatte, and 20 zng/mL denatured shead almon sperm DNA, followed by washing the filter in I x SSC at about 3 7.SWC. The skilled artisan will recognize horw to adjust the temnperUtre, ionic sftrenth etc. us necumrny to accommodate fra such as probe length and the like.

The tarm "epitopo tagge? when usedi herein refers tM a chimearic polypoptide, comprising a FZZ polypeptide fused toa ag polypetide?' The tag polypeptide has unough residues to provide an epitope against which an antibody can be made, yet in alort enough such that it doe not interfer with activity of! the polypeptide to which it is fused. The tag polypoptide preferably also isa fairly unique so that the antibody dose not substantiall cr-croureact with other epitopeo. Suitable tag polypieptides genaer ave at least six amino acdd residues and usuly between about S and 50 amino acid resiue. (preerably, between about and 20 amino acid residues).

Asa used herein, the term Imumdau designats jantibody iike molecules whigh combine the binding apecifloity of a heteroogous protein (an aadhuain) writh the effector functions of iinmunoglobulln contant -domins. Structurdy, the innwunoadhsui. comprise 301 a fisson of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding uite of an antibody is "heterologous), and the iuimunogkbulin constant domain sequene. Theo adlieufin part of an itammundhmin molecule typiemiy is at ontiguous auino acid sequence comprising at least the binding mte of a receptor or a ig and. The inimunoglobulin constant -dosaii sequence in the inmunaihin may bhe 3.6 obtained fin any icmnuneglobulin, such a. lgU-1, Wg-2, lgG-0. or igGA4 subt)es, IgA (inaluding IgA- I and IgA-2), IgH, laD or 1gM.

*reatment* refera to both therapeutic treatment and pwophylactic or preventative measure, wherein the object is to prevent or slaw down (lens.) the targete pathohei condition or disorder. Those in need of tretment include thou. already with the P1154R2 disorder as wll..s those prone to bavi the &BOTW or thou In whom the disorder is to be prevented.

"Chcnlv amiiaraimrefer to dwitsiof at he agent(s) in a COontiuouu mode as opposed to -an smite mode, so as tW maintain the initial theautic effect (activity) for an extended perod of time.

KMWmCa for purposes of treatment refers to any animal cmlasied aa a .mammal,. Including humans, domestic and farm animals, and zoo, sprs, or pet animnal., much ats dogs cats, cows, horse. shep, pigs, etc. Preferably, the mammal is huiman.

Admin istation 'in combination with" one or more further therapeutic agents imci oes uimUmnu (caz=cment) and consecutive administration in any ord-er.

The term lipopolyzaocharid?' or IYSM is used herein as a synonym of 'endotwdn." UpopolynAcearides 028S) are characteristic components of the outer memnbrane of Gram-negative bacteria, Rschtrichia cMJ. They onsist of.R polynacoharide part anid a fat called lipid A. The polysacearids, whichL varies from one bacteria species to another. is made up of the 0Qaecificchuin (builtfromn repeating units of three to eigtmugars)nad the twopartore LipiudA tirtualy, always includes two glucosatnin sugars modifie by phosphate and a varible number of fatty acids& For firther informaton see, for example, Rictathel =nd Bade, Anient~f I Anrian August 1992, 54-61.

The term ~Septic eboc? is used herein in the broadeut soe, including all definitions disclosed in Bone. Ann. Intern Mod, 114. 882-333 (1991). Spec&ifialy, septic sleek starts with a synteruic response to LUfection, a syndrome called seps-is. When this syndrome results in hypotensia and organ dysfunction, it is called septic sho& Septic shock may be initiate by graxa-poaiti" v. orgaw =d ftmt *s well as eadotondn-containing Gram-negative organisms. Accordiingly, the present defnition ig not limited to 'edouxj shok.

fl Compositioans and Mufthodim of the Inyontinn A. Pull-lenffth P11285- PROSEB and PRO358 Thu present invention provides newly idtibfied and iwla ted nucleotid sequene encoding pcbTwtdes refered to in the present apphocatn as PROU5 awl PRO286 In partulsri Applmicat have identified And isolated eDNAn encoding ?flO2S and PR10286 polypeptidee, am disclosed in fur-ther detai in tie Example. below. UEWn BLAST a"d ft" mequence alignment omputer program%, Applicants found that the oding mquee of FROMS and P10286 ore blgbly homolorus to DNA wjuewaes H5U8854_1 RBfuS8S78 1 HBUBOOYSJI, HSUSBSBOJI, and BSUOSBBLJ in the Genflank datatbase, The present invention futher provide newly identified and imola ucl*,tId uequenone encoding a polypeptide referred to in the present application ta PROM5. Ib particular, Applicant. have identified and isolated cl)hA encoding a novel human Tolli P*eptde (PWJS8). a r&sclwed in furthe detail in the Examples below. Uing BLAST and PatA sequence aligment computer progrms, Applicats found that the codin ainquone of PROBS ahow. Mgnfikat honmlog to DNA .eqfeias H816840h 8U8.1 HBUESBS7O PII&43 HEBSSSO, HBM1%3 and H'd2d i is the (len~ank databa With the exctoti of =809260_1, the noted proteins have been identfie husn toll-like reoptors Accordingly, it is presently believed that the POM85 PRO286 and P110358 proteins disclosed in the present appliation ars uiwly Identifed byumua homologize. of the Dnamphi~a protein Toll, and are likely to play an important role in adaptive inmmunity. Mbo specaly, P1102K5 PR10286 and PBO#58 Wday be involved in infloam matiena septic .hock, and response to pathogens, and play possile roles in diverse medica conditions that are aggravated by immune response, such as. for sampe diabetea, AJ4, cancer, rheumatoid arthritis, Ra ulcers The role of P3O05, PROS8S eond PR1385. KSpathome patter recognition remceptor, sensing the presenim of conserved molecular structures present on aicrobea, is fwtber supported by the data dislosd in the present application, shmming that a hnawn human Telllike receptor, TL 12 is a direct medator of LBS sinaing.

11 PRM 285. FE026 land PROMS8Vaiat In addition to the kUtl-length native sequence PR0OM, PR0288 andJ f0358 described herein 1 it is contemplated that variants of thes aemnm can be pwepared, M0O266, P30166 and PR10358 variants can he prepared by introducing appropriate nucleotide ehmane into the PR0286. PRO06 or P11035 DA, or by synthesis of the desired variat polypeptida Those skilled in the art wHi appreciate that amino acid changes may alter past-translational prwoesses of the PRO2US, P30288 or PR10858 polypeptides 1 suchL as changig the number or position of glycosylation sites; or altering the membrane anchoring characterisic.

Variation in the native fuldength sequence PROM8, PR0286 or PROM35, or in various domainis of the PROM8, PR0286, or P10358 descrcibed herein, can be ma-de, for example, usting any of the techniques aind guidelines for conservative and non-conservative mnutaitioset forth. for instance. in U1.S. Patent No. 5,364,0M. Variations may bea a 26 .abstition, deletion or insertin of ono or more codons enoding the P110285, PR0nG, or polypeptide that results in a chankge in the amio aci sequence as compared with the orresponding native sequence polype tdes. Optionally the variation is by substitution of at lea one amino acid with any other amino add in one or more of the domains of the PROM28, P302W6 or P11358. Gudance in determining which amino acid residue may be inserted, substituted or deleted without adversely affectig the dasied activty may be found by comparing the sequence of the PROMS, P3028G -or PRO358 with that of homologrous known protein moljecules and minirirm theonuber of amino acid sequence changes made in regions af 14lh haning. Amiro acid suutious can be the reault of replacng one amino acid with another amino acid having similar stnacurl andfar chemical properties, such as the Mt Toplacement *f a leuctue with a swrine, conservative ani acid replacements. kwsrtions or deletions may optinailly be In the rngep oI to 5 amino adds. The variation allowed mqay be detmidned by syfftemrna y maldug insertio, deletios or aubstiutiona of amin mais in the sequence and testing the Ireulting variate for activity in the in v4tro amy descibed in the Examples below.

P1154112 The variations cam be made using methods known in the art such us ollgonud*Moide-medited (sko-dfeW) MtagM~Nu eA10](320 anning, mud FOCR MUtageneuia Site-directed rnutageneuia ICarter at al., Nc diRe,fl$S31 (1988) Zollerc a hIML AddLHjA±407 (1987), cassete muwka (wels at ULh am% M;3l5 (I988), restriction meletlo mutaenesis fWell. et aL1 Thiaa Trans. R Sm~. LodonjCC SaA f415 (19M8] ac ther known techniques can be performd on the clone DNA to produce the PR10286 or PROUaO variant DNA, Scanning amino acid analysis can sac be employed to idoni1f' owe or moren amino acid.. along a contiguous sequee Among the preferred scanning amino atc6 mr relatively sma neutral amino acids. Such amino acids include alsne, glycine, seine, and cyateine. Alanino is typicaly a preferred scanning amino acid amiong this group because it eliminate. the udeachain beyond the bota-carbo and is less likey to alter th, main-chain onformatin of the variant Alanine is &la typicall preferred because it is the most common amino aid. VurthEr, it is frmaquntly found in both buried and empoged positons (Creighton flh hsmtint (W 1 11. Freeman Co., Chothia. IL)".LBoL. lfll (1976)]. If mianine substitution doe. not yield adequte amonts of varjant, an isoteric amino aoid can be used.

Vaiants of the PRD2OE, flW286 and M868S Toll protin &islosed herein include protein in which the flransmembrane doainsr have been delted or iactivated.

Tmnmsmbrane regons are highly hydrophobic or lipophilic domains that are the proper airs to span the lipid bilaye of the ceilndir membrane. They are believed to anchor the native, imatuie PROMS, PROMO and PROMO polypeptides in the cell mem2brat in PIR028:5 the tranamembrane domain skeatches train abut anniig acid poffition 840 to about awino acid position 864. In P110286 the transmembrane domain is between about amnino acid position M16 aZ nd about amino add poitn 840. n PRO M8 the tranamembrane domain is between about 2 amino aid puition 578 and amino aci position 59&.

Deeton or substitution of the tranamaembrane doma will facilitte recovey and provide a soluble -fornm of a PR0OM, PROM,88 and P110358 polypeptle by reducing its cellular or membrane lipid affinity and improving ita water aluhility. Ifthe trenamembram and cytoplasmic domain. are deleted onie avoids the introduction of potentially immunogenic epitopes, either by expomur of othierwjse intracellular polpeptdmu that might be recognized by the body as foin or by insertion of heterolongous polypeptide that am potentially immun rogenkc A principal advantag-e of a tranmmbxlan, domain deletc P11285 P10286 or PR0358 is that it is secrete into the culture medium of recombinant hosts. This vkriant is soluble in body fluids sfuch as blood and does not hayse an appreciable affinity fir cel inembame lipxi, thus onsiderably simplifying its recovery from reombinant cell culture.

It will be amply apparent fr-om the foregoin discuion that substtions, doletiona, insertions or any combination thereof are introduced to axnive at a final onstuct As a general proposition, Soluble variata wil not have a functinal benmmarne domnain a" prekmbly will =ot bav& a fanctonal cytopis sfqiioS. This is generall aoaoinplished by deletion of te relevant domain. athough adqtte inertional or substitutional vnints also are effectin for this purpose For -example, the tranamombrane -do-mink is ubstitufted by any amino acid sequmene e.g. a random or srdU ie equoeme of aboul 5 to 50 swime.

threonine, lyufime, argnn~re glutamine, aspartie acid and like hydropbilic redueo. whichL altovaier cihibit a hydrophiic hydropathy profiko ike the ddeatona (truncated) PROM28, P11286 and PHOS58 variants, these variant are secreted into the cultue medium or recombinant hoe to.

Further deletinul variants of the fuUIolngth mature P1102815, PROM88 and PBOSO p&lypptidim (or lrarnambhrsn domwau deetd to inactivated forms thereof) include variants from which the N-terminal wipa) pptid (?utivsly identifed a" amino acids I to 19 for P110285 and P110286. and as amin acids 1 to 26 for P11368) andfor the initiating methdonine has been deed The native signal sequence may also be substituted by another @sterolobous) signal peptide 1 which may be thatof anottr Toll-lIkprctein, or anuther humant or non-human (e.g.

7 backdeiaL~ yest or non-human mamnmalian mignal HequeDoe, It is believed that the intracllla domain, and especally its C-ter minal portion, is impotant for the biological function of these polypeptides. Accordingly, iflthe objretive is to make variants which retain the biological activil of a coresponding native Toll-like protein.

at least a substantial portion of these regions is retain, or the alterations, if any, involve conservative amino acid substitutionB and/or insertions or amino acids which are similar in character to these preient in the region where the amino acid is inserted. If, however, a substantial modification of ft biological function ofa native Toll receptor in required the objective is to prepare antagoniste of the resportive native Toll pojypeptides), the alterations involve the substitution and/or insertion of amino acids, wrhich diffior in character from the amn= acid at the targeted position. in the correeponing native Toll polypeptide.

Naturllyoccuning amin vads mr divid-ed fig* aroupe based on common Side chain properties: hydrophobic: norlucine, met, ala, vs] 1 lou, ii.; nutra hydrophobic; Mye ser. thr; acidic: asp, gin; hAusc, a0s hn his lIe Or residues that influence chain orientation! gly, pro: And aromatic: trp, tyrr, phe.

Conservativea aubslit" involve exchanging a mmber within one group for another member within the same group, wherea nenarvative substitutions v#11 entail exchning a member of one of thme. elans fort anther. Variant. obtained by non-ccuuerntive siubstitutions are expected to rerult i nx moe siificant changes in the biological properties/function of the obtained variant Amino acd inserton iMude amaino- aor crarbowyl-terml fusions ranging in length from one residue to pobypaplids coontaining a hundred or more reuldtee. as well au dP1154112 infrauequrnre insrton of aingle or muhltl amin acid residues Eutaeeueace bwrion (Lc.

inserion within die FROMS, PRO28O or PROB58 protein amino aci mequene) inmy range generally from about I to 10 resi. more preferably I to 5 residues more preferably I to 3 reiius. lj'umplj of twrrinitl insrwwa inede~ the PR025P MR28G swu1 PROME I polypeptideg with an N-seininal nmehionyl tei&e, ant Artifact of "t direct expresion mn bacterial recombinant cell culture and ftision of a heterologous N-terminal signal sequence to the N-trminus of the PRO2 85, PR0213 or PEL0358 moleomila to failita the secreton of the mature 1-ThAF protein from recombinant hoot cells Such signal sequences will generally be obtained from, and thus homologous to, the intended hoot cell species. Suitable seuences inolude EliI or Ipp for Lunj, alpha factor for yeast,, and vira 1 sijsls such as herpes gfl for mammalian cells.

Oter insertional variants of the native ToEL-like molecules disclosed herein include the hasfion of the N- or C-terinu of the native sequence molecule to immwnogenic palypeptides, e-g. bacterial polypeptideip auah e beta-Jacbman or an enzyme encoded by the L. 1i trp locus, or ye&at protein, amd C-terminal fusions with proteins harinag a long half-lWe ouch aa hnmnunoglcbulin regions (preferably immununoglobthn constant regions to yield immunoadheuins), albumin, or fenitin. as. described in WO 89/02922 published on 6 April 19w, For the- production of inionmoglobtin fusiona see also US Patent No- MZSJOO issfued June 27, Since it is often difficult; to vredit ini advante the characteristics of.a variant TOM-like protein, it will he appreciated that msening will be neede&d toD select the optimu varint. For this puarpose biochemical or other screening assays, such as thons dentied hereinbelow, will be readily available.

Covalent modifications of the MONS28, PHO2M6 and mRomS human Toil homolague. are included within the scope of this inventon- One type of wrmlent mnodifiaton irrAudm reatng targeted amino acid resfidue of the PROM8, PR0286 or PRO35S protein with an organic. deivaising agent that is apable of reacting with selected side chains or the N. or C. terninsi residlues Perivatization with bifunctional ats is useul for instance, for croalinking PR0285, PR02M, or PROS 58 to a water-inwohible support matix or surface for use in the method for purifying snt-PRO2SS -PR0286, or -PROMB antibodies, and vice-vers.

Commonly used crosslinkiug agents include 7 I, .hus(dl~anetyl).2.phenylstoe.

glutaraldehyde, N-hydrozyuucnuinimide eatce, for example, eafter with 4.arkbIdoaiylic acid, homoifuctionai imidoedters, including disuccininuidyl csters much as 3,W-dlthoebsg- (sueeinimldylpropint4) biuctioAl maleimides such as bim-N-mAteinmid-l,&-octane and agents such a methyl- 8-Ip-azuihenydiolprop~midate.

Other modifications include ctmkdanaghitmminyl and apeaginyl residlues to the correponding glumy and aspartyl residues, respectvely, hyroxylation of pralne and lyuine, phosphori-ton of bydrazyl groups of uuryl or threonl resdues, metbylation of theit- P1 154R2 amin grops of tmine, arffanne and histdin ohain Creighton. M= Sre J~ reeman Co., Ban Frarisoc, pp. 79-86 (19N3)], acetlsion of the N-terminal amine, and amidatloa of any C-terina carbozyl group.

Deriva~ztien with bifunctcna agents is nso"u for preparin intramolecuimr aggregats df the Tall-lIko receptor herein with pokyreptideu as wel as for eroe-likl the..

polypeptides to a water insoluble suyxort matrix ior surface for use in anya or affintity purifiation. In addition, a study of interchin cross-links will providef direct information on odormatiorwi ukuctbn. Commonly used cross-linkin agent. include 1J2-bis(dinoaetyl)-2.

0mudmie, lutradeldeN-hdestaera, homab ituctoal Lntidowetrs and bifianal maleimide.. Derivatizing agents much GO .methyl4.IUpazddophenyldithiojpropioimidate yield phutuactivutable intermediaxte, which are capa~ble of forming cross-lik in the presence of light Alternatively, raeactv water insoluble matrices such as cyanagen brwmide activated carbohydrate.g and the system reactive substrates dscribe in -US. Patent Nos, 3,9S9,f42; 8,9%9,287; Z,691,016; 4.1M.126: 4,24-4,42; 4,229.537; 4,055,636; and 4,380,440 are eimployed for protein immobilizatkn and crosa-linking.

Another type of covalent modification of the PROMBS, PR0flS and PROMS 1polypeptides included within the scope of this invention comprises satring the native glyoceylatien pattern of the polypoptide. 'Altering the native glyccaylatcn pauter, La intended for purpomes herein to mean deleting one or more carbohydrate moieties found in native sequstwe (eiher by reninrg the underlying glycosylation site or by deieting the glycosylation by chemical and/or enzymatic means) andlor ading one or more glyosylation mi%9 that are not p~resent in thea native sequence. In addition, the plinase includes qualitative changes. in the glyocylation of the native prate!=s, involving it change in the nature and proportions of the carbohydrates present.

The nativ, ful-ngtk PRO2O5 (wroded by DNA 40021) ha. potential Ntlnkod glycoslation sites at the following amino acid posfitaon: 689, 16'7p 202, 215. 981 o41%, 469p 523t, 53,590, 679, 720, 709 and 942. The native, fullIeugth PR088 (encoded by t)NA42668) ha potentia N-linked glyccaylaton sites at ths folflowing amino add positkon: 29, 42, go, W8 115, 150, 247, 2MS 293, 3W8, 182, 895, 416. 443, 811, 545. 582, 59W, W4, 680, 752. 937 and 10)26.

Addition of glycosylation sits to the PRO2Sa± Pit2ZO and PR0358 polpaddes may he accocpliahed by alterng the satn aci sequence. The alteration may be made, for sample, by the addition of, or substitution by, one or MOre norms or threonins rmsduu. to the naztive sequence (for Clinked glyconylation sites). The amino acid sequenc mhay optiunally be altered through change, at the DNA level, particularly by mnutating the DNA, enoding the FR0285, PRO28613 and PRWa58 polypptides at preselected base, much that cation. are penmratad that wrill translate into the desired amino acids.

Anothe means of inceasing the number of carbohydrate moietie on the FROMl8 f0285 and PRO 358 polypeptidee is by chemicual or =nzmatic couplin of glycoeids P1 154E2 to the palypepti&e Such methods ire a~tlb. 1h the art, in WOf 87M0330 published 11 Seteamber 1987, and in Aplin and Wriatog. PP. 2S-69- (1S81).

Removal. of carbohydrat zuoetic. present on the PROtS5, PROSBO and PRO08B polyepidu. may be accomplished chawaly or enzyatiolly or by mutational sufbtitutiOn-of C01de. enooding for amnino acid residues that serve LiR target for glyosgyiion.

Chemnical deglycosylation techniqlues sere known int the art and descrbed, for inance, by Hdkmudin e4ta]., A. Thrt Noathye fltS (1987) and by Edge at al., W iaim IMJ.*131 (1981). Enzymatclavage of carbohydrate inaekieaon polypeptides canbe achieved by the use of a variety of end&- and exo-glycouidasoe as described by Thotalwra at al., lfsh.

EnzmolLW-2350 (1987).

Another type of covalent modification comprises linki'ng te P11285, PRO28S and PROS5B polypeptidus to one of a variety of' nonprotetnceouu polymer., eg. .polythylee gbmy:a1 (PRO), polypropylene glycl, or pclynxalkylena in the imnnr aet forth in US. Patent NtA. 4,640h831. 4.496,68W 4,801,144, 4,670,417; 4,791,192 or 4,170,837.

The PROD2SS, PXO0BO and PROS58 polypeptde of the& preent invention mafy also be modifed in a way to forma a chinieric molecule comprising PR0285. PR02SS P10358, or a fragment thereof, fused to another, beterologou. polypeptide or amino acid e~juence In one embodiment, such a chimeric molecule comprises a fusion of the PRO2&, PR10286 or PROMS polypeptide with a tag polypeptide whic provides an epitops to which an enti-tag .atibody can selectively bind. The epitope tag in generally placed at the wino- or carboxylterminus of a native or variant P110285, P110286, or PRO(358 molecule. The presence of such epitope-tagged forx= can be detected using an antibody against the tag polypeptide. Ali*, provisin of the epitope tag eniabes the Ml0286, PR0286, or flOBw8 poLypeptides toube readily purified by iffirity purification usig an anti-tag antbo~dy or anote type of affinity matrix that binds to the epitope tag.

Varous tag pobtpepids and their respective antibodie are well known in the art. Emples include pc*y-bitii (Poly-his) or py-hixtizlie-glycie (poly-hi"Igy tagu, the flu HA tag polypeple ad it. antibody 12M5A [field et. aL, MoQa g 22W92165 (1988); the E!-skyt tag and thie W79, 307, MRO, 04, B?7 and 9110 antibodiu. thereto [Evan et al., bflaaazd RilnlUIihg fr86M10.5 (1980)3; and dhe Herpes Simplex virus gVpoproein D (SD) tag and its antibody [Paloreky ot al., 5()8758(1990)J. Othor tUS polypoptiea inclad* te flg-peptde [Hopp at a1. Maehemf: 1204. 1210 (IBM8]; the KTS epitope poptWi [Martinet -dw 3M: 192-1 94 uEm a-tubhn opiope peptid [Sinner at d1., J jgj, CeRL 2il456- 15106 (1991)]; WA thre 77 genm 10 protein peptids tag [Ltz.Freyermutb at al., Proc. NAtL Acad. ki. USd A 1:6393-6897 (19M)].

In.a further embodiment, the chfimrnei molele way comprise a fusion of the PROM8, PRO 286 or PRO3S8 polypeptides, Dr fragments thereo( with an immunoglobulin -or a pwrticuar reion of an immunoglobulin. Far a bivalmnt form of the otieric molecule, such a fusion could b6 to the Pu regin of an 4, such as, IgGl molecule. The Ig fusgions preferably P1154112 include the sukitilo of at oluble (-rnmembx-ane domain dauletwior inativated) form ofh PROM86 w PRO358 polyp eiIn ph=c of at leat mwe variable regio within an Ig motecule, For the production of immwnoglabnlln fion, see also US Patent No. SM110 isumi June 27. 1ISOS .EOIU fPOZ5 R28 n a8UM W The descritio beow reates primAy to product=o of PROM8. P11O2B a1d Toll beoiclcp by culturin cel transformed or trnxsbetd wMit vector containing intaeic Pad encding thes Vroein DNA4002I, flNA42608 and DNA47H81, recmdvl).

Idais of Ourae, contemplated that alternaive methods, which are well Loon in the r, may be employed to prepare PROISS5, PRO2dM, PROM5, or their variants, For insitande, the PRO2085, Pf0286 or PR0358 sequence 1 or portion thereof may be produced by direct peWtid syntheslis using sold-phase techique. [aee, eg., Stwart et al.gSbi-EhugaJnp~efMaLsia Win. Freemaan Co,. San Francisco, CA (196M; Merrifield, J. Am. 4 lQM. fif:2t49.21a4 (1983)J. In uiUroproteinasythesis maybe perfrmed usiig msuaI techniques or byautomation Automawted synthesis may be accmplished, for instance, usfing an Applied Bijosystema Pepiide Synthesizer (Poster City, CA) using mAnufacturer's instructions. Var~iotu paorof the PR0266, or P11085 may be chemkafly synthnirsd separately and conbined using chemical or enzymatic mnethods to produce the ful-lepgth PROM,5 P30286, or PROSSR, 1, luolation of DNAEncdingPR0285. P110288. or PROfl$ DNA enicding PR0285, PR~fO, or PR0358 may be obtained from a cl)NA libay prepared from tissujo belived to possess the P20285, PR0206. or PROS5R mRNA and to exprm it at at datectable level. Accordingly, human P30255, P02K6 or PRO 358 DNA mau be convvriienily obtaine from 5 EDNA library Prepared from hbuma ntiesue, such go described.

ina the Examples. The underlying gene may also be obtained fr-om a genoic, libraTjry or by oligmonuluide mynthsiR. In addition to the libraries dedorihed in the Examples, DNA eod~ing the human Toll proteis of the present invention can be isolatal, for example, from spleencell, or peripheral bWd leukocytes (P31.), Libraries can be screened with probe (suh a" antibodis to thes -pR285, P20286, or PROM5 protein or oligonueletidas of at least about 20-80 bun.) desipied to identify the gene of interet or the protein encoded by it. seening the cDNA or genmic libr with the se6lctd Probe may be coducte usig standard proedures, mcI as described in Samibrook et M~ajr QpI"_ gLiagI= Ifanmg (New York: Cold Sping Harbor Laboratry Pros, 1989. An alternative means to isolate the gene encoding Pk02u, flRt8K or P30358 is to use FOR ietbodology (ambrook et mpmu Dieffenbach et al., M Fim AabrtrManil(COld Spring Harbor laboratory Press, 1995)1.

The Examples below demcrbe tachniue for screenig a eDNA library. The 9lignuclotide sequences seected as probes should be of sufficient lengthi and .uffj~mnu fl~fb4UOUS that false positives are -"dmized The oiwnet is preferably labele such that it can be detected upon hybridizatio to DNA in the librimry being screened. Method of P1154R2 labeling are well known in the art. end include the use of radiolabu. like 32P-labeled ATP, bitinylation or essyms laheling. IjYbrldizato condition., includingf moderate stringency and higb stringency, are provided in Sambrook et An.

Sequences identfied i= such library screaing methiods can be compared and aligned to other bmwn nquee deposdW and available in public databaises such an Genhek or oier pirivate sequenc databases. Sequenc identt (wt either the amino acid or nucleotde level) within defined regions of the moecwule or across the fulfl-length sencae can be determined through oequence aligunent using computer software program smuch a. ALIGN, DNAatar, and INHERIT which employ various algorithua to measure homologylsequanCe identity7.

Nucleic acid having praotcoding bequence may be obtained by screening selected eDNA or genomnic libraries using thie deduce-d amin acid sequee disclosed hemen for the first time, and, if zaamuary, using conventional primer extension procedure a. described in Sambmuok et ul., ama to detest prenusor and processing intermediates of mRNA that may 16 not have lbeen reveneewtranucribed into eDNA.

2. Seletion and Transfomation of Bat Cells Hnst cells are transfeetad or transfomed with expression. or cloning vecto described herein for the production of the human Toll proteinii and cultured in conventional nutrient media modiied as appropriate for inducing promoters. selecting tranaformants, or amnplifyink the genes snroding the desired soqueneu. The culture conditions, sUet as media.

temperatuire, pH &nd the like, can be selecte by the sllead artisan without undue ezperimentation. In generial principles, praoaiZ and practical techniques far mnximizing the productivity of eel] cultures can be found in Mammalia (Jell Biotshnclpgy:.- a IFranticae Anuriach. M. Butler, ed. CMR Press, 1991) and Sambrook et at, sun Miethods of trsndeutvn ae known to the ordinarily silled artisan 1 for example, C&P0 4 and elcrprto.Depending an the host cell used4 t ormalion is performed using standard techniques appropriate to sfuch tells. The coalcum treatment employing calcium chloride, as descrbed in Sambrook et al.. wpn., or electroporation is generally used for prokaryota. or other cell. that contain sfubstantial cell-wall banrers. Infectin 'with SO A jra hariurn tumqlfncienr is ibad for bpxw~forrnation of certain plant cefla. aa described by Shaw et al., U&a,21:815 (1988) and WO 89/0589 rublished 2 9 June 1989, For maimuahan cells without such cell walls, the calciu phoerhate precipitation method of Graham and van der Fa. =~lgL~4W6457 (1975) can be employed. General aspects of mammalimn cell beet sysftem transformations have been deawrihed in UA9. Patent No, 4,399,21U, Transfortions into yeat are typicaly carie out acorwding to the methiod of Van Solingen et al., J Bad IaQ:946 (lfl7 and fliio et al., Ptor. Natt. Ag4. ad. (USA\, 16,382!9 (1979k- However, Other ntlods for introducing DNA into cells such as by nuclear icroinjction, slectporaliou, bacteril protoplast lusion with intact calls, or polycmtjon, polybrene, pot onidiinp, may also be -28- P116jR2 Used.. For various tecnquas forf trani~uoing =tlhmslian cell, wee Keown at al. Mulbiudin ,LW2m M:527-537 (1I9MO and Idansour et aL, tiskia fl134-3 (IM8:.

Sutable host cell for cloning or wxpreuing the WNA in the vector herei inclde prokaryotm, yeast or hIgher eukarycte cal.. Suitable prokaryoto include but are not limited to erubacteria, such as Gram-negtive or Gram-poaitive organisms, for eam pl, EnnvaLra a e much as K oh. Vaxiohs E, w4l strains are publicly available, such R. Cali K1 2 strain lMt94 (ATOC 31,446); B. celi Xf776 (ATOC 3t,5a7); wUh stain WS 11o (ATC 27,325) and K5 772 (ATOC 53,635).

in addition to proazyotes. eu ati microbe. such As filamentous fungi or ywet are sfuitable cloning or exprewim on ets for human Toil-encoding vectors. Sacharomycn eeerae ns a commonly used lower eukuryotic bout tnicrocrgunizm.

Suitable host cells for the expression of gtycosylate human Toil proteins are dsiye fr-OM multioular orgaisma. Examplee of invertebrate cell. include insect cells such an Droophila 82 and Spodoptera Sf. as wrell as plant cell.. Examplesa of useful mammain bost call linea include Chinese hamster ovary (CHO0) and Cos cells. More specifc examples include monkey kidney MVI line trantormed by 8V40 (005-7, ATOC CRL 1651k; huwaum embryonic kidney line (20l or 293 cell sitbdoned for growth in suspension culture, Graham et at, JJ, aVenL D6lK59 (19M);h Chinese hamser ovary cefls/-DHFR (CH0, Urlaub and Chasmr, Proc. Nail. MAd, Sd. USA, fl4210 (1960)); mouse .ertoli cellsa (fl4, Matber. Dial R., 2&9243-251 (1980)): human lung calls (WI 38, AICO M4T 75); human liver cello (ap G2, ItS 8065); and mouse mammar tumor (1047 060562, ATCO COLE The selection of the appropriate host cell is deemed to be within the skil in the A.

3. Selction and Use of at Repiabla Wetn The nucleic acid oDNA or genomic DNA) encoding PRO 285, FFLO28 or PR0358 may be inserted into a repliable vector for cloning (amplification of the DNA) or for expea~m.Variou vectm orae publicly available. The vector may, for be in the form of ja plaamid cosmid, viral particle, or phage. The appropriate nucleic acid Muenee- may be inserted into the vector by a variety of pmedurea. Ina general, DNA im inserted into an aOpriate6 restriction endonucluase site(S) using techniques known in the art. Vector components generally include, but are wt. limited to,, mne or more of a- signa beqnetie am origin oftrplcation one or more marker Mmnes, an enhancer element, a prmmoter and a traumnapion termination sNequePe Construction of sultalo vectors containing wne or mnore of these components employs standard ligation techniques which are ktton to the skilled jartisan, The PR0285, PRO2M6 and MOMS5 protein. may be produced recombinantly 36 mt. only diretly, but also ms a fusion pelypeptide with a lieteralogous polypeptide, which May be a signal sequence or other polypeptid having a upecifie cleavage site at the N-terminus pf the maure Prutai or polypeptde. In general 1 the signal uequence may be a component of tha vector, or it masy be it part of the PRO=,5 PR02-90 orr PROWlS D-NA that ii inserted IntO the vector. The signal m&euenmay be a proisryotic sinel sequence meleected, for example, from -24.

P114R the group of the alkaine pho~hatae% peniillinaze, lpp, or heat-.table Eafteti ff H leadn.

For yeast wSeceto tUM pal sequence rway be, eat. the yeast invert... leader, alpha facor leader (Including &zcharomyc and Kaweromyc as-fator= lea ders, the latter demribed in U.S. Patent No. 5,010.182, or acid phomphatase leader. the U afldoon glucamylaee leader (EP 382,1 79 publihed 4 April IggO), r the dina deribed in WI) 90i16 published IS November L990. In marmalian cell expression dsin~nalian signal sequences may be used to direct secretion of the protein, such as signal sqac from secreted pbypptidea of the same or related specie. as nei as viral secretory leaders Both .qwm nd eloning vecrs ontainsa nueei acid sequence tht enables thea vector to repIkate in one or more selected host ce. Such sequences are well ICIwwD for a variety of bwaria, yeast, and viruses. The origin or replication from the plAsmid pIBRS2. is suitable for most Gram-negative bateia, the 2p plaumnld origin is sfuitable for yeast. and vaious viral origins (SV4O, polyoma, admncviruu, WYV or BPV) are modali for cloning vector in maImmalian cells.

1s Expresio and cloing vectors will typicAlly contain a s~election gene, also termed a selectable marker. Typical selection genes encode proteins that (a coulfer resistance to antibiotic. or other toxins, ampiilin, neotnycin, ruethotexcate, or tetracycline, (b) compykwanet auxotrophic deficiencies, or supply critiWa nutrients not available from complex imedish ag., the gone encoding D-anwine racemae for Bacii.

An example of suitable electable markers for zmammialian cells ars those that enable the identifiaton of cells competet to take up the PROMS, FROMS, or PROSIS nucleic acid, such as DHFE Dr thyinidine kinase. An appropriate boat cell when wild-tye DHFR is smtpkysd Le the OH() cel line deficnt in DHFR activity, prepared and propagated.to described by Urraub et al., Proe..NatI. &Ad. SQL USA, U,42l16 {190). A suitable selection se~o for um in yeat is the brs1 gone present in the yeast plasmid YRp7 [Stinchcomb et al., Nfaur =-89D (1979); ingifman at al..8hisl:141 (19M9); Twhlemper etn) aLSMnID:157 (1080]L The trpl gene provide. a selection maarker for a mutant strain of yeasft lacking the ability to grow in tryptophan, for example, ATCC No. 440F76 or PEN-I. Jones, &512 (197T12 Expression and cloning vector usually contain a promoter opeably linked to 00 the nucleic aci sequence ecoding the PRO2SB, PR0238 or PROBBO proten to direct mIINA synthesis Promoters recognize by.a varity of IateTtM1 hot cells are well known. Promoter suitable for use with prnksryotic hosts include the P.Lanttnae mad lactose promoter sysitems (Chang at al., liAgs M.6815 (197); Gonddel et al., NaluJ =Jd644 (1979)), allkwin Rhosphate.., a Utyptohan (tip) prmoter system (GoddelkAd R.1:4057 (1980); EP K6776], and hybrid promoters much a. the tea promoater [deBoer a.t o. M Had. Atari. Rni, 118± W.21-25 (198a. Promoters for us in bacterial systems mis will contain a Shine- Ulgarno eqnenca operably linked to the DNA encoding PRl0285, PROM.80 or Examples of sutable promoting sequecee for use with yeast hosts inuluule the prootmforS-pospbglcerto kinase itmman et al., J.a~h Bi, Uo t 2078 (190) or *P1154R2 odm 0ykr&MM~s FM e IAftdMkjC 1!149)(1968)Hollm4~c 11~4M0 (1078)], gach ao enoan., glyceroldehyde-3.phosphat. dehydrogeme. hezokinns, pyruvat. decarbozylase, pbskfutknsghce--heht om erase, 8phoaplioslycerate antitame, pyruvate kin...

7 triosephosphmte msom erse, phosphoglucome 18 omerase, and glucokinast, Other yeast promoters, which are inducible promoters htaving the additional advantag of tranwnpto controled by growth onditions, are the promoter region for alcohol dehyrogeaae2, lsocytochrcine C. acid phogphataus. hgradativ. enzymes aniociated with nibrngsn metabolism, mcatellathmicnin, glyoenlehyde-3-phoephate deywae* and erymes responsible for maltose and gulactous utilization. Suitable vectors and promoters for use in yeamt expresala km further described in BP 73,657.

P110285, P110286 or PROM 86 ranscripton. from vectors in mammalian host cells ia controlled, for exampla, by promoter obtained from the gnomes of viruses auU ae polyoma virus, fowipox virus (LYK 2,211,5G04 published 5 July 109M, adenovirua (such s Adenovirus bovine papilloma virus, avian onrcma virus 7 cytonxealvirus, a rutrvirue, hepatitia-B virus ad Simian Virus 40{5SV4a), from hetenLous mamnmalian promoter., e., the actin promoter or an imnnnmoglobulln promoter, HIXI from heat-shock promoters, provided such promotere are compatible with the host cell systems.

Trasciption of a DNA encodinkg the P110285, P110288, or FROM35 polypeptide by higher eukaryotes may be increaed by inserting an enhance sequence into the vector.

Enhancers are -cis-acting elements of DNA, usually about from 10 to 300 bp, thait act on a promoter to increase its transcription. Many enhancer sencs are now known from mammalian genes (globin, elastae, albumin, rc-fntcpven, and insuln). Typiclly, however.

one will use an. enhancer fr-om a eukaryotic cell virus. Examplesa include the $V40 enhancer on the late side of the replication origin (bp 100-270), the cytornalovir u aly promoter enhaner.

the polyoma enhane n the late sid of the replic atingin, and adenovru enhaers. The embanner may be spliced into the vector at a positin 5' or 8'to the PROM28, P1102K6 or coding sequence. but is preferably lod at a site 8' from the p romoter.

Expression vectors used in eukaryotic host vel.a (yeast, fungi. insect plant.

anm lhuman, Or Mucleated yule frMM other multicelulAr organisms) wil alo contagin aquerra necessay for the terminatio of trascriptio and for stabilizng the nSNA. Such sequences are citimonly available frDm thep 5' and, occasionally untranslated. regions of eukaryotic or viral PNo or ct4As, These region contain nucleotide segments transcribed as polyadezzyated frnginezM in the untralate portion ofthe mRNA encding P1102M3 P10288, or P30C358.

Still other method, vectoro, and hcat cell. suitable for adaptation to the synthesis of P110288, P110286, or PR10858 in recombina vertebra ell culture are descrbed in Usding at Naun MEUSo-SIS 0981); M aet Nalc=140-46 (197): E? 117,080; an EP 117,Q05K -26- P1154R2 C Dtlaad Gone Gone amplificaton. and/or evo mway be masured in a sample directy, for eample, bpy dventionul Soutbnm blotting Norther blotting to ipiantitabe the trani.. o of inRNA Muhmas, Pr.c NW-att Aad, IN& T].&wl20520S (1990), dot bltting (D)NA analyls), or in &ue hybridination, using an apprnpriately labele probe, bused On tii wpzOmEe provided herein. Altiernatinldy, antdie. may be employed that can recognjie specific duplexes, includig DNA duplexe. RM dupleiss, mud DNA-UNA hybrid duplexee or DNA-1protein duplex... The antbodies in turn may be labeled and tho assay may be carrie out wher the duplex is bound to atsurface, wo diet upon the formation of dupex on the suisae, the prwenoe of antibody bound to the duplex can be deeced Gene expression I alternatively, may be measured by immunological method., atrh a lnun K~Uaoksohemical swunkw of cell, or tisme sections and &my ao col culture or body fds, to quantitate directly the expression. of gene product. Antibodie usfl for immunohkstohemleal staining amlkfr any of naple fli~ds may be either monoclonal or polycalona and may be prepared in anky mammal. Conveniently, the antibodies my be prepared against a native sequence PR0285 PRO286 or PROMSS pclypepfldnh or against a synthetic poptide based on the DNA bemos provided herein -or against exogenou sequaence fused to PRO2SSL. PHRf28Oor PROS5S DNA ad encoding a se&c antibody epitope.

PuriiainLfEcinnfid Forms of PRCI285, P1102.6 or PRtO&58 may be recovered fron culture edium or from host veil lyuates. i meambrane-bound, it can be released from the membran, using a suitable detergent solution (ej. Triton-X 100) or by enzymatic clavge. Cells employed i:4 erpressiun at PROW8, VRO2SO or PRO358 can be disrupted by various physIcal or chemical means, much as freeze-thaw cycling, sonication mechianical disrupton, or cell lyuing agents.

maybe dsired to purif PR0285, M102K6 or PR10358 from recmbinant cell proteins or polypoptid em. The following procedures are exemplar of suialble punricaton procedures: by fractionation on an ion-exchange column; ethanol precipitation; revers phase HPLC; chromatography on. s ilica or on a cation-exchange resin muh ts DEAR; cluomatofoening; SDS-PAGE: aimaninin sulfte precipitaton; ge] Nilka&mik using. for example, Sephadex 0-76; protein A Sepharcee columnsg to remove contaminas such a. IgG- "nd M"to helatng columns to bind epitope-tUaged forns of the TeDl proteins. Various methods Of protein purificato may bO employed and muck methods axe known Wn the art and deucriled for example in Deutecher, (1090);Scope.

Pb bc pzinu-Verlng, New York Thepurifiction step) .eleted wil depend. for example, on the nature of the production prune.. cued and the particular Tol prti produced, Ue o h Toil prtensan ancdn uclei adds Ntcletide Baqnennef (or their complement) encoding th-e Toil proteins of the present inrontion have various appliains in the art of molecular biolog, including ume as Pt 15R2 hybridizaton probes, in chromosome and gene maNppng and in the generatio of f&mntss RNA and DNA. Toll nuclei adid will also be usefu kor the preparation of PROflD. PR0286 and PROMS Polypeptdee b* the recombimant techniques described herein..

The Mizlenhgth native asqveno DNA4002I, DNA42068 and DNA47Isi genes, PRO2M, PROISE, and P10858 respectively, or portion thea may be used as hybridization probes for a cDNA librart to isolate the fulesngt gone or to isolate stil other genes (for istace, those enoding natually-occurring nariant of PROM28, P80286, or PROWS8 or ther fur-ther human hoznologuee 1 or homologus from other pecicu) which have a desired sequence identity to the PROM6, PRtOM8, ai PRO058 sequence disclosed 1n Pip, 2, io 3 and 12A-B, reapectv~y. Optionally, the length of the prbes wil be about 20 to about bws The hybridizatio probes may be derived fro~m die nucleotde sequence of Figue 2 (SEQ ID NO, or Figun 4 (SEQ ED1 No- or Figure IIA-B (SEQ MD NO- 14). or from genonuc sequenes including promoters, enhancer elements and introns of native sequence By way of example, a screening method will omrprise isolating the coding region of lb. P10285, or PRO2MS, or FRO358 gene using the known DNA nequenoe to synthesize a selected probe of aba~t 40 bases. Hybridization pras may be laboled by a -variety of labels, including radionucleotdeas such au ,s or Wor enzymatic labels such a alkaline phosphutaoe coupled to the probe via evidin/biotin coupling uystemt, Labeled probes having a see complenientry to that of the P10286. PRO2SO, or PROMS8 gene (DNA. 40021, 42W8 an 47301) of the present invention can be used to inreen libraries of humn eDNA, genomnic DNA or rnENA to determine which xnember of such librarie, the probe hybridizes to. Hybridization techniques are dscribed im fiurther detail in the Examples bsiowh The probe. may also be empLoyed in PUB techniquesa to generate a pool Df sequences for identifian of closely related MAlI nequences.

Nu-cleotide equence. encoding a Toll protein herei% can ealso be used to amnstruct hybridiralion probes for mapping the gene which ennods that Toll protein and for the genetic analyss of indivda with genetic disorders Ikhenucleotde sapzeir provded heren muay be moapped to a chromosome and specific wegins of a chromosome usfing known technique, sruch am in sium hybridization, linkag analysis againat knownx claroamoomal markers, and hybridiation screening with librarin The human Toll proteins of the present invention can also be used in mgus to identify other proteins or molecule. involved in Tol-matated signa trsnsdrtbm. For example, PRO2Sb, PROM8, and PROWS8 are usefi in identifying the as of yet unknown natuiral fganda of buman Tolls or other fators that partiipaf* (directly or indirecty) inathe ctivaticn of and/or signaling though aL human Toll rsepqtor, such as potantia Toll receptor associated king$",. In addition, inhibitors of the rsaspor'ligmnd binding interautioxn can be identified. Proteins involved in such binding Interactons can also be used to screen for peptide or small molecul inhdibtrs oragonist, odie bindinginterwacton Screeningassays can he designed to find lead ompounds that maimic the biological activit nativ Toll polypeptid. or a ligand for&a native P1l'lf To7l polypeptide. Such reening assays will inolude ssways amenable to high-throughput screening ohamical librarie making them partinularly suitable fIr idmntiing amal molecule drug candidates. Small moleculea contemplated include synthetic rganic or inorganic compounds. The asays an be performed in a variety of format. including prein-proteia binding asays, biochemical screening assaysa, immunoassays and cell bae assays, which are well characterized in the art.

In viro asys employ a mixture of components including a Toil receptor polypaptide, which may be part of nsion product with anather peptide or polypeptide, a tag for detecting or anchoring, etc. The assy mixtures may further comprise (for binding assays) a natural intra- or ertrellular Toll binding target a Toll ligand, or another molecule knwn to activate and/or signal through the Toll receptr). While nadve binding targets may be used, it is frequently preferred to use portion of such native binding targets (e.g peptides), so long as the portion provide. biiding affnity and vidity to the subject Toll protein conveniently measurable in the assay. The assay mixture also contains a candidate pharmacological agent Candidate agents encompass numerous chemical classes, through typically they are organic compounds, preferably small organic compounds, and are obtained om a wide variety of sources, incluling libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture, snmh as, salta, buffers, neutral proteins, e.g. albunmin, detergenta, protasae inhibitors, nuclease inhibitors, antimirobial agents, etc.

In in utr binding assays, the resultant mixture is incubated under conditions whereby, but for the presence of tbhe candidate molecule, the Toll protein specifically inds the cellular binding target, portion or analog, with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facihtates optimal binding. Inubation periods are 26 likewise selected for optimal binding but also minimized to facilitate rapid high-throughput sCr&Offug' After incubation, the agentbiaed binding between the Tol protein and one or more binding targets Is detCted by any convenient technique. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may S0 be efeted by precipitation(.g. TCA preipitation, immunoprecipitation, etc.), immobilization (e.g on a solid substrate), etc., followed by washing by, for exmple, membrane filtration (e.g.

Wbattmass P-18 ion echange paper, Polyfiltronic's hydrophobic GPOC membrane, etc.), gel chromatography gel filtration, affinity, etc.). For Toll-dependent tranriptian assays, binding is detected by a change in the expression of a Toll-dependent reporter.

Detection may be effected in any convenient way. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optic or electron density, etc., or indirect detection, such as, an epitpe tag, an nayme, etc. A variety of methods may be used to detect the label depending an the nabature of the label and other nasny omponela, e.g. through optical F 11542 or elctro density, rdiative emmudons nohmAkIative ennrgy transfer., etc. or indirect] detected with anibody conjugates, etc.

Nucleic acids which encode PRO25, PROM8, or PROM, or their modified forms can also be USed to generate either trangnic animal or 'knock out t animals whiclz in turn, ae uful in the development and screening of theraputally useful reagent A tngmnic animal a ouse or rats is an animal having cell, that contain a transgene, which tranagene was introduced into the sninal or an ancestor of the ahjmal at a prenatal, e.g., an embryonic stage. A trangenee is a DNA which is intepated into the genome of a cell frm which a ensgenwic animal develop. in one embodiment eDNA encoding PR0285 or PR0286 can be ued to clone genomic DNA encoding PRO28, PRO286, or PRO858 in accordance with established techniques and the gera& sequ-uens used to generate rmnasgoic animals that contain coils which express DNA encoding P028, P802D6, or PR0U8. Mthods for generating tranagenic animals, parbtularly animals guch as mine or rats, have become conventinal in the art and are deribed, for example, in US.. Patent Noa. 4,736,866 and 4,870$)O9. Typically, particular cells would be targeted for franagene inorporation with tissue- .peffic enhaners. Tranagenic animals that include a copy of a trmag.no encoding PRO285, PRO2RO, or PROM58 introuced into the germ line of the animal at a embryonic stage can be used to examine the effect of increased expression of DNA encoding PROMSb, P802S, at PR08. Such animals can be used a tester animals for reagents thought to confer protetion from, for example. pathological conditions assciated with its overereseirm In accordance with this facet of the iavetknr4 an animal is treated with the reagent and a reduced incidence of the patholocal codition, compared to untreated animal bearing the tranagene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human vrtebate mammalian) homologues of PRO 2-6 or P88 or FRO58 can he used Wa constuct a 'knock out" animal which has a defectiv, or atered goe encoding PE0285 or PM026 or P80358, as a resut of homologous recombination betweein the endogenoe gene encoding PR0285, PR0286, or P8038 proein and altered genomic DNA encoding P0285, P0286I or P0358 introduced into an embryonic 0ll of the animal. For example, ODNA encoding P80285, P0286. or P1R358 can be used to clone gennmic D3NA encding PRBM, P02K86, or PR0858 in acodace with established techniques. A portion of the geomic DNA encoding PROM,5 PEOZUE, or P0858 can be deleted or replaced with another gene such as a gene encoding a seectabl, marker which can he used to monitor integration. Typically, meral klobaes of unaltered flankin DNA (both at the 5' End 3' ends) are imluded in the votar lee. ecg, Thomas and Capemhi, M:BO (1987) for a deectiption ofhomologous reambination vetosj. The vuctor is introduced into an embrynic item cUll line by euctroporation) and cels in which the introduced DNA has harnolgoly reombined with the endogenos DNA are selcted (see Li a1 at fdL gag (1992)]. The melected cells ae then injected into a batoyt ofan animal a mouse or rat) to form aggrgatio cuhimeras [ae eg., radley, in 21. ~wri ~wr" and £mb'ycn Sim Cd& P1154H2 AA-racicdApprooA. IM.J. RabSMtOn,4L(4 Oktbrl, 198n7), 11 i3-152j. A chimeric embryo can than be impisated into a suitable, paudoprqsnant female touter- aia md the embryo brought to torm to ornate a 'knock out' animal Progeny harboring the komologouuly recombinod DNA In their gem ce&l can he identfid by itandard technigu and used to breed mnimml. in which all cells ofte animal contain the hornologouay recombined DNA. Knockout animals can be characterized for istanoe for their ability to defcnd igainat certain pathologica onditions and for their development of Mtholoical onditioms due to absence of the PRO285, PRO2t or PROWS5 polypeptide.

Nuclei acid encoding the Toil polypeptide dirlomed heren my also be used in gene therapy. In gene theapy applicatnsa gene are lnlroditcd into cell. in order to achie ins wia synhis of a fherapomtically effective genetc produt, for uxnmplo for replacement of a defective gene. 'wUene therapy includes both onventioal Setke therapy whore aL Jesting effet is achieved by a singe treatment, and the admrinistratkon of gene therapeutic igenta, which rnvolyrs the a=e time or repeated adminisetration of a therpeutically effective DNA or mRNA.

Antjuonie RNAis and DNAa can be used s therapeutic scent. for blocking the expression of certain genes in vime. It has already been shown that short antiasnee olgonucleutides can be imported into cells where they act au inhibitors, desfpite their low inatracellular concentrations caused by their restricted uptake by the cell membrane. (Zamacnik et al., Pro. Nail. Acad.&S9d.

U8A ial 414&.4146 [.19M6). Thet olgomiclotdes can be modified to enhance their uptake, eg.

by substituting their negatively charged phoaphodiester groups by uncharged groups.

There are a vaiety of techniques availble for introducing nucleic acids into viable cels. The techniques vary depenudirtg upon whether the nucleic adid is transfered into cultured cells in uWro 1 or in ivo in the cells of the intended host. Tecniques uitable for the transfer of nucleic acid into annsli cell in vitr include the use of liposomes, 2Z electopoatimn, mkrinjtion, cell fuuion, DEAE4-ders, the cakitun phosphate precipitaton methodL etc. The currently preferred in vv gene trasfer techniques inclde frvnnsftn with viral (typicaly retoviral) vectors and viral oat protein-iposotn mediated transfection (Dzan ei u Tap& n Bak~b~k~g 1. 2052 1O (1993]). Ina sme sitizatina it is desirable to provide the nucleic acd soure with an agenat that targets the target cells, ich as "n antibody specific W0 ices cell uuttwa membranke protein or the target celk, a ligand furea receptor on the target cell, etc. Where lipaomes are employed, proteins which bind to a cell suraIce membrane protein anointed withi endeytosia may be used for targeting andic t*o facilitate uptake, ejg. Capaid proteins or rraginent. thereof trpic for a particular oell type, antibodies for proteing which undergo intsnRIJizatio in cyng 1 s protein that target intacelula locaization and enhance intracellular half-life. The technique of receptor-meated. endocytosis in descrbed, for enample, by Wueti, 'j 2flJ. ~g.M 4429.44=2(1987); and Wegner et GL, Proc. Nat!. Acad. Bd. IJSA AL 3410-8414 (19W,) For review of the currently known gene mrkig an~d gene therapy protocol. see Anderson el at. Scinn M&L WB-813 (1992).

P1154R2 The variou uses lited in connectio with the Toi protein herein, are also available for agonist. of the native Toll receptor, which mimic at last owa biological funcrtio ofta native Toll receptr.

r. AtiToll u~dj The presenrt invention farther provides aaWiToll protein antibodie. Ezempim antibodies include polycristd monoclonal, humanized, bispeifie, and haturoconjugats antibodies.

1.wgnlAn~~ The mnti-Tchil protefin mmlibodies may comprise polyclothl antibodies Methds of preparing polyclonal antibodfies ane known to the skilled artiuan. Polycionl antibodies can be ratsed in a mammal, for example, by one or more injections of an immnunizing aget mi-d, if desired, an adjuvant- flTpiculLy. the immumizing agent and/or ad~juvant will be injected in the marnmaI by multple subcutaneous or intrapennealM injections. The immunixiug afgent my include the PROM28 and PROM28 polypeptides or a fusion protein therse It may be useful to 16 B onjugate the immunizing agent to a protein known to be immunogeni c in the mammal being immunized. Examples of such huwnmogwnk proteins include but are not limited to keyhole limpet hemccynin serum albumain, bovine thyroglobuln =a soybean typin inhibitor.

Ersnpicsk Of adjiUVants which may be employed include Freand's complete adjuvsnt and MM,- TD)M adjuvant (maophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one tliled in the A without undue sxpeimsntation.

2. aoag nbde The anti-Toil protein antibodies may, alternatively, be maoona antibodie.

Moona antibodies may be prepared using bybridom methods, such as those described hy Kohler and Miletein. Nture =f495 (1975). In a bybridoma method, a mouns, banter, orP ether appropriate hoot Animal, is typieally immunized with on immunizing aget to elict lymphocytes that produce or are capable of producing antibodies that will specificalLy bind to the immunizing agent Alternatively, thie kymphocte may be immunized in Wiro.

The immuniing agent wil typically Include the PROM,86 PROM8G or P11028 polypeptide. or a fusion protein tharsot Generally 1 either pEriphnal blood lymphocyte.

PBLa) are used if cells of buman. origin are desired, or spleen cells or lymph rnds cells axe used if non-bumaul mammaimn sourcew are desired. The lym-phocytes ame then fused with on immortalized cell line using a suitable fueing agect such an polyethylene glycol, to form a hybridoma cell ((lig o kstodu Academic Free, (19M6) pp. 5U-10SJ. lmmortmllzed cell lines are usally tranufbrmed maznnelian telparticularly myaoma cells of rodent, bovfine and human origin- Usually, rat or mouse wrioma cell lies ame employed. The hybrido=An cells may be cultured in a suitable culture medium that preferably omtaa cm or ~m ghff~w~wthat inhibit the growth iyr survival of thke utjaed, immortalized cells. For example, if the parental ea& sck the enzyme hypexanthine guanine phospborhouyl In amters. (MGflT or HFR the culture medium for the hybridoona typialy will irluda -32- P115432 hypoxanthine. aminopterin, and tiiymidi no CHAT imadiumj, wich substances prevent the gfrowth of HOPRT-deficient calls.

Preferred uimalihzed *ell lis axe those thiat fum efficiently, suppor stable high level epsimof antibody by the selected atibody-producing DallS, and are menailive to a medium such as HAT medium. Mnre ipreferrd Immnortl&izd cell lines are murin. myslonia lines, which can be obtined, for instami, Emm the Salk Intitute Cell Distriin Center, San DieMo Calfrnia and the American Type. Culture Cgllection, Rcckvifl, Miryland.r Human mycom and mnouse-human heteromyeloa elllnes also have been described &or the producion of hpuman monoclonal antibodie.~xN I~rbr dminoL-, 1i3(X)1 (19M4); Brodeur et aL ~c"A~d rduto elnge arlDkeLm o York, (1987) pp. 51-68].

The culture medium in which the hybzidjxa cell. are cultured can then be assayed for the presxeneof juanclonal antibodies directe agmimit PROMS5, PRONG6. or Preferably, the binding specific-ity of monocloral antibodies produced by the hybdoma cells is determined by immuriopracipitatian or by an ina iiro binding assgay, snub us radinimmunoana~y (RMl) or enzymerlinked inamunoabeorbent sway (SLIM) Such tslniquee and assays are known in the art. The binding affinity of the monoclonul antibody can, for example, be determined by the Scatokard anmais of Munson and Pollard,Anl ohe.

1Ql:220 (198).

2D After the desind hybridoma cells are idenified, the chaos may be aubcloned by limitin dilution procedures and grown by stwaard methods ((lading aupal. Suaitable culture media for this purpose include, for example, Dulhecco's Modifie Eagles Medium and RM.

1641 medium. Alternatively, the hybridma cels rmay he grown in viva as acitem in a mammal.

The monxzlonal antibodien secreted by the Mibdona may be isolated or purified from the culture medium or acitem fluid by conventional immrunoglobulin purification procedures much as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel eietrophorsls, diaysis, or affinity chromtogrmphy.

The monoelonal antibodies may also be made by renombinant RNA methods, such an thoe descibed in U.S, Patent No. 4016,567. DNA encoding the mdnmlonal antibodies of the invention can be readily isolated and sequenced ageing cotbvagtional procedure. es by usiag olinnudeotide probes that are capable of binding upecifcly to genes encoinmg the heavy and light chains of murin antibodie). The hybrid*=&n cell. of the invention mre as a prefered mnnce of such DNA. Once isolated the DNA may be placed into expression vectors, which are then tranufeated into host cells much as sinmn COS ell. Chess bsnmtur ovary (CEO) sells, or tnyeloma cells that do not otheri produce imniunoglobulin protei, to obtan the synthesis of monoclonal antibodies In the recombinant beet cells. The DNA also may be modified, for excample, by ultituWiu the coding sequence for human heavy and light chain constant domains in plate of the homologous murine sequencs MU.S. Patent NW 4381O0.867; Morrison Atat, Mwj or by Moval joining to the iinmuncobuln codig mequence al t& P11fl4I2 part of the coding sequence &or a n-mmunogkbuli polypeptide. Sch a non-immunogLoblm polypeptide cn be uubsutMW for the constanit domains of an antibody of the invention, or COn ho substituted for the varible domains of ome anis-ccnbining ffite of an anfibody of the invention to crate a Abimeic biralent antibody.

6 Thea antbodies may be monkovalent antibodies. Mdethods for preparing monorvalcnt antibodies amm well knwtr in the art. For example, one mrethod involves recombinant expression of immuno~globulin light chain and modified heavy chain. The heavy chain is truncated gonerally at any point i the Fr, region eo a to prevent heavy chain crosabaldug. Alternatively, the relevant cyateine residues are mubetitted with another amtiw( acid residue or arn deleted so so to prevent crossliniu I Oiro methods are also suitable ror preparing monovalent antibodies.

Digestion of antibodies to produce fragmentis thee partaicuarly, Fab fraumsn anta bIMe accomplished psipg routim tOAOWcq kago in the art, Uumn adELM&ua~afl lb The anti-Toll antibodies of the invention my ftmjier comprise humanized antibodies or human antibodiem. Haumnized fbrms of non-human (e4g., murinie) antibodies a~re chimeic immwtoglobulius immunoglobulin chains or fragments thereof (sucah an Fv, Fab, FMb', F(sb') 2 or other antigen-binding subsequenes of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies incude human 2D immunoglobulineq (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a ODII or a non-human spaee (donor antibody) such as mouse, rat or rabbit having the duired specificity, affinity end capacity. In same instances Nw framework residue. of the huiman Linmiungbulin are replaced by corrsponding sun-human residues. Humanized antibodies may alma comprise tniduee which are fond neither in the recipient antibody nor in the imported CDRI or framework sequences. In general, the humnize iatibody, will compris eubeta-ntally al of at letat one, andl typically two, nriable domiains, in which all or iubstsntWaly all of the CDR regions corrampond to those of a non-human imwunoilobulMn and all or subsftantialy all of the FE region are those or a human immunogiobulin consensus mequemce The humanized a~ntbody optimally also will comprise at least AL portion of mn immunoglobPlin constant region (FA), typiallythat cia hu man immunoglbulin [Jones at al., Natur, m:522-528 Rlsehm et a.,hIIfM2, =82t3'fM (1i8"; and Frosfts, C=u.!20. Sun. Bio., Z:.593-5f (1992)].

Methods far humanizing non-human antibodies an, well known in Lhe art.

Genally, A kunied antibody has one or more amino acid resi~due. introduced into it frotn a sources Which is non-humani Thes. non-human amino aicid ruuidues are often referred to as 'import' residues, wich are typicall taken fromn anm Iinpcr variable domain, Rumantiml can be essentially performed folowin the method of Whiter and co-workers [Jones At aL.

Nakaza M2t522-625 (t986); Riechnrenn et =U B-27 (IOW8; Verhosyem at ali, Sa 21584*1538 (1985)1 by substitutin rodent CU~s or CDR3 equence for the PI11541M.

0XV~ago a equomos60 of.j human 4jLapIy. Amordjjtngy, aw manime antodies are chie Qatibodim Paltet N1o, 4,816, 1 M7), wherein arubstantiafly lass tan an intat human variable domain has been wubetitUted by- the correpondin bequses fom a nnbsunain .fpecia In practice, humanized antiboies are typialy human antibwaie in which nom.e CDR residues and posibly wso FR residuew are subttuted by reisidues from analogous .it. in rodene antibodies.

Human nbbntADa can Elso be pnxltrad using various techniques known in the art, hwludimg phage display Ubra rim [IHoogmnboom and Winter, J.MABivL- =L7381 (1991); Marks it a0., £,hW C=581 (190L)]. The techniques of Ccl. and Doerar et al. ane also availble fk& tho preparation of humtan monoolonal antibodies (Cole et MWnalj AntI~q$.~~jCancer Theranv. Alan R. Lism. p. 77 (1985) and Soernar it al,. JjnMUngL 2311M:86-95 (1991)J. Similarly, human antaiodies can to made by introducing of humain lennunoglobulin loci into train genic animals, mice in which the endogenous immunogobuln gme have been parialy or completely inactivated. Upon challenge, humain 1 5 antibody pfoduction is olbserved, which closely resembles +hat seen in hiumans in a11 respwte including gene rerrangenment, jasmbly, and antiboy repertoire. This approah is descibed.

for exaumple, irk U.S. Patent No& 5,545,807, 5,545,8W6; 5,569,W25; N6G25,126; 5,633.425;, 6,"61016, and in the folwn cetfcpubications: Marks etaL, RkN&l~gV 0 779-788 (1992); Loxnbmrg -4 aL, SAtine M 856-859 (1994); Morrison, Na&.ur HL 812-IS (1994)-p Fish wild d aL, Naue kf.4lJ, 84B.51 (1996); Neuberger,NaueBccho I 828 (1996); Thnberg end Eusozar, Jntarun.Eav. Immvn$. Id465-93 (1995).

4, Bumpecifi Aritilb~l Biapeciflc antibodies ore monoclonaL, preferably human or hunmnisd.

antibodies Eihat ave binding .peci~fii for at leant two different antigens. In th.present cm, 26 ao dthe binding eecfitius maybe for tbe PR0285, P11028E, or PROMS protein, the other ome for any other antige, and preferably for a cell-surface protein or recepbor or recetr subunit It is al" possble to prepare hispecifc antibodies, having epecifcitims to two different Toll-like proteins, much as, any two of thie Toll homologuee diwcloed in the present application, or a Toll protein disclosed herein, and a Toll protein known in &h wt, e~gh, TflM. Suph bispecitzc antbodies could blck the reoopiitinm .fdiffernt~pathogen pattrnn b6y Toll receton, and are, therefore, erpadad to have significant benefits in th-e treatmant Of sepsis and septi shoA Methfods fur mmun bispecific antibodias are, known In. the art Traditionally, the recombinant productio of biapecific antibdies is bused on the co-exprmi of two iununoglbulin heavy-chainflght-chah pair, where the two'heavy c a have differen sOlficities [MiNAt mad nUOIe. *isfln It5-539 aeeaj)]h Because of &he random awwrtment of imruunoglobulin heavy and light chains, the hybridomas (quadromaa) produce apotential mixture of ten different antibody moleulMs of which only one has the correct bispe3cil strut. The pVri~Alic of th~e correct molecule is uually amcmplished by affinity PIIM&R2 chromatography atops& Similar proce~urea Ar" ciscleed in WO OSBh published 18 May L993, and in Traunocer at XU" fl35-B659 (1991).

Antibody variable detrains wit the deered binding epeofficitie. (anfibodyantigen combining sites) can be fused to immunogCkbuln constant domain sequemae. The fiion preferably in with "n izmunoglobulin heavy-chain cortant domain, comprising at least part of the binge, 0H2 and CES regiont It isi preferre to Lava the first heavy-chsi constant region (CHII) containing the site necmaxy fig light-chain bhiding present in at leant one of the Atiafl% DNAu envodiv& tho iwvwncgko~hu heavy-chain fusions and, if desired, the immunoglobuli light chain are inserted into separate expessfion vectors "n are cotamected. into a suitable host orgaim. For further detakils of generating biapecih antibodies r, far example4 -Suresh et M. atbpi mLm ly. Uhgonmufate Antiie Heteroconjugate antibodies are also itii the mope af the presnt invention.

Hateroeotagate antibodie are compose of two covalmntly joined antibodies. Such antibodie have, for example, been proposed to target immune system cells to unwanted oslis [US. Patent No, 4,676.9MOt and for teatment of lilY infection [WO 9 110030; WO 92F2037a; EP Q3089], It is contemplated thAt the antibodies may be prepared in vi"r using known mehiods in nmthetie protein cbemistry, including those invoking crosslinking agant.. For exmpyle, icumunotina may be constructed usirg a distdfide exchange reaction or by forming a thiceter bon&L Examipla of suitable reagents for W&i purpose include iminohiolateaend nisthyl-4mercaptobutytimidate and theme disclosed, for exiample, in Patent No. 4,876 98O.

Cl. Mien ar ani-Tal protein mnttqjW The anti-Toll antibodies of the invention have various utilitie. For example, mnti-PR0285, anti-PRO25 anti-PRQ.358, andl .nti-TLR2 antibodies may be uied in diagnostic 26 as for PROM8, P1R0286. PROMO or TLLRZ detecting it. expresson in ipecific ells dmimes or seru. Variou diagnostic assay thniques known Ln the art may be uizad, much au competitive binding assay, direct or indirect sandwich ays and imnuanouprecipitatioa eays conducted in either. heterogeneous or homogeneous phases (ZoloL =a.lA m Manual of Techniques. CRC Freom, ln41. (1987) pp. 147-1W8. The antibodin used in the diagnostIc assays can be labeled with a detectable mnoiety. The detectable moiety shoiVl be capable of producing, either directly or indirectly, a detectable signsl For example, the dletectable moiet may be a radiootope 1 such a 5 S, 10hI.sw i, a fnsoraeent or chsinilumineacent omtpound, such a flucroem isodziocyanat., rhodamine, or luciterin, or an enzyme, such as alkalin. phosphate, teta-galactcuidsse or hoarseradish peroxidue Any methoad known in the art fuir onjugating the antibody to the detectable moiety ty be employed, including those methods described by Huntar et aL., titz, 1"%S4 (192); Da~viA at al., flumat 110l4 (1974), Pakn et at, 4&219 (1981); -and Nygrnu HiMtehR, alyteu. M;:407 (1982).

P115432 AnWaPROZSS, anti-PRO286, anti- .PROSOS or nti-TLR2 antibodfieu Also are useful for the affixity purifiation of thus. protein froxm renmnnt eelL culture or natural inoins. In this prooes, the antibodies against thes Trol proteins are immobilized an a sitable support, such a Sephadex resin or filkt paper, using methods well known in the eart The inimohilisod antibody then in contacted with a sample containing the to ha purified, and thereafter the support is washed with a *mible solvent that will remove substantially all the ;natonal in the sample except the PROMS5 FROM3, PROS35% or TLR2 protein which is boun-d to the inunobiized antiody. FInally, the supot is washed with another muitable solvent that will release the protein from the antibody.

Anfl.Thll receptor anti-flOISS anti-PR0286, anti-FBOUA or anti-TLIL antibodies) may also be useful in blocking the biological ativitie of the respective Toil receptors. The primary fwin of the family of.Toll receptors is believd to be to aet "a pathogen pattern meognition raceptorD sensing the presence of connved molecuA r pattern present on mic robes. Lipopolysacchawides OLI'S, als kimown as endotknns), potentially lethal nxlecule, produced by nrious bacteria, bind to the 11popolysacoharide binding protein (LDP) in the blood. The complex frm-ed then activatee a receptor known as 0D14, There is no consensus in the art about what happens next. Accordihg to a hypothesis, 01)14 does not direty instruct moacrophages to produce cytokines, cell adhesion proteins and anhymas involved in the productiob of lower molecular weight prcnnlarnmator mediators, rather enables LAPS to natvate a secnd receptor. Alternatively, it has bee= suggested that LP8 may activate certainf receptors directly, without help fron LBP or 01)14, The data disclosed in the prent applncaton indica that the humm atll-like receptor are signaling receptor that are activated by LPS in an fLEP and CD114 rewponsive nmnner. As this mnechanism, under pathopbyuiologie conditions can lead to an oftean fastal syndromem cal septic shock, anti-Toil receptor antibodiesa 2- (just as other Toll receptor antagonists) might be useful in the treatment of septic shck. It is fotesen that the different Toll receptors uiigbt recognize 4ifferent patboguens, eag., various strains of Giramn-negative or Gram-positive bacteria- Accordingly in certain situations, combinatiocn therapy with a mixtur, of antibodies gsecficall binding different Toil receptors, ox the use of bispecific anti-Toll antibodies maybe desirable.

so Rlis apovfiaily dmonatrated herein &hat .ni-huTLL2 antibodies ame belied to be specifically useful in blockig the inductio of thi. remnpto by LI'S. Au it has boen shownL that LI'S exposun csa lead to septic shock (Purrdll, N.LW.g L Mfl, 1471 -1477 [1093]), ani-huTLltP antibodies are potentially useful in the treatment of map ti c shock.

The therapeUM13Uti and diagnostic uses liste in onetion wit the anti 36 Toll receptor antibodies are also applicable to other Foi antagoims. other moleules (proteins, poptdes, smal orgmic molecule. etc.) tat hiock Toll receptor mctivation sndlot signul trasmduction mediated by Toll receptor.

In view of their therapeutic potential. the Toll protein (ncuding variant. of the native Toll hoznologuea), and their agonist. and anlarniuts (inluin but rt imited to anti-Tll .ntlioe) Re icorported in compoeldeni sitable for therapeutic uee. Therapeutcc composiion Aft prepaeuiw ccstage by iin th. e ingredi~ent having the deaiwi degme of purity with opional physloleiaall acceptable efrisre, esciints or stabilines (Remingtcad.

Phannaceutcal Science I"t Eition, Oso, K Ed. 1IBM in the form of lyvhuse formulations or aqusom ilulions. Atoeplable carrisrs6 sicipienta ot sabilizere are nontmei UP rcipients at the dosages and coscentrmtiona employed, and include buffera isuch a.K phosphate, citrte and ~othier organic acids; antioxdamts including scorbic acid, low molecular weight (lems the about residues) polypspthl~e, proeins, such ansaruim albumin, gelatin -or immunoglobulna hydrophflic polymers such as polyinypyrrlidone, amino acids sfuch as glycine, glutarnine, asparaginep arpuine or lyaine; maonaebrid~e, disaccharides and other carbohydrate.

iIudiug glucose, mannosa, or dextrins; chelating agents muh so WETA; suma alcohols. such a mannitol. or arbitol; atlonuaing counterions such as sodium; and/or nonionic surfactants suoh na Tween, Plurcncu or PEG.

The active ingredents may .lso he entrapiped in microcepsulas )roarsd, for -exarnpie, by coacervation techniques or by intmarfcal pclywerimation, for example, hydroxynxetbylceiluae or gelati-micrccapaulo. an poly-(methylmetlaqyLate) microcapaules respectively), in colloidl drug dlelivery systems (fur example, liposomes albumin mkrngpllsre%, microomulsiona nano-particle and nsnocapeules) or in tacroemulsiona. Suth techniques are disclosed in &emkutb~a.Thmacudtial ciencu, .rupra The formulations to be used for in vivo sdnhinistraticn must be Hierije. This i.

rail~y %ccomplished by filtratin through sterile filtration membranes, prior to or Molowing lycpbilzaticn id reconstitution.

Therapeutic compositions herein generally a~re placed into a container having sterile acom port, for example en intravenous solutio bag or vial having. stopper piefteeble by a hypodermic injetin needle.

The route of administrution is in accord with known methods, eg. ijection or Inuion by intLavu, intraperitoneal, intrcereinL intraiusculur, intraocuar 1 intraarteia or intraleslonal routs,, topical adminiatrition, or by sustained release ar~s.irp Suitable examplea of suaned reise preparaton incude usmipsrmeable polym~er matrice in the Aknn of taped articles, ekg. films, or naroouulm. Sustained releas matrices Inclue pelyes, hydrogein, polylactides Patent 8,77a,919, ElP 56,481), copolymers of L-hatmnk acdsdpmma ethyl-Lrglutaeta Sidmamn a sL. nam (IX 547-556 [1983]), pol* 2hdoyty-ehcyae Langer, 1al, Be. fl 187-277 (1981] and IL Lanter. ChemSh. It 98-105 (1Q82]) ethylene viuyl acotate i-angr Al AL, Ld.) or poly-D.-8--hydroxybuOtyr acid (El' 133,966). SaUstaied releaset compoionsa also include liposomes. Lipownnes containing a molecule within the moaps of the preet invention are prop"tw by methods kon Maa DE 8,218.121; Eptain CLJO, E=a Nal Aeu Sc USA~t 8688-8692M(19W5) Hwangggj2, Pint Nati. $eAd. SUL L14 M 4034 (19W); Xl' 52922; EP' MA;A EFP 8846; EP 143949; EP' 142641; Japanese patent I1154N22 application 3&-118008 U.S, patents 4A485P0i5 and 4.54-4.545; and E? 102.324. Ordinarly the liposmes are ofth smeoll (about 2DD0800 Anggtwota) uniameAw type in which the lipid qontat is rester than about 30 mot %6 cnhlesterol, tho soeete proportion being adjustad far the optimnal NT-i therapy.

An effective amount of th ed cilngrent will depend, for example, upon the.

th'erapeutic objetives. th, route or Oadministration, and the conditio of the patient.

Accurdingly, it will be nwssary for the therapiat to titer thei dosag and modify the route of administration au required to obtain the optimal therapeutic effect A tykmsl daily dosag might range from about 1 igikto up to 100 mg/k or more depending on the factors menidoned above.

Typially the clinica will administer a mnolecule of the p resent invention un til a dosage is reaed that provides the required biological efftC 'The prov-es of tUs therapy ie easily monitored by conventional asayn.

The follwing example. are offered for illustrative purpos only, and are not intended to limit the soope of the premoat invetokn in an way.

All patent and literature refere. cithd in the present specification are herey incorporated by reference in their entirety.

Commercially available reagets refirred to in the exampiles ware used according to manufacturer's instructions unless otherwie. indicated. The sourm of thou. cells identified ki the following eamples, and throughout the specification, by ATCO accesonol n""per is the American Type Culture Colletion, Rockvle, Maryland.

EXAMPLE 1 b-datagiL H&canes aw Huan- EQ2l A proprietary expressed sequerce tug DNA database (LUflEQTh lmyt Pharmaceuticals, Palo Alto, CA) was searched and an RM (#22432M9 wan idetified which showed homology to the Lirowphila Toll protein.

Based on the EST, a pair of POR primer (forward and revene.z TAACACCAGCTGTGACQG (SEQ MD NO:M ATCCATGAGCCTCflATGGU (SEQ ID) NO- "Ad a probe: AflFATGTUCGAGLA"GGGACTGGTTACCAtG(3(A mCAGTrC (SEQ ID NO:?7) were uynthesized.

.,RtA for coTWuntuionof the cDNAWlirares wias isoated frmhuman placentaR tisue. The cDNA libr.Anm unit to isolate the cDNA clone. worn caosruted by standard method. using comnmerially available ragenks such as those fr-om Invibrogen, San Diego, CA (Fast Track The eDNA ne& primed with achgo dT containing s Notd site, linked wit blunt to Sai mla ad apnrs cleaved with NotL size appzopritely by pel elmctroporeia amd cloned In a dufned Orieationm int the clonig vecto p032.1 (Invitrogen, hnc. using reagets -aW -I 'P1154R2 and protocls from ife Technokogi&m. (klAbbfl Wi Wper script Plumid System). The double stranded vDHA was sized to greater than IO0 bp and the eDNA wan cloned into BainHj'Not cleaved vector 1 pCBI2.1 is a commercial available plaumid, designed easy closin of FOM fragments, thak maim AmpR and Ranl generselection, and Largene for blue-white seletion, In order to screen neveral libraies~ for aL mvurc of a hill-lengthi clone, DNA from the libraries wias scrend by FOR amnplification with the PCR pritaer patir identified aba. A poutiv. Ulrary was then used to isolate clones encoding the PR0288 gene using the prbb olwonualeotide and one of the POR primers A cDNA clone was sequenc~ed mn entirety. The entire nucleodde isqueng of DN&40021 (encoding9 PRL1285) ig shown in F~gure 2 (SEQ ID NO:2). Clone DNA4OO21 Contains a single open reading fr me withenaprntanlinaiitainmeatncoidpstos 61463(fg. The predicted polypeptideu precursor isa 10149 amino acds long, Including at putative gnal peptide at amino acdd positions 1-29 a pawa tranenmbranedOmain between Amino acid positionis 9374MI, and a leucine zipper pattern at amino aci position 132.156B and 704-725, respectivel. It is noted that the inilicted boundarie are appoxmat 1 and the ,pacta limits of the indicated regions night differ by a few amino adids. Clone DNA40021I ha, been deposited with ATMC deeignation DNA40021-U54) and is aasigne ATCC deposit no.200389.

liased on at BLAST and PaatA aequence alignment analysis (asing the ALIGN computer program) of the full-length sequence is it human analogue of the Drosophila Toll protein, and is homologous Wo the olOwing human Toll ps~oaits: TOMl (DNAX# fISU8854O-l' which is identical with the rao sequenced full-length aDNA #RIHUMRCVS& ToU12 (DNAX#HRSUSS7S-); Toll (DNAXOflSU8887sq> ay nd Toll4 (DNAXA HSUSSRSOj).

EXAMAPLE 2 A proprietary expressed sequenc tag (EM2 DNA database (LlflSQM- Thcyts Pharmaceauticafl., Plob Alto. CA) wan searched. and at EST Q#694401) was identified which showed homology to the Dnisophila Toll protein.

flamed on the EST, a 1pair of KR1 primex. (forward and reverse): GCGAOAACAAAAACGTrTCC (IEQ ED) NO:$) OATCCAGflCTCATCCAflAGCC (SEQ ID) NO: and a probe: TC0AGAAOCTCATGAGAGCATCAACCAAC"GA4J&CMAnAI (SEQ 01010) were syntheuized.

'uRNA for consructiuon of the eDNqA lirarie ws isolated from human placenta tissue. This RI4A naa used to geneate an oligo dTl primd aDA library in the vector pRXZD using eagentS and protool from Wie Technologies, Gaithereburg. WD (Super Scfrp Plasmid SYstem). pRUb is a cloning vector that has anmp WOanscripio isitiado Rile followed by an MO restrico OnsYme gito preeding thieXhoVlotl eDNA domg mites The cUNA W"a primed with align dT ontainin a Not! site, linked with btunt to Sil hemikinsed adaptors, claved with NotT sized to grear than 1000 bp appropriately by gel cft *8 and clowd in a defned orientation into XhaVNotl-eleaved p3KB]).

In order to anreen several libraries for a incwc of a full-lengthi cane, DNA hrum the libraries wa nvrened by ['CR amplification with the POR primer pair identified above. A positiva library won then used to isolate clontes encoding the PROISO6 gene using the probe oligonuclootde identified shove and one of the PCR primer.

A aDMA clone was sequepoed int etiety. The eatire utloidAe sequene of DNA426S (encoding P30286)1. shownin Fgure4 (SE D NO4). CloneDNA4266B ontain.

a singl open reading frame with an apparent franidional initiation site ait nucleotide position 57.59 (Pig. The predicted polypeptide precuraor is 1041 amino acids long, includinig a putative signal popti-de at amtiw acid positions 1.26, a potential transmsmbrane domain at axnino acdpos~itions826-84, and leucine tippet pattrsa mino acidsl W0151, 206-227, 562- 684, 689-690 and 693-614, respectvely. It is note~d that the indicated borun-daries are 1BS approximate, and the actua Emits of the indicated regin. might differ by a few amino aids.

Clone flNA42668 has been deposited with ATCC (deaignation DNA4268S-1154) and i assigned ATCC deposit no. 209386.

Based on a BLAST and liafft sequence alignme nt analysis (using the ALIGN computer program) of the full-length sequence of P1102K6 it is a human janskgue of the Drosohia Toll protein, and ig homologous to the following human Tol proteinst Toil (DNAX# HSU88640 1, which is idlentical with thie random sequencedM fl-lngthcfNA #HUMRSC7B6-1); Td11 fDNAX.# HSU8-1); ToDS (DNAX# 113L88879-1l; and ToU4 (DNAX# H9S8OSO 1).

EXAMPLE a Emltionmuc N coesLagdj mnRQf The sxlnoelluler domain (ECD) seuences (Incuding th. secetion signal sequence, if any) from known members of the human Toll receptor family wa used to search EST databas. The EWT databases included public EST databases Genflhnk) and a proprietary EST databae (ESEQrM *lncte Plarn uticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST [Altschul et &iat. o SnXmghngz 2M:460-480 (19M]) ass compariso of the E4XJ protein sequencee to a 6 fr-ame trsmeltion of the EST seuences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustee and. assembled into consensus DMA sequences withL the program "phrap' (Phil Green, Univeraity df Wasingon.Seaffle Wahington).

An XNS' was identified in the Incyts database (114C81 15949).

Based on tbm EST seqene, oligonueleotides were synthesized to identify by FOR a zDNA library that contained the sequene of interest and for use as probe. to isolato a clone of the Ball-length coding bequeo fou [PROM, A pair df [CR primers (orward "n revers) were syntheuisci .P1IS54R2

TCOCACCAGGTATCATALACTGAA

(SEQ ID NO: rrATAAGCATCTGrrCTCAkTCAGAGA (RQ W) NO-.e1) A pe wamsosynhizeb AAAAAGCATACTTOGAATGGCCCAAUUATAGGiTGTAAATG (SEQ ID 140!17) In order to neen seeral libraA"e for ansoin of a fufll-Ieth olone DNA fom the lihrieu wats creeed by PCR amplification with the P01 primer Pair idenified above. A positve library was then iWed to iSolate clansf nOUdg the P30358 gut. using-thu probe oligonmolotide and one of the FOR pitners.

RNJA for constuetiOn of the oDNA FiRurks was isoate fr-om human bone marrow (LIRt5O. The eDNA librresused to isolate the eDNA clons were constructed by standard methoeds using cummn rcaly available reag w&c as9 thou. frnm lnvitragen, Sim 1 5 Diemo CA. The oDNA nas prime& with dlig dT containing a Not] sits, linked withi blunt to3 Sail h~emWkiaed adapters, cleaved with Nod, sized apptoprimtely by gel dst .and cloned in a defined orientaton ino asuitable cloning vector (s-uch me pUjM or pEIl; pRKL5B is a precursor of p31(60 that doe not contain the Sifil site; see 4 Holmes et atM 28 M28 (199 in the unique XhaoI and Hofi sites.

2fl DNA Dequencing of the clones isolated aw described above gave th. full-length DNA sequence for PROS58 (Figtzeu IaA and 1IM SEQ ID NO:14)snd the -derived protein sequence for PR0868 (Figures 12A end Ilo SEQ ED NO., 1).

The entire nurdeWe aequence of the clone identified (DNA47SO ig shown in Pigure iaA-B (SEQ 13D NO:14). Clone IDNA4YaBIl contains -a single open reading frame with en aPParenut tratwlational Intiation site (ATO otart signal) t nucleotids positions underlined in Figure IRA and 158 The predied polpeptid incureor is,811 amino acds long, inchling a Putative signal sequene (&mina acidsg I to 19), &n extraoelluln domain (amino acids go to 57g, including leucine rich repeats in the regio froze posRton 55 to position 578), a putative tran~mbrmne domaidn (amino acdsa 576 to 695). Clans DNA473BI (designated DNA4II 1249) has been deposited with ATOC gad is assine ATOC deposit no. 209481 Band an aL BLAST and FasL4 sequence alignment anjalss (using the AL(IN computer program) of the full-1length sequence of PR0 2S it ig a humain analogu* of the Dnawphae Tall protein, a"d is homologus to the folowig h6uman Toll pruon± Till (DNAX# HSUSSS4O-1 1 which is identical with the random sequenced AI-length eDNA #HM1JSC7A&Q); 96 TollS(DNAX#HSUoeeMs-); TollS(DNPflflSU88793.t); and Tol4 (DNAX#IffSUsssae1).

-42-.

EXAMPLR 4 Um. of PROIM. PH 2M mad PROSMI DNA at hiddugml2M rrhe The fbllowicg method dscrlbes uwe of.a nudwoide aequenm encoding PRO=,i PR0286 or PROSS as a hybridixaljon probe, In the Following desciption. these proteins Are ollectvel referred to us "Oll homoogi.a.

DNA comprising tha coding sequence ofta Toll hazenlogue is employed as a probe to icreen fior homologous DNAs (such a. those encoding naturslly-ocrrring variantsof tdhepe particular Toll proteins in human timssu 6DNA libiraries or hunm as genomic libraries.

Hybridiato and washing of flitms contain either irary DNMe is perrmd 1D under the follwing high stringency ouditlous. Hybridizatio of radioabele Toll hotnologue.

derived probe to the filter is performed in a solution of 50% formamide, 8tx SSC. 0.1% SDS, CU% sodim prophahata, 50 mM sodUmn pwhospae, pH Zzrsnhardt' sohitica, and 1011 daxtran sulate at 42 0 C for 20 hoir. Washing ofthe filters is perrmd in an aqueou solutio of 0.lx BBC and 0. 1% 8DB ut 4200.

DNAe having a desired sequence identity with the DNA encoding fu~flftj native sequence ToN Immologne can then be irnetiafie win tdard technique. nwnr in the EXAMPLE Bxnressian 2f FR0295, E9A028. Rod PRO35O in EK caN This example illustrate. preparation of an unglyooelated formn or P30285.

PR0Z85 or FPRO358 M r11 bomologuefl by recombinant expreusion in Ea coIL The DNA sequence encoding a. Toll homolopr is initially amplified using 901ected PCE priman 7he9 Primers shul~d contain redriotioan enzyme mite Which Orrepond to the retictiranwym sites on thie sielected axpenin vetor A variety of expresio vetor.

2b may be employed. An example ofh SltaNe vector is p33*22 (derived from e014 Bee Bolivar et al., 2,95 (1977)) which contains gonea for empidfln and tetraccln rniuan. The vector is digeated ith reafrition enzyme anid depmhocrylatsd. The PCI ainpldhequonces a then ligated into the vector. The vector will preferably include peuwh ono&fj am antibiotic resistance gene, a tiP promoter, a polyhis leader (including the ELMs six &ME oodons polyhis sequence, and entorokinAs. clevag mite), the PR0288 coding region. lamb&a trasriptional terafinatr, and ex .rgU gone.

The ligation mixture ii then ua] to transorm a aelected R oo zLMM stainsig thie meithods desribed in &mbrook et al, Tranuformt. are identified by their ability to TrOw (m LB jilatee And antibiotic resistant cWOoie are then seloted Plsmd. DNA can be isolated and confimed by restricton analyss and DNA sequencing.

Selected do~ne can be pown overnigh in liquid culture medium sur-h ita LB broth supplemented with antibiotics. The overnight culture =Vy subsequently be used to ilatilte a larger wale culture. The ells wre tMe grown to a dlesired optca density, during which U. exrrem promoter is turned on.

After Culturng the cells for sevea? nm hours the cells iun be harveste by conikgatnn The eel pelle obtained by the centrifugation can be a:hiblikad using vario agents known in the art, and the uolublhss Toll howologue oan then he puriid Using a meta chelating oiun. under oondition that allow tight bindin of the protein.

ZEXMPM

B

Kxnirion of PR0285. P%1028 and PRCJ35S in m=m Kagiiu cl This example illust'atea preparatio ofta glyocaylated form of PRO2SB, PRO2l6 and PROMl M ol honmnngus') by recombinant aprussion in mammalian" cells.

The vectoDr, pME (se EP 307.247, publihed March 15, 319), is employed as the expression vectar. Optinly, the Toll homologue-emdnj DNA is ligted into pOM with selected restrictio enzymes to allow insertion of the Toil homologua-eaccxhng DNA usin ligation methods such as described in Santhrook st aL, The rsulting vector is called PBK5*PR0285, -PROSSO or -P110368, as the case may be.

In one embodiment, the selected host cells may be 203 cells. Human,293 cells 16 (ATCC, CCL 1573) are erown to confluence in tissue cultUre plates in meilun, ouch as DIQM spplemented with fata calf met-urn and optionally. nutrient components andir antibiotcs.

About 10 Pg p1WFRO255 -PR0286, or .FROB5S DNA ia mixed with about I #g flNA encoding the VA RNA gene Timmappaya et al., fdL jj-543 (1982)] and dissoved in So0 A oft1 MM TMA-MI, 0. 1 caM EDTA, 0.227 M CsCI 2 To this mixtue is adde-d, drcpwise, SWO Vd of 50 mM HBIPM (pH 7.U6), 280 mnM NaCa, 1-5 mM NaPO 4 t and a precipitte in allawed to form for minutes at 25"C. The precipitate is suspended =nd added to the 293 calls and allowed to settle for abnt four hours at 37DC. The culture medium is aspirated off and 2 Ml of 20% glycerol in flS is added for 30 seconds, The 298 cells- are Chen washed with serum fr~ee medium, fresh mnedium is added and the cells are incubated for about 5 days.

Approximately 24 hours aftr the kranelatko, the cultare medium is removed and replaced with culture medium (alone) or cultur medium aoutaining 200 jiglinl 858cysteie and 2W0 W0VI 3 '-methionine. After a 12 hur incuhtion, the condiiond medium is collected, *wentrated on -a spin fitter, and loaded onto a 15% SDS gel. The poesed gel my he drie and exposed. tic Slm for a selected perio of tinme to ±revsal the presefne of P110285 DO polptde. The cultures containig transfixted cells m~ay undergo fither incubationM (Lu serum fiee medium) and "h medium is tesfted in selected bkoasay.

In an alternative technique, Toll homologue DNA may be introdued into 293 cell transiently using 4ze aeun sulate method dux~icd by Somparyrac et at, £rWa.istL M. 12:7575 (1981). 298 celUa anq grown to maximal density insa spinner fLsk and 700) PgfpRKZ-PRO 2 Y(fl58) DNA Ls addedL The cells ame firHt concentrated from the spiner flask byr osufriffieon and washed with PBS. The I)NA-dextzan preaitate is inubated on the cell pelhl foyr four hours. The cells a trated with 20% glycerol for 90 seconds, washe with tissuew Oulue medium, and re-Intrduced into the spinner flask containing tdome culture medium, 5 $gfml bovine insulin and 0. 1 pgWm bovine transerrin. After about four da~ys the Ft Th482 oonditiond media incmtrifused and flltard to tmumvu cells and debris. The sample containing Lie corraponding sxprued Toll homologue can then be conceninted nd purified by any selected method, such -a dialysis andlor olumn chromategraphy.

In another embodim ent the Toll homologues can be expressed in COG cell.

The pEE5 5vector can bo frwaece into 0CH0 cels using known reagent. much as 00104 or DEAR-dextran. Ain described above, the cell cultures cen be incubated, and the mediumn replaced with culture! medium (alone) or medium containg a radiolabel ouc-h as 38 tuethinine. After determining the preene of PROMS, PR0286 or PRO 358 polypqptide, the culture medium may be replaced with smri free medium. Prefierably, the cultures aw incubate for stout 6 days, and then the conditioned medium is harvested. The medium contining the expressed Toll huirwiogue can then be cwrentrnted and purified by any selecte method.

Epitope-tagged Toll homologues may also be ezpreuoed in host 0CH0 ce'lls. The Toll homklgue DNA may be muboloned out of the p1LK vector. The .ubclo insert can undergo POCt to fuse in frame wit a select e pitope tag such a a poly-his tag into aL Baculovirus epedmvector. The poly-hia tagged insert: can then be snbckoned into at SV40 driven vector contain~ing a selection marker wunh a DHFR for selecton of stable clans.: Flinally the CHO cell. can be trauiseeted (as described above) with the 5V40 driven vector. Labeling may be performd, as described mbove, to verify expression. The culture miedium containing the epa dpo-ly-i tagged Tollhomologue can then be concentrate and purified by any selected method such a by Ni 2 tohelate affinity chromatography.

EXAMPLEZ 7 j~zprssaion of P.0205. f02& gnd FR0358 inYat The following method describes recombinant exprsmion of PROM.S FROM28 or PROI5S (-Mol homologize?) in yeast Finkt yeast exprsio vewco are antnetsd for intrscA-elltdr production or macradon of a Toll homalagu from the ADHS/GAFDRpromcter. DNA encoding the desiredToll homologe, a selected signal peptide and the promoter ie inerted into suitable restriin enzyme sitAn in the selected plasmid to diret intracellular expreson. For secreton, DNA s0 encoding the Belected Nil h0=010gu Can bhe loned into the selected iplaami together with DNA encoding the ADH2IGAPDH promter, the yea at alpha-actor acretmry slgnsaltdur .ecPaene. and linker nequenoes (if needed) for expression.

Yeast cells, such es yast strain ARt1O0, can then be transformed with the expression plaamids described above and cultured in seedw fermsntation media. The izandmoxud yeast uuperma can be amslmed by preciptation with 10% trichlroaceti acdd and paration by 8)8-PAGE, follwed by staining of the gels wit Coomasule Blue stain.

Recombinant Toll homuLogni can subsequently he islated and purified by removing the yinst cells from the fennentation medium by centrifugation and then oncentrating the Medium wingr Selected cartridge ifiters The concentrate containing the* TuB homolgkze may further be purifie using seleted olun dnummtgaphy remanA.

EXAMPLE 8 Tflu following method describe recombinant expression ofPFROMB, PR02l6 and CToll homologu4) in Daculovir ne infected insocteelle.

The Toll homiologue coding sequwnce is fiud upstrem of an epitepe, tag contained with a 4aculovirus expresmon vector. Such epitape tVg icude poWy-his ta4s ad imnmunoglobulin tags (like Ae regions of IgG). A variety of plannid. ma" be employed.h Incudng plaamidas derived from cminercielly available plmmmide such as pYLISG3 (Novagen). Briely, the Toll homologiz coding muncefrCi e desired portion of the coding sequmnc %wuch as the sequence encoding the extratelkula doinaiz) Pa auplied by 2CR with primers complementary to the V rid 8'regions. The 51 primer may inorporate flanking (saeeted) resftriction enzyme sites. The product is then digested with thous selcted restriction mnzymes and muboloned iatc IE the expi-esiort vector.

Recombinant baoultwini is generated by vc-tranfectin the above plammi and BaculoGol 9 7M virus DNA (Pharmnigen) ito Spod~ptera fnqiperda ("5MD cells (ATPCC CHlL 1711) usfng lipofecutin (commercially available from GIBCO-flL). Altr 4 -5 day. of incubation at 280C. tho released viruse are harvestd and used for further ampliictions. Viral infectio and protein expressio is performed as deiscribed by fVfeitley et al., Baculariras expressin vectort. A labora tory Manupl, Ohxod Oxford University Press (1904).

Expressd poly-hia tagged Toll honmoog can then be purified, for exampie, by Ni-chelate affinity ahromatogrphy as follows. Extracets are prepared from recombinma viru-infecte MS cels as described by Rupei-t at al.. N&aM.1 2B:76-l1D (199a). Brefly, M1 cells are wauhed, resuspended in isonication buffer (2b at Hopes, pH 7.B; 12,5 r=M Mg01 2 0.1 mM EDTM; 10% Glycero 0. 1% NP-40; 0.4 M KOI, and sordcated twice for 20 aneond on in- The sonicatee are cleared by centrifugatloat, and the supernatant is dihitad 80-fold in loading buffer (80 mk plmoat, 3W mM NaC 7 10% Glycerol. pK 7.8) and filtered trough A 0.45,um filter. A N] 2 t NTA agarcefe cohumm (conneaerzly available froam Qiae) iw prepared with a bW volume of 5 ml., washed with 26 mL of water and equilibrate with 25 tnt of loading buffer.

The filtered cell extract ii loaded onto the column at 0.5 nmL per nilnute. The olumn is washed to letelliie A28 0 witL loai buffer, at which point frstion collection is started Next, the calumn ia washed with a secondary wash buffer (50 mM phosphate; 800 aM N&aI Glycerol, pH which eluteg nonspecifically bound protein, After reaching A baseh"line again Urns column is devekoped with a aI to 800 InM Imidazuis gradient in the secondary wash bxfMer. One niL fractions are aollocte and anatlyzed by SDS-?AGS and silver staining or western blot with Ni 2 t NTA-conmugated to alkaline phollphatass (Qiagenj Fractins octuining the eluted 11s 10 -tgged PM1285 are pooled .nd iaywed against loding buffer.

IP1164112 Alternaively, purification oflde IgG tonged (or Pc taged) ToEl homnologue can be perfomed using known chromatography techniques, including for inAutnes, Protein A or protein a column chromatography.

EXAUM 9 As the Toll proteins signal through the NF-xB pathway, their biological activity emn be teste in an NP-KB assay. In this assay Jurkat cells are transietly fransfeetd using Upofecta min reagent (Gibes ER111 according to the manufacturee. instructions. bIgt pB2XLuc platid, containing NP-xB-driven luciferase gem is conkeansfixted with lgg ~pBRNmi tO vector with or without the uacit encoding PR0285 or FROM.8 For a ponitive ontrol cells ame fted with FMA (phorbol myristyl nwtter 20 ngll and PHAl (phytohem~glutnu, 2PgOnl) for three to four hours. Cells ae Lysd 2cor 3 dAys later for menemarem ant of luciferam. activity usi~ng reagnt. from Promega.

EXAMPLEM1 B PO FQ 'WI This example illustr-ates preparation of monoclonal antibodie whici -cam speifically bind PROM28, PR102850or PROS58 (Toll homAoluesl Techniques for pradumng the nionoconal antibodis are known in the art and are downribed, for instance in Goding. snag. Immunogens that way he emph*yed include purified Toll homologues, fusion proteins containing thea deewl Toll hozolgue, and cell exprosung recombinant Toll hamalogues an the cel surface. Selection of the immunogp csr4 be made by the skcill-ed artisa without undue experimentatbn.

Mice, such as Balbk 1 are innnizad with the Toll homologu imumunogor emulsifid in complete Frend. adjtznnt and inected uubcutaneoudly or inkrapertonemlly in an amount from 1- 100 micrograma. Alternatively, the Imuzegmn is emulsified in MFL-TDM adjuvant (Rihi Immunchernicial Research, Hamilton, MT) and injected into the omanl' shind foot pads. The immunized ice are then boosted 10 to 12 clays later with additiona iinmuncgs emuluifld in the selectd adjuvant. Thereafter, for several weeks, the mice may also be boasted with -adtional immunization injections. Serum samples may he periodically obtained from the mice by retro-orbital bleeding for testng in ELISA amayu to detec FW1285 svtijbe.

After a suitable, antibody titer has been detected the antimuls Rpste for antibodies can be injeeted with a final intravenous je~fcof ea Toll homologze. Three to kwu day. later, the mica are sacrificed and the spleen oe are harmeted. The spleen cells are then fted (using M5 polyethylene glycol to a selected murine mycloma cell line such a.

PSXBgU. available from ATC. No. (fL 1597T The fusios generate hybridoma cells which tan themr be plated in 95 well tissu culture plate ontaining EAT (hyponntLhue, aminopterin, ad thynhdine) nedium th inhiibit proliferatio Of Dnn-fused ell myeoama hybrds, and spleen cell hybrid&.

I PIt54R2 'The hybridonta cobh will be meed in an HLIS fa reacthity against the Ce*wnding Tel homooue. Determination of "pouitiven hybridoma oa secreting the desired monoelonl antibodies aganit a Toll hornologfue ai within thg W in the ard.

The positive hybridoma cell can be injected intraperitoeaLly into syngeneic Baib/c mic to produce incites containg the anti-Toll komobgue monodona anibodi.

Alternatively, the hybridoma cells can be grown in tissue culture faks or roller bottles.

Purifaion of the monoeonAl ahdbcdies produced in the urnitos can be accomplished usin ammonlunm suitte precipitation, follwed by gel excluion chrmatography. Alternav*l, affinity chromatography based upon binding of antibody to protein A or protein G enm be employed& Msehnds Rmgenie [f]Lebeled unlabeed 7 LCD2 "ndS. minestRSO5JPS wor from List Biochenicals (Campbell, CA) and .ll other Li'S were from Sigma Chemical Co. (HL Louis, MO). LP wan suppled as conitioned media from 293S cons. tjranfted With a butas LBP express OD vector. The TLlt2Jc fint protein wan produced by baculovirs isyata, and purified as described. Mark it at. L.JioL Chrnn 4 2M, 10720-10728 (1994).

Consructin of Expression Plan ais A cDNA encoding human TIA2 wrao clond from human r Ma lug irary. The predicted amino acid siequenc matched that of the previously published sequence Rock et 4L, supra), with the exeptaon of a to map substitution at aimino acid 726. The amino acid ternAnl epitope tag version of ThR2 (dG.TLIU) was constructed by adding an XhoI resturit mie immediately 'upsteam of lennine at position 17 (the first amino acid of thr predicted mature form o(TLR2) and liking this to amino aids 1-68 of herpes simplex virus type I glyoprotei D) as described. Mark et aL, supra PME produats wert sequenoed and suboloned into a mammalian exprwssio vector that contins the puwrmycin res~istance geneo. C-terminmi truncation variants .f gD.TLR2 w"r constucted by digestion of thie eDNA at either a Bipi (vadrat AX1) or Mil (variant A2) site prent in the *Wding soquenoc of the intracellular domain and at at Not! site preent in the 3' polyheker of the expressio 3D vecr followed by ligation of ollgoucoedde linkers.

Al1: 5W-TCA GM2 CYFA AGC-8 (REQ MI NO: 18) anid COGO TTA CCXI C-8' (SEQ ID NO: A2: 5'-TAA OT TAA CG-X (SEQ ED) NO: 20) and W.GC C TTM AOC rrA ThC A-a 4 (SBQ ED NO! 21).

The CD4ffrLR2 chimera was onstrtd by FOR and contained atmin acds 1- 205 (the sigoni peplide pnd two immunoglobulin-lik. domains) of human CD4 fused to amino acids 508-704 (the franmmmbrane and intracellular domain) of human ThUS with a linker.

encoded vuline at the junction of the CIDt and ThEL2 aequewaf The pGL.EIAMtk reporter plasmd contained the sequence PLI1dR2 5r4-3rfAUC TIGA CAT CATT rr T fT~ AAO C!AT COT (lA TAT TCC CGGG (AAGT T" TM ATO OCA TI'S 0W AT? TOO TOT flTA OAT CIO aai COO TOO Cfl GTC CAC TrC UCA TAT TAA GilT GAO GCC TGT 000 OTO GA&A CAC MiA GM AG0O C' AG COA CCC OCA AGOC TI-8r (SEDQ MD NO! 22), inserted betwee the Sal and Indifl nit. of the luelferass rqwvtd plasmld pGtS (Promg).

The C-terminal epitiops tug verson ofCLBP (LBP-FLAG) was constructed by POR trough the addition of an Aec 1 site in place of the tti slop nodon and the suheloning of this fragment into pliS-FLAG resulting in the. C-terminal addition of amino adids (IRA DYK DI)] DE (SEQ 7D NO: 28).

Stable ceU lnft/pol 293 1humant embryonic kidney cells were grown in LGDMEMIHAW. f12 (SOhif) media supplemented with 10% flS, 2 mM glutamine, and penicillin/streptomycin For stable ezpression of gD.TLR2, cells were trsnsfeated with the gD.TL 4 RI expression vector and selected for puromyein resistance at a final concentration ofI ttnfl A stable po of calla (23-TIA2 pop 1) wan isolated by FACS using an antibody to the gD 165 tag, Both the pool and the uiirgls cmUl clone (293-TLt2 clone 1) were characemrzd by FACE ard western blot analyses as descrbed in Mark el at, suspna.

Lutiferan rmpofler assay and eletrophorgi inob~ty ishif awsay (EMA)W 29332 pergntal or stable celIE (2 x I-CD 5 cells per well) were seeded inato six-well pla and transfected on the Mlowing day with the -expressi-on Plasmid. together with 0h5 p~g of the luciferase reporter plasmidpGUL.-RLAMLk and 0.05 p~g of the &nilla luciferase reported vector as an interal ontrol. After 24 hours 1 cells were treated with either TIPS 1 LBP or both LPS and LISP and reporter gene actvity was measured Datae exprwessed as relative luciferane motivity by dividing firefly ludfersae activty with thatt of Rn$Ue luciferase. For EMA nclear extract were prepared and used in a DNA-binding reaction with a 2 Pj-radiohabelled oligonucleotidea containng a consams NP-icB binding site (Sunta Cruz Biotechnology, sc-251 1).

The idenity of NF-xfl in the complex wiea confirmed by uupenhift with antibodies to NF~cE (data not shown).

RA expresion The tissue norther blt wan purchased from Oluntech and hybridized with a probe encompaning the extracellular domain of TLU± Polyadenylatad S0 mitlA was iMoated from M9 cells or 293-TER2 cal and Norther hintm wa r ite with human 11-8 cONA fragment. TLR2 espreesic wa detuiminad usig quntitmtrve POR tumin rea time &taqnmnl* techm~ogy mad analyzed an Model 770 Sequence Detetor (Applied Biospatems, Forward and reverse primers, 5"-GOG OA AGO ATD 'DPG GOT AA-Sr SEQ MD NO 24, anmd CCC AAC TAG AGA AAG ACT GOT C-31 SEQ MD NO: wore used witha a hybridhation probe, r- njA GAG GUI OA TAA ACT CT AUG TIC COA TAT-a' SEQ ED NO: 26 P1154R2 labeld on the 5' uueotide, with a reporter dye FYAX hhd tb. a' nucleoti with a qusirhin dye TMIRA. Maaophagemaoncytms were treated 16 h with 1 It"m of LI'S.

certowr binding OEMa To determine the diret binding, 20 Wi of 13MJ 1LP was mixed with 600 ng of TLE2-Fc in 100 #1 ofT binding bufifer (150 raM tied 20 mM Hepes, 0.08% RSA) containiing 15 ptl pratein A eephare... After Sb-incubation at room temperature, protein A aepharose samples were washed twice with cold PBS/0.1% NP-40 and resuspendend in binding buffer including 1% $DS end 25 mM HIMA, and counted.

in Dromqhaiia tb. Toll receptor wi requred for embryoni dorac-venfral pattern formation and aso participated in an anti~ual immune response in the adult kl. Bhlvin na Anderson, Ann. Rey. Cell. Bid. 1L 89S*416 (1906): Lemaltre gi a4,Qa 2u 169n-983 (LOW,) Toll is a type! Itransniimbrane protein contuainng an extracalkilar domain with multiple ]eucine-rich repeats (IU) Ra a cytophamic domain with sequence homology to the itrleuk-in recEptor (EL-IR), and several plant dismas-reaistance protein. Activation of Toll leads to induction of gemies through the acetivation of the NVP-ic pathway. Ao noted before, there are severa human honiologues that have been cloned, aoms of which are dismlosed as novel protein. in the press* application. These human proteins minror the topographic structure of their Drosophila counterpart. Overexpresslo of a constitutivsly active mutant of one humn TEAR (TLR4) has been showgn t* lead to thie acivtion ofrNF-cB and induction of the inflammatory cytokins end constimulatory molecules QMddhitov ei al., and Rock f aL, sup ni.), T examine if h="a flR.s mght he involved. in U'S-induced call activation, we firat investigated the expression of TEaR. in a variety or iwtmune tissuee. One of the TlEl.

T~LRS, was found to be expressed in all byinhcoid tinsme examined with the highest expression in peripheral bluod leaukcytas (Figure 5a). llxpresbin of TLR2 is enrched i 2Z mnoaaznaariphAges, the pimzary CDlt-expreuing and LPS-responsfive cells. [nterestingly, tUM is up-regulated upon stimulation of isolated ma aghacopoes with Li'S (Fire similar to what has been reported for CT) 14 (hatauuraet ao., Bur,. Titimmunol. SL 1663-166b D02]; Crouton es a. gLBilflOXi IM, 16514-16517(15T Tis result promp~d um ta deernino., W TLR2 is involved in LPB-niediated cellular signaling. We engineered hmumn embryonic kidney 293 eqils to express a versio of TERS (gD-TLR2 ontaiin an amn-emnlepitope-tag. A stabie poo1 of clanes me well a n individual clope we.. isolated and shown to express a novel protein of about 105 kD;t (Figure 6b), conustoat with tre predicted sigm of TLkL2 89 kDa) and the presnco of 4 potntal ites for N-inlW glyvcsylation. We exzamined the response of 298 or 203-TLR2 culls and IP by 38 meawming the expression efa reported gone driven by the NY-xS responsive =hiarmer of The E-aolcti gene (Cre~es ak, SWru). While neither LI'S nor LUP treatment alone resuted in .ignilcst gone mctkvation, addition of both LI'S and LAB? resulted in substantial inducton Of reporter gene activity in cells expreaging TLtS> but not in contol 203 cll. (Figure Wa.

rurahomors, usin an sctohrlcmobty shift nmy (ENMA), weD ound that LPS, in I 1154fl2 combination with LBP 1 induced NK-xD activity ina ThUS exunwing cell (*isure 6c). The kinetic Of 1LPS-inDuced NF-XD activty in. 2fl.TLR2 celta truhied flat of inysloid and non myslold cells (Delude staU, L.BilChem, 2& 222M32220 11M)43: Lee PA al., Prue,4$jLd Acad fki. UA i 993049834 [19981) in that nuclear lolleatOn of NF.4 is maximal witin S 80 minutes following expoisure to U'S. Ativation of NV-rxE by [YSILBP in 293TLR2 cels do" not require de nave protein synthas Onc preteatment w.ith cyoloheximide (Figume GC) -or actincunycin D (not shown) doe. not inhibit NF-iD aativation.

Both the membrane-boud form of C014 (mOD14), which is preset on myeloid cels, and soluble CDV14 (ACMl4) which is present in plasm (RazIl es at.. Eunidw=MsuL JA 1685Mh IBD have been shown to wibhame they Tsnciveneof cells to LP& We obwwrve that 293 cells gexres little or no 01D14 on their aurbc (data not shown). However, transient twngectio of 293 cells which mOD 14 increased the sensitivity and. magnitude of TLR2mediated LPS reap onslyones (Fgure 6d).

The diata presented ambove suggested that TL412 might -funonm ass aignaling tandume for LI'S. To examine th. role of the intxaceliular omain cit TLX2 in mediating the LPS response, we detemi~ned if Th7R2 variant, with C-terminal tvmunctin Cf either 13 CILR Al1) or 141 amino acids (TLRZ-2) could regulate the KUAM reporter in transiently transfec ted 293 cale.p We observed that both C-terminal trcation variants were defective for aictivation of the reporter gene although we could detect expression of these receptors at the cells sufce by FACS analysis (not shown) and by Westrn blot (Fgure 7c). The regio or the intracellular domaxin delete in TLR2-*AI bears striking similarity to a region of the IL-lB intracellular domain that is required fro Aaociation with tha lIE Iasociated kinase lEAK (Croutonv Ota, supre) (Figure We also demonstrated that the axtraceliular domain of TLR2 is requred for LFS-niiponeene in that a ThB2 variant in which the LMD of TiltS waB replaced with a portion of the BCD) of 0114 also failed to respod to LPS (Figure 7a and 7b), LPS in a complex glycolipid onsising of the proximal hydrophobic lipid A nwiety the distal hydrwphihc 0-antign polysecaharids region and the core oovxaiett joins lipi A and 06antige strcures. kn contrast to the lipid A portion, there is a comMeawbla diversity in tbe 0-antige uturea from different Gram-negative bactera. Lipid A is requied for LPS responsMs and treatments that remove the raty acid side ejains ot lipi A inactivate UPS. We ozpn-d the potenc of bPS proposed. from various Gram-negative bacteria, a. well as LPS which Lhad been 9atoxified" by alkaline hydrolysis. We observed that LPS isolated igvi EU~whEia coU i erotype LOD25 Wats nearl two orders of tuagnitude more potent that the Oeroogcaly distinct .Emweihia coi 055:B5 UPS for activatin TUR2 (Figure 8a). bPS prepared from mbtneata R595 LPS h. als a potent inducer of TLJI2 activity, while T~lt! failed to respond to -detoxied bPS'.

We examined. if flitS bind. UPS by determining if. soluble form of the TLRt2 acullular domain (2732Fc) bound 3 H-Ltled LPS in an sn vatr assy. We obseved that 3 H-LCDIS LPS bound the fiBS-P fusion pnotin, but did not bhi either Fe alonak or fiusio PI 154Ri proteins containing the ED] of aeveral othier receptor, (Figre Sb). This binding was specifically compete with col 14C025 IFS but wot with detoxifed UPS. Preliminary aziiayui of the binding of LI'S to TLR2-Fe suggest that the Ed is relatively low (50047W aM and that the kinetics of bindig are very slow (data not Whown). We speculate that otherr protehn, such as ISP, might set to cuhaos the Waudin rLPS t TiRm in tiva 1 much like IMP acts to transfe IM from its: fre aggregated (iellar form) to OD14. This is consistent with owr in vu results .howin that LII? is required to obtain a mensitive rspoaa of TLR2 to LI'S (Piguo SOa), ELS treatment of macrophags lWad to expression of a number of inflammaor cytokines. Similorly expression of TLHS in 293 cells re sulted in ak >10 tol d-induction of LbA t0 uiRNA in response to LPSJLP, while detoxified IS iis inactive in this assay (Figure 9).

These data sfuggest that TUCZ plays a sentinel role in the innate immune response, the first line of idefense againkst microbial pathog-ons. TL11 and 01D14 are both expressed on myeloid calls, and their inductioia is coordinately induced upon [PS treatment, Expressin of TLR2 in noa-inyelid cells confers LIPS responsveness to normally non- rosponive cths by it mechanism that is dependent on LBP and is enhalneed by the expression -of mOD 14 LX'S treatment of T.LR2 expressng cells results in activation of N-F-xB =nd aubsequntn induction.

of genes that initiate the adaptive resfponse such as 1L4 (Figure Our data suggest that TLR2 participates in both sensing the presnce of LI'S and tranamitting this information across the pNlama membrane becae intact dxtracellular and intracellular domains are required for UPS reeponsos Moreovr at region in thie C-ternaul taEl of ThUS that has homology to a portion of theshiLR that ia required for. association with lMEAs nocessary for NV-rM activatlont Drosophila TbU and the Toll related-receptor 1a Wheele play and important role int he induction of antimicrbial peptideg in rsfponse to bacteria and fung, respectively.

Medzhitovai at, supi-a Genei dAa han implicated Spitzle as a ligand for Toll in darsoentra] patterning and has led to speculation that a homnologue of Spitzle might he important for regfulatio of human TLP& in the immune response. Our obseintions that activation of TLR2 by LI'S is not blacked by vcycloheximide and that the extrac~lluar -domafin of flRt is a low affimity receptor for LI'S in vitr is consistent with a model in whic TLR2 participated in LI'S recognition. Ouir dlata does not exclude the possbility tat other protei (such as A spitzle humulupw) may modify the response of ThR2 to LI'S. We note that whi e straUlular doma of TLR2 and Droaophila Tol] both ontain ER~s 1 they share ls than M0 amino "ci identity.

Secondly, LER protein are Fatten Recognitio Receptor (Flit) for a variety of types i4 molecules, such..s proteins, peptde.m, and carbohydrates. Da&4iel at, Q&iL 17-24 (19D97).

Thirdly. the requirement for Spitee in the Drosophila immune response is lesscdear than that Of ToiL Unlike defects in TOL Spittle mutant. induce normal levela of the antimicrobial peptidft flefemain and Atacein aind are only partially defective in Cecoin A expression follwing funga] challenge 1 and are not defcive in activatin of DrsaJ in response t'0 lgurY.

Lensaitre a at, 0gl ft 97U-983(IM9), Lemaite i ENE"j JA, aae-55 (1905).

Pi1 54Al2 Ain noted before, TlRt2 ius a member of a large tasil of human Toil-related receptors, incuding dthee novel receptor. (encoded by DNAIooSi, DNA42SB, and DNA473GI, respecively) specfically isclosd in the paunt .ppliaion. The data rsented in this5 exmple its well..s evidence for the Involvewmt of TLR4 in activation of F-rx bresponsive genes, auggast that a. primEary function of ths family of receptors; is to act ae pathogeOn pattrn ropiffition receptor sensing thie presenme of conserved molecular stuctres present on mrobes 6 OriiAfly suggested by Janeway and collsruea (Mecditov el uL, wtyrn).

The human TiLt famly way be target. for therapeutic strategies for the treatment of septic EXAMPLE 12 InAsib hybridi2at~i ha powerfu and versatile technique far the detecton and localization of nucleic ad eequenoeu within ell or tissue preparsdons. it may be usefil for ennpie. to identify sites of gene .xprmion analyze the tissue distribution of trbiptio, idetify and localiz, viral infection tMlcw dbangms int specifc mUNA aynth enls andJ aid in chrmosome mapping.

In situ hybridization was performed folowing =n OPtiumized verion cf thea protocol by Lu an Gfilk ell V.ian 1: 169-176 (1994, using PORgenerited 3 FP.abele ribaprobos. Briefly, forialindlxed, paramfn-emb~ded human tissues were sectoned deparalflniaed, deproteknated in proteinae K (20 g/nd) for 165 minutes at 37'C, and further processed for in 44wt byhrM iztion as descibed by Lu and Giflstt suprc. A 3 3 Ut-J.abeled audsn.. riboprobe was generated from POR prOdU4C and hybridized at 58C5C overnightL The slides were dipped in Kodak ?fl'21nuclear track emtdsion and exposed for A wews., 6.0 VI (12S moi) of 3 3 P-UTp (Amersbai 1002. SAc2000 Cl/mind) wee speed noe drietdL To emeb tube ontainingf dried 3 3 P-UTP, the folloing ingredient. were added: jil fx transcription buffer (100inM) pd NTW mix (2.5 mM: 10 p, each of 10 mU GTE', CIT ATP-i+ 10 0 H 2

O)

1L0pi UTF (O pM) pI RDuad pI DNA template (lgg) PIl 120 A RNA palymerase (for PM~ products T8 AS. T7 w8, usually) Thes tubes were inmubated at SI*C fr hOurb. 1.0 p] RQ1 DNmee were added, followed by incubationl at 37S C for 35 minutesa. DO Ai TE (10 mU Trim pH 74'lwmM EDTA pH were added& and th. mixture was petted onto DEAL paper. The remaining moluticn 'was loaded in a Microooa-50 ultrafitration unit, and spu using program 10 (8 minute). The P11E4R2 filtratio unit was inverted over at second tube and spcn uuing program 2 (8 minutes). After the fine! rwezy spin. lOD p1 IT wae added. I Id of the Anal prodzC was pipetted on DE&i pa and Counted in aMIl of Biofluor [H.

The probe was run on at TEE/urea gA. 14-3 of the probe or 5 S iof RNA Aft M~ ntre added to 3 plof lading buffer. After hteating -on a 95C heat blook for three minute, the ge] wan immediately plated on lce. The wells of gel were flushed, the isample loaded, and run at 180-250 volts for 45 minute.. The gel was wrapped in saran wrap and exposed to XAR film with an intbnsifying een iW -70 freezer one hoaxr to overnight.

IPreuia~ment afrm sealions The slide. were removed fr-om the freezer, planed on aluminium tr-ays and thawed at room temperature for 5S minutes. The tray. were placed in 0 incubator for five minutes to reduce condensatin. The 'lidsa were fixed for W- minuats in 4% pararormaldehyd on ice in the fame hood, and nashed in 0.5 xSSC for 5 minute, at room temperature (25 ml 20 x SSC +9T Mi m)Q H 2 After depraoteination in 0.5 pg/al proteins. K for 10minutes at 7rc (It&1 of 10 ifelsto~tck in 250 ml prewarmedflNase~fre RNAse buffa4) the sectorwa were washed in 0.5 x SSC far 10 minutes at room temperature. The sections were dehydrated in 7D%, 95%, 100r% ethanol. 2 minutes eack PtetreWdmtnl of paniffis-m bedded sections The slides wer deparafflnied, placed in SQ RI2O, and rinsed twioo in 2Zx 88G at room temperature, for 5 midnutes each time.

Thehotionwere de tdeitAin 2Ogmlproteie K Iofa0mgmin 25 ml i~as fEme RMaae huff-er; 37'0, 15 minutesw burnn embryo,, or 8Bx protoinase K (100 Al in 2150 nil Ram. buffr, 37'C, 30 minutes) farmalin tissues, Subsequent rinsing in 0,6 z 550 and dehydration were performed as doseeibed above.

Prehybridization The slide. were laid out in plastic box lined with Box 26 buffer (4 x SSC, M0% formamide) saturated fiter paper, The tissus wa~s covered with 50 pi of hybridization buffer (3.75g Dortran SWlfte 6 ml SQ H120). vortexed and heated in the micrwave fig 2 minutes with the cap loosened. After ooling on ice. 18. 75 'at foramide, 3.75 ml 20rz SSC and 9.ml SQ H2 were added, the tissue was vortexed well, and incubated at, 42C for 1-4 hours ffybrdfration l1r O6 0 pa probe and 1.0 Fil RNA0(0 mWg/msok) per slide were boated at W50 for 8 mainutes. The slides were cooled manus and 48 pI~ hybridizatin bufe wereadded persUli After vortaxing, 5 p 33 p mix were added to 50 jd prahybridintion on slide The slides were incubated overnight at 56C.

Wasfhes Washing wpm done 2:1 m' oinute. wit UBfSO, J UA at room temperature (40D ml 20 x R80 16 ml O.25M EPTA, Vf=4L), folowed b6y B~aaA treatment at 8SIT for 30 minutes (SWOE1 lot 10 mg/ni in 250 mil Rn buffer 20 pg/ni), Thea s~ide. were waihed 2Yr10 mainute. with 2 x SSC, EDTA at room temperature. The striagency wak P115432 conditions were am follows: 2 hour. at 55'O 1 0. 1 x SSC, EIUA (2O ml 20 x580C+ 16 =1 EDTA, The expression pattern of PR0285 (DNA4X)2 1) in human adult and fetal tissues was examined. The following probes ware used, .ynthueuized bused upon thB full-length DNA4IO21 eequencet Oligo I~ -GA TIC TAA TAC GAC TCA CTA TAG GGC AAM CTc TOO OCT OTO ATO TOS (8XQWMNO:$2,7 Oligo OVA TOA AAT TAALCCC TOA CTA AAG O-GA AMO AG OC 0.4 AIM ACT TAG (SEQ ID NO. 28) In thLis. experiment low levels of expression. were obmerred in thes placenta and ov~er homatopojedo cells in the moupe feta liver. No expression was detacted in either human &Wu1 adult or cimip lymph nde and no expression was detected i hwinan feta or human adult spleen. These data are no fulfly consistent with Northernblot or FOR data, probably due to the Lack or sensitivity in the in sffu hybri~ditto assay. It is possible that further tissue. would show same expression under more nensitive conditions.

ENO Meu (pNA4SGI The expreuion patien of PR358 (DNA4VSOI) inhwinn adult and fetal inues wa.R examined. The following probes were usyed, syndieslzed based oipon the fulli-length flNA47361 uequsncie Oligo 1±00.4A TTC TAA TAC GAO TCA CPA TAG COO TOO CAA TALA ACT GOA G;AO ACT {SEQ In O-29) Oligo 2: CPA lilA AIT TAA COO TCA CIA AAO UGlA TUG AGT TOT TOT TGG GTT GTT (EQ ID NO- In thiasexpmhnesnt ~rwk we foud i= gut-amandated lymphoid tisue and developing splenic white pulp in the fetus low levels apreeuin wason m in te pALS rgion oflnornml adult speen. Although &U1 other tissues were tteptve. it is possile that low levels of expression could be oasned in other tissue Lypes under more sensitive condition.

Tb. "*wing materals En.e Won depsted with the American Type Cultu= Collction, 12301 Parklawn Drive, Rockvill, MP, USA (ATCO)! Matidd AITQ emN. agi Pl DNA40021-1154 20938S Octber 17, 1997 (wmcofrg POBO P1 154R2 DNA42663-l114 20938 October 17, 1991? (encoding PR0286 DNA47361-1249 20943 November 7, 1 97 This deposit was made =nder the provsios of the Budapest Treaty on the International Reognition of the Depowit ofivrplm for the Purpose of Patent Procedure and the Reguwlton theeuder (Buiapeat Treaty). Thl. assure iitenance of a viable culture of the depost for So ya humn the date of deposit. The deposit will be made avOail by ATCC under the terms of the Budapat Treaty, tand subject to gm agreement between tiensutech, Inc, and ATCC, which assumee pftramet and unronirictad availbiity af the progeny of the culture of the deposit to the public upon issni of the paetient U"S. patent or upon Laying open to the publi of any US. or foreign patent Application, whichever cornea fti and swures availability of the progeny to one deterniined by the U.S. Conunisaioner of Patents and Trademark@ to be entitled thereto according to 85 USC 1112 and th s Comcniuuonr 4 rules pursuant thereto (inaluding 37 CF'R 1.14 with particuarx reference to W6SO 638), The aignee of the prement application has agreed that if a c'4tuxe of the matterials on deposit sh~ould Aie or be lost or destoed when cultivated under- suitable conditions, the inatarials will be promptly replaced on notification with another of then same.

Availailty of the deposfited materal in not to be consti-uwi a a licene to practice tie invention in tokbravenfion of the rights granted under the Puthority of any gorvernment in accordance with its patmntilaws, The foregoing written specifiction is considered to be sufficient to enable one skiled wn the art to practie the invention. The preent invention is not to be limited in scope by the consrucmt depoisited, sinoe the deposite embodiment is intended mu a single illustration of certain aiect of the lnveniwtaM my constructs that mrs fwxtdnaiy equyivn arm within the ax"p af this invetion. The deposit of mterAtl herein does not constittem admission that the written description harein containd is inadeayate to enbie the practice of any spect of the invention 1 inaluding the beet mode thereoL nor is it to be construed as limiting the aoope of the clairm to the aped& illusftration that It represents, hndeed, varoua modification Of the invention in addiio to &ose shown and describe herein will become apparent to those skille in the art from the foregoing description. and fal withiin the s"oe of the appended clis.

The entEM disclosure in the omplete specfication of ouw Awtalian patent Application NW.10703/99 is by this crau.-refu~nce incorpmored into the prcwnt specification.

Claims (28)

1. An isolated nucleic acid comprising a polynucleotide sequence having at least a sequence identity to a DNA molecule encoding a PRO285 polypeptide having amino acid residues 27 to 839 of Fig. I (SEQ ID NO:1); or the complement of the DNA molecule of(a).

2. An isolated nucleic acid according to claim 1, comprising DNA having at least a sequence identity to a DNA molecule encoding a PRO285 polypeptide having amino acid residues 1 to 839 of Fig. I (SEQ ID NO: or the complement of the DNA molecule of(a).

3. An isolated nucleic acid according to claim I or claim 2, comprising DNA encoding a PR0285 polypeptide having amino acid residues I to 839 of Fig. 1 (SEQ ID NO: I).

4. An isolated nucleic acid according to claim I or claim 2, comprising DNA encoding a PRO285 polypeptide having amino acid residues I to 1049 of Fig. I (SEQ ID NO: 1). An isolated nucleic acid according to claim 1, comprising DNA encoding a PR0285 polypeptide having amino acid residues 1 to 839 and 865 to 1049 of Fig. I (SEQ ID NO: 1).

6. An nucleic acid according to claim I, wherein said DNA comprises the nucleotide sequence starting at nucleotide position 85 of Figure 2 (the sequence of SEQ ID NO:2), or its complement.

7. An isolated nucleic acid comprising DNA having at least a 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the human Toll protein cDNA in ATCC deposit No. 209389 (DNA40021-1154), or the complement of the DNA molecule of

8. A nucleic acid according to any one of claims I to 7, wherein the polypeptide has the ability to induce activation of NF-KB.

9. A vector comprising a nucleic acid according to any one of claims I to 8. A vector according to claim 9, operably linked to control sequences recognized by a 2 5 host cell transformed with the vector. II. A host cell comprising the vector according to claim 9 or claim

12. A host cell according to claim I wherein said cell is a CHO cell.

13. A host cell according to claim I, wherein said cell is an E. coli.

14. A host cell according to claim 1 wherein said cell is a yeast cell.

15. A process for producing a Toll polypeptide, comprising culturing a host cell according to any one of claims I 1 to 14 under conditions suitable for expression of a polypeptide encoded by a nucleic acid according to any one of claims I to 8, and recovering said polypeptide.

16. An isolated polypeptide having at least 80% sequence identity with the amino acid sequence set out in Figure 1 (SEQ ID NO: wherein the polypeptide has the ability to induce the activation of NF-KB.

17. A polypeptide according to claim 16, having amino acid residues I to 839 of Figure 1 (SEQ ID NO: 1). 57 N:\Melbourne\Case\Patent\37000-3799\P37785 AU I\Specia\2003203679 amended speci pages doc 14/03/07

18. A polypeptide according to claim 16, having amino acid residues 1 to 1049 of Figure 1 (SEQ ID NO: 1). S 19. A polypeptide according to claim 16, having amino acid residues 1 to 839 and 865 to 1049 of Figure 1 (SEQ ID NO: 1).

20. A polypeptide according to claim 16, wherein the polypeptide is encoded by the nucleotide sequence starting at nucleotide position 85 of Figure 2 (SEQ ID NO: 2).

21. A polypeptide according to any one of claims 16 to 20, wherein the polypeptide has the k\ ability to induce the activation of NF-KB. M 22. A chimeric molecule comprising a PR0285 polypeptide according to any one of claims ci 10 16 to 21 or a transmembrane-domain deleted or inactivated variant thereof, fused to a heterologous amino C acid sequence. C 23. A chimeric molecule according to claim 22, wherein said heterologous amino acid sequence is an epitope tag sequence.

24. A chimeric molecule according to claim 22, wherein said heterologous amino acid sequence is an Fc region of an immunoglobulin. An antibody which binds specifically to a polypeptide according to any one of claims 16 to 21.

26. An antibody according to claim 25, which binds specifically to a polypeptide having the amino acid sequence set out in Figure I (SEQ ID NO: 1).

27. An antibody according to claim 25, which binds specifically to a polypeptide encoded by DNA 40021.

28. An antibody according to any one of claims 25 to 27, wherein said antibody is a monoclonal antibody.

29. An antibody according to any one of claims 25 to 28, which is capable of blocking the recognition of a Gram-negative or Gram-positive organism by said polypeptide. A composition comprising an effective amount of an antibody according to any one of claims 25 to 29, in admixture with a pharmaceutically acceptable carrier.

31. A method of treatment of septic shock, comprising administering to a patent an effective amount of an antibody according to any one of claims 25 to 29.

32. Use of an antibody according to any one of claims 25 to 29 in the manufacture of a medicament for the treatment of septic shock.

33. An agonist of PR0285 polypeptide, selected from the group consisting of antibodies to PRO285 or fragments of said antibodies, and fragments or sequence variants of native PRO285.

34. An antagonist of PRO285 polypeptide, selected from the group consisting of antibodies to PRO285 or fragments of said antibodies, fragments or sequence variants of native PRO285, and anti- sense nucleic acid, or triple-stranded DNA specific for a nucleic acid encoding PRO285. A method of screening for agonists or antagonists of PRO285 polypeptide, comprising 58 N:\Melboune\Cases\Patent\37000-37999\P37785.AU I\Specis\2003203679 amended speci pagendoc 14/03/07 the step of assessing the ability of a candidate compound to modulate a biological activity of PR0285 polypeptide, in which mimicking or enhancing the biological activity is indicative of agonist activity, and blocking, preventing, inhibiting or neutralizing the biological activity is indicative of antagonist activity.

36. A method according to claim 35, in which the biological activity is ability to induce activation of NF-KB and/or expression of NF-KB-controlled genes for IL-I, IL-6 or IL-8.

37. A nucleic acid according to claim I, a polypeptide according to claim 16, an agonist according to claim 33, an antagonist according to claim 34, or a method according to claim substantially as herein described with reference to any one of the examples or drawings. 59