CA2263830A1

CA2263830A1 - T1 receptor-like ligand i

Info

Publication number: CA2263830A1
Application number: CA002263830A
Authority: CA
Inventors: Jian Ni; Reiner L. Gentz; Craig A. Rosen
Original assignee: Individual
Current assignee: Human Genome Sciences Inc
Priority date: 1996-08-23
Filing date: 1996-08-23
Publication date: 1998-02-26
Also published as: JP2002514051A; EP0958374A4; EP0958374A1; AU7235396A; WO1998007881A1

Abstract

The present invention concerns a novel T1R-like ligand I protein. In particular, isolated nucleic acid molecules are provided encoding the T1R-like ligand I protein. T1R-like ligand I polypeptides are also provided, as are recombinant vectors and host cells for expressing the same.

Description

W O98/07881 PCTrUS96/13777 Tl Receptor-Like Ligand I

Background of the Invenfion Field of tl2e Invenfion The present invention concerns a novel Tl receptor (TlR)-like ligand I
protein. In particular, isolated nucleic acid molecules are provided encoding the TlR-like ligand I protein. TlR-like ligand I polypeptides are also provided, as are recombinant vectors and host cells for expressing the same.

Rela~ed Art Inferleukin-l (IL-l). Interleukin-1 (IL-la and IL-1~) is a "multi~nctional" cytokine that affects nearly every cell type, and often in concert with other cytokines or small mediator molecules. (Dinarello, C.A., Blood 87:2095-2147 (March 15, 1996).) There are three members of the L-l gene farnily: IL-I a, IL-l ~, and IL- I receptor antagonist (IL-lRa). IL-1 a and IL-l ~ are agonists and IL-lRa is a specific receptor antagonist. IL-lo~ and ~ are synthesized as precursors without leader sequences. The molecular weight of each precursor is 31 kD. Processing of IL-l a or IL-l ~ to "mature" forrns of 17kD requires specific cellular proteases. In contrast, IL-1 Ra evolved with a signal peptide and is readily transported out of the cells and termed secreted IL-lRa (sIL-lRa).
n-l Recep~or and Ligands. The receptors and ligands of the IL-l pathway have been well defined (for review, see Dinarello, C.A., FASEB ~
8:1314-1325 (1994); Sims, J.E. et al., Interleukin-l signal transduction:
Advances in Cell and Molecular Biology of Membranes and Organelles, Vol. 3, JAI Press, Inc., Greenwich, CT (1994), pp. 197-222). Three ligands, IL la, IL-l ~, and IL-l receptor antagonist (IL-lra) bind three forms of IL-l receptor, an 80-kDa type I IL-l receptor (IL-lRl) (Sims, J.E. et al., Science 241:585-589 (1988)), a 68-kDa type II IL-1 receptor (IL-lRII) (McMahan, C.J. et al., ~MBO

W O ~t~7aBl PCT~US96tl3777 J. 10:2821-2832 (1991)), and a soluble form of the type II IL-lR (sIL-lRII) (Colotta, F. et al., Science 261:472-475 (1993)).
The interactions between the Il,- 1 ligands and receptors play an essential role in the stimulation and regulation of the IL-l -m~ te~1 host response to injury and infection. Cells ~ es~ g IL- 1 Rl and treated with IL- 1 a or IL- I ,B respond in several specific ways, including stimulating nuclear loc~li7~tion of the rel-related transcription factor, NF-lc~ (for review, see Thanos, D. & ~flni~ti.~, T., Cell 80:529-532 (1996)), activation of protein kinases of the mitogen-activated protein kinase superfamily that phosphorylate residue threonine 669 (Thr-669) of the epidermal growth factor receptor (EGFR) (Guy, G.R. et al., J. Biol. Chem.267: 1846-1852 (1992); Bird, T.A. et al., J Biol. Chem. 268:22861 -22870 (1991);Bird, T.A. e~ al., J. Biol. chem. 269:31836-31844 (1994)), and stimulation of transcription of the IL-8 gene (Mukaida, N. et al., ~ Biol. chem. 265:21128-21133 (1990)).
IL~ likefamily. Many proteins from diverse systems show homology to the cytoplasmic domain of the IL-lRI. This e~cr~n~lin~ IL-lRI-like farnily includes m~mm~ n proteins, Drosophila proteins, and a plant (tobacco) protein.
(Gay, N.J. & Keith, F.J., Nature 351:355-356 (1991); Hashimoto, C. et al., Cell 52:269-279 (1988); Schneider, D.S. et al., Genes & Dev. 5:797-807 (1991); Edon, E. ct al., Development 120:885-899 (1994); Mitchan, J.L. et al., J. Biol. Chem 271:5777-5782 (March 8, 1996)).
The m~mms~ n IL-lRI-like receptor family members include a murine protein MyD88 (Lord, K.A. et al., Oncogene 5: 1095- 1097 (1990)) and a human gene, rsc786 (Nomura, N. et al., DNA Res. 1:27-35 (1994)). Another murine receptor member, Tl/ST2, was previously characterized as a novel primary response gene expressed in BAL/c-3T3 cells (Klemenz, R. et al., Proc. Natl.
Acad. Sci US~ 86:5708-5712 (1989); Tomin~ S., FEBSLett. 258:301-304 (1989); Tominga, S. et al., FEBSLett. 318:83-87 (1993)). The tr~n.cmemhrane protein mulL-lR AcP (Greenfeder, S.A. et al., J. Biol. Chem. 270: 13757-13765 (1995)) has homology to both the type I and type II IL-lR. IL-lR AcP has recently been shown to increase the affinity of IL-lRI for IL-l~ and may be involved in meAi~tin~ the IL-1 response.
Tl Receptors. Tl/ST2 receptors (hereinafter, "Tl receptors"), as a memberoftheIL-l receptorfamily(Bergers,G.,etal., EMBO~ 13:1176(1994)) has various homologs in different species. In the rat, it is called Fit-1, an estrogen-inducible, c-fos-dependent transmembrane protein that shares 26% to 29% amino acid homology to the mouse IL-lRI and II, respectively, In the mouse, the Fit-l protein is called ~T2 and in the human it is called T1. The organization of the two IL- 1 receptors and the Fit- 1 /ST2/T1 genes indicates they are derived from a common ancestor (Sims, J.E., et al., Cytokine 7:483 (1995)).
Fit-l exists in two forms: a membrane form (Fit-lM) with a cytosolic domain similarly to that ofthe IL-lRI and Fit-lS, which is secreted and composed of theextracellular domain of Fit-M.
In many ways, these two forms of the Fit-1 protein are similar to those of the membrane-bound and soluble IL-lRI. It has been shown that the IL-lsRI is derived from proteolytic cleavage of the cell-bound form (Sims, J.E., et al., Cyto/ane 7:4~3 (1995)). On the other hand, the Fit-1 gene is under the control of two promoters, which results in two isoforms coding for either the membrane or soluble form of the receptor. Two RNA transcripts result from alternative RNA
splicing ofthe 3' end ofthe gene. Although IL-l,B binds weakly to Fit-1 and doesnot tr~n.~ ce a signal (Reikerstorger, A., et al., ~ Biol. Chem. 270:17645 (1995)), a chimeric receptor con~ ting of the e~ctracellular murine IL-lRI fusedto the cytosolic Fit-I tr~n~ducçs an IL-I signal (Reikerstorger, A., et al., J. Biol.
Chem. 270:17645 (1995)). The cytosolic portion of Fit-1 align with GTPase-like sequences of IL-I RI (Hopp, T.P., Protein Sci. 4: 1851 (1995)) (see below).
IL-l prod~ction in various disease states. lncreased IL-I production has been reported in patients with various viral, bacterial, fungal, and parasitic infections; intravascular coagulation; high-dose IL-2 therapy; solid tumors;
lellk~ h~im~r's rii~ç~e; HIV-1 infection; autoimmune disorders; trauma (surgery); hemodialysis; ischemic ~ e~cs (myocardial infarction); noninfectious hepatitis; ~thm~; UV radiation; closed head injury; pancreatitis; periodontitis;

W O 98/07881 PCT~US96113777 graft-versus-host ~ e~e; transplant rejection; and in healthy subjects after strenuous exercise. There is an association of increased IL-1,B production in patients with ~17heimPr's disease and a possible role for IL- 1 in the release of the amyloid precursor protein (Vasilakos, J.P., et al., FEBS Lett. 354:289 (1994)).
However, in most conditions, IL- 1 is not the only cytokine exhibiting increasedproduction and hence the specificity of the IL-l findings as related to the pathogenesis of any particular disease is l~e~ing. In various disease states, IL- 1,B
but not IL-1 a is detected in the circulation.
lL-l in T/terapy. Although IL-1 has been found to exhibit many important biological activities, it is also found to be toxic at doses that are close to therapeutic dosages. (Dinarello, C.A., Blood 87:2095-2147 (March 15, 1996)). In general, the acute toxicities of either isoform of IL-l were greater after intravenous compared with subcutaneous injection. Subcutaneous injection was associated with signi~lcant local pail, erythema, and swelling (Kitamura, T., &
Takaku, F., Exp. Med. 7:170 (1989); T ~-lghlin, M.J., ~nn. Hematol. 67:267 (1993)). Patients receiving intravenous IL-l at doses of 100 ng/kg or greater experienced significant hypotension. Patients receiving IL-1 ~ from 4 to 32 ng/kg subcutaneously, there was only one episode of hypotension at the highest dose level (I .al-ghlin, M.J., Ann. ~Iematol. 6~:267 (1993)).
Contrary to IL-1-associated myelostimulation in patients with normal marrow reserves, patients with aplastic anemia treated with 5 daily doses of IL- 1 a (30 to 100 ng/kg) had no increases in peripheral blood counts or bone marrow cellularity (Walsh, C.E., et al., Br. J. Haematol 80:106 (1992)). IL-1 has been ~lmini~tPred to patients undergoing various re~;...~..l.~; of chemotherapy to reduce the nadir of neutropenia and thrombocytopenia.
Daily tre~tm~nt with 40 ng/kg IL-la from day 0 to day 13 of autologous bone marrow or stem cells resulted in an earlier recovery of n~ul,openia (median, 12 days; P < .001) (Weisdorf, D., etal., Blood84:2044 (1994)). After 14 days of tre~tm~nt~ the bone marrow was significantly enriched with committed myeloid progenitor cells. Similar results were reported in patients with AML
receiving 50 ng/kg/d of IL-l ~ for 5 days starting at the time of transplantation W O 98/07881 PCT~US96/13777 with purged or nonpurged bone marrow (Nemunaitis, J., ef al., Blood 83:3473 (1994)). Injecting humans with low doses of either IL~ or IL- 1 ~ confirms the ples~ive pyrogenic and hypotension-inducing properties of the molecules.
Ameliorafion of Disease Using Soluble II~l Receptors. Administration S of murine IL-lsRI to mice has increased the survival of heterotopic hear allografts and reduced the hyperplastic lymph node response to allogeneic cells (Fanslow, W.C., e~ al., Science 248:739 (1990)). In a rat model of antigen-in-lul~ec~ arthritis, local instillation ofthe murine IL-lsRI reduced joint swelling and tissue destruction (Dower, S.K., el al., Therapeutic Immunol. 1:113 (1994)).These data suggest that the arnount of IL-lsRI ~mini~tered in the normal, contralateral joint was acting systemically. In a model of experimental autoimmllne encephalitits, the IL-lsRI reduced the severity of this disease (Jacobs, C.A., et al., J. Immunol. 146:2983 (1991)).

Summary of the Inven~ion The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a human T1 receptor-(TlR-)like ligand I polypeptide having the amino acid sequence in FIG. I (SEQ ID N 0:2). The TlR-like ligand I contains an open reading frame encoding a polypeptide of about 217 amino acid residues including an N-t~rmin~l methionine, a leader sequence of about 27 amino acid residues, an extraceilular mature domain of about 151 residues, a potential tr~n.~m~mhrane domain of about 23 residues and an intracellular domainof about 16 amino acid residues, and a deduced molecular weight of about 25 kDa. The 151 amino acid sequence ofthe expected mature extracellular TlR-like ligand I protein is shown in FIG. I and in SEQ ID NO:2 (residues 28-178).
In another aspect, the invention provides isolated nucleic acid molecules encoding an TlR-like ligand I having an amino acid sequence encoded by the cDNA of the clone deposited as ATCC Deposit No. 97656 on July 12,1996.
Preferably, the nucleic acid molecule will encode the mature polypeptide encodedby the above-described deposited cDNA.

The invention is further directed to nucleic acid fragments of the nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figure I (SEQ ID NO: 1) is inten(led fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are usefulas diagnostic probes and primers as discussed herein. Of course, larger fr~gm~nt~
50-738 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figure 1 (SEQ ID NO: 1). By a fragment at least 20 nt in length, for example, is intended fr~gment.~ which include 20 or more contiguous bases from the nucleotide se~uence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 (SEQ ID NO: 1). Since the gene has been deposited and the nucleotide sequence shown in Figure 1 (SEQ ID NO:1) is F~rovided, generating such DNA fragments would be routine to the skilled artis~n. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, suchfragments could be generated synthetically.
Preferred nucleic acid fragments include nucleic acid molecules which encode: a leader sequence of about 27 amino acid residues (amino acid residues from about I to about 27 in Figure 1 (SEQ ID NO:2); an extracellular mature domain of about 15 lresidues (amino acid residues from about 28 to about 178 in Figure l (SEQ ID NO:2); a tr~n.~memhrane domain of about 23 amino acids (amino acid residues from about 179 to about 201 in Figure 1 (SEQ ID NO:2);
an intracellular domain of about 16 amino acids (amino acid residues from about 202 to about 217 in Figure 1 (SEQ ID NO:2); and a polypeptide comprinsing the extracellular and intracellular domains having all or part of the transmembrane region deleted.
Further embodiments of the invention include an isolated nucleic acid molecule having a nucleotide sequence that is at least 90% identical and, more preferably, at least 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of any of the nucleic acid molecules described herein.
The present invention also relates to recombinant vectors which include the isolated nucleic acid molecules ofthe present invention, host cells cont~ining the recombinant vectors, and the production of TlR-like ligand I polypeptides orfragments thereof by recombinant techniques.
The polypeptides ofthe present invention include the polypeptide encoded by the deposited cDNA, the polypeptide of Figure 1 (SEQ ID NO:2) (in particular the mature polypeptide), as well as polypeptides having an amino acid sequence with at least 90% similarity, more preferably at least 95% similarity to the amino acid sequence of the polypeptide encoded by the deposited cDNA, the polypeptide of Figure 1 (SEQ ID NO:2), or a fragment thereof. Further polypeptides of the present invention include polypeptides having an amino acid sequence at least 80% identical, more preferably, at least 90% or 95% identical to the amino acid sequence of the polypeptide encoded by the deposited cDNA, the polypeptide of Figure 1 (SEQ ID NO:2), or a fragment thereof.
Preferred polypeptide fr~gment.c according to the present invention include a polypeptide comprising: the mature polypeptide, the extracellular domain, the transmembrane domain, the intracellular clom~in, or the extracellular and intracellular domain with all or part of the transmembrane domain deleted.
An additional embodiment of the invention relates to a polypeptide or peptide having the amino acid sequence of an epitope-bearing portion of an Tl R-like ligand I described herein. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of an Il,-1-like polypeptide of the invention include portions of such polypeptides with at least 6-30 or 9-50 arnino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described herein also are included.
In another embodiment the invention provides an isolated antibody that binds specifically to an TlR-like ligand I polypeptide having an amino acid sequence as described herein.

.

wo 98/07881 PCT~US96/13777 It is believed that biological activities of the TlR-like ligand I of the present invention are similar to the biological activities of the T1 R ligand and IL-1. Significantly, higher or lower levels of TlR-like ligand I may be detected intissues or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having a TlR ligand- or IL-l-related disorder, relativeto a "normal" TlR-like ligand I gene expression level, i.e., the expression level in tissue or bodily fluids from an individual not having the Tl R ligand- or IL- 1-related disorder. Thus, ~letecting ~ cs~ion of TlR-like ligand I gene expressionaccording to the present invention is a diagnostic marker.
In a further embodiment, the invention is related to a method for treating an individual in need of an increased or decreased level of TlR-like ligand I
activity in the body, comprising a~imini~tering to such an individual a composition comprising a TlR-like ligand I polypeptide or an inhibitor thereof.
The invention further provides methods for isolating antibodies that bind specifically to an TlR-like ligand I polypeptide having an amino acid sequence as described herein. Such antibodies may be useful diagnostically or therapeutically as described above.

Brief Descrip~ion of fhe Figures FIG. I shows the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ ID N 0:2) sequences of the TlR-like ligand I protein determined by sequencing the cDNA clone contained in ATCC Deposit No. 97656. Amino acids from about 1 to about 27 represent the signal peptide (first underlined sequence); amino acids from about 28 to about 178 the extracellular domain (sequence between the first and second underlined sequences); amino acids from about 179 to about 201 the transmembrane domain (second underlined sequence);
and amino acids from about 201 to about 217 the intracellular domain (the rem~inin~ sequence).

W O98/07881 PCTrUS9~/13777 FIG. 2 shows the regions of similarity between the amino acid sequences of the TlR-like ligand I and the protein sequence of GenBank accession No.
U41804 (SEQ ID NO:3), showing an overall 33% identity.
FIG. 3 provides an analysis of the Tl R-like ligand I amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;
Amphir~thic regions; flexible regions; antigenic index and surface probability are shown.

De~ailed Description of fhe Invention The present invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding a TlR-like ligand I protein having an amino acid sequence shown in Figure 1 (SEQ ID NO:2), which was determin.o~l by sequencing a cloned cDNA. The TlR-like ligand I protein of the present invention shares sequence homology with the TlR ligand (Figure 2).
The nucleotide sequence in FIG. 1 (SEQ ID NO:l) was obtained by sequencing the HEMEM90 clone, which was deposited on July 12, 1996 at the Arnerican Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession number 97656. The deposited clone is contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).

Nucleic Acid MolPculP~

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA
sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of peptide, polypeptides or proteins encoded by DNA molecules dPt~nnin~d herein were expected by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence det~ ed herein can contain some errors. Nucleotide sequences d~tennined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA
molecule.
The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the expected amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
Unless otherwise indicated, each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T).
However, by "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T) in the specified deoxynucleotide sequence in is replaced by the ribonucleotide uridine (U). For instance, reference to an RNA molecule having the sequence in SEQ ID NO:1 set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxynucleotide A, G or C in SEQ ID NO: I has been replaced by the corresponding ribonucleotide A, G or C, and each deoxynucleotide T has been replaced by a ribonucleotide U.
By "isolated" nucleic acid molecule(s) is int~n-1erl a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolatedDNA molecules include recombinant DNA molecules m~int~ined in heterologous host cells or purified (partially or substantially) DNA molecules in solution.

WO 9~ 8I PCT/US96/13777 Isolated RNA molecules include in vivo or in vitro RNA ~ SCliylS of the DNA
molecules of the present invention. Isolated nucleic acid molecules according tothe present invention further include such molecules produced synthetically.
Using the information provided herein, such as the nucleotide sequence in FIG. l (SEQ ID NO:1), a nucleic acid molecule of the present invention encoding an TlR-like ligand I polypeptide can be obtained using standard cloningand screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIG. I (SEQ ID NO:I) was discovered in a cDNA library derived from human endothelial tissue. Further, the gene was also found in cDNA
libraries derived from the following types of human cells: adult heart, TNF
induced amniotic cells, chondrosarcoma, fetal kidney, fetal heart, hippocampus, Jurkat T-cells, Jurkat membrane bound polysomes, microvascular endothelial, smooth muscle, salivary gland, tonsils, thymus, activated T-cells, fetal liver spleen, and infant brain.
The TlR-like ligand I cDNA contains an open reading frame encoding a protein of about 217 amino acid residues whose initiation codon is at positions 88-90 of the nucleotide sequence shown in FIG. I (SEQ ID NO. I); a predicted leader sequence of about 27 amino acid residues and a d~ r.ed molecular weight of about 26 kDa. The amino acid sequence of the mature TlR-like ligand I
protein is shown in FIG. l (SEQ ID NO:2) from amino acid residue 28 to residue 217. The mature TlR-like ligand I protein has three main structural domains.
These include the extracellular domain, from amino acid residue about 28 to about 17~ in FIG. 1 (SEQ ID NO:2); the tr~n.cmçmbrane domain, from amino acid residue about 179 to about 201 in FIG. 1 (SEQ ID NO:2)); and the intracellular domain, from amino acid residue about 202-217 in FIG. I (SEQ ID NO:2)). The TlR-like ligand I protein of the present invention in Figure I (SEQ ID NO:2) is about 33 % identical and about 52 % similar to the TIR ligand, which can be ~ccç~sed on GenBank as Accession No. U41804.
As one of o~di~l~uy skill would appreciate, due to the possibilities of sequencing errors ~iiecl1~cecl above, as well as the variability of cleavage sites for W O98/07881 PCTrUS96113777 leaders in different known proteins, the actual TlR-like ligand I encoded by thedeposited cDNA comprises about 217 amino acids, but can be anywhere in the range of 200-225 amino acids; and the dedllcecl leader sequence of this protein is about 27 amino acids, but can be anywhere in the range of about 15 to about 40 amino acids. Further, for example, the exact locations of the T1 R-like ligand Iprotein extracellular, intracellular and transmembrane domains in Figure l (SEQ
ID NO:2) may vary slightly (e.g., the exact amino acid positions may differ by about 1 to about 5 residues coll,pa~ed to that shown in Figure 1 ) depending on the criteria used to define the domain.
As indicated, nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, or in the forrn of DNA, including, for instance, cDN~ and genomic DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA
can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
Isolated nucleic acid molecules of the present invention include DNA
molecules comprising an open reading frame (ORF) with an initiation codon at positions 88-90 of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1 ) and further include DNA molecules which comprise a sequence subst~nti~lly dirre,~nt that all or part of the ORF whose initiation codon is at position 88-90 of the nucleotide sequence in FIG. I (SEQ ID NO: 1) but which, due to the degeneracy of the genetic code, still encode the TlR-like ligand I protein or a fragment thereof. Of course, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate the degenerate variants describedabove.
In another aspect, the invention provides isolated nucleic acid molecules encoding the TlR-like ligand I protein having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No.
97656 on July 12, 1996. Preferably, this nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA clone.

wo 3~,~7a~l PCT/US96/13777 The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIG. I (SEQ ID NO:1) or the nucleotide sequence of the TlR-like ligand I cDNA contained in the above-described deposited clone, or having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the TlR-like ligand I gene in human tissue, for instance, by Northern blot analysis. As described in detail below, detecting altered TlR-like ligand I gene expression in certain tissues may be indicative of certain I 0 disorders.
The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figure 1 (SEQ ID NO. 1) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1200 nt in length are also useful according to the present invention as are fir~gment.~ corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figure l (SEQ ID NO. 1 ) . By a fragment at least 20 nt in length, for example, is intended fr~gm~nt.c which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 (SEQ ID NO. 1). Since the gene has been deposited and the nucleotide sequence shown in Figure 1 (SEQ ID NO 1 ) is provided, generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage or ~he~ring by sonication could easily be used to generate fMgm~ntc of various sizes. Alternatively, such fragments could be generated synthetically.
Preferred nucleic acid fragmPnt~ of the present invention include nucleic acid molecules encoding: a polypeptide comprising the TlR-like ligand I
extracellular domain (amino acid residues from about 28 to about 178 in Figure WO ~ hl PCI/US96/13777 1 (SEQ ID NO: I); a polypeptide comprising the TlR-like ligand I
transmembrane domain (amino acid residues from about 179 to about 201 in Figure I (SEQ ID NO 1)); a polypeptide comprising the TlR-like ligand I
intracellular domain (amino acid residues from about 202 to about 217 in Figure S 1 (SEQ ID NO. 2)); and a polypeptide comprising the TlR-like ligand I
extracellular and intracellular domains having all or part of the transmembrane domain deleted. Further preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the T1 R-like ligand I protein. In particular, isolated nucleic acid molecules are provided encoding polypeptides comprising the following amino acid residues in Figure 1 (SEQ ID NO:2), which the present inventors have determined are antigenic regions of the TlR-like ligand I protein: a polypeptide comprising amino acid residues from about 20 to about 53 in Figure 1 (SEQ ~D NO:2); a polypeptide comprising amino acid residues from about 82 to about 98 in Figure 1 (SEQ ID
NO:2); a polypeptide comprising amino acid residues from about 106 to about 134 in Figure I (SEQ ID NO:2); and a polypeptide comprising amino acid residues from about 155 to about 184. Methods for determining other such epitope-bearing portions of the Tl R-like ligand I protein are described in detail below.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC
Deposit 97656. By "stringent hybridization conditions" is inten(led overnight incllb~tion at 42~C in a solution comprising: 50% formamide, 5x SSC (150 mM
NaCl, 1 5mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ~lg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65DC.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is int~n~le~l a polynucleotide (either DNA or RNA) hybridizing to at least about 15nucleotides (nt), and more preferably at least about 20 nt, still more preferably at W O 98/07881 PCT~US96/13777 least about 30 nt, and even more preferably at least about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide (e.g., the deposited cDNA clone), for instance, a portion 100-750nt in length, or even to the entire length of the reference polynucleotide, alsouseful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in FIG. I (SEQ ID NO:1). By a portion of a polynucleotide of "at least 20 nt in length," for exarnple, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide, (e.g, the deposited cDNA or the nucleotide sequence as shown in FIG. I (SEQ ID NO: 1)). As indicated, such portions are useful diagnosticallyeither as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (P~R), as described, for instance, in Sambrook, J. et aL, eds., Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Since an TlR-like ligand I cDNA clone has been deposited and its dete~nined nucleotide sequence is provided in FIG. 1 (SEQ ID NO: 1), generating polynucleotides which hybridize to a portion of the TlR-like ligand I cDNA
molecule would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication of the T1 R-like ligand I cDNA
clone could easily be used to generate DNA portions of various sizes which are polynucleotides that hybridize to a portion of the TlR-like ligand I cDNA
molecule. Alternatively, the hybridizing polynucleotides of the present invention could be generated synthetically according to known techniques.
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3 ' terminal poly(A) tract of the T1 R-like ligand I cDNA shown in FIG. I (SEQ ID NO: 1)), or to a complementary stretch of T (or U) resides, wouldnot be included in a polynucleotide of the invention used to hybridi~ to a portion , W 03~~'C/~&l PCT~US96113777 of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule contain a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
As indicated, nucleic acid molecules of the present invention which encode the TlR-like ligand I can include, but are not limited to, those encodingthe amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the about 27 amino acid leader sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding S' and 3' se~uences, such as the transcribed, non-tran~l~ted sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, e.g., ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, the sequence encoding the polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embo~liment~ of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are publicly and/or commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. US~ 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of thefusion protein. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza h~m:~gglutinin (HA) protein, which has been described by Wilson et al., Cell 3 7:767 (1984). Other such fusion proteins include the TlR-like ligand I protein or a fragment thereof fused to Fcat the N- or C-termin~
The present invention further relates to variants of the nucleic acid molecules ofthe present invention, which encode portions, analogs or derivativesof the TlR-like ligand I protein. Variants can occur naturally, such as a natural CA 02263830 l999-02-23 W O 98/07881 PCT~US96/13777 allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an org~nism Non-naturally occurring variants can be produced, e.g, using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions can involve one or more nucleotides. The variants can be altered in coding or non-coding regionsor both. Alterations in the coding regions can produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially plc~ d among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the TlR-like ligand I or portions thereof.
Also especially pl~r~lled in this regard are conservative substitutions.
Further embodiments of the invention include isolated nucleic acid molecules compr-~ing a polynucleotide having a nucleotide sequence at least 90%
identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical to (a) a nucleotide sequence encoding the full-length TlR-like ligand I having the complete amino acid sequence (including the leader) sho~vn in FIG. 1 (SEQ ID
NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656;
(b) a nucleotide sequence encoding the mature TlR-like ligand I (full-length polypeptide with the leader sequence removed) having the amino acid sequence at positions from about 28 to about 217 in FIG. 1 (SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656; (c) a nucleotide sequence çncof~ing the TlR-like ligand I extracellular domain having the amino acid sequence at positions from about 28 to about 178 in FIG. 1 (SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656; (d) a nucleotide sequence encoding the TlR-like ligand I transmembrane domain having the amino acid sequence at positions from about 179 to about 201 in FIG.
1 (SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656; (e) a nucleotide sequence encoding the T1 R-like ligand I intracellular domain having the arnino acid sequence at positions from about 202 to about 217 in FIG. 1 (SEQ lD NO:2) or as encoded by the cDNA clone contained in ATCC

.

W O98/07881 PCT~US96/13777 Deposit No. 97656; or (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e).
By a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a TlR-like ligand I polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five mutations per each 100 nucleotides of the reference nucleotide sequence encoding the TlR-like ligand I polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those termin~l positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the nucleotidesequence shown in FIG. 1 or to the nucleotide sequence of the deposited cDNA
clone can be determined conventionally using known computer programs such as the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. BESTFIT uses the local homology algorithm of Smith and Waterman,,4dv. Appl. Math. 2:482-489 (1981), to find the best segment of homology bet~veen two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total nurnber of nucleotides in the reference sequence are allowed.
The present application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98%, or g9% identical to a nucleic acid sequence described above irrespective of whether they encode a polypeptide having T1 R-like ligand I protein activity. This is because, even where a particular nucleic acid molecule does not encode a polypeptide having Tl R-like ligand I activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptidehaving TlR-like ligand I activity include, inter alia, (1) isolating the TlR-like li~and I gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TlR-like ligand I gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting TlR-like ligand I mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence described above which do, in fact, encode a polypeptide having TlR-like ligand I protein activity.
By "a polypeptide having Tl R-like ligand I protein activity" is int~nr~ed polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TlR-like ligand I protein of the invention as measured in a particular biological assay. TlR-like ligand I activity can be assayed using known receptor binding assays (Mitcham, J.L. et al., J. Biol. Chem. 271:5777-5783 (1996); and Gayle, M.A. et al., J. Biol. Chem. 271 :5784-5789 (1996)).
These assays include an NF-KB gel shift assay, an in vitro Thr-669 kinase assay,and an IL-8 promoter activation assay.
To pe.r~ - these assays, it is first necessary to transfect m~mm~ n cells with an expression vector containing the cDNA for a suitable receptor. ~or W O 98/07881 PCTrUS96/13777 example, an expression vector c~ the cDNA for the T1/ST2 receptor can be used. This cDNA can be obtained as described (Klemenz, R. et al., Proc, Natil. Acad. Sci. U.S.A. 86:5708-5712 (1989); Tomin~g~, S., FEBSLett. 258:301-304; Bergers, G. et al. EMBO J. 13:1176-1188)). Alternatively, Tl/ST2 cDNA
can be amplified using the polymerase chain reaction. A commercially available cDNA library, prepared from mRNA from a suita1ole tissue or cell type (such as NIH-3T3 cells (Klemenz, R. et al., Proc, Natl. Acad. Sci. U.S.A. 86:5708-5712 (1989)), can be used as template. Using any of several transfection methods wellknown to those of ordinary skill in the art, a suitable cell line (e.g., COS 7cells) can be transfected with the Tl/ST2 expression plasmid. Expression of the receptor can be verified by radioimmunoassay (see Mitcham, J.L. et al., ~ Biol.
Chem. 271:5777-5783 (1996)). One to three days post-transfection, confluent transfected COS7 cells are stimulated with 1 - 10 ng of T1 R-like ligand I protein for 15 minutes to 20 hours. Duration of stim~ tion by T1 R-like ligand I proteinwill vary, depending on which assay is used, and can be determined using only routine ~ entation.
To perforrn the NF-lcB assay, nuclear extracts from transfected cells are prepared immediately after stim~ tion (Ostrowski, J. et al., J. Biol. Chem. 266:12,722-12,733 (1991)). A doùble-stranded synthetic oligonucleotide probe (5' TGACAGAGGGACTTTCCGAGAGGA 3') cont~inin~ the NF-KB enhancer element from the immunoglobulin lc light chain is 5'-end labeled by phosphorylation with ~y-32P]ATP. Nuclear extracts (10 ,ug) are incubated with radiolableed probe for 20 minutes at room ten,l)~.dlulc, and protein-DNA
complexes are resolved by electrophoresis in a 0.5X TBE, 10% polyacrylamide gel.
To perform the in vitro Thr-669 kinase assay, cytoplasmic extracts of transfected eells are prepared immediately after stim~ tion (Bird, T.A. et al., Cytokine 4:429-440 (1992)). 10 ~1l of cell extract is added to 20 ~ of reactio~,..ixl.~.e eu~ ing 20 mM HEPES buffer (pH 7.4), 15 mM MgCI2, 15 ~M ATP, 7S ,uCi/ml [y-32P]ATP, and 750 ~M substrate peptide (residues 663-673 of EGFR). Blands are incubated with distilled HsO in place of the peptide.

W O 98/07881 PCT~US96/13777 After inr.uh~tion at 30~C for 20 minlltes, the reactions are termin~tecl by addition of formic acid. Reactions are cleared by centrifugation, and 30 ,ul of supernatant are spotted on phoshocellulase paper discs. After washing (three times with 75 mM orthophosphoric acid) and drying, peptide-incorporated counts are determin~d bymonitoring Cerenkov counts. Results are expressed as the ratio of Thr-669 kinase activity detected in unstimulated cells compared to activity detected in stimulated cells.
To perform the IL-8 promoter activation assay, COS7 cells (1 x 105 cells per well in a 12-well tissue culture plate) are cotransfected with the T1/ST2 receptor e~ ession vector and the pIL8p reporter plasmid (Mitcham, J.L. et al., J. Biol. Chem. 271:5777-5783 (1996)). One day post-transfection, the medium is changed and and cells are either stimulated with l ng/ml IL-la or are left stimulated. 12-16 hours post-stimulation, cells are washed twice with binding medium co.~ g 5% (wtv) non-fat dry milk (5% MBM) and blocked with 2 ml of 5% MBM at room temperature for 30 minutes. Cells are then incubated at room temperture for 60-90 minlltes with 1.5 ml/well of 5% MBM cont~ining 1 llg/ml of an anti-IL-2Ra antibody (R&D Systems, Minneapolis, MN) with gentle rocking. Cells are washed once with 5% MBM and incubated with 1 jl/well of 5% MBM c~ g 1:100 dilution of l25I-goat anti-moust IgG (Sigma, St. Louis, MO) for 60 mimltes at room te~llpe~ re. Wells are washed four times with 5%
MBM and twice with phosphate-buffered saline. Wells are stripped by the addition of 1 ml of 0.5 M NaOH, and total counts are determined. Results are e~ cssed as total cpm averged over two duplicate or three triplicate wells.
Thus, "a polypeptide having Tl R-like ligand I protein activity" includes polypeptides that exhibit T1 R-like ligand I protein activity in the above-described assay.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence described above will encode a polypeptide "having TlR-like ligand I protein activity." In fact, since degenerate variants of WO ~I'C /~81 PCTIUS96113777 these nucleotide sequences all encode the sarne polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a S polypeptide having TlR-like ligand I activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
For exarnple, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J.U. et al., Science 247. 1306-1310 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that m~int:~in functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino aeid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For exarnple, most buriedarnino acid residues require nonpolar side chains, whereas few features of surface side ehains are generally eonserved. Other sueh phenotypieally silent substitutions are deseribed in Bowie, J.U., et al., supra, and the references eited therein.

Vectors and Host Cells The present invention also relates to veetors which include the isolated DNA moleeules of the present invention, host cells whieh are genetically ~nginP.ored with the reeombinant veetors, and the production of Tl R-like ligandI polypeptides or fragments thereof by reeombinant teehniques.

W O98/07881 PCT~US96/13777 Recombinant constructs may be introduced into host cells using well known techniques such as infection, transduction, transfection, transvection, electroporation and transformation. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Rekoviral vectors may be replication S competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides may be joined to a vector cont~ining a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in aprecipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate p~r~eing cell line and then transduced into host cells.
Preferred are vectors comprising cis-acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
In certain preferred embodiments in this regard, the vectors provide for specific ~ ci,~ion, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as telll~eldlllre and nutrient additives.
Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
The DNA insert should be operatively linked to an a~lo~iate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, t.?l~nin~tion and, in the kanscribed region, a ribosome binding site for kanslation. The coding _, ., .. . ... ~ .... ..

portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginnine and a termin~tjon codon a~plu~liately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistancegenes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture media and conditions for the above-described host cells are known in the art.
Among vectors pl~felled for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
Among ~)rere.l~d eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Ph~ Other suitable vectors will be readily a~)palellt to the skilled artisan.
Among known bacterial promoters suitable for use in the present invention include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the ~pt promoter, the lambda PR and PL promoters and the trp promoter.
Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-m~ tPrl transfection, electroporation, transduction, infection or other methods.

Such methods are described in many standard laboratory manuals, such as Davis e~ al., Basic Methods in Molecular Biology (1986).
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer se~uence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, al)yf~yliate secretion signals may be incoryorated into the e~ylessed polypeptide.
The signals may be endogenous to the polypeptide or they may be heterologous 1 5 signals.
Thus, the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged arnino acids, may be added to the N-terrninus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent h~nt~ling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are f~qmili~r and routine techni~ues in the art. A preferred fusion protein comprises a heterologous region from imrnunoglobulin that is useful to solubilize proteins. For exarnple, EP-A-O 464 533 (C~n~ n co~lt~lyall 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein isthoroughly advantageous for use in therapy and diagnosis and thus results, for . . .

W O 98107881 PCTrUSg6/13777 example, in improved ph~ cokinetic properties (EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL5- has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).
The TlR-like ligand I can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and m~mm~ n cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

Polypepfides and Peptides of f/le TlR-like ligand I

The invention further provides an isolated Tl R-like ligand I polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in FIG. I (SEQ ID NO:2), or a peptide or polypeptide comprising W O 98107881 PCT~US96/13777 a portion of the above polypeptides. The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two arnino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.
By '~isolated" polypeptide or protein is intended a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the invention as are native or recombinant polypeptides and proteinswhich have been substantially purified by any suitable technique such as, for example, the one-step method described in Smith and Johnson, Gene 67:31-40 (1 988).
It will be recognized in the art that some amino acid sequence ofthe TlR-like ligand I can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which detçrmine activity. In general, it is possible to replace residues which form the tertiarystructure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.
Thus, the invention further includes variations of the TlR-like ligand I
which show substantial TlR-like ligand I activity or which include regions of TlR-like ligand I such as the protein portions discussed below. Such m~lt~nt~
include deletions, insertions, inversions, repeats, and type substitutions (for example, subslilulillg one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral"amino acid substitutions will generally have little effect on activity.
Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids ~la, Val, Leu and Ile; interchange of the W 03~ 1 PCTrUS96/13777 hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
As indicated in detail above, further guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on a function) can be found in Bowie, J.U., et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
The polypeptides ofthe present invention include the polypeptide encoded by the deposited cDNA including the leader sequence, the polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein), the polypeptide of FIG. 1 (SEQ ID NO:2) including the leader, the polypeptide of FIG. 1 (SEQ ID NO:2) minus the leader, the TlR-like ligand I extracellular domain, the TlR-like ligand I transmembrane domain, and the TlR-like ligand I intr~cell~ r domain as well as polypeptides which have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%,97%, 98%, or 99% similarity to those described above. Further polypeptides of the present invention include polypeptides at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98%, or 99% identical to a polypeptide described herein, and and also include portions of such polypeptides with at least 30 arnino acids and more preferably at least 50 amino acids.
By "% similarity" for two polypeptides is intçnded a similarity score produced by c~ g the amino acid sequences of the two polypeptides using the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) and the default settings for det~rmining similarity.
BESTFIT uses the local homology algorithm of Smith and W:~t~ n, Adv. Appl.
Math. 2:482-489 (1981), to find the best segment of similarity between two sequences.

W O 98/07881 PCTrUS96/13777 By a polypeptide having an amino acid sequence at least, for exatnple~
95% "identical" to a reference amino acid sequence of a TlR-like ligand I
polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference arnino acid sequence of the TlR-like ligand I polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95%
identical to a reference amino acid sequence, up to 5% of the arnino acid residues in the reference sequence may be deleted or substituted with another arnino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy termin~l positions of the reference amino acid sequence or anywhere between those terminal positions, intclspGl~ed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in FIG. l (SEQ ID NO:2) or to the amino acid sequence encoded by deposited cDNA clone can be dettqrmined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. When using Bestfit or any other sequence aligntnent program to detçrmine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of arnino acid residues in the rer~lence sequence are allowed.
As described in detail below, the polypeptides ofthe present invention can be used to raise polyclonal and monoclonal antibodies, which are useiùl in gnostic ~says for detecting TlR-like ligand I expression as described below .. . . ..

or as agonists and antagonists capable of enhancing or inhibiting T1 R-like ligand I protein function. Further, such polypeptides can be used in the yeast t~vo-hybrid system to "capture" TlR-like ligand I binding proteins which are also candidate agonist and antagonist according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989).
In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the imrnunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to whichan antibody can bind is defined as an "antigenic epitope." The number of imrnunogenic epitopes of a protein generally is less than the nurnber of antigenic epitopes. See, for instance, Geysen, H.M. et al., Proc. Natl. Acad. Sci. USA
81:3998-4002 (1984).
As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserumthat reacts ~,vith the partially mimicked protein. See, for instance, Sutcliffe, J.G.
et al., Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl termin~l~ Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those col-tiqi.,i~-g proline residues, usually are effective. Sutcliffe et al., supra, at 661.
For inct~n~e 18 of 20 peptides designed according to these guidelines, Cont~ining 8-39 residues covering 75% of the sequence of the influenza virus h~ glutinin W O 98/07881 PCT~US96/13777 HAl polypeptide chain, in~ ced antibodies that reacted with the HAI protein or intact virus; and 12/12 peptides from the MuLV polymerase and 1 8tl 8 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bindspecifically to a polypeptide of the invention. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein. Sutcliffe et al., supra, at 663. The antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes posttranslation proces.cing The peptides and anti-peptide antibodies may be used in a variety ofqualitative or quantitative assays for the mimicked protein, for in~t~nce in competition assays since it has been shown that even short peptides (e.g., about9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for in.~t~nce, Wilson, I.A. et al., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography using methods well known in the art.
Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 arnino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, cont~ining about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for in~ .in~ antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected to provide . _ . ,, substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided);
and sequences cont~ining proline residues are particularly preferred.
Non-limiting examples of antigenic polypeptides that can be used to generate TlR-like ligand I specific antibodies include: a polypeptide comprisingamino acid residues from about 20 to about 53 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 82 to about 98 in Figure l (SEQ ID NO:2); a polypeptide comprising arnino acid residues from about 106 to about 134 in Figure 1 (SEQ ID NO:2); and a polypeptide comprising amino acid residues from about 155 to about 184.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies.Epitope-bearing peptides also may be syntheci7~d using known methods of chemical synthesis. For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 dirr~relll 13 residue peptides re~ sellling single amino acid variants of a segment of the HAlpolypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. Houghten, R.A., Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985). This "Siml-lt~neous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Patent No. 4,631,211 to Houghten et al.
(1986). In this procedure the individual resins for the solid-phase synthesis ofvarious peptides are contained in separate solvent-permeable packets, enabling the optimal use of the many identical ~elili~e steps involved in solid-phase methods. A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously. Houghten et al., supra, at 5134.
Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. ,4cad.
Sci. USA 82:910-914; and Bittle, F.J. et al., ~ Gen. Virol. 66:2347-2354 (1985).Generally, animals may be immllni7P~l with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides cont~ining cysteine may be coupled to carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are irnmunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intr~(lerm~l injection of emulsions cont~ining about 100 ~lg peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for inct~nre at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detecte~l for exarnple, by ELISA assay using free peptide adsorbed to a solid surface. T~e titer of anti-peptide antibodies in serum from an immuni~d animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
Tmmllnogenic epitope-bearing peptides of the invention, i.e., those parts of a protein that elicit an antibody response when the whole protein is the immllnogen, are identified according to methods known in the art. Por instance, Geysen et al. (1984), supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art. For instance, the immlmologically illlpOl ~ll epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Then, a conl,~,lcte repl~rernent set .. .. . . .

WO 98tO7881 PCTrUS96/13777 of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method. U.S. Patent No. 4,708,781 to Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.
Further still, U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or deterrnining the sequence of monomers (amino l O acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complennent~ry to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complçnnen1~ry to the ligand binding site of a particular receptor of interest.
Similarly, U.S. Patent No. 5,480,971 to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide Mixtures discloses linear Cl-C7-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for (1et~rmining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest. Thus, non-peptide analogs of the epitope-bearing peptides of the invention also can bemade routinely by these methods.
The entire disclosure of each document cited in this section on "Polypeptides and Peptides" is hereby incorporated herein by reference.
As one of skill in the art will appreciate, TlR-like ligand I polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immllnoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g.,for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of m~mmz~ n immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84- 86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TlR-like ligand I protein or protein fragment alone (Fountoulakis et al., JBiochem. 270:3958-3964 (1995)).

TlR-like Ligand I Related Disorder Diagnosis For TlR-like ligand I related disorders, it is believed that substantially altered (increased or decreased) levels of TlR-like ligand I gene expression canbe detected in tissue or other cells or bodily fluids (e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder,relative to a "standard" TlR-like ligand I gene ~ ssion level, that is, the TlR-like ligand I gene ~ s~ion level in tissue or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method usefulduring diagnosis of an TlR-like ligand I-related disorder, which involves measuring the ~ e;,sion level of the gene encoding the TlR-like ligand I in tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard TlR-like ligand I gene ~ur~ssion level, whereby an increase or decrease in the gene ~xp~s~ion level compared to the standard is indicative of an Tl R-like ligand I related disorder.
TlR-like ligand I-related disorders are believed to include, but are not limited to, lellk~?mi~ Iymphoma, arteriosclerosis, autoimmune ~liee~ee, infl~mm~tory disease, Alzheimer's ~liee~ee~ ophth~lmic disease, apoptosis, hll~aul.,line growth retardation, preeclampsia, pemphigus and psoriasis.
By individual is int~n-lçd m~mms~ n individuals, preferably hllm~n.e. By "me~e~lring the expression level of the gene encoding the TlR-like ligand I" is interlded qualitatively or qual~ ely measuring or estim~ting the level of the T1 R-like ligand I protein or the level of the mRNA encoding the T1 R-like ligand I protein in a first biological sample either directly (e.g., by detçrmining or estim~ting absolute protein level or mRNA level) or relatively (e.g., by comparing to the TlR-like ligand I protein level or mRNA level in a second biological sample). Preferably, the TlR-like ligand I protein level or mRNA
level in the first biological sample is measured or estim~ted and compared to a standard Tl R-like ligand I protein level or mRNA level, the standard being taken S from a second biological sample obtained from an individual not having the disorder or being determin~.d by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard T1 R-like ligand I protein level or mRNA level is known, it can be used repeatedly asa standard for comparison.
By "biological sample" is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains TlR-like ligand I protein or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) whichcontain secreted mature TlR-like ligand I, or tissue sources found to express TlR-like ligand I protein. Methods for obtaining tissue biopsies and body fluidsfrom m~mm~l~ are well known in the art. Where the biological sarnple is to include mRNA, a tissue biopsy is the preferred source.
Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem.
162:156-159 (1987). Levels of mRNA encoding an TlR-like ligand I are then assayed using any app,~,~,;ate method. These include Northern blot analysis, S In~lcle~e mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
Northern blot analysis can be p~lrolllled as described in Harada et al., Cell 63:303-312 (1990). Briefly, total RNA is prepared from a biological sample as described above. For the Northern blot, the RNA is denatured in an applup,iate buffer (such as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected to agarose gel electrophoresis, and transferred onto a nitrocellulose filter. After the RNAs have been linked to the filter by a W linker, the filter is prehybridized W O 98/07881 PCTrUS96/13777 in a solution cont~ining formamide, SSC, Denhardt's solution, denatured salmon sperm, SDS, and sodium phosphate buffer. TlR-like ligand I cDNA labeled according to any ~pplol,l;ate method (such as the 32P-multiprimed DNA labeling system (Amersham)) is used as probe. After hybridization overnight, the filter is washed and exposed to x-ray film. cDNA for use as probe according to the present invention is described in the sections above and will preferably at least 15 bp in length.
S l mapping can be performed as described in Fujita et al., Cell 49:357-367 (1987). To prepare probe DNA for use in Sl mapping, the sense strand of above-described cDNA is used as a template to synthesize labeled antisense DNA. The antisense DNA can then be digested using an appropriate restriction endonuclease to generate further DNA probes of a desired length. Such antisense probes are useful for visu~li7ing protected bands corresponding to the target mRNA (i.e., mRNA encoding the TlR-like ligand I). Northern blot analysis can be performed as described above.
Preferably, levels of mRNA encoding the TlR-like ligand I are assayed using the RT-PCR method described in Makino et al., Technique 2:295-301 (1990). By this method, the radioactivities of the '~amplicons" in the polyacrylamide gel bands are linearly related to the initial concentration of the target mRNA. Briefly, this method involves adding total RNA isolated from a biological sarnple in a reaction mixture cont~inin~. a RT primer and appropriatebuffer. After incubating for primer ~nne~ling, the mixture can be supplemented ~vith a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather than labeling the primers, a labeled dNTP can be included in the PCR reaction mixture. PCR
amplification can be performed in a DNA therrnal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of the ~pl(3pfiate bands (corresponding to the mRNA encoding the TlR-like ligand I) is quantified using ~ . .

an im~ging analyzer. RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art.
Variations on the RT-PCR method will be appalcnt to the skilled artisan.
Any set of oligonucleotide primers which will amplify reverse transcribed S target mRNA can be used and can be de~ign~cl as described in the sections above.
Assaying TlR-like ligand I levels in a biological sample can occur using any art-known method. Preferred for assaying TlR-like ligand I levels in a biological sample are antibody-based techniques. For example, TlR-like ligand I expression in tissues can be studied with classical immunohistological methods.
In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological staining of tissue section for pathological e~c~min~tion is obtained. Tissues can also be extracted, e.g., with urea and neutral detergent, for the liberation of T1 R-like ligand I for Western-blot or dotlslot assay (J~lk~nen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol.
105:3087-3096 (1987)). In this technique, which is based on the use of cationic solid phases, quantitation of T1 R-like ligand I can be accomplished using isolated TlR-like ligand I as a standard. This technique can also be applied to body fluids. With these samples, a molar concentration of T1 R-like ligand I will aidto set standard values of TlR-like ligand I content for different body fluids, like serum, plasma, urine, synovial fluid, spinal fluid, etc. The normal appearance of TlR-like ligand I amounts can then be set using values from healthy individuals,which can be compared to those obtained from a test subject.
Other antibody-based methods useful for detecting TlR-like ligand I
levels include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, TlR-like ligand I-specific monoclonal antibodies can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify the TlR-like ligand I. The amount of TlR-like ligand I present in the sample can be calculated by referenceto the amount present in a standard p~ aldtion using a linear regression computer W O 98/07881 PCT~US96/13777 algorithrn. Such an ELISA for detecting a tumor antigen is described in Iacobelli et al., Breast Cancer Research and Treatment 11:19-30 (1988). In another ELISA assay, two distinct specific monoclonal antibodies can be used to detect TlR-like ligand I in a body fluid. In this assay, one of the antibodies is used as the irnmunoadsorbent and the other as the enzyme-labeled probe.
The above techniques may be conducted es.eenti~lly as a "one-step" or "t~o-step" assay. The "one-step" assay involves contacting TlR-like ligand I
witl1 immobilized antibody and, without washing, contacting the mixture with thelabeled antibody. The "two-step" assay involves washing before contacting the mixture with the labeled antibody. Other conventional methods may also be employed as suitable. It is usually desirable to imrnobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide forrned by the enzyme-labeled antibody/substrate reaction. Besides enzymes, other suitable labels include radioisotopes, such as iodine (125I, '2'I), carbon (14C), sulfur (35S), tritiurn (3H), indium ("2In), and technetiurn (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
In addition to assaying TlR-like ligand I levels in a biological sample obtained from an individual, TlR-like ligand I can also be detected in vivo by im~ging. Antibody labels or markers for in vivo im~ing of TlR-like ligand I
include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detect~hle characteristic spin, such as CA 02263830 l999-02-23 W O98/07881 PCTrUS96/13777 \deuterium, which may be incorporated into the antibody by labeling of nutrientsfor the relevant hybridoma.
A TlR-like ligand I-specific antibody or antibody fragment which has been labeled with an appropliate detectable im~in~ moiety, such as a radioisotope (for example, '3'I, "2In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the m~mm~l to be examined for a disorder. It will be understood in the art that the size of the subject and the im~ging system used will determine the quantity of im~ging moieties needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then pre~relltially accumulate at the location of cells which contain TlR-like ligand I. In vivo tumor im~ging is described in S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments"
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S.W. and Rhodes, B.A. eds., Masson Publishing Inc., (1982)).
TlR-like ligand I specific antibodies for use in the present invention can be raised against the intact Tl R-like ligand I or an antigenic polypeptide fragment thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at leastabout 25 amino acids), without a carrier.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, forexample, Fab and F(ab')2 fr~gment~) which are capable of specifically binding toTlR-like ligand I. Fab and F(ab')2 fragments lack the Fc portion of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J: Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the TlR-like ligand I or an W O 98/07881 PCTrUS96/13777 antigenic fragment thereof can be ~-lmini.ctered to an animal in order to induce the production of sera cont~ining polyclonal antibodies. In a preferred method, a preparation of TlR-like ligand I protein is l)lcl~a~,d and purified as describedabove to render it substantially free of natural cont~min~nt~. Such a pre,uaralion is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or TlR-like ligand I binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology (Colligan, Current Protocols in Immunolo~y, Wiley Interscience, New York ( 1990-1996);
Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (19B8), Chapters 6-9, Current Protocols in Molecular Biology, Ausubel, infra, Chapter 11, entirely incorporated herein by reference).In general, such procedures involve immunizing an animal (preferably a mouse) lS with an TlR-like ligand I antigen or, more preferably, with an TlR-like ligand I-~x,ures~ing cell. Suitable cells can be recognized by their capacity to bind anti-TlR-like ligand I antibody. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serurn (inactivated at about 56~C), and suppleJnente~l with about 10 ~g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about l 00 ~lg/ml of streptomycin. The splenocytesof such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection (ATCC) (Rockville, Maryland, USA). After fusion, the resulting hybridoma cells are selectively m~int~ined in HAT medium, and then cloned by limiting dilution as described by Wands et al., Gastroenterology 80:225-232 ( l 98 l ); Harlow & Lane, inSra, Chapter 7. The hybridoma cells obtained through such a selection are thenassayed to identify clones which secrete antibodies capable of binding the Tl R-like ligand I antigen.

., W O98107881 PCTrUS96113777 Alternatively, additional antibodies capable of binding to the TlR-like ligand I antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, TlR-like ligand I
specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the TlR-like ligand I-specific antibody can be blocked by theTlR-like ligand I antigen. Such antibodies comprise anti-idiotypic antibodies tothe TlR-like ligand I-specific antibody and can be used to immunize an animal to induce forrnation of further T1 R-like ligand I-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fr~gment~ are typically produced by proteolytic cleavage,using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Alternatively, TlR-like ligand I-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
Where in vivo im~ging is used to detect enhanced levels of TlR-like ligand I for diagnosis in humans, it may be preferable to use "hl~m~ni7ed"
chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).
Further suitable labels for the T1 R-like ligand I-specific antibodies of the present invention are provided below. Examples of suitable enzyme labels W O 98/07881 PCT~US96113777 include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, aspar~in~e, glucose oxidase, beta-galactosidase, ribonuclease, urease, c~t~l~se,glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.
Examples of suitable radioisotopic labels include 3H, "'In, '25I, '3'I, 32p, 35S, 14C, 5~Cr, 57To, 58Co, 59Fe, 75Se, ~52Eu, 90Y, 67Cu 2l7c; 2~At 2~2pb 47Sc ~ospd etc. " 'In is a preferred isotope where in vivo im:~gin~ is used since its avoids the problem of dehalogenation of the '25I or ~3~I-labeled monoclonal antibody by theliver. In addition, this radionucleotide has a more favorable gamnla emission energy for im~in~ (Perkins et al., Eur. J. Nucl. Med. 10:296-301 (1985);
Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)). ~or example, "'In coupled to monoclonal antibodies with l-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumorous tissues, particularly the liver, and therefore enhances IS specificity of tumor localization (Esteban et al., J. Nucl. Med. 2~:861-870 (1 987)).
Examples of suitable non-radioactive isotopic labels include '57Gd, 55Mn, s2Tr~ and 56Fe.
Examples of suitable fluorescent labels include an '52Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluolesc~ ine label.
Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.
Examples of chemilurninescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.
Fx~mrles of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and Fe.

.. . . , , . . , . . . ~ .

CA 02263830 l999-02-23 W O 98/07881 PCTrUS96/13777 Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al. (Clin. Chim. Acta 70:1-31 (1976)), and Schurs et al. (Clin. Chim. Acta 81:1-40 (1977)). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.

Chromosome Assays The nucleic acid molecules of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome m~rking reagents based on actual sequence data (repeat polymorphisms) are presently available for m~rking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
In certain plef~lled embodiments in this regard, the cDNA herein disclosed is used to clone genomic DNA of an TlR-like ligand I gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available col~ lcl~;ially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose. Typically, in accordance with routine procedures for chromosome mapping, some trial and error may be nt cess~ry to identify a genomic probe that gives a good in situ hybridization signal.
In some cases, in addition, sequences can be mapped to chromosomes by pl~p~;ng PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untr~n~!~ted region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids cont~ining individual human chromosomes. Only those hybrids cont~ining the human gene corresponding to the primer will yield an amplified portion.
PCR mapping of somatic cell hybrids is a rapid procedure for ~igning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels ofportions from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in si~u hybridization ("FISH") of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from the cDNA as short as 50 or 60 bp. For a review of this technique, see Verma et al., Human Chromosomes: ~ Manual of Basic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is n~cecs~ry to detPrmine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with , , .

wo 3n,~ lo~l PCTrUS96/13777 the disease could be one of between 50 and 500 potential causative genes. (This assumes I meg~b~e mapping resolution and one gene per 20 kb).

Treatment of TlR-like Ligand I Disorders It is believed by the present inventors that TlR-like ligand I polypeptides S ofthe present invention share biological activities with interleukin-l ~IL-1) and the TlR ligand. Thus, the TlR-like ligand I (particularly the mature form) can be exogenously added to cells, tissues, or the body of an individual to produce a therapeutic effect. In particular, disorders caused by a decrease in the standard level of TlR-like ligand I protein activity can be treated by ~mini.stering an effective amount of a TlR-like ligand I polypeptide of the invention. Preferably, a ph~rnl~ceutical composition is ~lmini~tered comprising an amount of an isolated T1 R-like ligand I polypeptide of the invention effective to increase the TlR-like ligand I protein activity. Disorders where such a therapy would likely be effective are discussed above and below.
One of ordinary skill will appreciate that effective arnounts of a TlR-like ligand I polypeptide can be determined empirically for each condition where ~lmini~tration of a such a polypeptide is indicated. The polypeptide having T1 R-like ligand 1 activity can be ~-lmini~tered in pharmaceutical compositions in combination with one or more pharmaceutically acceptable carriers, diluents and/or excipients. It will be understood that, when ~lmini~t~red to a human patient, the total daily usage of the pharmaceutical compositions of the presentinvention will be decided by the attending physician within the scope of sound medical judgment. ~he specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the type and degree of the response to be achieved; the specific composition an other agent, if any, employed; the age, body weight, general health, sex and diet of the patient;
the time of ~lmini~tration, route of ~-lmini.~tration, and rate of excretion of the composition; the duration of the treatment; drugs (such as a chemotherapeutic agent) used in combination or coincidental with the specific composition; and like factors well known in the medical arts.
The TlR-like ligand I composition to be used in the therapy will also be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of tre:~tment ~vith T1 R-like ligand I alone), the site of delivery of the T1 R-like ligand I composition, the method of a-lmini~tration, the scheduling of 7~1minictration, and other factors known to practitioners. An "effective amount"of a TlR-like ligand I polypeptide for purposes herein is thus (let~rmine-l by such 1 0 considerations.
As a general proposition, the total ph~ ceutically effective amount of a TlR-like ligand I polypeptide ~ inictered parenterally per dose will be in therange of about 0.01 ng/kg/day to 10 ~g/kg/day of patient body weight, although, as noted above, this will be subject to the~ ,culic discretion. More preferably,this dose is at least 1.0 ng/kg/day, and most preferably for hllm~n.~ between about 1.0 to 100 ng/kg/day for the hormone. If given continuously, the T1 R-like ligand I is typically ~riminictered at a dose rate of about 0.01 ng/kg/hour to about 100 ng/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
A course of TlR-like ligand I polypeptide trc~tment to affect the immllne system appears to be optimal if continued longer than a certain minimum number of days, 7 days in the case of the mice. The length of tre~tmtont needed to observe changes and the interval following tre~tm~nt for responses to occur a~pe~ to vary depending on the desired effect.
The TlR-like ligand I polypeptide is also suitably ~ministered by sllcl~ined-release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sl.ct~ined-release matrices include polylactides (U.S.
Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glllt~mzlte (U. Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-PCT~US96113777 WO ~ ,,Oal hydroxyethyl methacrylate) (R. Langer et al., J: Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988). Sustained-release TlR-like ligand I compositions also include a liposomally elltrd~l)ed TlR-like ligand I polypeptide. Liposomes cont~ining a TlR-like ligand I
polypeptidearepl~cdbymethodsknownperse: DE3,218,121;Epstein,etal., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;
EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal TlR-like ligand I therapy.
For parenteral ~-1ministration, in one embodiment, the TlR-like ligand I polypeptide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a ph~ eutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
Generally, the formulations are prepared by contacting the TlR-like ligand I polypeptide uniformly and intim~tely with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired fo~ ti- n. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein,as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are W O 98/07881 PCT~US96/13777 non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosacch~rides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
The TlR-like ligand I is typically formulated in such vehicles at a concentration of about 0.001 ng/ml to 500 ng/ml, preferably 0.1-10 ng/ml, at a pH
of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the forrnation of TlR-like ligand I salts.
TlR-like ligand I to be used for therapeutic a~1mini~tration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic TlR-like ligand I
compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
TlR-like ligand I ol.lhl~;ly will be stored in unit or multi-dose conlainers, for example, sealed ampoules or vials, as an aqueous solution or as a Iyophilized formulation for reconstitution. As an example of a Iyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous TlR-like ligand I solution, and the resulting mixture is Iyophili7.~?~1 The infusion solution is prepared by reconstituting the lyophilized TlR-like ligand I using bacteriostatic Water-for-Injection.
For example, sati~f~ctQry results are obtained by oral ~(1mini~tration of a polypeptide having TlR-like ligand I activity in dosages on the order of from 0.05 to 5000 ng/kg/day, preferably 0.1 to 1000 ng/lcg/day, more preferably 10 .. , . .. " .
. .... , . . . . --WO ~8~ DI~1 PCT/USg6/13777 tolO0 nglkg/day, ~-lmini.~t~red once or, in divided doses, 1 to 4 times per day. On ~lmini~tration parenterally, for example by i.v. drip or infusion, dosages on the orderoffrom 0.01 to 500 ngAcg/day, preferably 0.05 to 100 ng/kg/day and more preferably 0.1 to 50 ng/kg/day can be used. Suitable daily dosages for patients S are thus on the order of from 2.5 ng to 250 ~g p.o., preferably 5 ng to 50 llg p.o., more preferably 50 ng to 12.5 llg p.o., or on the order of from 0.5 ng to 25 ,ug i.v., preferably 2.5 ng to 500 llg i.v. and more preferably 5 ng to 2.5 ~g i.v.

TlR~ e Ligand I An~ibody T/terapy By the invention, disorders caused by enhanced levels of TlR-like ligand I protein activity can be treated by ~-lmini~tering an effective amount of an antagonist of a TlR-like ligand I polypeptide of the invention. Therefore, antibodies (preferably monoclonal) or antibody fragments that bind a Tl R-like ligand I polypeptide ofthe present invention are useful in treating TlR-like ligand I-related disorders as are soluble TlR-like ligand I proteins, such as the extracellular domain, which competes with the intact protein for binding to the TlR-like ligand I receptor. Such antibodies and/or soluble TlR-like ligand I
proteins are preferably provided in pharmaceutically acceptable compositions.
The pharmaceutical compositions of the present invention may be ~imini~tered, for example, by the parenteral, subcutaneous, intravenous, intramuscular, inLld~eliloneal, tr~n~-lçrm:~l, or buccal routes. Alternatively, or concurrently, ~1mini~tration may be oral. The dosage ~lmini.~tered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.Compositions within the scope of this invention include all compositions wherein the antibody, fragment or derivative is contained in an amount effectiveto achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of theart. The effective dose is a function of the individual chimeric or monoclonal antibody, the presence and nature of a conjugated therapeutic agent (see below), W O 98/07881 PCTrUS96113M7 the patient and his clinical status, and can vary from about 10 ng/kg body weight to about 100 mg/kg body weight. The preferred dosages comprise 0.1 to 10 mg/lcg body wt.
Pl~aldlions of an TlR-like li~and I antibody or fragment for parenteral ~ministration, such as in detectably labeled forrn for im~ing or in a free or conjugated form for therapy, include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propyleneglycol, polyethyleneglycol, vegetable oil such as olive oil, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/a~ueous solutions, emulsions or suspensions, including saline and buffered media, parenteral vehicles including sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. See, generally, Remington's Pharmaceutical Science, 16th ed., Mack Publishing Co., Easton, PA, 1980.
The antibodies described herein may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with Iymphokines or hemopoietic growth factors, etc., which serve to increase the number or activity of effector cells which interact with the antibodies.

~re~t~d Pleiotropic biologic effec~s of TlR-like ligand I

The T1 R-like ligand I polypeptides of the present invention are expected to have pleiotropic biological effects including many of those shown in Table 1 below. Similar biological effects have been shown for IL- I, particularly those associated with pancreatic endocrine tissue (Mandrup-Poulsen, T., et al., Cytohne 5:185 (1993)), thyroid glands (Rasmussen, A.K., Autoimmunity 16:141 (1993)), hypothalamic-pituitary-adrenal axis (Fantuzzi, G., & Ghezzi, P., Mediator Inflamm. 2:263 (1993); Rivier, C.,Ann. NYAcad. Sci. 697:97 (1993); Rivier, C., .. , . . . . . ~

PCT~US96/13777 & RiVCSt, S., Ciba. Found. Symp. 172:204 (1993)), fever (Coceani, F., "Fcver:
Basic Me~h~ni~m~ and MarlagemeIlt", New York, NY, Raven (1991) p. S9), bone metabolism (Tatalcis, D.N., ~ Peridontol 64:416 (1993)), deStrllCtiOII of cartilage in the pathogenesis of rh~um~toid arthlitiS (AIend, W.P., & Dayer, J.M., Arthritis Rh~um 33:305 (1990); Krane, S.M., et al., Ann. NYAcad. Sci. 580:340 (1990)), uterinc implalltation (LeWtS, M.P., et al., Placenta 15: 13 (1994)), and IOSS of leatl bodymass(Roubenoff,R., etal., J. Clin. Invest. 93:2379(1994)).

TABLE 1. POSSIBLE BIOLOGIC EFFECTS OF T1R_LIKE LIGAND I
Effects of sys~emicaily injected Tl R-like ligand I
Fever; increased slow wave sleep; sociai d~ ;on; anorexia Hypotension; myocardial suppression; tachycardia; lactic acidosis Increased circulating nitric oxide; hypn~ ninn~idPn i~
Hyperinc~linPmi~ hyperglycemia; hypoglycemia Si Istinn of hypothalamic-pituitary-adrenal axis Release of hypothalamic l~ n~ ,5 and r u~ ides Neutrophilia; increased marrow cellularity; increased platelets Increased hepatic acute phase protein synthesis Hypoferremia; hyl,o,; .- ~ increased sodium excretion Hyperlipid~P~ increased muscle protein breakdown Hypo~ a; decreased drug mPt~ oli~m Increased ~
Increased non~e~ irlc resistance to infection (pl~ allll~lll) Learning defects in offspring after maternal IL- I treatment Effects of locally injected TlR-like ligand I
Infiltration of m ~ ul lic into rabbits knee joint Increasedprot~ Iy . 1,. Làki~ inrabbitkneejoint Induction of uveitis following intravitreal injection Ar.6iot,cll~ s;s in anterior chamber of eye Cellular infiltrate and cytokine induction in cerebral ventricles Neutrophil and albumin influx into lungs aRer hl~dtla~ hedl ll ~~inn W O 98/07881 PCTrUS96/13777 Changes in immunologic responses Increased antibody production (adjuvant ef~ect) Increased Iymphokine synthesis (IL-2, -3, -4, -5, -6, -7, - 10 and - 12) Increased IL-2 (,B) receptor ~ De.~lLr of ~pe 2 human T-cell clones Inhibition of tolerance to protein antigens .-- ~.1 of spleen cell mitogenic response to LPS
Ef~ects of Tl R-like ligand I on cultured cells or tissues Increased expression of ELAM- I, VCAM- I, ICAM- I
Cytotoxicity (apoptosis) of insulin-~,.u~ue,-,~ islet ,B cells Inhibition of thyroglobulin synthesis in thyrocytes Canilage l,.~kd~)...., release of calcium from bone Increased release of arachidonic acid, prostanoids, and eicos~ c Increased mucus 1,.- '~. ~;c l and chloride flux in intestinal cells Fn~ 1 in chloride nux (GABAA receptor) in brain Syn~rto~o~c Proliferation of fibroblasts, smooth muscle cells, m,~c~ igJ cells Growth inhibition of hair follicles Increased corticosterone synthesis by adrenals Increased HIV-I e"~ ,-ul.
Assays used: pancreatic endocrine tissue (Iv' ' .1~, Poulsen, T., e~ aL, Cytokine 5:185 (1993)), thyroic gland (~ r~ cil~n, A.K., ~ 16: 141 (1993)), h~ul~ -pituitary-adrenal axis (Fantuzzi, G., & Ghe~zi, P., Mediator Inflamm. 2:263 (1993); Rivier, C., Ann. NYAcad. Sci. 697:97 (1993); Rivier, C., & Rivest, S., Ciba. Found Symp. 1 72:204 (1993)), fever (Coceani, F., "Fever: Bscic Me,l.~ ...c and Md~ ", New York, NY, Raven (1991) p. 59), bone ~ ' (Tatalcis, D.N., J. Peridon~ol 64:416 (1993)), destruction of cartilage in the pathogenesis of rll~Jllldtu:d arthritis (Arend, W.P., &
Dayer, J.M., Arthritis Rheum 33:305 (1990); Krane, S.M., et al., Ann. NYAcad. Sci. 580:340 (1990)), uterine irnrlqrt~ion (Lewis, M.P., e~ al., Placenta 15:13 (I994)), and loss of lean body mass (Roubenoff, R., et al., J. Clin. Invest. 93:2379 (1994).

Having generally described the invention, the same will be more readily understood by reference to the following exarnples, which are provided by way of illustration and are not int~n~ecl as limiting.

Examples Example 1: Expression and Purif cation oSTIR-like ligand I in E. coli The DNA sequence encoding the mature T1R-like ligand I in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the arnino tennin~l sequences of the TlR-like ligand I and to vector sequences W 03~ 1 PCT~US96/13777 3' to the gene. Additional nucleotides cont~ining restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
One of ordinary skill in the art will understand that the full-length, mature TlR-like ligand I protein (amino acid about 28 to about 217) can be expressed inE.coli using suitable 5' and 3' oligonucleotide primers.
The cDNA sequence encoding the extracellular domain of the full length TlR-like ligand I in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. The 5' oligonucleotide primer has the sequence 5' CGC CCA TGG AGC TCA CCT
TCG AGC TG 3' (SEQ ID NO:4) containing the underlined Nco I restriction site followed by 17 nucleotides (nucleotides 170 to 186) of the TlR-like ligand I
protein coding sequence in FIG. 1 (SEQ ID NO: 1 ) beginning immediately after the signal peptide.
The 3' primer has the sequence 5' CGC AAG CTT TCA TCG GCT ATT
AAG GTC TTC 3' (SEQ ID NO:5) cont~inine a Hind III reskiction site followed by a stop codon and 18 nucleotides complementary and reverse to nucleotides 604-621 ofthe TlR-like ligand I coding sequence in FIG. 1 (SEQ ID NO:1).
The restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE60, which are used for bacterial expression in M15/rep4 host cells in these examples. (Qiagen, Inc., Chatsworth, CA, 9131 1).
pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterialorigin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), a 6-His tag and reskiction enzyme sites.
The amplified TlR-like ligand I DNA and the vector pQE60 both are digested with NcoI and HindIII and the digested DNAs are then ligated together.
Insertion of the T1 R-like ligand I DNA into the restricted pQE60 vector places the TlR-like ligand I coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG
appropriately positioned for translation of TlR-like ligand I.
The ligation mixture is transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al., _55_ Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described here. This strain, which is only one of many that are suitablefor expressing TlR-like ligand I, is available commercially from Qiagen.
Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA was confirmed by restriction analysis.
Clones cont~ining the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 llg/ml) and kanamycin (25 llg/ml).
The O/N culture is used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250. The cells are grown to an optical density at 600nm("OD600") of between 0.4 and 0.6. Isopropyl-B-D-thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lacl repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested bycentrifugation and disrupted, by standard methods. Inclusion bodies are purifiedfrom the disrupted cells using routine collection techniques, and protein is solubilized from the inclusion bodies into 8M urea. The 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X
phosphate-buffered saline ("PBS"), thereby removing the urea, exch~n~ing the buffer and refolding the protein. The protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered. The sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 ~l/ml.Analysis of the pltpaldlion by standard methods of polyacrylamide gel electrophoresis reveals that the ple~aldlion contains about 95% monomer TlR-like ligand I having the expected molecular weight of approximately 18 kDa.

.

PCTrUS96tl3777 Example 2: ~loning and Expression of TlR-like ligand I in a Baculovirus Expression System The cDNA sequence encoding the full length TlR-like ligand I in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' CGC GGA TCC GCC ATC ATG GGC
AGC ACT GTC CCG 3' (SEQ ID NO:6) containing the underlined BamHI
restriction enzyme site followed by 18 nucleotides (nucleotides 88-105) ofthe TlR-like ligand I coding sequence in FIG. 1 (SEQ ID NO:1). Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding TlR-like ligand I provides an efficient signal peptide. An e~ficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., Mol. Biol. 196: 947-950 (1987) is a~pro~liately located in the vector portion of the construct.
The 3' primer has the sequence 5' CGC GGT ACC TCA CTG CTC CAG
CCT GGG GC 3' (SEQ ID NO:7) cont~ining the underlined Asp 718 restriction site followed by a stop codon and 17 nucleotides complementary and reverse to nucleotides 771-787 of the TlR-like ligand I coding sequence set out in FIG. 1 (SEQ ID NO:1).
The cDNA sequence encoding the extracellular domain of the full length TlR-like ligand I in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' CGC GGA TCC GCC ATC ATG GGC
AGC ACT GTC CCG 3' (SEQ ID NO:6) con~inin~ the underlined BarnHI
restriction enzyme site followed by 18 nucleotides (nucleotides 88-105) of the TlR-like ligand I coding sequence in FIG. 1 (SEQ ID NO:1). Inserted into an c~p,cs~ion vector, as described below, the 5' end of the arnplified fragment encoding TlR-like ligand I provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) is apl)lopliately located in the vector portion of the construct.
The 3' primer has the sequence 5' CGC GGT ACC TCA TCG GCT ATT
AAG GTC TTC 3' (SEQ ID NO:8) co~ g the underlined Asp 718 restriction site followed by a stop codon and 18 nucleotides complementary and reverse to nucleotides 604-621 of the Tl R-like ligand I coding sequence set out in FIG. I
(SEQ ID NO:l).
The amplified fragments are isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with BarnH I and Asp 7 18 and again is purified on a 1 %
agarose gel. This fragment is ~lecign~te~l herein F2.
The vector pA2 is used to express the TlR-like ligand I full length and extracellular domains of an TlR-like ligand I in the baculovirus expression system, using standard methods, as described in Summers et al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The vector pA2 is used to express the TlR-like ligand I full length and extracellular domains of an TlR-like ligand I in the baculovirus ~x~le~sion system, using standard methods, as described in Summers et al., A Manual o~
Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). The pA2 vector does not contain a signal peptide coding region. Thus, the TlR-like ligand I
signal peptide is relied upon (nucleotides 88 to 168 in Figure 1 (SEQ ID NO: 1 );
arnino acids 1-27 in Figure 1 (SEQ ID NO:2)).
If the TlR-like ligand I signal peptide does not result in efficient expression of the TlR-like ligand I protein, the pA2-GP vector may be used instead of the pA2 vector. The signal peptide of AcMNPV gp67, including the N-tt?rrnin~l methionine, is located just ~ of a BamHI site. One of ordinary skill in the art will understand that if the pA2-GP expression vector is used, the 5' oligonucleotide used should not contain sequence coding for the TlR-like ., .. , . _ ....

W O 98/07881 rCT~US96113777 ligand I signal peptide. Instead, the S' oligonucleotide should begin at nucleotide 169.
Both the pA2 and pA2-GP expression vectors contain the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. The signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For an easy selection of recombinant virus the beta-galactosidase gene from ~. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
Many other baculovirus vectors could be used in place of pA2 or pA2-GP, such as pAc373, pVL941 and pAcIMI provided, as those of skill readily will appreciate, that construction provides appropriately located signals for lr~lscl;~,lion, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required. Such vectors are described in Luckow et al., Virology 170:31-39, among others.
The plasmid is digested with the restriction enzyme XbaI and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is tlesi~n~ted herein "V2".
Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase. E. coli HB 101 cells are transformed with ligation mix and spread on culture plates Bacteria are identified that contain the plasmid with the human TlR-like ligand I gene by digesting DNA from individual colonies using XbaI and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA seqllenrin~ This plasmid is design~tecl herein pBacTlR-like ligand I.

CA 02263830 l999-02-23 W O 98/07881 PCTrUS96/13777 _59_ 5 ~g ofthe plasmid pBacTlR-like ligand I is co-transfected with 1.0 ~lg of a commercially available linearized baculovirus DNA ("BaculoGoldTM
baculovirus DNA", Ph~nin~en, San Diego, CA.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. US~l 84: 7413-7417 (1987).
1 llg of BaculoGoldTM virus DNA and 5 ~,lg of the plasmid pBacT1 R-like ligand I are mixed in a sterile well of a microtiter plate cont~inin~;50~1l of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~1 Lipofectin plus 90 Ill Grace's medium are added, mixed and incubated for 15 minutes at room le"~l)cldlule. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27~C. After5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect mediurn supplemented with 10% fetal calf serum is added. The plate is putback into an incubator and cultivation is continued at 27~C for four days.
After four days the supern~t~nt is collected and a plaque assay is perforrned, as described by Surnmers and Smith, cited above. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-e,~ es~ g clones, which produce blue-stainedpla~ues. (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
Four days after serial dilution, the virus is added to the cells. After apL)lopliate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar cont~ining the recombinant viruses is then resuspended in an Eppendorf tube cont~inin~ 200 ~1 of Gracels medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the sUpprn~t~nt~ of these culture dishes are harvested and then they are stored at 4~C. Clones cont~ining properly inserted hESSB I, II and III are .

W 03~ 7O81 PCTrUS96/13777 identified by DNA analysis including restriction mapping and sequencing. This is flesign~ted herein as V-TlR-like ligand I.
Sfg cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-TlR-like ligand I at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3).
Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg). 42 hours later, 5 ~Ci of 35S-methionine and 5 ,uCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation, Iysed and the labeled proteins are vicl-~li7~d by SDS-PAGE and autoradiography.

Example 3: Cloning and Expression in Mn~nntn~ Cells Most of the vectors used for the transient expression of the TlR-like ligand I protein gene sequence in m~mm~ n cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g. COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other m~mm~ n cell line can also be utilized for this purpose.
A typical m~n~m~ n expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of trancription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long t~rrnin~l repeats (LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
However, cellular signals can also be used (e.g. hurnan actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), W O 98/07881 PCTr~S96/13777 pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC
67109). ~mm~ n host cells that could be used include, human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, African green monkey cells, quail QCl-3 cells, mouse L cells and Chinese hamster ovary cells.
Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) is a useful marker to develop cell lines that carry several hundred or even several thousand copies ofthe gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem ~ 227:277-279 (1991); Bebbington et al., Bio/Technology 10: 169-175 (1992)). Using these markers, the m~tnm~ qn cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the arnplified gene(s) integrated into a chromosome. Chinese h~m.~ter ovary (CHO) cells are often used for the production of proteins.
The t;~ es~ion vectors pC l and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Brology, 438-4470 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g. with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest The vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.

, CA 02263830 l999-02-23 W O 98/07881 PCTrUS96/13777 F.~rntr/e 3(a): Cloning and Expression in COS Cells An ~A~lession plasmid is made by cloning a cDNA encoding TlR-like ligand I into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: (Dower, Colotta, F., ef al., Immunol Today 15:562 (1994)) an E.coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (Greenfeder, S.A., et al., J.
Biol. Chem. 270:13757 (1995)) an ampicillin resistance gene for selection of plasmid-cont~ining prokaryotic cells; (Polan, M.L., et al., Am. J: Obstet. Gynecol.
170:1000 (1994)) an SV40 origin of replication for propagation in eukaryotic cells; (Carinci, Mora, M., et al., Prog. Clin. Biol. Res. 349:205(1990)) a CMV
promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged sothat a cDNA conveniently can be placed under expression control of the CMV
promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
A DNA fragment encoding the entire TlR-like ligand I precursor and an HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell 37: 767 (1984). The fusion ofthe HA tagto the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is as follows.
The TlR-like ligand I cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of ~ s~ion vectors for ~xl"ession of T1 R-like ligandI in E. coli. To facilitate detection, purification and characterization of the expressed TlR-like ligand I, one of the primers contains a hemagglutinin tag ("HA tag") as described above.

W O 98/07881 PCT~US96/13777 One of ordinary skill in the art will understand that the full-length TlR-like ligand I protein (amino acid about I to about 217) can be expressed in COS
cells using suitable 5' and 3' oligonucleotide primers.
The cDNA sequence encoding the extracellular domain of the full length TlR-like ligand I in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
The 5' primer has the sequence: 5' CGC GGA TCC GCC ATC ATG GGC
AGC ACT GTC CCG 3' (SEQ ID NO:6) having the underlined BamH1 site plus 18 nucleotides corresponding to nucleotides 88-105 in SEQ ID NO:l.
The 3' primer, cont~ining the underlined Xba I site, a stop codon, 9 codons thereafter forrning the hemagglutinin HA tag, and 18 bp of 3' coding sequence, has the following sequence:
S' CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG
GTA TCG GCT ATT AAG GTC TTC 3' (SEQ ID NO:9) as 604-621 of the reverse complement of SEQ ID NO: 1.
The PCR arnplified DNA fragment and the vector, pcDNAI/Arnp, are digested with BamH I and Xba I and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and e~mine~l by restriction analysis and gel sizing for the presence ofthe TlR-like ligand I encoding fragment.
For expression of recombinant TlR-like ligand I, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of Tl R-like ligand I by the vector.
Expression of the TlR-like ligand I HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for exarnple Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media cont~ining 35S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with d~elge,lL-co~ ,g RIPA buffer: 150 mM NaCl, 1%
NP-40,0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are ,~)leci~ d from the cell lysate and fromthe culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell Iysate, which is not seen in negative controls.

Example 3(b): Cloning and Expression in CHO Cells The vector pC4 is used for the e~ cssion of Tl R-like ligand protein.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No.
37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese h~m~ter ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing thecells in a selective mediurn (alpha minus MEM, Life Technologies) supplemented ~vith the chemotherapeutic agent methotrexate. The amplification of the DHFR
genes in cells resistant to methotrexate (MTX) has been well docl-mente(l (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Biol.Chem. 253:1357-1370, Hamlin, J.L. and Ma, C. 1990, Biochem. et Biophys.
Acta, 1097:107-143, Page, M.J. and Sycl~nh~m, M.A. 1991, Biotechnology Vol.
9:64-68). Cells grown in increasing concentrations of MTX develop resi.ct~nGe to the drug by overproducing the target enzyme, DHFR, as a result of arnplification of the DHFR gene. If a second gene is linked to the DHFR gene it is usually co-amplified and over-~r~ssed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the W O 98/07881 PCTrUS96/13777 methotrexate is withdra~vn, cell lines contain the arnplified gene integrated into the chromosome(s).
Plasmid pC4 contains for the expression of the gene of interest a strong promoter of the long te~ninAl repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular biology, March 1985, 438-4470) plus a fragment isolated from the enhancer of the irnmediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BarnHI, Pvull, and Nrul. Behind these cloning sites the plasmid contains translational stop codons in all three reading frarnes followed by the 3' intron and the polyadenylation site of the rat preproinsulin gene. Other highly efficient promoters can also be used for the exples~ion, e.g., the human ~-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g. G418 plus methotrexate.
The plasmid pC4 is digested with the restriction enzyme BarnHI and then dephosphorylated using calf intPstinAl phosphates by procedures known in the artThe vector is then isolated from a 1 % agarose gel.
The DNA sequence encoding TlR like ligand I protein is arnplified using PCR oligonucleotide primers specific to the arnino t~.nnin:~l sequence of the T1 R
like ligand I protein and to vector sequences 3' to the gene. Additional nucleotides contAining restriction sites to fAcilit~te cloning are added to the 5' and 3 ' sequences respectively.
The cDNA sequence encoding the full length TlR-like ligand I in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. The 5' primer has the sequence 5' CGC

, .... . .

wo ~ ,8l PCT/US96/13777 GGA TCC GCC ATC ATG GGC AGC ACT GTC CCG 3' (SEQ ID NO: 6), cont~ining the underlined BamH I restriction enzyme site followed by 18 nucleotides (nucleotides 88-105) of the sequence of TlR-like ligand I in FIG. 1 (SEQ ID NO:l). Inserted into an expression vector, as described below, the S' end ofthe amplified fragment encoding TlR-like ligand I provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) is ap~lo~l;ately located in the vector portion of the construct.
The 3 ' primer has the sequence 5 ' GCG GGT ACC TCA CTG CTC CAG
CCT GGG GC 3 ' (SEQ ID NO: 9), cont~ining the underlined Asp 718 restriction site followed by a stop codon and 17 nucleotides reverse and complementary to nucleotides 771-787 ofthe TlR-like ligand coding sequence in FIG. 1 (SEQ ID
NO: 1). The restriction sites are convenient to restriction enzyme sites in the CHO
expression vector PC4.
The cDNA sequence encoding the extracellular domain of the full length TlR-like ligand I in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the S' and 3' sequences of the gene.
The 5' primer has the sequence 5' CGC GGA TCC GCC ATC ATG GGC
AGC ACT GTC CCG 3' (SEQ ID NO:6) cont~ining the llnclerlined BamHI
restriction enzyme site and 18 nucleotides (nucleotides 88-105) ofthe TlR-like ligand I coding sequence in FIG. I (SEQ ID NO:l). Inserted into an ~ ession vector, as described below, the 5' end ofthe amplified fragment encoding TlR-like ligand I provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:
947-950 (1987) is appl~p.iately located in the vector portion ofthe construct.
The 3' primer has the sequence 5' CGC GGT ACC TCA TCG GCT ATT
AAG GTC TTC 3' (SEQ ID NO:8)co~ g the underlined Asp 718 restriction site followed by a stop codon and 18 nucleotides reverse and complement~ry to nucleotides 604-621 ofthe TlR-like ligand I coding sequence set out in FIG. 1 (SEQ ID NO:l).

W O 98/07881 PCT~US96/13777 The amplified TlR-like ligand I DNA are digested with BarnH I and Asp 718. The vector pC4 is digested with BamHI and the digested DNAs are then ligated together. The isolated fragment and the dephosphorylated vector are thenligated with T4 DNA ligase. Insertion of the TlR like ligand I protein DNA into the BamH I restricted vector places the TlR like ligand I protein coding region downstream of and operably linked to the vector's promoter. E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid pC4 inserted in the correct orientation using the restriction enzyme BamHI. The ligation mixture is transformed into competent E. coli cells using standard procedures as described, for example, in Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and ex~mined by restriction analysis and gel sizing for the presence of the Tl R-like ligand I-encoding fragment. The sequence of the inserted gene is confirmed by DNA sequencing.

Transfection oSCHO-DHFR-cells Chinese h~ ovary cells lacking an active DHFR enzyme are used for transfection. S llg of the expression plasmid C4 are cotransfected with 0.5 ~g of the plasmid pSVneo using the lipofectin method (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the gene neo from TnS
encoding an enzyme that confers resi~t~n~e to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplem~nted with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM).

. ~

W O~t~7~1 PCTrUS96tl3777 Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates cont~ining even higher concentrations of methotrexate (500 nM, 1 ~lM, 2 ~lM, ~ IlM). The same procedure is repeated until clones grow at a concentration of 100 IlM.
The ~ression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE. Expression ofthe TlR-like ligand I fusion protein is ~letect~(1 by radiolabelling and immnnnprecipitation, using methods described in, for example Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media cont~ining 35S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-cont~ining RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody.
The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An ex~lession product of the expected size is seen in the cell lys~te, which is not seen in negative controls.

Example 4: Tiss~e distribution of TlR-like Ligand I gene expression Northern blot analysis was carried out to examine ex~lession levels ofthe TlR-like ligand I gene in human tissues, using methods described by, among others, Sambrook et al., cited above. A cDNA probe cont~ining the entire TlR-like ligand r nucleotide sequence (SEQ ID NO:1) was labeled with 32p using the rediprimeT~ DNA labelling system (Amersham Life Science), according to manufacturer's instructions. After labelling, the probe was purified using a CHROMA SPIN-100TM column (Clontech Laboratories, Inc.), according to m~nl~f~rlllrer's protocol number PT1200-1. The purified labelled probe was then W O 98/07881 PCT~US96/13777 used to e~zlmine various human tissues for ~ cs~ion of the TlR-like ligand I
gene.
Multiple Tissue Northern (MTN) blots cont~inin~ various human tissues (H) and human immune system tissues (IM) were obtained from Clontech and are examined with labelled probe using ExpressHybTM Hybridization Solution (Clontech) according to manufacturerls protocol number PT1190-1. Following hybridization and washing, the blots were mounted and exposed to film at -70~C
overnight, and films developed according to standard procedures. An approxi-mately 2.0 kb TlR-like ligand I signal was detected in lanes cont~ining mRNA
from spleen, Iymphnode, thymus, appendix, bone marrow, fetal liver and peripheral blood leukocytes. No signal was detected lanes cont~ining mRNA
from non-immune tissues.
An approximately 2.0 kb TlR-like ligand I signal was detected in lanes containing mRNA from spleen, Iymphnode, thymus, appendix, bone marrow, fetal liver and peripheral blood leukocytes. No signal was detected lanes cont~2ining mRNA from non-imml-ne tissues.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in light of the above te~ching~ and, therefore, are within the scope of the appended claims.
The disclosures of all patents, patent applications, and publications referred to herein are hereby entirely incorporated by reference.

CA 02263830 l999-02-23 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Human Genome Sciences, Inc.
9410 Key West Avenue Rockville, MD 20850 United States of America APPLICANT/INVENTORS: NI, JIAN
GENTZ, REINER L.
ROSEN, CRAIG A.
(ii) TITLE OF INVENTION: Tl RECEPTOR-LIKE LIGAND I
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN h FOX, P.L.L.C.
(B) STREET: 1100 NEW YORK AVENUE, SUITE 600 (C) CITY: WASHINGTON
(D) STATE: D.C.
(E) COUNTRY: USA
(F) ZIP: 20005-3934 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To be assigned (B) FILING DATE: 23-AUG-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:Goldstein, Jorge A.
(B) REGISTRATION NUMBER:29,021 (C) REFERENCE/DOCKET NUMBER: 1488.040PC00 (ix) TELECOMMnNICATION INFORMATION:
(A) TELEPHONE: 202-371-2600 (B) TELEFAX: 202-371-2540 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1401 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

W O ~ Al PCTAUS96/13777 -7l-(A) NAME/KEY: CDS
(B) LOCATION: 88..738 (ix) FEATURE:
(A) NAME/KEY: sig_peptide ~B) LOCATION: 88..166 (ix) FEATURE:
(A) NAME/KEY: mat_peptide (B) LOCATION: 169..738 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Met Gly Ser Thr Val Pro Arg Ser Ala Ser Val Leu Leu Leu Leu Leu Leu Leu Arg Arg Ala Glu Gln Pro Cys Gly Ala Glu Leu Thr Phe Glu Leu Pro Asp Asn Ala Lys Gln Cys Phe His Glu Glu Val Glu Gln Gly Val Lys Phe Ser Leu Asp Tyr Gln Val Ile Thr Gly Gly His Tyr Asp Val Asp Cys Tyr Val Glu Asp Pro Gln Gly Asn Thr Ile Tyr Arg Glu Thr Lys Lys Gln Tyr Asp Ser Phe Thr Tyr Arg Ala Glu Val Lys Gly Val Tyr Gln Phe Cys Phe Ser Asn Glu Phe Ser Thr Phe Ser His Lys Thr Val Tyr Phe Asp Phe Gln Val Gly Asp Glu Pro Pro Ile Leu Pro Asp Met Gly Asn Arg Val Thr Ala Leu Thr Gln Met Glu Ser Ala Cys Val Thr Ile His Glu Ala Leu Lys ., . ~ _ .. . . . . ... .. . .

CA 02263830 l999-02-23 W O 98/07881 PCT~US96/13777 Thr Val Ile Asp Ser Gln Thr His Tyr Arg Leu Arg Glu Val Gln Asp Arg Ala Arg Ala Glu Asp Leu Asn Ser Arg Val Ser Tyr Trp Ser Val Gly Glu Thr Ile Ala Leu Phe Val Val Ser Phe Ser Gln Val Leu Leu Leu Lys Ser Phe Phe Thr Glu Lys Arg Pro Ile Ser Arg Ala Val His Ser C~ lC ATGATGCATG GGTCATTTGT CTTGGGTGTC CTATCCCATA TGGAGAAGAA 1148 AGGGGCTCTA AGTTCTGGCT ~ CTT TGGGGTTCTC TGTACCTGAG GAAACCAGGC 1208 GGACAGAATG GTGACTGGGT GCCCTTGGTG AG~ ~lAT TTCCTAGGAG GTAGAAAACT 1328 GTGGGAAACT GTGGCTAATA AAAACTAAGT GTGAGCGTCC TGGAAAAAAA PAAVU~LAAA 1388 PAU~UUUAAAA AAA 1401 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 217 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~Q~N~ DESCRIPTION: SEQ ID NO:2:

Met Gly Ser Thr Val Pro Arg Ser Ala Ser Val Leu Leu Leu Leu Leu Leu Leu Arg Arg Ala Glu Gln Pro Cys Gly Ala Glu Leu Thr Phe Glu ~eu Pro Asp Asn Ala Lys Gln Cys Phe His Glu Glu Val Glu Gln Gly ~al Lys Phe Ser Leu Asp Tyr Gln Val Ile Thr Gly Gly His Tyr Asp Val Asp Cys Tyr Val Glu Asp Pro Gln Gly Asn Thr Ile Tyr Arg Glu Thr Lys Lys Gln Tyr Asp Ser Phe Thr Tyr Arg Ala Glu Val Lys Gly Val Tyr Gln Phe Cys Phe Ser Asn Glu Phe Ser Thr Phe Ser His Lys ~hr Val Tyr Phe Asp Phe Gln Val Gly Asp Glu Pro Pro Ile Leu Pro ~sp Met Gly Asn Arg Val Thr Ala Leu Thr Gln Met Glu Ser Ala Cys Val Thr Ile His Glu Ala Leu Lys Thr Val Ile Asp Ser Gln Thr His Tyr Arg Leu Arg Glu Val Gln Asp Arg Ala Arg Ala Glu Asp Leu Asn Ser Arg Val Ser Tyr Trp Ser Val Gly Glu Thr Ile Ala Leu Phe Val Val Ser Phe Ser Gln Val Leu Leu Leu Lys Ser Phe Phe Thr Glu Lys ~rg Pro Ile Ser Arg Ala Val His Ser (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 227 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein CA 02263830 l999-02-23 W O 98/07881 PCTrUS96l13M7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Met Ala Ala Gly Ala Ala Leu Ala Leu Ala Leu Trp Leu Leu Met Pro Pro Val Glu Val Gly Gly Ala Gly Pro Pro Pro Ile Gln Asp Gly Glu Phe Thr Phe Leu Leu Pro Ala Gly Arg Lys Gln Cys Phe Tyr Gln Ser Ala Pro Ala Asn Ala Ser Leu Glu Thr Glu Tyr Gln Val Ile Gly Gly Ala Gly Leu Asp Val Asp Phe Thr Leu Glu Ser Pro Gln Gly Val Leu Leu Val Ser Glu Ser Arg Lys Ala Asp Gly Val His Thr Val Glu Pro Thr Glu Ala Gly Asp Tyr Lys Leu Cys Phe Asp Asn Ser Phe Ser Thr Ile Ser Glu Lys Leu Val Phe Phe Glu Leu Ile Phe Asp Ser Leu Gln Asp Asp Glu Glu Val Glu Gly Trp Ala Glu Ala Val Glu Pro Glu Glu Met Leu Asp Val Lys Met Glu Asp Ile Lys Glu Ser Ile Glu Thr Met Arg Thr Arg Leu Glu Arg Ser Ile Gln Met Leu Thr Leu Leu Arg Ala Phe Glu Ala Arg Asp Arg Asn Leu Gln Glu Gly Asn Leu Glu Arg Val Asn Phe Trp Ser Ala Val Asn Val Ala Val Leu Leu Leu Val Ala Val Leu Gln Val Cys Thr Leu Lys Arg Phe Phe Gln Asp Lys Arg Pro Val Pro Thr (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRAMDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

W 0~8~'~7~1 PCTrUS96/13777 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) S~QU~:N~ DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Claims

What Is Claimed Is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a nuceleotide sequence at least 95% identical to a sequence selected fromthe group consisting of:
(a) a nucleotide sequence encoding the full-length T1R-like ligand I polypeptide having an amino acid sequence shown in FIG. 1 (SEQ ID
NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656;
(b) a nucleotide sequence encoding the mature T1R-like ligand I polypeptide having an amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656;
(c) a nucleotide sequence encoding the T1R-like ligand I
extracellular domain having an amino acid sequence shown in FIG. 1 (SEQ ID
NO:2) or as encoded by the cDNA contained in ATCC Deposit No. 97656;
(d) a nucleotide sequence encoding the T1R-like ligand I
transmembrane domain having an amino acid sequence shown in FIG. 1 [SEQ ID
NO:2] or as encoded by the cDNA contained in ATCC Deposit No. 97656;
(e) a nucleotide sequence encoding the T1R-like ligand I
intracellular domain having an amino acid sequence shown in FIG. 1 [SEQ ID
NO:2] or as encoded by the cDNA contained in ATCC Deposit NO. 97656; and (f) a nucleotide sequence complementary to the nucleotide sequence of any one of the polynucleotides of (a), (b), (c), (d), or (e).

2. The nucleic acid molecule of claim 1, wherein said polynucleotide has a nucleotide sequence at least 95% identical to a sequence encoding the T1R-like ligand I polypeptide extracellular domain and intracellular domain wherein all or part of the transmembrane domain is deleted.

3. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), (d), (e), or (f) of claim 1 wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

4. An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a T1R-like ligand I polypeptide having an amino acid sequence in (a), (b), (c), (d), or (e) of claim 1.

5. The isolated nucleic acid molecule of claim 4, which encodes an epitope-bearing portion of a T1R-like ligand I polypeptide selected from the group consisting of: a polypeptide comprising amino acid residues from about 20 to about 53 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 82 to about 98 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 106 to about 134 in Figure 1 (SEQ
ID NO:2); and a polypeptide comprising amino acid residues from about 155 to about 184.

6. A method for making a recombinant vector comprising inserting the isolated nucleic acid molecule of claim 1 into a vector.

7. A recombinant vector produced by the method of claim 6.

8. A method for making a recombinant host cell comprising introducing the recombinant vector of claim 7 into a host cell.

9. A recombinant host cell produced by the method of claim 8.

10. A recombinant method for producing a polypeptide comprising culturing the host cell of claim 9 under conditions such that said polypeptide is expressed and recovering said polypeptide.

11. An isolated T1R-like ligand I polypeptide having an amino acid sequence at least 95% identical to a sequence selected from the group consistingof:
(a) the amino acid sequence of the full-length T1R-like ligand I polypeptide shown in FIG. I (SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656;
(b) the amino acid sequence of the mature T1R-like ligand I
polypeptide shown in FIG. 1 (SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit No. 97656;
(c) the amino acid sequence of the T1R-like ligand I
polypeptide extracellular domain shown in FIG. 1 (SEQ ID NO:2) or as encoded by the cDNA contained in ATCC Deposit No. 97656;
(d) the amino acid sequence of the T1R-like ligand I
polypeptide transmembrane domain shown in FIG. 1 (SEQ ID NO:2) or as encoded by the cDNA contained in ATCC Deposit No. 97656;
(e) the amino acid sequence of the T1R-like ligand polypeptide intracellular domain shown in FIG. 1 (SEQ ID NO:2) or as encoded by the cDNA contained in ATCC Deposit NO. 97656;
(f) the amino acid sequence of the T1R-like ligand I
polypeptide extracellular and intracellular domains shown in FIG. 1 (SEQ ID
NO:2) or as encoded by the cDNA contained in ATCC Deposit NO. 97656 wherein all or part of the transmembrane domain is deleted; and (g) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e), or (f).

12. An isolated polypeptide comprising an epitope-bearing portion of the T1R-like ligand I protein, wherein said portion is selected from the group consisting of: a polypeptide comprising amino acid residues from about 20 to about 53 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 82 to about 98 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 106 to about 134 in Figure 1 (SEQ

ID NO:2); and a polypeptide comprising amino acid residues from about 155 to about 184.

13. An isolated antibody that binds specifically to a T1R-like ligand I polypeptide of claim 11.

14. A method for treating an individual in need of an increased level of T1R-like ligand I activity comprising administering to said individual a composition comprising an isolated polypeptide of claim 11.

15. A method for treating an individual in need of an decreased level of T1R-like ligand I activity comprising administering to said individual a composition comprising an isolated antibody of claim 13.

16. A method useful during the diagnosis of a disorder, comprising:
(a) measuring T1R-like ligand I gene expression level in cells or body fluid of an individual;
(b) comparing the T1R-like ligand I gene expression level of said individual with a standard T1R-like ligand I gene expression level, wherebyan increase or decrease in the T1R-like ligand I gene expression level over saidstandard is indicative of a T1R-like ligand I-related disorder.