AU2001264889B2 - Human receptor proteins; related reagents and methods - Google Patents

Description

WO 01/90151 PCT/US01/16766 HUMAN RECEPTOR PROTEINS; RELATED REAGENTS AND METHODS FIELD OF THE INVENTION The present invention relates to compositions and methods for affecting mammalian physiology, including morphogenesis or immune system function. In particular, it provides nucleic acids, proteins, and antibodies which regulate development and/or the immune system. Diagnostic and therapeutic uses of these materials are also disclosed.

BACKGROUND OF THE INVENTION Recombinant DNA technology refers generally to techniques of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.

WO 01/90151 PCT/US01/16766 For some time, it has been known that the mammalian immune response is based on a series of complex cellular interactions, called the "immune network". Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the immune response does, in fact, revolve around the networklike interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally hold the opinion that soluble proteins, known as lymphokines, cytokines, or monokines, play critical roles in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which will lead to significant advancements in the diagnosis and therapy of numerous medical abnormalities, immune system disorders.

Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and/or differentiation of pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages which make up a complex immune system. Proper and balanced interactions between the cellular components are necessary for a healthy immune response. The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.

Cell lineages especially important to the immune response include two classes of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal), and T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T- WO 01/90151 PCT/US01/16766 cells) making up the immune network. These lymphocytes interact with many other cell types.

Another important cell lineage is the mast cell (which has not been positively identified in all mammalian species), which is a granule-containing connective tissue cell located proximal to capillaries throughout the body.

These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows: when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators, histamine, serotonin, heparin, and prostaglandins, which cause allergic reactions, anaphylaxis.

Research to better understand and treat various immune disorders has been hampered by the general inability to maintain cells of the immune system in vitro.

Immunologists have discovered that culturing many of these cells can be accomplished through the use of T-cell and other cell supernatants, which contain various growth factors, including many of the lymphokines.

The interleukin-1 family of proteins includes the IL-la, the IL-1P, the IL-1RA, and recently the IL-ly (also designated Interferon-Gamma Inducing Factor, IGIF). This related family of genes have been implicated in a broad range of biological functions. See Dinarello (1994) FASEB J. 8:1314-1325; Dinarello (1991) Blood 77:1627-1652; and Okamura, et al. (1995) Nature 378:88-91.

In addition, various growth and regulatory factors exist which modulate morphogenetic development. This includes, the Toll ligands, which signal through binding to receptors which share structural, and mechanistic, features characteristic of the IL-1 receptors. See, Lemaitre, et al. (1996) Cell WO 01/90151 PCT/US01/16766 86:973-983; and Belvin and Anderson (1996) Ann. Rev. Cell Devel. Biol. 12:393-416.

From the foregoing, it is evident that the discovery and development of new soluble proteins and their receptors, including ones similar to lymphokines, should contribute to new therapies for a wide range of degenerative or abnormal conditions which directly or indirectly involve development, differentiation, or function, of the immune system and/or hematopoietic cells. In particular, the discovery and understanding of novel receptors for lymphokine-like molecules which enhance or potentiate the beneficial activities of other lymphokines would be highly advantageous. The present invention provides new receptors for ligands exhibiting similarity to interleukin-1 like compositions and related compounds, and methods for their use.

WO 01/90151 PCT/US01/16766 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic comparison of the protein architectures of Drosophila, Caenorabditis, and human DTLRs, and their relationship to vertebrate IL-1 receptors and plant disease resistance proteins. Three Drosophila (Dm) DTLRs (Toll, 18w, and the Mst ORF fragment) (Morisato and Anderson (1995) Ann. Rev. Genet. 29:371-399; Chiang and Beachy (1994) Mech. Develop. 47:225-239; Mitcham, et al. (1996) J. Biol. Chem. 271:5777-5783; and Eldon, et al.

(1994) Develop. 120:885-899) are arrayed beside four complete (DTLRs 1-4) and one partial (DTLR5) human (Hu) receptors. Individual LRRs in the receptor ectodomains that are flagged by PRINTS (Attwood, et al. (1997) Nucleic Acids Res. 25':212-217) are explicitly noted by boxes; 'top' and 'bottom' Cys-rich clusters that flank the C- or N-terminal ends of LRR arrays are respectively drawn by opposed half-circles. The loss of the internal Cys-rich region in DTLRs 1-5 largely accounts for their smaller ectodomains (558, 570, 690, and 652 aa, respectively) when compared to the 784 and 977 aa extensions of Toll and 18w.

The incomplete chains of DmMst and HuDTLR5 (about 519 and 153 aa ectodomains, respectively) are represented by dashed lines. The intracellular signaling module common to DTLRs, IL-1-type receptors (IL-IRs), the intracellular protein Myd88, and the tobacco disease resistance gene N product (DRgN) is indicated below the membrane. See, Hardiman, et al. (1996) Oncogene 13:2467-2475; and Rock, et al. (1998) Proc. Nat'l Acad. Sci. USA 95:588-.

Additional domains include the trio of Ig-like modules in IL-IRs (disulfide-linked loops); the DRgN protein features an NTPase domain (box) and Myd88 has a death domain (black oval).

Figures 2A-2C show conserved structural patterns in the signaling domains of Toll- and IL-1-like cytokine receptors, and two divergent modular proteins. Figures 2A-2B show a sequence alignment of the common TH domain.

WO 01/90151 PCT/US01/16766 DTLRs are labeled as in Figure 1; the human (Hu) or mouse (Mo) IL-1 family receptors (IL-1R1-6) are sequentially numbered as earlier proposed (Hardiman, et al. (1996) Oncogene 13:2467-2475); Myd88 and the sequences from tobacco (To) and flax, L. usitatissimum represent

C-

and N-terminal domains, respectively, of larger, multidomain molecules. Ungapped blocks of sequence (numbered 1-10) are boxed. Triangles indicate deleterious mutations, while truncations N-terminal of the arrow eliminate bioactivity in human IL-1R1 (Heguy, et al.

(1992) J. Biol. Chem. 267:2605-2609). PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (King and Sternberg (1996) Protein Sci. 5:2298-2310) secondary structure predictions of a-helix -strand or coil are marked. The amino acid shading scheme depicts chemically similar residues: hydrophobic, acidic, basic, Cys, aromatic, structure-breaking, and tiny.

Diagnostic sequence patterns for IL-1Rs, DTLRs, and full alignment (ALL) were derived by Consensus at a stringency of 75%. Symbols for amino acid subsets are (see internet site for detail): o, alcohol; 1, aliphatic; any amino acid; a, aromatic; c, charged; h, hydrophobic; negative; p, polar; positive; s, small; u, tiny; t, turnlike. Figure 2C shows a topology diagram of the proposed TH P/a domain fold. The parallel 1-sheet (with P-strands A-E as yellow triangles) is seen at its Cterminal end; c-helices (circles labeled 1-5) link the Pstrands; chain connections are to the front (visible) or back (hidden). Conserved, charged residues at the C-end of the P-sheet are noted in gray (Asp) or as a lone black (Arg) residue (see text).

Figure 3 shows evolution of a signaling domain superfamily. The multiple TH module alignment of Figures 2A-2B was used to derive a phylogenetic tree by the Neighbor-Joining method (Thompson, et al. (1994) Nucleic WO 01/90151 PCT/US01/16766 Acids Res. 22:4673-4680). Proteins labeled as in the alignment; the tree was rendered with TreeView.

Figures 4A-4D depict FISH chromosomal mapping of human DTLR genes. Denatured chromosomes from synchronous cultures of human lymphocytes were hybridized to biotinylated DTLR cDNA probes for localization. The assignment of the FISH mapping data (left, Figures 4A, DTLR2; 4B, DTLR3; 4C, DTLR4; 4D, DTLRS) with chromosomal bands was achieved by superimposing FISH signals with DAPI banded chromosomes (center panels). Heng and Tsui (1994) Meth. Molec. Biol. 33:109-122. Analyses are summarized in the form of human chromosome ideograms (right panels).

Figures 5A-5F depict mRNA blot analyses of Human DTLRs. Human multiple tissue blots (He, heart; Br, brain; P1, placenta; Lu, lung; Li, liver; Mu, muscle; Ki, kidney; Pn, Pancreas; Sp, spleen; Th, thymus; Fr, prostate; Te, testis; Ov, ovary, SI, small intestine; Co, colon; PBL, peripheral blood lymphocytes) and cancer cell line (promyelocytic leukemia, HL60; cervical cancer, HELAS3; chronic myelogenous leukemia, K562; lymphoblastic leukemia, Molt4; colorectal adenocarcinoma, SW480; melanoma, G361; Burkitt's Lymphoma Raji, Burkitt's; colorectal adenocarcinoma, SW480; lung carcinoma, AS49) containing approximately 2 gg of poly(A) RNA per lane were probed with radiolabeled cDNAs encoding DTLR1 (Figures 5A-5C), DTLR2 (Figure 5D), DTLR3 (Figure 5E), and DTLR4 (Figure 5F) as described. Blots were exposed to Xray film for 2 days (Figures 5A-5C) or one week (Figure at -700 C with intensifying screens. An anomalous 0.3 kB species appears in some lanes; hybridization experiments exclude a message encoding a DTLR cytoplasmic fragment.

SUMMARY OF THE INVENTION In a first aspect there is provided an isolated Santigenic polypeptide comprising the amino acid sequence of SEQ ID NO:12. In one embodiment, there is provided a fusion protein comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:12.

00 In another aspect there is provided an isolated 00 antibody or antibody fragment which specifically binds to 10 a polypeptide comprising the amino acid sequence of SEQ ID NO:12.

SIn yet another aspect, there is provided an isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:12. In one embodiment, there is provided an expression vector comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:12. In another embodiment there is provided a host cell which comprises this expression vector. Also provided is a process for recombinantly producing a polypeptide, comprising culturing the -host cell under conditions in which the polypeptide is expressed.

Also described herein is nine novel related mammalian receptors, e. primate, human, DNAX Toll receptor like molecular structures, designated DTLR2, DTLR3, DTLR4, DTLR5, DTLR7, DTLR8, DTLR9, and DTLR10, and their biological activities. It includes nucleic acids coding for the polypeptides themselves and methods for their production and use. The nucleic acids are characterized, in part, by their homology to cloned complementary DNA (cDNA) sequences enclosed herein.

Described herein is a composition of matter selected from the group of: a substantially pure or recombinant DTLR2 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 4; a natural sequence DTLR2 of SEQ ID NO: 4; a fusion protein comprising DTLR2 sequence; a substantially pure or recombinant DTLR3 protein or peptide exhibiting identity U over a length of at least about 12 amino acids to SEQ ID O NO: 6; a natural sequence DTLR3 of SEQ ID NO: 6; a fusion Sprotein comprising DTLR3 sequence; a substantially pure or recombinant DTLR4 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 26; a natural sequence DTLR4 of SEQ ID NO: 26; a fusion protein comprising DTLR4 sequence; a substantially 00 pure or recombinant DTLR5 protein or peptide exhibiting 0 identity over a length of at least about 12 amino acids to SEQ ID NO: 10; a natural sequence DTLR5 of SEQ ID NO: a fusion protein comprising DTLR5 sequence; a Ssubstantially pure or recombinant DTLR6 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 12, 28, or 30; a natural sequence DTLR6 of SEQ ID NO: 12, 28, or 30; a fusion protein comprising DTLR6 sequence; a substantially pure or recombinant DTLR7 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 16, 18, or 37; a natural sequence DTLR7 of SEQ ID NO: 16, 18, or 37; a fusion protein comprising DTLR7 sequence; a substantially pure or recombinant DTLR8 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 32 or 39; a natural sequence DTLR8 of SEQ NO: 32 or 39; a fusion protein comprising DTLR8 sequence; a substantially pure or recombinant DTLR9 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 22 or 41; a natural sequence DTLR9 of SEQ ID NO: 22 or 41; a fusion protein comprising DTLR9 sequence; a substantially pure or recombinant protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 34, 43, or 45; a natural sequence DTLR10 of SEQ ID NO: 34, 43, or 45; and a fusion protein comprising DTLR10 sequence. Preferably, the substantially pure or isolated protein comprises a segment exhibiting sequence identity to a corresponding portion of a DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10, wherein said identity is over at least about Samino acids; preferably about 19 amino acids; or more Spreferably about 25 amino acids. In specific embodiments described herein, the composition of matter: is DTLR2, which comprises a mature sequence of Table 2; or lacks a post-translational modification; is DTLR3, which comprises a mature sequence of Table 3; or lacks a post- 00 translational modification; is DTLR4, which: comprises a

OC

j00 mature sequence of Table 4; or lacks a post-translational S 10 modification; is DTLR5, which: comprises the complete sequence of Table 5; or lacks a post-translational; is 0 DTLR6, which comprises a mature sequence of Table 6; or lacks a post-translational modification; is DTLR7, which comprises a mature sequence of Table 7; or lacks a posttranslational modification; is DTLR8, which: comprises a mature sequence of Table 8; or lacks a post-translational modification; is DTLR9, which: comprises the complete sequence of Table 9; or lacks a post-translational; is which comprises a mature sequence of Table 10; or lacks a post-translational modification; or the composition of matter may be a protein or peptide which: is from a warm blooded animal selected from a mammal, including a primate, such as a human; comprises at least one polypeptide segment of SEQ ID NO: 4, 6, 26, 12, 28, 30, 16, 18, 32, 22, or 34; exhibits a plurality of portions exhibiting said identity; is a natural allelic variant of DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10; has a length at least about amino acids; exhibits at least two non-overlapping epitopes which are specific for a primate DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or exhibits sequence identity over a length of at least about amino acids to a primate DTLR2, DTLR3, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9 or DTLR10; further exhibits at least two non-overlapping epitopes which are specific for a primate DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10; exhibits identity over a length of at Sleast about 20 amino acids to a rodent DTLR6; is O glycosylated; has a molecular weight of at least 100 kD Swith natural glycosylation; is a synthetic polypeptide; is ;Z attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from natural sequence; or is a deletion or insertion variant from a natural sequence.

00 Also described herein is a composition comprising: a 00 sterile DTLR2 protein or peptide; or the DTLR2 protein or IND 10 peptide and a carrier, wherein the carrier is: an aqueous Scompound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; a sterile DTLR3 protein or peptide; or the DTLR3 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; a sterile DTLR4 protein or peptide; or the DTLR4 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; a sterile DTLR5 protein or peptide; or the protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; a sterile DTLR6 protein or peptide; or the DTLR6 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; a sterile DTLR7 protein or peptide; or the DTLR7 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; a sterile DTLR8 protein or peptide; or the DTLR8 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, \D including water, saline, and/or buffer; and/or formulated 0 for oral, rectal, nasal, topical, or parenteral administration; a sterile DTLR9 protein or peptide; or the SDTLR9 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; a sterile 00 protein or peptide; or the DTLR10 protein or peptide and a 00 carrier, wherein the carrier is: an aqueous compound, 10 including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral Sadministration.

In certain fusion proteins described herein, there is a fusion protein comprising: mature protein sequence of Table 2, 3, 4, 5, 6, 7, 8, 9, or 10; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another receptor protein.

Also described herein is a kit comprising a DTLR protein or polypeptide, and: a compartment comprising the protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.

Binding compounds described herein include those comprising an antigen binding site from an antibody, which specifically binds to a natural DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10 protein, wherein: the protein is a primate protein; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide of Table 2, 3, 4, 5, 6, 7, 8, 9, or 10; is raised against a mature DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10; is raised to a purified human DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10; is immunoselected; is a polyclonal antibody; binds to a denatured DTLR2, DTLR3, DTLR4, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10; exhibits a Kd to antigen of at least 30 4M; is attached to a solid

I

substrate, including a bead or plastic membrane; is in a 0 sterile composition; or is detectably labeled, Sincluding a radioactive or fluorescent label. A binding ;Z composition kit often comprises the binding compound, and: a compartment comprising said binding compound; and/or instructions for use or disposal of reagents in the kit.

Often the kit is capable of making a qualitative or 00 quantitative analysis.

OC

i00 Methods are described, e. of making an antibody, comprising immunizing an immune system with an immunogenic amount of a primate DTLR2, DTLR3, DTLR4, DTLR5, DTLR7, SDTLR8, DTLR9, or DTLR10, thereby causing said antibody to be produced; or producing an antigen:antibody complex, comprising contacting such an antibody with a mammalian DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or protein or peptide, thereby allowing said complex to form.

Other compositions described herein include a composition comprising: a sterile binding compound, or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.

Nucleic acids described herein include an isolated or recombinant nucleic acid encoding a DTLR2-10 protein or peptide or fusion protein, wherein: the DTLR is from a mammal; or the nucleic acid: encodes an antigenic peptide sequence of Table 2, 3, 4, 5, 6, 7, 8, 9, or encodes a plurality of antigenic peptide sequences of Table 2, 3, 4, 5, 6, 7, 8, 9, or 10; comprises at least 17 contiguous nucleotides from Table 2, 3, 4, 5, 6, 7, 8, 9, or 10; exhibits at least about 80% identity to a natural cDNA encoding said segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a mammal, including a \U primate; comprises a natural full length coding sequence; O is a hybridization probe for a gene encoding said DTLR; or Sis a PCR primer, PCR product, or mutagenesis primer. A ;Z cell, tissue, or organ comprising such a recombinant nucleic acid is also provided. Preferably, the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse 00 cell; a primate cell; or a human cell. Kits are described 00 comprising such nucleic acids, and: a compartment \O 10 comprising said nucleic acid; a compartment further comprising a primate DTLR2, DTLR3, DTLR4, or DTLR5 protein or polypeptide; and/or instructions for use or disposal of reagents in the kit. Often, the kit is capable of making a qualitative or quantitative analysis.

Also described herein is a nucleic acid which: hybridizes under wash conditions of 30° C and less than 2M salt to SEQ ID NO: 3; hybridizes under wash conditions of 300 C and less than 2 M salt to SEQ ID NO: 5; hybridizes under wash conditions of 300 C and less than 2M salt to SEQ ID NO: 7; hybridizes under wash conditions of 300 C and less than 2 M salt to SEQ ID NO: 9; hybridizes under wash conditions of 300 C and less than 2 M salt to SEQ ID NO: 11, 13, 27; or 29; hybridizes under wash conditions of 300 C and less than 2 M salt to SEQ ID NO: 15, 17, or 36; hybridizes under wash conditions of 300 C and less than 2 M salt to SEQ ID NO: 19, 31, or 38; hybridizes under wash conditions of 30° C and less than 2 M salt to SEQ ID NO: 21 or 40; hybridizes under wash conditions of 30° C and less than 2 M salt to SEQ ID NO: 23, 33, 42, or 44; exhibits at least about 85% identity over a stretch of at least about nucleotides to a primate DTLR2; exhibits at least about identity over a stretch of at least about nucleotides to a primate DTLR3; exhibits at least about identity over a stretch of at least about nucleotides to a primate DTLR4; or exhibits at least about identity over a stretch of at least about nucleotides to a primate DTLR5. Preferably, such nucleic s acid will have such properties, wherein: wash conditions O are at 450 C and/or 500 mM salt; or the identity is least and/or the stretch is at least 55 nucleotides.

More preferably, the wash conditions are at 550 C and/or 150 mM salt; or the identity is at least 95% and/or the stretch is at least 75 nucleotides.

Also described are methods of producing a 00 ligand:receptor complex, comprising contacting a

OC

0 substantially pure primate DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10, including a recombinant or synthetically produced protein, with Scandidate Toll ligand; thereby allowing said complex to form.

Described herein is a method of modulating physiology or development of a cell or tissue culture cells comprising contacting the cell with an agonist or antagonist of a mammalian DTLR2, DTLR3, DTLR4, DTLR6, DTLR7, DTLR8, DTLR9, or DTLR10. Preferably, the cell is a pDC2 cell with the agonist or antagonist of DETAILED DESCRIPTION

OUTLINE

I. General II. Activities III. Nucleic acids A. encoding fragments, sequence, probes B. mutations, chimeras, fusions C. making nucleic acids D. vectors, cells comprising IV. Proteins, Peptides A. fragments, sequence, immunogens, antigens B. muteins C. agonists/antagonists, functional equivalents D. making proteins V. Making nucleic acids, proteins A. synthetic B. recombinant C. natural sources VI. Antibodies A. polyclonals B. monoclonal C. fragments; Kd D. anti-idiotypic antibodies E. hybridoma cell lines VII. Kits and Methods to quantify DTLRs 2-10 A. ELISA B. assay mRNA encoding C. qualitative/quantitative D. kits VIII. Therapeutic compositions, methods A. combination compositions B. unit dose C. administration IX. Ligands I. General Described herein is the amino acid sequence and DNA sequence of mammalian, herein primate DNAX Toll like receptor molecules (DTLR) having particular defined properties, both structural and biological. These have been designated herein as DTLR2, DTLR3, DTLR4, DTLR6, DTLR7, DTLR8, DTLR9, and DTLR10, respectively, and increase the number of members of the human Toll like WO 01/90151 PCT/US01/16766 receptor family from 1 to 10. Various cDNAs encoding these molecules were obtained from primate, human, cDNA sequence libraries. Other primate or other mammalian counterparts would also be desired.

Some of the standard methods applicable are described or referenced, in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.

(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates, Brooklyn, NY; or Ausubel, et al.

(1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York; each of which is incorporated herein by reference.

A complete nucleotide (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 2) of a human DTLR1 coding segment is shown in Table 1. See also Nomura, et al. (1994) DNA Res. 1:27-35. A complete nucleotide (SEQ ID NO: 3) and corresponding amino acid sequence (SEQ ID NO: 4) of a human DTLR2 coding segment is shown in Table 2. A complete nucleotide (SEQ ID NO: and corresponding amino acid sequence [SEQ ID NO: 6) of a human DTLR3 coding segment is shown in Table 3. A complete nucleotide (SEQ ID NO: 7) and corresponding amino acid sequence (SEQ ID NO: 8) of a human DTLR4 coding segment is shown in Table 4; see also SEQ ID NO: 25 and 26. A partial nucleotide (SEQ ID NO: 9) and corresponding amino acid sequence (SEQ ID NO: 10) of a human coding segment is shown in Table 5. A complete nucleotide (SEQ ID NO: 11) and corresponding amino acid sequence (SEQ ID NO: 12) of a human DTLR6 coding segment is shown in Table 6, along with partial sequence of a mouse DTLR6 (SEQ ID NO: 13, 14, 27, 28, 29, and 30). Partial nucleotide (SEQ ID NO: 15 and 17) and corresponding amino acid sequence (SEQ ID NO: 16 and 18) of a human DTLR7 coding segment is shown in Table 7; full length sequence is WO 01/90151 PCT/US01/16766 provided in SEQ ID NO: 36 and 37. Partial nucleotide (SEQ ID NO: 19) and corresponding amino acid sequence (SEQ ID NO: 20) of a human DTLR8 coding segment is shown in Table 8, with supplementary sequence (SEQ'ID NO: 31, 32, 38, and 39). Partial nucleotide (SEQ ID NO: 21) and corresponding amino acid sequence (SEQ ID NO: 22) of a human DTLR9 coding segment is shown in Table 9; see also SEQ ID NO: and 41. Partial nucleotide (SEQ ID NO: 23) and corresponding amino acid sequence (SEQ ID NO: 24) of a human DTLR10 coding segment is shown in Table 10, along with supplementary sequence (SEQ ID NO: 33, 34, 42, and 43) and rodent, mouse, sequence (SEQ ID NO: 35, 44, and wo 01/90151 WO 0190151PCT/USOI/16766 Table 1: Nucleotide and amino acid sequences (see SEQ ID NO: 1 and 2) of a primate, human, DNAX Toll like receptor 1 (DTLRI) ATG ACT AGO Met Thr Ser -22 -20 ATO TTC CAT TTT GCC ATT ATC TTC ATO Ile Phe H-is Phe Ala Ile Ile Phe Met ATA CIT CAG Ile Leu Gin ATC AGA Ile Arg AlA CAA TTA TCT GAA OAA ACT GAA TTT TTA OTT OAT AGG TCA Ile Gin Leu'Ser Glu Oft Ser Olu Phe Leu Val Asp Arg Ser AAA AAC GOT CTC ATO CAC OTT C AAA Lys Asn Oly Leu Ile His Val Pro Lys CTA TOO CAO AAA Leu Ser Gin Lys ACA ACA Thr Thr ATO TTA AAT ATA TOO CAA AAT TAT ATA TOT GAG OTT TGG Ile Leu Asn Ile Sec Gin Asn Tyr Ile Sec Oiu Leu Trp ACT TOT GAO Thr Ser Asp ATO TTA TCA Ile Leu Ser 010 ICA AAA OTO Leu Ser Lys Leu ATT TIG ATA AlT TOT CAT AAT AGA Ile Leu Ile Ile Sec His Asn Arg ATO CAG Ile Gin TAT OTT OAT ATO Tyr Leu Asp Ile OTT TIC AAA TTC Val Phe Lys She CAG GAA TTO OAA Gin Oiu Leu Glu

TAC

Tyr 75 ITO OAT ITO TOO Leu Asp Leu Ger AAC AAG TIG GTO AAG AT? TOT TOO CAC Asn Lys Leu Val Lys le Ser Cys His ACT GTG AAC OTO Thr Val Asn Leu CAC TO GAO CTG His Leu Asp Leu TIT AAT OCA TTT Phe Asn Ala Phe OAT 000 Asp Ala 105 010 OCT ATA Leu Pro Ile AAA GAG ITT 000 Lys Olu She Oly ATO TOT OAA CIA AAA ITT 010 Met Sec Gin Leu Lys She Leu 120 000 ITO AGO Gly Leu Ser 125 ACC ACA CAC Ilk Thr Thr His Leu AAA TOT AOT 010 Lys Sec Sec Val OCA ATT OCT Pro Ile Ala OAT ITO His Leu 140 AAT ATO AGO AAG Asn Ile Sec Lys TO 010 010 ITA GGA GAG ACT TAT GGG Leu Leu Val Leu Gly Giu Thc Tyr Oly 150

GAA

Oiu 155 AAA OAA GAO OCT GAO 000 OTT OAA GAC ITT AAO ACT GAG Aol Lys Olu Asp Pro Oiu Gly Leu Gin Asp She Asn Ihr Oiu Sec CAO AlT 010 TIC His Ie Val She ACA AAO AAA OAA TIC CAT TIT ATI Thc Asm Lys Oft Phe His Phe Ile 180 ITO GAT 010 Leu Asp Val 185 wo 01/90151 WO 0190151PCT/USOI/16766 TCA GTC AAG ACT GTA GCA AAT CTG Ser Val Lys Thr Val Ala Asn Len 190 CTA TCT AAT ATC AAA TGT GTG Glu Leu Ser Asn 195

CTA

Len

CAA

Gin

ACT

Thr 235

GTA

Val

TTC

Phe

CAC

Hius

GA

Giu

CGC

Arg 315

TTG

Leu

GGG

Gly

AAA

Lys

CAA

Gin

GAA

Giu

ACA

Thr 220

TGG

Trp

TGG

T rp

AGA

Arg

CAA

Gin

ATC

Ile 300

ATG

Met

GAT

Asp

CAC

His

GAA

GIlu

CAA

Gin 380

AAA

Lys

AA(

Lys

TTC

Phe T CA Ser 255

GAT

Asp

AGC

Ser

AAT

Asn

ATG

Met

AAT

Asn 335

GAG

Gin

AAA

Lys

ATT

Ile

CTA

Leu

ACC

Thr

CAA

Gin

AAG

Lys 260

TCC

S er rTTT Phe

AAT

Asn

AAA

L ys

GAC

Asp 340

ATT

Ile

ACT

Thr

GTA

Val

AGTMATT

Ser Ile TTA AAC Len Asn 230 CTA GTT Leu Val 245 CTA CAG Len Gin TTG AAG Leu Lys CCG CAA Pro Gin TTC ACA Phe Thr 310 ATT AC TIP Ser 325 ACG GTT Thr Val TTA CAA Leu Gin ACA CAG Thr Gin AGC TAT Ser Tyr 390 Ile Lys 200 CTG GCG Leu Ala 215 AAC ATT Asn Ile TGG CAT Trp His GGT GAG Giy Gin GCC TTG Ala Leu 280 ACT TAT Ser Tyr 295 GTG TCT Val Ser CCG TTC Pro Phe TTT GAA Phe Gin ATG AAT Met Asn 360 ATG AAG Met Lys 375 CAT GAA Asp Gin 672 720 768 816 864 912 960 1008 105C 11OZ 115, 1201 124' GGA GAG TGT TCT TGG ACT AAA ACT TTA TTA AGT TTA AAT ATG TCT TCA Cly Asp Cys Ser Trp Thr Lys Ser Len Len Ser Len Asn Met Ser Ser 395 400 40c; 410 wo 01/90151 WO 0190151PCT/USOI/16766 AAT ATA CTT ACT Asn Ile Leu Thr ACT ATT TTC AGA Thr Ile Phe Arq TTA CCT COO AGG Leu Pro Pro Arg ATO AAG Ile Lys 425 1344 GTA CTT GAT Val Leu Asp GTA AAA CTG Val Lys Leu 445 ACT GAO CTT Thr Asp Leu 460 CAC AGO AAT AAA His Ser Asn Lys AAG AGC ATT CCT Lys Se Ile Pro AAA CAA GTC Lys Gin Val 440 AAT TCT TTA Asn Ser Leu GAA GOT TTG CAA Glu Ala Leu Gin GAA OTO Giu Leu 450 AAT GTT GOT Asn Val Ala 1392 1440 1488 OCT GGA TGT Pro Gly Cys AGO TTT AGO AGO Ser Phe Ser Ser TOT GTA TTG ATO Ser Val Leu Ile

ATT

Ile 475 GAT CAC AAT TOA Asp His Asn Ser TOO CAC OCA TCA Ser His Pro Ser GAT TTO TTC CAG Asp Phe Phe Gin 1536 TGO CAG AAG ATG AGG TOA ATA AAA Oys Gin Lys Met Arg Ser Ile Lys GOA GGG Ala Giy 500 GAC AAT OCA TTC Asp Asn Pro Phe CAA TGT Gin Cys 505 ACC TGT GAG Thr Cys Giu OTO GGA GAA TTT GTO AAA AAT ATA GAO CAA Leu Gly Giu Phe Val Lys Asn Ile Asp Gin 510 515 GAG GGO TGG COT GAT TOT TAT IkAG TGT GAO Giu Gly Trp Pro Asp Ser Tyr Lys Oys Asp 530 5-35 GTA TCA AGT Val Ser Ser 520 TAO COG GAA Tyr Fro Giu 1584 1632 1680 1728 GAA GTG TTA Giu Val Leu 525 AGT TAT AGA Ser Tyr Arg 540 GGA ACC OTA Gly Thr Leu AAG GAO TTT CAC ATG TOT GA.A TTA TOO Lys Asp Phe His Met Ser Giu Leu Ser 550

TGC

0 ys 555 ~AO ATA ACT CTG Asn Ile Thr Leu ATO GTO ACC ATO Ile Val Thr Ile GOC ACC ATG OTG Ala Thr Met Leu TTG GOT GTG ACT Leu Ala Val Thr ACC TOO OTO TGO Thr Ser Leu Cys TAO TTG GAT OTG Tyr Leu Asp Leu COO TGG Pro Trp 585 1776 1824 1872 1920 TAT OTO AGG Tyr Lell Arg GTG TGO CAG TGG Val Cys Gin Trp OAG ACC OGG OGO Gin Thr Arg Arg AGG GOC AGG Arg Ala Arg 600 OAT GOA TTT His Ala Phe AAC ATA COO Asn Ile Pro 605 TTA GAA GAA OTO Leu Giu Giu Leu AGA AAT OTO CAG Arg Asn Leu Gin wo 01/90151 WO 0190151PCT/USOI/16766 ACT TCA Ile Ser 620 TAT AGT GGG CAC GAT TCT TTC TGG Tyr Ser Gly His Asp Ser Phe Trp GTG AAG Val Lys 630 AAT GAA TTA TTG Asn Clii Leu Leu 196S 2016

CCA

Pro 635 AAC CTA GAG AAA GAA CCT ATG CAG ATT TGC *CTT CAT GAG AGA Asn Leu Ciu Lys Glu Gly Met Gin Ile Cys Leu His Ciu Arg TTT GTT CCT Phe Val Pro CCC AAG Cly Lys AGC ATT GTG GAA AAT ATC ATC ACC TC Ser Ile Val Ciu Asn Ile Ile Thr Cys ATT GAG Ile Clu 665 AAC ACT TAO Lys Ser Tyr C2AA TGC TGC Glu Trp Cys 685 TCC ATC TTT GTT Ser Ile Phe Val TCT CCC AAC TTT Ser Pro Asn Phe CCC CAd ACT Val Gin Ser 680 CTC TTT CAT Leu Phe His CAT TAT GAA CTC His Tyr Giu Leu TTT CCC CAT CAC Phe Ala His His 2064 2112 2160 2208 2256 2304 GAA GGA Clu Cly 700 TCT AAT AGC TTA ATC CTC Ser Asn Ser Leu Ile Leu -705 ATO TTG CTC Ile Leu Leu CCC ATT CCG CAG Pro Ile Pro Gin

TAC

Tyr 715 TCC ACT CCT AGC Ser Ile Pro Ser TAT CAC AAG CC Tyr His Lys Leu ACT CTC ATC GCC Ser Leu Met Ala ACG ACT TAT TCC Arg Thr Tyr Leu TCC CCC AAG GAA Trp Pro Lys Giu AGO AAA CdT CCC CTT TTT Ser Lys Arg Gly Leu Phe 745 TGG GCT AAC TTA Add OCA Trp Ala Asn Leu Arg Aia 750 GCC ATT AAT Ala Ile Asn 755 ATT AAG CTC ACA Ile Lys Leu Thr GAG CAA CCA dlu Gin Ala 760 2352 AAG AAA TAGTCTACA 2367 Lys Lys MTS IFHFAIIFMLILQIRIQLSEESEFLVDRSKNiGLIHVPKDLSQKTTILNISQNYISELWTSDILSLSKLRILI ISHNRIQYLDISVFKFNQELEYLDLSHNKLVKISCHPTVNjLEHLDLSFNAFDALPICKEFGNMSQLKFLGLSTTH LEKSSVLPIAHLNI SKVLLVLGETYGEKEDPEGLQDFNTESLHIVFPTNKEFHFILDVSVKTVANLELSNIKCVL

EDNKCSYFLSILAKLQTNPKLSSLTLNNIETTWNSFIRILQLVWHTTVWYFSISNVKLQGQLDFRDFDYSGTSLK

AL SIHQVVSDVFGF PQ SYIYEIF SNMIKNFTVSTRIMVMLCP SKIS PLHLDF SNNLLTDTVFENCGHLTELE TLILQMNQLKELSKIAEMTTQMKSLQQLDISQNSVSYDEKKGDCSWTKSLLSLNMS SNILTDTIFRCLPPRIKVL DLHSNKIKSIPKQVVKLEALQELNVAFNSLTDLPGCCSFSSLSVLI IDHNSVSHPSADFFQSCQKMRSIKACDNP

FQCTCELGEFVKNIDQVSSEVLEGWPDSYKCDYPESYRGTLLKDFHMSELSCNITLLIVTIVATMLVLAVTVTSL

CIYLDLPWYLRMVCQWTQTRRRARNI PLEELQRNLQFHAFI SYSGHDSFWVKNELLPNLEKEGMQICLHERNFVP

GKSIVELNIITCIEKSYKSIFVLSPNFVQSEWCHYELYFAHHNLFHEGSNSLILILLEPIPQYSPSSYHKLKSLM

ARRTYLEWPKEKSKRGLFWANLRA-AINIKLTEQAKK

wo 01/90151 WO 0190151PCTIUSOI/16766 Table 2: Nucleotide and amaino acid sequences (see SEQ ID NO: 3 and 4) of a primate, human, DNAX Toll like Receptor 2 (DTLR2).

ATG CCA CAT ACT S Met Pro His Thr -22 TTG TCG ATG Leu Trp Met TGG GTC TTG GG Trp Val Leu CGly ATC ATC AC Ile Ile Ser CTC TCC Leu Ser AAG GAA CAA TCC TCC AAT CAG GCT TCT CTG TCT TGT GAC Lys Giu Glu Ser Ser Asri Gin Ala Ser Leu Ser Cys Asp 1 AAT GGT ATC TCC AAG CCC AGC TCA CGA Asn Gly lie Cys Lys Gly Ser Ser Cly TTA AAC TCC ATT Leu Asn Ser Ile CCC TCA Pro Ser CCC CTC ACA GAA GCT GTA AAA AGC CTT GAC CTC TCC AAC Gly Leu Thr Glu Ala Val Lys Ser Leu Asp Leu Ser Asn AAC AGG ATC Asn Arg Ile CTC CAG CCT Leu Gin Ala ACC TAC ATT Thr Tyr Ile AGC AAC AGT GAC Ser Asn Ser Asp CAG ACG TGT CTG Gin Arg Cys Val CTG GTC CTG ACA TCC AAT Leu Val Leu Thi: Ser A~n ATT AAC ACA ATA Ile Asn Thr Ile GAA GAT TCT TTT Glu Asp Ser Phe

TCT

Ser TCC CTC GGC ACT Ser Leu Gly Ser CAA CAT TTA CAC Clii His Leu Asp TCC TAT AAT TAC Ser Tyr Asn Tyr TOT AAT TTA TCC Ser Asn Leu Ser TOC TCC TTC AAG Ser Trp Phe Lys OTT TCT TCT TTA ILeu Ser Ser L-eu ACA TTC Thr Phe 105 TTA AAC TTA Leu Asn Leu GGA AAT CCT TAC Cly Asn Pro Tyr ACC CTA CCC CAA Thr Leu Cly Clii ACA TCT CTT Thr Ser Leu 120 AAT ATC CAC Asn Met Asp TTT TCT CAT CTO ACA AAA TTC Phe Ser His Leu Thr Lys Leu 125 ACC TTC ACT AAC ATT CAA AGA Tbr Phe Thr Lys Ile Gin Arg MTC CTC ACA GTG Ile Leu Arg Vai AAA CAT TTT GCT Lys Asp Phe Ala CTT ACC TTC CTT Leii Thr Phe Leu

GAG

Ciii 155 CAA CTT GAG ATT Ciii Leu Glii Ile GCT TCA GAT CTA Aia Ser Asp Leu ACC TAT GAG OCA Ser Tyr Ciii Pro ACT TTC AAG TCA ATT GAG AAC CTA ACT CAT CTG ATO CTT CAT Ser Leu Lys Ser Ile Gin Asn Val Ser His Leu Ile Leu His ATC AAC 624 Met Lys 18 WO 011901511 CAG CAT Gin Nis GAA TOT Gin Cys GAA CTA Gln Leu 220 AGA AAT Mrg Aen 225 TTG AAT Len Asri CTT AMT Len Asn GAT CCA Asp Pro AGO TTT Arg Phe 200 AGA OTT Arg Val 315 TOT TTA Cys Lou GAA AMT Gin. Asn GCC TG Ala Trp TCA TTG Ser Leu 280 PCT/US01/16766 ATT TTT Ile Phe 195 ACT, GAT Thr Asp 210 AAT TCA Asn Ser GAA AGT Giu Ser TTA GAA Leu Giu.

AGA SCA Arg Ala 275 TTA ACA Lou Thr 290 CTG AGO Leu. Ser GAA AAC Ciu Asn AAA TCA Lys Ser TAC TTG Tyr Len 355 TTA ATT Le Ile 370 ACT TTG Thr Leu GTA GAT OTT ACA AGT Val Asp Val Thr Ser 200 TTG GAC ACT TTC CAT Leu Asp' Thr Phe His TTG ATT AAA AAC TTT Leu Ile Lys Lys Phe 230 TTG TTT CAG OTT ATG Leu ?he GIn Val Met 245 TTA GAG TTT GAT GAC Len Giu. 1he Asp Asp 260 TCT GAT AAT GAC AGA Ser Asp Asn Asp Arg 280 ATC CGC ACG CTG CAT Ile Arg Arg Lou His1 295 ACT TTA TAT TCA CTT Thr Leu. Tyr Ser Leu 310 ACT AAA OTT TTT CTG Ser Lys Val Phe Len 325 TTA GAA TAG TTG GAT Lon Gin Tyr Len Asp 340 AAA AAT TCA CCC TOT Lye Asri Ser Aia Cys 360 TTA AGO CAA AAT CAT Len Arg Gin Asn Fis 3-75 CTC ACT CTG AAA AAC Len Thr Len. Lys Asa 390

TOCGOTO

Ser Vai TTT T CA Phe Ser ACA TTT Thr Phe A.AA CTT Lys Len 250 TOT ACC Cy5 Thr 265 OTT ATA Vai Ile ATT CCA I Ie Pro ACA CAA Thr Gin C'T CCT Vai Pro 330 CTC ACT Leu Ser 345 GAG OAT Gin Asp TTO OCA Leu. Aia TTG ACT Len Thr 1008 1056 1104 1 152 1200 1248 1296 AAC ATT CAT MTC ACT AAO AAM ACT TTT CAT TCT ATO CCT OAA ACT TOT Asn Ile Asp Ile Ser Lys Asn'Ser Phe his Ser Net Pro Gin Thr Cys 395 400 405 410 wo 0119011-1 WO 0190151PCT/LISOI/16766 CAG TGG CCA GAA AAG ATG AA-A TAT TTG AAC TTA TCC AGO ACA CGA ATA 1344 Gin Trp Pro Glu Lys Met Lys Tyr Leu Asn Leu Ser Ser Thar Arg Ile 415 420 425 CAC AGT GTA ACA GGC TGC ATT CCC AAG ACA CTG GAA ATT TTA GAT G-T 1392 His Ser Val. Thr Cly Cys Ile Pro Lys Thr Lela Clu Ile Leu Asp Val.

430 435 440 AGO AAC AAC AAT CTC PAT TTA TTT TCT TTm AAT TmG CCG CAA CTC AAA 1440 Ser Asn Asn Asn Leu Asn Leu Phe Ser Leu Asn Leu Pro Gin Leu Lys 445 450 455 GAA OTT TAT ATT TCC AGA AAT AAG TTG ATG ACT CTA CCA GAT GCC TOO 1488 Guu Leu Tyr Ile Ser Arg Asn Lys Leu Met Thr Leu Pro Asp Ala Ser 46D 465 470 CTC TTA CCC ATG TTA CTA GTA TTG AAA ATC ACT AGG AAT GCA ATA ACT 1 36 Leu Leu Pro Met Leu Leu Val Leu Lys Ile Ser Arg Asn Ala Ile Thr 475 480 485 490 ACG TTT TCT AAG GAG CAA OTT GAO ICA TTT CAC ACA CTG AAG ACT TTG 1584 Thr Phe Ser Lys Giu Gin Leu Asp Ser Phe His Thr Leu Lys Thr Leu 495 500 505 GAA GOT GGT GGC AAT AAC TTC ATT TGC TOO TGT CAA TO OTO TOO TTO 1632 Clii Ala Giy Gly Asn Asn Phe Ile Cys Ser Cys Clii Phe Leu Ser Phe 510 515 520 ACT CAG GAG CAG CAA GCA OTG GOT- AAA GTC TTG ATT CAT TGC OCA GCA 1680 Thr Gin Glu Gin Gin Ala Leu Ala Lys Val Leu Ile Asp Trp Pro Ala 525 530 535 AAT TAO CTG TGT GAO TOT OCA TOO OAT GTC CT GC CAG CAC GTT CAG 1728 Aen Tyr Leu Cys Asp Ser Pro Ser His Val Arg Gly Gin Gin Val Gin 540 545 550 GAT GTO CCC 010 TOG GTG TCG GAA TGT CAC ACG ACA GCA CIG GTG TOT 1776 Asp Val Arg Leu Ser Val Ser Ghu Cys His Arg Ihr Ala Leui Val. Ser 555 560 565 570 CCC ATG TGC TGT GCT CTG TO CIG CTG ATC CTG 010 AOG GGG GIC CTG 1824 Cly Met Cys Cys Ala Leu Phe Leu Leu Ile Leu Leu Thr Gly Val Leu 575 580 585 TGO CAC CT ITO OAT GGC OTG TCC TAT ATG AAA ATC ATG TGC CO TGC 1872 Cys His Arq Phe His Cly Leu Trp Tyr Met Lys Met Met Trp Ala Trp 590 595 600 OTO CGGCCC AAA AGG AAG 000 AGG AAA GOT CCC AGO AGC AAC ATO TGO 1920 Leu Gin Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys 605 610 615 wo 0119011-1 WO 0190151PCT/LISOJ/16766 TAT OAT Tyr Asp 620 OCA TT OTT TOT TAO ACT GAG COG CAT Ala Phe V~al Oar Tyr Ser Gin Arg Asp 625 TAOC TCC COG GAG Tyr Trp Vei Gin

AAC

Asn 635

OT

iO Cys OTT ATO STO CAG Len Met Val Gin OTT CAT AAC CCC Leu f-li Lys Arg 655 OTG GAG AAC TTC Leo 0 u Asn Phe MAT 000C Asn Pro 6415 CCC TTO MAG Pro Phe Lys SAC TTC ATT COT Asp Phe Ile Pro RAG TOG ATO ATO Lys Trp Ile Tip GAO AAT Asp Asn 665 ATC AOT GAO Ile Ile Asp ATO CMA fAG AC Ile Gin Lys Ocr AAA ACT OCO TTT Lys Thr Vai Phe CTGCOTT TOT Vai ten Ser 680 GAO TTO TOO Asp Phe Ser GMA AO TUT GTC flAG ACT GAG Gin Asn Phe Val Lye Ser Gin 685 TOO MOG TAT Cys Lys Tyr GMA CTS Gin Len 695 1958 2016 2064 2112 2160 2208 2256 2304 2352 2355 CAT TOO His Phe 700 CT OTT TTT CMA GAO MOC AAT CAT GOT Arg Len Phe Gin Gin Asn Asn Asp Aia '705 ATT CTC ATO OTT Le Len Ile Leu 2S OTO Len 715 GAG COO ATT GAO Gin Pro Ile Gin A A LYS Ly5 720 0CC ATO CCC Ala Ile Pro 0CO TOO TOO flAG Asp Phe Cys Lys COG MCG ATA ATO MAC ACC AAC ACC TAO OTG GAO TGG COO ATG Asp Lye Ile Met Aen Thr Lys Thr Tyr Len Gin Orp Pro Met GAO GAG Asp Ciu 745 CT CAC CG Aia Gin Asp GAA GOA Gin Sly 750 TOT TCC STA MAT OTO AGA OCT 000 Phe Trp Val Aen ten Arg Ala Ala ATA MCG TC Tle Lye 760

TAG

MPHTLWtVWVLGVII SLSKzEES SMCASLSCDRNGICI{GS SGS LN SI PSGLTEAVKSLOLSNNRITYISNSE1LQRO

VNQLLSGNIESSLSELLYYSLSWKLSTLLGPKLESFHT

LO-ILRVCNMDTFTEIQRKDFACLTFLEELEIDASDLQSYfEPKSLKS IQNVSMLILKNKQHILLLEIFVDVTSSVE

CLELRDTDLDTFHFSELSTGEC-NSLIKKFTFRNVK(ITDESLFQVMKLLNQISGLLELEFDDCTLNGVGNFRASDN

DRVIDPCKVETLTIRRLHI PRFYLFYDLOTLYOLVERVKRIT'VENSKVFLVPCLLSQHLKSLEYLDLSENLMpJEE YLKNSAC EOAWAPSLQOLILRQN-LASLEXTGETLTmx JLTNTDIT OHS FHMPEOOQWPEKMKYLNLS STRING VTGCCIPKTLEILDVSNNLNLF SLNLPQLICELYI 9P KLMTL PDASLL PMLLVLKI SRNAITTFOKGQLDS POOL KOLEAGGNNFIOBOEPLSFTQEQQAzLAKVLIDWPANYLOS PSHVRCQQVQ DVRLSVSECHRTALVSGMCCALmT, LILLTGVLOHRFHGLWYMKWIMAWLQAKRKPPXPSRNOYDAPVSYSERAY7gVENLMVQELENFNPPFKLCLH KR DFIPGKWIIDNIIDSIEKSKVFVLSENFVXSEWCKYELOFSHFRLFEENNDAAILILLEPIERKAIPQRFC KLRKISO4TKTYLE4PMDEAQREG?MLRAATKS WO 0119011-1 PCT/USOI/16766 Table 3: Nucleotide and amino acid sequences (see SEQ ID NO: 5 and 6) of a mammalian, human, Toll like Receptor 3 (DTLS)3 ATS AGA CAG ACT TTG CCT TGT ATC TAC TTT TGG GGG GGC CTT TTG CCC 48 Met Arg Gln Thr Leu Pro Cys Ile Tyr Phe -21 -20 TTT GGG Phe Gly -5 GAA GTT Glu Val OTA CCC Leu Pro AGA TTA Arg Leu GAT GTA Asp Val AAA CTT Lys Leu CAA CTT Gin Lou CAT CTC His Leu AAG CAG Lys Gin 125 TCT ACA Sec Thr 140 CTA TTA Lou Lou ATC TTT Ile Phe ATG CTG Met Leu GCT GAC Ala Asp ACA AAC Thr Asn CCA GCC Pro Ala GGA TTT Gly Phe CCC ATG Pro Met TCT GAT Ser Asp ATG TCC Met Ser 110 AAG AAT Lys Asn AAA TTA Lys Leu TCA AAC Ser Asn GCC AAT Ala Asn 175 -15 GCA TCC TCC Ala Ser Ser 1 AGC CAC CTG Ser His Lou ACA GTG TTG Thr Val Lou 35 AAC TTC ACA Asn Phe Thr 50 ACC ATC TCA Thr le Ser 65 AAA GTT TTG Lys Val Lau ACC TTT GCC Thr Phe Ala TCA ATC CAG Ser lie Gin 115 ATC ACA TTA Ile Thr Lou 130 ACT CAG GTT Thr Gin Val 145 AAA ATT.CAA Lys Ile Cln TCT TTA AAA Ser Leu Lys Trp. Gly AAG TGC Lys Cys ACT CAG Thc Gin ACC CAT Thr His AGC CAG Ser Gin GAG CCA Glu Pro CAG CAC Gin His ACG AAT Thr Asn AAA AAT Lys Asn TCT CAT Ser His 135 GAA AAT Glu Asn 150 AAA AGT Lys Ser GAG TTG Glu Leu Gly Lou Lau ACT GTT AGC Thr Vol Ser GTA CCC GAT Val Pro Asp AAT CAA CTC Asn Gin Lau CTA ACT AGC Leu Thr Ser GAA TTG TGC Glu Lou Cys AAT GAG CTA Asn Glu Lou TTG ACT GAA Leu Thr Glu 105 AAT CCC TTT Asn Pro Phe 120 AAT GGC TTG Asn Gly Lou CTC CAA GAG Leu Gin Glu GAA GAA CTG Glu Glu Lou 170 TCA TCG AAT Ser Ser Asn 185 wo 011901-1-1 WO 0190151PCT/US01I16766 ATT AAA GAG Ile Lys Giu 1.90 S GGC CTC TTT Gly Leu Phe 205 CTA TCT TTC Leu Cys Leu 220 AAC ACC CAC Asn Ser Gin TGC ACA AAT Trp Thr Asn GTT GCT AAC Vai Gly Asn 270 CTA GAG TAT Leu Glu Tyr 285 CTT TTC AAT Leu Phe Aqn 300 AGT ATT TCC Ser Ile Ser TOG CTA AAA Tr-p Leu Lys GCC ATA AAA Gly Ie Lys 350 AGT CTA TCC Ser Leu Ser 365 TTT GTA TCA Phe Val Ser 380 ITT TCT CCA GGG TCT ITT CAC GCA ATT GGA AGA Phe Ser Pro Giy Cys Phe his Ala Ile Giy Arg 195 200 CTG AAC Leu Asn CAA TTIA Oila Leu CTG TCC Leja Ser 240 CTC ACT Leu Thr 255 CAT TCC Asp Ser AAT AAT Asqn Asn G AGC Vai Arg CTT GCC Leu Aia 320 TCT TTC Cys Leu 335 ACC AAT Per A.

AAC TCC Asn Ser CTI' GCT Leu Ala

GGT

Cly

ATT

Ile

ACA

Thr 245

TCC

S ez

CCA

Pr o

TTT

Phe

AAA

Lysa

ATT

Ile 325

ATG

Met

TTC

Leu

CGA

Arg

CAC

His CCC -ACC CT Pro Ser Leu 1215 CGG AAT CTG Arg Asn Leu 230 ACT TTC TTO Ihr Phe Leu TAC AAC AAC Tyr Asn Asn CAA CTA CAA Gin Leu Glu 280 TCT CAC TCT Ser His Ser 295 CCC TCT TTT Arg Ser Phe 310 OAT CAT ITTT Asp Asp Phe GAA CAT AAT Glu Asp Asn ATA AAC CTC Ile Asn Leu 360 ACT TIC ACA Thr Leu Thr 375 ATA CTC AAC Ile Leu Asn 390

TA

Leu

GAG

Glu

CTG

Leu

CIA

Leu 250

AAT

Asn

TIC

Phe

CAC

His

A-AA

Lys

TIT

Phe 330

ATT

Ile

TAC

Tyr

GAA

Gic

ACC

Thr

C

6-72 720 768 8iG 864 912 960 1008 1056 1iO4 1252 1200 1248 1296 AAT AAA ATC TCA A.AA ATA GAG ACT GAT C TTC TCT TCC TTG Asn Lys Ile Ser Lys Ile Glu Ser Asp Ala Phe Ser Trp Leu Gly His wo 01/90151 WO 0190151PCT/USOI/16766 CIA GAA GTA CTT GAO GIG GGC CTT AAT GAA ATT GGG CAA GAA CTC ACA 1.344 Len Giu Val Len Asp Leu Gly Leu Asn Gin Ile Gly Gin Gin Len Thr 415 420 425 GGC CAG GAA TGG AGA GGT OTA GAA AAT ATT TTC GAA ATC TAT CTT ICC 1392 Gly Gin Gin Trp Arg Gly Leu Gin Asn Ile Phe Gin Ile Tyr Leu Ser 430 435 440 TAO AAC AAG TAO CTG CAG CTG ACT AGG AAC TCC TTT GOC TO GTC CCA 1440 Tyr Asn Lys Tyr Leu Gin Leu Thr Arg Asn Ser Phe Ala Len Vai'Pro 445 450 455 AGO CTT CAR OGA CTG ATG CTC CGA AGO GTG G00 CTT AAA AAT GTG GAT 1488 Ser Len Gin Arg Leu Met Len, Arg Arg Val Ala Leu Lys Aso Val Asp 460 465 470 475 AGO TCT CCI TCA CCA TIC GAG 001 OTT CGT AAO TTG ACC ATT CTG GAT 1536 Ser Ser Pro Ser Pro Phe Gln Pro Len Ar Asn Len Ihr Ile Len Asp 480 495 490 CIA AGO AAO AAO AAO ATA G00 AAO ATA AAT GAT GAO ATG TTC GAG GGT 1584 Leu Ser Asn Asn Asn Ile Ala Asn Ile Asn Asp Asp Met Len Glu Gly 495 500 505 CII GAG AAA CIA GAA AlT 010 GAT TTG GAG OAT ?AOC AAO TTA GCA COG 1632 Len Gin Lys Len Gin lie Len Asp Leu 0Gin His Asn Asn Len Ala Arg 510 515 520 010 IGG AAA CAC GCA AAC OCT GGT GOT CCC All TAT TIC CIA AAG GGT 1680 Len Trp Lys His Aia Asn Pro Gly Oly Pro Ile Tyr Phe Len Lys Oly 525 530 535 CIG TOT CAO 010 CAO ATC CTT AAO TIC GAG TOG AAO GGC TTT GAO GAG 1728 Len Ser His Len His Ile Len Asn Len Gin Ser Asn Gly Phe Aisp Gin 540 545 550 555 ATO CCA GTT GAO GIG TIC AAG GAT TTA TT GAA CIA AAG AIC AIC GAT 1776 Ile Pro Val Gin Val Phe Lys Asp Len Phe Gin -Len Ly Ile Ile Asp 560 565 57C TIA OGA TO AAT AAT TIA AAC ACA OTT OCA OCA TOT GIG ITT AAI AAT 1824 Len Oiy Len Asn Asn Len Asn Thir Len Pro Ala Ser Val Phe Asn Asn 575 580 585 GAG GIG TOT CIA AAO ICA T AAO CIT GAG AAG AMT 010 AlA ACA ICC 1872 Gin Val Ser Len Lys Ser Len Asn Len Gin Lys Asn Len le Thr Ser 590 595 600 OTT GAG A.AG AAG OTT TIC GO OCA OCT TO AGG AAO GIG ACT GAG TIA 1920 Val Gin Lys Lys Val Phe Gly Pro Ala Phe Arg Asn Len Thr Giu Len 605 610 61.5 WO 01190151

GAT

Asp 620

TTT

Phe

AGC

Ser

AGA

Arg

TTT

Phe

CTT

Leu 700

TCA

Ser

CAG

Gin

TGG

Trio

AAA

Lys

GAA

Glu 780

ATA.

Ile

CAT

His

TTG

Leu PCT/US01/16766 CGC TTT AAT CCC TTT GAT TOO ACG TGT SPA AGT Arg Phe Asn Pro 625 Phe Asp C4s Thr Cys Slu Ser ACC CAT Thr His CCA CCT Pro Pro 660 TGC AAA Cys Lys 675 ATO CTG Ile Leu TGG AGO Trp Arg TTC AAA Phe Lys ATA ATT Ile Ile 740 TCA ATG Ser Met 755 GAC TTT Asp Phe AAA AGA Lys Arg GAC CCA Asp Pro ATT GAA lie Glu 820 OCA GAT Pro Asp 835 630 AAC ATC CCT Asn lie Pro TAT OAT GGG Tyr His Sly AGT GCC CCC Ser Ala Pro 680 ATT TTT ATO Ile Phe Ile 695 TCT TTT TAT Ser Phe Tyr 710 ATA GAC AGA Ile Asp Arg GCC TAT AAA Ala Tyr Lys AAG GAA GAO Lys Glu Asp 760 GCG GOT OTT Ala Gly Val 775 AGA AAA ATT Arg Lys Ile 790 TGO AAA AGA Cys Lys Arg AAT CTG GAT Asn Leu Asp AAA CTG AAC Lys Leu Asn 840 GCC TGG Ala Trp 635 CTG TCA Leu Ser 650 OCA CTG Pro Val OA A CTC Glu Leu ATT GTA Ile Val AAT GTT As Val 715 ACA GAA Thr Glu 730 AAG GAT Lys Asp TCT CTC Ser Leu GAA OTA Glu Leu TTT OTT Phe Val 795 AAG GTA Lvs Val ATT ATA Ile Tie GCA CTC Ala Leu 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2544 2592 wo 0119011-1 WO 0190151PCT/LISOI/16766 TGT TTG CGA AGA GGA ATG TTT AA.A TCT CAC TGC ATC TTG AAC TGG CCA 2640 Cys Leu Arg Arg Oly Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro 845 850 855 OTT CAG AAA GAA CGG ATA GOT GCC TTT CGT CAT AAA TTG CPA GTA GCA 2688 Val GIn Lys Glu Arg Ile Gly Ala Phe Arq Hi's Lys Leu Gin Val Ala 860 865 87 0 875 CTT GGA TCC AAA AAC TCT GTA CAT TAA 2715 Leu Gly Ser Lys Asn Ser Val His 880 MRQT LPC IYFWGGLL PFGMLCASSTTKCTVS HEVADCSHLKLTQVP DL PTNITVLNLT'HNQLRRL PANFTRYS QLT SLDVG ENT I SKLEELCQKLPMLKVLNLQHNELSQLS DKTFAFCTNLTELHLMSNS IQKTKNNPFVKQKJLI TLLHGS XGQQELELSNIAKELD ASSKLLSQIX SG HZGLF LFLNNVQLGPSLTEKLCLELANTS IRNLSLSNSQ7 STTSNTTFLGL~ICTNLTMLDLSYNNLNVVGNDSFAW4LPQL EYFFLEYNNIQHLFSHSLHGLFNVRYLNLKRSFTKQSISLASLPKI 00FSFQWT1<CLEHLN14EDNDIPGIKSUMF TGLINLKYLSLSNS FTSLRTLTNET FVSLAHS PLHILNLT KNKI SKIES DAFSWLOHLEVLDLOLNEIGOELTGO

FWCLNFILYKLLPSAVSQLLRVLNDSSFPRLIDSNINN~

LEGLHILEILDLQHNNLARLWKHANPGGPIYFLKGL3HLHILNLESNGFDEIPVEVFKDLFELKII7LGLNNLNT

LPASVFNNQVSLKSLNLQKNLITVEKKVFPAFRNLTELDRFNFCTCESIAWFVNW~INETHTNIPELSSHY

LCNTPPHYHGFPVRLFDTSSCKDSAPFELFFMINTSILLI FIFIVLLIHFEGWRIS FYWNVSVHRVLGFKEIDRQ TEQFEYA-AYIIHAYIDKDWVWEHSSMEKEQSL<FCLEERDFEAGVEELEAIVNS IIRSRKII FVITHHLLKDP LCKRFKVH HAVQQAI EQNLS I ILV LEEP D YKLNALC LRROMESHC U NWFVQKERI GAFRHKLQVALGSK

NSVH

wo 0119011-1 WO 0190151PCT/LISOI/16766 Table 4: Nucleotide and amino acid sequences (see SEQ ID NO: 7 and 8) of a mammalian, primate, human, DNAX Toll like Recpptor 4 (DTLPR4) ATG GAG CTG AAT TIC TAG AAA ATG CCC GAC AAC CTC CCC TIC TCA ACC 48 Met Clu Lou Asn Phe Tyr Lys le Pro Asp Asn"Leu Pro Phe Ser Ihr 1 5 10 AAC AAC GIG GAG GIG AGC TTT AAT CCC CTG AGG CAT TTA GGCC AGC TAT 96 Lys Asn Leu Asp Leu Ser Phe Asn Pro Leu Arg his Leu Gly Ser Tyr 20 25 AGC TIC TIC ACT TIC CCA GAA GIG GAG GIG CTG GAT TTA ICC AGG TGT 144 Ser Phe Phe Ser Phe Pro Glu Leu Gln Val Leu Asp Leu Ser Arg Cys 40 GAA AIG CAG AGA AlT GAA CAT CCC GGA TAT GAG AC CTA ACCGAG GTC 292 Gin Ile Gin Ihr Ile Gin Asp Gly Ala Tyr Gin Sex Leu Ser His Leu 55 ICT ACC ITA ATA TTG AGA GGA AAG CCC ATC GAG AGT TTA GCC GIG GG;A 240 Ser Thr Leu Ile Leu Ihr Gly Asn Pro Ile Gin Ser Leu Ala Leu Cly 70 75 CCC TTT IGI GA GTA TGA ACT TIA GAG AAG GIG GIG GCI GTG GAG AGA 288 Ala Phe Ser Gly Leu Ser Ser Lou Gin Lys Len Val Ala Val' Gin Thr 90 MAT CTA GCA ICI CIA GAG AAG TIC CCC AlT GCA CAT GIG AMA ACT TIG 336 Asn Leu Aia Ser Len Gin Asn Phe Pro Ile Gly His Len Lys Ihr Len 100 105 110 AAA CAA CIT AAT GIG CTCAC GAG M CIT AIC CAA ICT TIC AAA ITA CCI 384 Lys Gin Leu Asn V~al Ala His Asn Len Ile Gin Ser Phe Lys Len Pro 115 120 125 GAG TAT IIT TOG AAT GIG ACC AAI CIA GAG GAG TIC GAG CIT ICC AC 432 Gin Tyr Phe Ser Asn Len Thr Asn Len Gin His Len Asp Leu Ser Ser 130 135 140 AAG AAG All CAA ACT ATT TAT ICC ACA GAG TIGCGGG GIT CIA CAT CAA 480 Aen. Lys Ile Gin Ser Ile lyr.Cys Thr Asp Len Arg Val Len His Gin 145 150 155 160 AIG CCC CIA GIG AAI GIG ICT ITA GAG GIG ICC GIG AAC CCI AIC AAC 528 Met Pro Len Len Asm Len Ser Lou Asp Len Ser Leu Asn Pro Met Asn 165 170 175 TTT ATC CAA CCA CCI CI ITT AAA CAA All ACG CIT CAT AAG GIG ACT 576 Phe Ile Gin Pro Gly Ala Phe Lye Giu Ile Arg Len His Lye Len Thr 180 185 190 ITA AGA AAI IAIT III CAT AGI T2IA AT CIA AIG AMA ACT TGI All CAA 624 Leu Arg Asn Asn Phe Asp Ser Len Asn Vai Met Lys Thr Cys Ile Gin 195 200 205 WO 01/90151 PCT/USOI/16766 GCT CTG GCT GGT TTA GAA GC CAT CGT TTC CTT OTO GGA GAA! TTT AGA 672 Gly Leu Ala Gly Leu Glu Val His Ary Leu Val Leu Gly Giu ?he Arg 210 215 220 AAT CA.A GCA AAC TTG GAA AAG TTT CAC TCT -CT CTA GAG CCC CTC 720 Asii Giu Gly Asn Leu Ciu Lys Phe Asp Lys Ser Ala Leu Glu Gly Leu 225 230 235 240 TGC AAT TTG ACC ATT GAA GAA TTC OGA TTA GOA TAO TTA GAC. TAO TAC 768 Cys Asri Leu Thr Ile Giu Glu ?he Axg Leu Ala Tyr Leu Asp Tyr Tyr 245 250 255 CTC GAT GAT ATT ATT GAC TTA TTT AAT TGT TTG ACA AAT GTT TCT TCA 816 Leu Asp Asp Ile Ile Asp Leu Phe Asn Cys Leu Thr Asn Val Set Ser 260 265 270 TTT TOC CTG CTG ACT GTG ACT ATT GA.A AGG GTA AAA GAO TTT TOT TAT 864 Phe Ser Leu Val Sex Val Thr Ile Glu Arg Val Lys Asp Phe Set Tyr 275 280 AAT TTC GGA TCC CAA CAT TTA GAA TTA GTT AAO TGT AAA TTT GGA CAG 912 Asn Phe Gly Trp Gin His Leu Giu Leu Val Asn Cys Lys Phe Cly Gin 290 295 300 TTT CCC ACA TTG AAA OTC AAA TOT OTO AAA AGG OTT ACT TTO ACT TOO 960 Phe Pro Thr Leu Lys Leu Lys Set Leu Lys Arc Leu Tnx Pne Thr Ser 305 310 31E 320 AAO AAA GGT CCC AAT CT TTT TOA GAJT GTT GAT OTA OCA AGO OTT GAG 100B Asn Lys Gly Gly Asn Ala Phe Sex Gin Val Asp Leu Pro Set Len Glu 325 330 335 TTT OTA CAT OTC ACT AGA AAT GGC TTG ACT TTO AAA GGT TC TGT TCT 1056 Phe Lau Asp Leu Ser Arg Asn Gly Leu Ser Phe Lys Cly Cys Cys Set 340 345 350 CAA AGT CAT TTT CCC ACA ACC AC COTA AAG TAT TTA CAT CTG AGO TTC 1104 Gin Ser Asp She Gly Thr Thr Set Leu Lys Tyr Len Asp Leu Sex Phe 355 360 365 AAT GGT CTT ATT ACC ATG ACT TOA AAO TTO TTG CCC TTA GAA CAA OTA 1152 Asn Ciy Val Ile Thr Met Sex Sex Asn Phe Len Cly Leu Gin Gin Len 370 375 380 GAA OAT CTC CAT TTO CAGCOAT TOO AAT TTG AAA CAA ATG ACT GAG TTT 1200 Gin His Leu Asp Phe Gin His Ser Asn Leu Lys Gin Met Set Gin Phe 385 390 395 400 TCA GTA TTC OTA TOA OTO AGA AAO OTO ATT TAO OTT GAO ATT TOT OAT 1248 Sex Val Phe Len Sex Leu Ax g Asn Len Ile Tyr Leu Asp :ile Sex His 405 410 415 ACT CAC ACC AGA GTT GOT TWO AAT CCC ATO TTO AAT CCC TTG TOO AGT 1296 Thx His Thx Arg Val Ala She Asn Gly Ile She Asn Gly Leu Sex Set 420 425 430 WO 01/90151 PCT/USOI/16766 OTO GAA GTC TTG AAA ATG GCT GGC A-AT TOT TTC CAG GAA AAC TTC OTT 1344 Leu Gin Val Len Lys Met Ala Gly Asn Ser Phe Gin Gin Asn Phe Len 435 440 445 CCA GAT ATO TTC ACA GAG CTG AGA AAG TTG ACC TTC CTG GAG CTC TCT 1392 Pro Asp Ile Phe Thr Gin Leu Arg Asra Leu Tht Phe Leu Asp Leu Ser 450 455 460 CAG TGT CAA CTG GAG CAG TTG TOT COA ACA GOA TTT AAC TOA GTC TOG 1440 Gin Cys Gin Leu Gin Gin Leu Ser Pro Thr Ala Phe Asn Ser Leu Ser 465 470 475 480 AGT OTT CAG GTA CTA AAT ATG AGO CAC 0AC AAO TTO TTT TOA TTG GAT 1488 Ser Len Gin Val Len Asn Met Ser H-is Asn Asn Phe Phe Ser Leu Asp 485 49C 495 AG TTT GOT TAT AAG TGT OTO AAO T -G OTO GAG GTT CTT GAT TAO AGT 1536 Thr Phe Pro Tyr Lys Gys Leu Asn Ser Let Gin Val Len Asp Tyr Ser 500 505 510 OTO AAT GAO ATA ATG ACT TOO AAA AAA GAG GAA OTA CAG CAT TTT OGA 1584 Leu Asn His Ile Met Thr Ser Lys Lys Gin Gin Leu Gin His Phet Pro 515 520 525 AGT AST OTA GOT TTO TTA AAT OTT ACT CAG AAT GAO TIT GCT TGT ACT 1632 Ser Ser Lou Ala Phe Lou Asn Leu Thr Gin Asn Asp Phe Ala Cys Thr 5930 53 5 540 TGT GAA GAG GAG AGT TTO OTG CAA TGG ATO AAG GAO CAG AGG CAG CTO 1680 Gys Gin H4is Gin Ser Phe Lou Gin Trp Ile Lys Asp Gin Arg Gin Leu 545 550 555 560 TTG GTG GAPA GTT GAA OGA ATG GAA TOT GOA ACA OCT TCA GAT AAG CAG 1728 3 5 Leu Vol Gin Val Giu Arg Met GLu Gys Ala Thr Pro Ser Asp Lys Gin 565 570 575 GGO ATG GOT GTG GTG AST TTG AAT ATO ACO TGT GAG ATG AAT AAG AGO 1776 Gly Met Pro Val Lou Ser Leu Asn Ile Thr Cys .Gin Met Asn Lys Thr 530 595 590 ATO ATT GGT GTG TOG GTO OTO AGT GTG CTT GTA GTA TCr STT GTA GCA 1824 Ile Ile Giy Vol Ser Val Len Ser Val Lou Val Val Ser Val Vol Ala 595 600 605 GTT CTG GTO TAT AAG TTO TAT TTT CAG OTG ATG OTT GTT GOT GGO TGO 1872 Vol Leu Val Tyr Lys Phe Tyr Phe His Lou Met Lou Leu Ala Gly Cys 610 615 620 ATA AAG TAT GGT AGA GGT GAA AAO ATO TAT GAT GOC TTT OTT ATO TAG 1920 Ile Lys Tyr Giy Arg Gly Giu Asn Ile Tyr Asp Ala Phe Vai Ile Tyr 625 630 635 640 34 wo 01/90151 WO 0190151PCT/USOI/16766 TCA AGO CAG GAT Sezr Ser Gin Asp GAG CAC Giu Asp 645 TCC GTA AGG AAT GAG OTA Trp Val. Arg Asn Giu Leu 650 CIA AAG A-AT ITTr Val Lys Asn Leu 655 GAA CAA GGC Glu Glu Gly AlT CCC CT Ile Pro Gly 6 AAA AGC CGA Lys Ser Arg 690 CCT CCA TTT Fro Pro ?he CAC CTC Gin leu 665 TCC CTT CAC TAC Cys Leu Eib Tyr AGA GAO TTT Arg Asp Phe 6,70 CCT TTC CAT Gly Phe His 19E8 2016 2064 2112 GIG GCC ATT GCT Val Ala Ile Ala AAC ATC ATC CAT Asn Ile Ile His AAG GTG ATT Lys Val Ile GIG GTG TCC CAC Val Val Ser Gin TTC ATO CAG AGC Phe Ile Gin Ser TCC TGT ATC TTT Trp Cys Ilie Phe CAA TAT Giu Tyr 710 GAG ATT GCT Glu Ile Ala ACC TO-G CAG TTT Thr Trp Gin Phe AGO AGT CGI Ser Ser Arg ACC CTG CTC Thr Leu Leo ACT TAC CTG lhr Tyr Leo 755 CCT 'GGT Ala Gly 725 ATC ATC TIC All Ile Ilie he Ile CIG CAG AAG GIG Leo Gin Lys Val CAG CAG GIG GAG Gin Gin Val Glu CIG TAC CGC CIT CTC AGC AGG AAC Leo Tyr Arg Leu Leu Ser Arg Asn 745 750 GTC CTG GGG CGG CAC AIC TO ICC Val Leu Giy Arg his Ile Phe Trp 765 GAG ICC GAG GAO Glu Trp Gin Asp 2160 2208 2256 2304 2352 2397 2400 AGA OGA CIC AGA AAA GC Arg Arg Leo Arg Lys Ala CTG CAT GGT AAA Leo Asp Gly Lys TGG AAT OCA CAA Irp Asn Pro Glu GGA ACA Giy Thr 785

TGA

CTC CCI ACA Val Giy Thr TGO AAT TGG CAG Cys Asn Trp Gin GCA ACA TOT ATC Ala Thr Ser Ile MELNFYKI PDNLPFSTKNLDLSFNPLRHLGSYSFFS FPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNP

IQSLALGAFSGLSSLQKLVAVETNLASLENFPIGHLKLKELNVAENLIQSFKLPE-FSNLNLE{LDLSSNK

IQSIYCTDLRVLEQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLILRNNFDSLNVNKTCIQGLAGLEVHRLV

LGEFRNEGNLEKFDKSALEGLCNLTIEEFRLAYLDYYLDDIIDLFNCLTNVSS FSLVSVTIERVKDF'SYN'GW

QHLELVNCKFGQFPTLKLKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFEKCCSQSDFGTTSLKYLD

LSFNGVITMSSNFLCLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVANT FNCLSSLEVLKM AGNS FQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVLNMSHNNFFSLDTFPYKCLNSLQVLDY SLNH4INTSKKQELQHFPSSLAFLNLTQNDFACTCEHQS FLQWIKDQ?QLLVEVERMECATPSDKQGMPVLSLN

ITCQMNKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLACIKYGRGENIYDAFVIYSSDEDWVRNELVCNLE

EGVPPPQLOLHYRDFIPCVAIAANI IHEGFHHSRKVI VVVSQHFIQCRWCIFEYE IAQTWQELSSRACIIFIV LQKVEKTLLRQQVELYRLLSRTYLEWIEDSVLGRHT FWRRLRF(ALLFIGEStNPEGTVGTGCNWQEATSI wo 011901-1-1 WO 0190151PCT/US01I16766 supplemented primate, humar, DTLR4 sequence (SEQ ID NO: 25 and 2S); note that nucleotides 81, 3144, 3205, and 3563 designate6 A, each may be A, C, G, or T; nucleotides 3132, 3532, 3538, and 3553 designated G, each may be G or T; nucleotide 3638 designated A, may be A or T; and nucleotides 3677, 3685, and 3736 designated-C, each may be A or C: AAAATACTCC CTTGCCTCAA AAACTGCTCG GTCAAACGGT GATAGCAAAC CACGCATTCA CAGGGCCACT GCTGCTCA4CA AAACCAGTGA GGATGATGCC AGGATG ATG TCT GCC 115 .0 Met Ser Ala -22 TCG CGC CTG Sec Arg Leu GCT GCC Ala Gly ACT CTG ATC CCA Thr Leu Ile Pro GCC ATG Ala Met -10 GCC TTC CTC Ala Phe LeU TCC TC Ser Cys GTG AGA CCA GAA AGC TGG GAG CCC TGC GTG GAG GTT COT AAT ATT ACT Val Arg Pro Glii Ser Trp Glu Pro Cys Val Glu Val Pro As Ile Thr TAT CAA Tyr Gin TGC ATG GAG CTG AAT TTC TAG AAA ATC CCC GAC A.AC CTC CCC Cys Met Giu Leu Asn Phe Tyr Lys Ile Pro Asp Asn Leo Pro

TTC

EThe TCA ACC AAG AAC CTG GAC CIG AGO TTT AAT CCC CTG AGG CAT Ser Thr Lys Aon Lou A~sp Leu Ser Phe Asn Pro Leu Arg His GGC AGC TAT Gly Ser Tyr AGC TTC Ser Phe GAA ATC Glu Ile TTC ACT TTC CCA SAA CTG CAG GIG CTG Phc Sec Phe Pro Giu Leo Gin Val Leu 55 GAT TIA Asp Leu ICC AGG TGT Sec Arg Cys AGO CAC CTC S§er His Leu CAG ACA ATT GAA GAT GGG GCA TAT Gin Thr Ile Giu Asp Gly Ala Tyr 70 CAG AGO CTA Gin Sec Leo TCT ACC TTA ATA TIC ACA GGA AAC CCC AIC CAG AGT TTA Ser Thr Leu Ile Leu Thr Gly Asn Pro Ile C-in Ser Leu CCC CTG Aia Leu GGA GCC TTT TCT Gly Ala Phe Ser CTA TCA ACT TTA CAG AAG CTG GIG GC Leu Sec Ser Leo Gin Lys Leo Val Ale

GTC

Val1 110 GAG ACA AAT CTA GCA TCT CTA GAG AAC TIC CCC ATT GGA CAT Giu Thr Asn Leu Ala Sec Leu Glu Asn Phe Pro Ile Gly His AAA ACT TIC A-AA Lys Thr Len Lys CTT AAT GTG CI Leu Asn Val Ala AAT OTT ATC CAA ICT TIC Aen Leu Ile Gin Sec Phe 140 AAA TTA CCI GAG TAT ITT TCT AAT Lys Leu Pro Glu Tyr Phe Sec Asn 145 ACC AAT CTA GAG Thr Asn Leo GJlu CAC TTC GAGC His Leu Asp 155 wo 01/90151 WO 0190151PCT/USOI/16766 CTT TOO AGO AAC AAG ATT CAA AGT ATT TAT TGC ACA GAO TTG CGG OTT 591 Leu Ser Ser Asn Lys Ile Gin Ser Ile Tyr Cys Thr Asp Leu Arg Vai 160 165 170 CTA CAT CAA ATG CCC CTA CTC A.AT CTC TOT TTA GAG CTG TOO OTG AAC 739 Len Fis Gin Met Pro Leu Leu Asn Leu Ser Leu Asp Lou Ser Len Asn 175 180 185 OCT ATG AAC TTT ATO CAA OCA GOT OCA TTT AAA GAA ATT AGG OTT CAT 787 Pro Met Asn Phe Ile Gin Pro Giy Ala The Lys Glu Ile Arg Lou Nis 190 195 200 205 AAG OTG ACT TTA AGA AAT AAT TTT GAT AGT TTA AAT GTA ATG AAA ACT 835 Lys Leu Thr Len Arg Asn Asn Phe Asp Ser Leu Asn Val Met Lys Thr 210 215 220 TGT ATT CAA GOT OTO GOT GGT TTA GAA GTC OAT COT TTG GTT OTG GO-A 883 Cys Ile Gin Gly Len Ala Gly Len Gin Val His Arg Leu Val Len Gly 225 230 235 GAA TTT AGA AAT GAA OGA AAO TTG GAA AAG TTT GAO AAA TOT GOT CTA 931 Gin Phe Arg Asn Gin Gly Asn Len Gin Lys ?he Asp Lys Ser Ala Len 240 245 250 GAG 000 OTO TGO AAT TTG ACO ATT GAA GAA TTO OGA TTA GCA TAO TTA 979 Giu Gly Len Cys Asn Lau Thr Ile Gln Gin Phe Arg Len Ala Tyr Len 255 260 265 GAO TAO TAO OTO GAT GA: ATT ATT GAC TTA TTT AAT TGT TTG ACA AAT 1.027 Asp Tyr Tyr Len Asp Asp Ile Ile Asp Len Phe Asn Cys Len Thr Asn 270 275 280 285 GIT TOT TdA TTT TOO OTG OTO AOT OTG ACT ATT GAA AGG GTA AAA GAC 107 Val Ser Ser Phe Ser Len Val Ser Val Thr Ile Gin Arg Val Lys Asp 290 295 300 TTT TOT TAT AAT TO GOA TOG CAA OAT TTA GAA TTA OTT AAO TGT AAA 1123 Phe Ser Tyr Asn Phe Gly Tro Gin His Len Giu Len Val Asn Oys Lys 305 3--0 315 TTT OGA GAG TTT COO ACA TO AAA CTC AAA TOT CTC AAA AGO OTT ACT 1271 Phe Gly Gin Phe Pro Thr Len Lys Len Lys Ser Len Lys Arg Len Thr 320 325 330 TTC ACT ICC AAO AAA GOT 000 AAT GOT TTT TCA GAA GTT GAT CIA COA 1219 The Thr Ser Asn Lys Giy Giy Amn Aia The Ser Gin Val Asp Len Prc 335 340 ,345 AGC OTT GAG TTT CIA OAT 010 AGl AGA AAT 000 TTG AGI TO AAA GOT 1267 Ser Len Gin The Leu Asp Len Ser Arg Asn Gly Leu Ser Phe Lys Giy 350 355 360 365 WO 01/90151

TGC

Cys

CTG

Lveu

GAA

Glu

AGT

Ser AlT Ie 430

TG

Leu A~c As n

GAC

Asp T CA S ex

TCA

Se r 510

-AT

Asp

CAT

His

GCT

Ala PCT/USOI/16766 GAT TTT GGG Asp Phe Gly GTT AT" ACC Val Tie Thr CTG GAT TTC Leu. Asp Phe 405 TTC CTA TCA Phe Leu Ser 420 AGC AGA TT Thr Arg Val 435 GTC TS AAAh Val Leu Lys ATC TTC ACA Ile Phe Thr CAA CTG GAG Gin Leu. Glu.

485 CAG CIA CTA Gin Val Leu S0o CCT TAT AAG Pro Tyr Lys 515 CAC ATA ATG His Ile Met CTA GCT TTC Leu. Ala Phe CAO GAG AGT His ClIn Ser 565 ACO CTA Sex Leu

TCA..APAC

Sex Asnl TOO AAT Ser Asn AAC CTO Asn Leu 425 AAT GG0 Asn Gly 440 GGC AAT Gly Asn AGA ARC Arg Asn TOT CCA Sex Pro AGO CAG Ser His 505 AAC TOO Asn Ser 520 AAkA AAA Lys Lys OTT ACT Leu Thr CAA TGG Gin Trp TTA CAT Leu Asp 380 GCC TTA Sly Leu CAA ATO Gin Met OTT SAC Lau Asp AAT 550 Asn Gly 445 OAG SA Gin Giu 460 TTG CG Phe Leu TTT AAO Phe Asn TIC TTT Phe Phe GTT OTT Val Leu 525 CTA CGS Leu Gin 540 GAO TTT Asp ?he GAC CAG Asp Gin 2315 1363 1411 1459 15 07 1555 1603 1653.

1699 1747 1795 1843 1891 1939 ASS CG GTG TTG GTG CPA GTT SPA CA ATG GPA TST SCA ACA OCT IGA Arg Gin 575 Leu Leui Val Giu. Val 580 Siu Arg Met Glu Ala Thr Pro Sex WO 01/90151 PCT/USOI/16766

GAT

Asp 590

AAT

Asn

GTT

Val

GCT

Ala

CT

Va1

AAG

Lys 670

AGA

Arq

GGT

Gly

ATC

Ile

CAG

Gin

GTG

Val 750

AGC

Ser AAG GAG GGC AIG CCT GTG CTG ACT TTG AAT ATC ACC TGT GAG ATG Lys Gin Cly Met Pro Val Leu Ser Leu Asn Ile Thr Cys Gin Met 595 600 605

ACC

Thr

GCA

Ala

TGC

Cys 640

TAG

Tyr

TTA

Leu

TTT

Phe

CAT

His

AGC

Ser 720

CTG

Leu

AAG

Lys

AAC

Asn

GTG

Va1

CAC

His

ATC

ile

AGG

Arg 665

CTG

Lau

AAC

Asn

GTG

Va1

ATT

Ile

ATT

Ile 743

CTG

Leu

GTC

Val

CTT

Len

CTG

Len

TAT

Tyr 650

AAT

Asn

TGC

Gys

ATC

Ile

TCC

Ser

GCT

Ala 730

GTC

Val

TAG

Tyr

CTG

Leu GTA GTA Val Val 620 ATG CTT Met Len 635 CAT GCC Asp Ala GAG CTA Glu Lau GTT CAC Leu His ATC CAT Ile His 700 GAG CAC Gin His 715 GAG ACC Gin Thr CTG CAG Len Gin CGC CTT Arg Leu GGG CGG Gly Arg -780

TCT

Ser

CTT

Len

TTT

Phe

GTA

Val

TAG

Tyr 685

GAA

Clu

TTC

Phe

TGG

Irp

AAC

Lys

CTC

Len 765

CAC

His 1987 2035 2083 2131 2179 2227 2275 2323 2371 2419 2467 2515 2563 ATC TTG TCG AGA CGA CTC AGA AAA CCC CTG CTG GAT CGT AAA TCA TGG Ile Phe Irp Arg Leu Arg Lys Ala Len Lou Asp Cly Lys Ser Trp 39 wo 011901-1-1 WO 0190151PCT/US01I16766 MAT CCA GMA GGA ACA GTG GGT ACA GGA TGC AMT TGG CAG GAA GCA ACA Asn Fro Glu Gly Thr Val Gly Tim Gly Cys Asn Trp GIn Glu A la Thr 800 805 810 TCT ATC TGAAGAGGAA AAATAAAAAC CTCCTGAGGC ATTTCTTGCC CACCTGGGTC Ser Ile 815

CAACACTTGT

TGAGTAATTC

ACAGGGAATA

AGTCAAGGAA

GACAGAGAAA

TAT GTTATAG

TCCTTTTTGA

TGATGCA.AGA

CTAATTCCTA

TGTTCTATTT

TTTTTTTACG

AATATCCATA

TCACTCGATG

TGAAATAAT

TCTTATCATG

CCTTTTCTTG

TTGCAGCAGA

TAGGGAGACA

CGTGVGAAGG

TGACCACATT

GGGGTTCTTA

GGAACATTCA

AT GAGAT AT C

ATATAGAGAA

TCAGTTAATA

CAT GGT GCAC

AATGCTAGAC

CCCATGACAA

AGTATTAAAT

TAGATATGCA

TAAAATACAG

AGAAAGTCAT

ACAGAAAGAG ACATTGTTCT CCA'TCATAAA ACCATTT7GG TT'GAATACAA TTTAAATTCT TGCCCCTTCC ATTTTAACTC AGGAAACCTG ATTA.ACACAT TTTAACTAAT CACCCCTGAT TCTTGCCTAT AAGCTAATAT TTAACCACTA TTTTTCAAGG TCATTCCAAA GTTATTGCCT TfGTTTAAACGG GGGCACTCTT GACAATTTGG GCTAGAGGCA ATTCCAGAAA CATATGGGC:-

AGTTTATTTT-TTTCAGAACA

CAGATGGCTG GGATCCCTCC TATTCAAGGC AGGGAGTATA TTGGGAAGAG TGGATGTTAT TAAAGAAGGT TCCCAGAAAA AGGAAAAGGA CAATCAGGAT ACCTTATACC AGGTAGATGG ATTGGAACCC TTCTTCACTG

GCTGCCACAT

GGGCTGCTAA

AGTCTTCCAG

TTCAACTCTT

TTT CCC GAGT

TAGTTTTGAC

ACTTGATGAC

TGTCTCCTTA

GCTCACAACC

ATATTTTTAT

CATAAATAAG

AAGTATGGAA

ACTAAGTAAT

TTAAACGGGA

GGAAGGAAGT

GATAAACCCG

ACT GATGTTT

CCTGTACCCT

CATTCCTGTT

CATTGAGAAA

GAATGTTCAT

GTCATCAGGG

CTACTATAAA

CTGGAGGGA.A

GT CAGGCCTT

TCTCAAGGAG

GTGGGCATTT

ACCTCATCAA

CTTTTGAATG

TGAACTGGGT

TGCAGTCGTC

CAGAGGTTAA

ATCCTGGTCA

TTTTATATAT

GTTGTTTAAG

AAGTACACTC

GACTGTCATG

ACAAANAT TTC

GGGATGACCT

GGGTGACCTC

CAT GGACCT C

TCTCACTGCC

TCCTGTTGGG

ACAATGTGTC

TCCAGCTTCT

AAATGAAAAT

AAAATGAAGT

TGGAAAATGG

ATGCTAAGGG

CTTCCAGTGC

CAACCZAACTC

GTTGAATAAA

GAAATTGTAT

GTTCACTTTT

A.AGGGGCTCC

AGTCTA-ATGG

TTCTCGAACA

CCAGTTTTCA

ACSTGCTTCA

TGTCACTTTG

AAAGCAGCAT

CGCTTCCTGG

CAGGAAGTCA

ATGAAATGAG

TGAATCTCTT

AGGAGAACTA

CAATGCTCCT

TGGAATTAAT

TCAGGAAACA

AAAAACCACA

GTCATCAAGG

TGTAGCCGTT

2611 2667 2 727 2787 284-7 2907 2 967 3027 30837 3147 3207 3267 3327 ,337 3447 3 507 356'7 3627 3687 3747 3807 3867 3927 3987 4047 4107 wo 0119011-1 WO 0190151PCT/LISOI/16766

ATCAAAAACP.

ATCTCAC-TTC

ACTCCCATGT

CA.AATTTCCA

ATTATTCAGC

GGCCATTATG

TATATGAGGT

GGAA&AGGAG

GTATGCAAGA

TACTGTATTA

CATATACATA

GGTGATGGTT

TAT GCAGT TT

GTACGGAGGT

TGTATATATA

TCATTGTGGC

TTGGAAAATA

CTA-MAAAG

CTATGTAAA

TCTAAAATAG

GAAGGGAGAA

TGAATTAGCT

TGCACTTAAC

TACACAAGGA

TGACAGGTAT

TAT.AATATCA

TTCTCAAAAA TTA'\AAAAT.

CCCMAATAA TTC-AAATC ACTCTTCACA ATCACTGT AATGGACAAA GGAAAI GT GGGGGAC'CCT GTTATTTA TGAGCAAGTA ACAGAAAG; TCAM2CTCAT AGAAGCAG ATGAGGAAAT AGGGAGTT CTAAAGATCA GCTGTATA ATTTTGTTPJ\ GAGGGTAC( AGCTTTTCGA GGTGATGGi GTGACTATGT CTAAACTCI

~AAAAA~AAAAAAAAA

AG

AG

TT

GC

TG

AC

AG

GT

GC

CT

kT

W

AACTGCTATA TGATCCAGCA AATTTCAAGA AJAATAT'TTAC CCAAAG7TAT GGAP.ACAACC

ATATAACGTA

ACAACATGAA

AAATACTGCC

A.ATAGAACAG

CTAATTGGTA

AGAGTTCGTA

CP-CATGTTAA

ATATTTATTA

CAAATTGTAT

CAATGGGGAT

TAAA;CCCGGA

TGATTTCATT

TGGTTCCTAG

TAAAkATTATA

TAATGAACAA

GTGTTCTTAC

CCTTGATTGT

ACATTAAATA

4167 4227 4287 4347 4407 4467 4527 4587 4647 4707 47E7 4827 43E5 MSASRLAGTLI PAMAFLSCVRPESWEPCVEVPNITYQCMELNFYKI

PDNLPFSTKNLDLSFNPLRHLGSYSFFSF

PELQVLDLSRCEIQT IE DGAYQS LS HLSTLILTGNPI QS LALGAPS GLS SLQKLVAVETNLASLENFF IGI-LKT L KELNVAHNLIQSFKLPEYFSNLTNLI-LDLSSNZIQS I YCTDLRVLHQMLLNLSLU)LSLNPMNFIQPGAFKE:R LIEKLTLNNFDSLNVMKCIQLAGLEVERVLGERNGNLEKFDKSALEGLCNLTISEFRLACYLDYYLDDI I C LFNCLTNVSS FSLVSVT I ERVKDFS YNFGWQHLELVN CKFGQFPTLKLKSLKRLT FT SNKGGNAFSEVDL PSLE F LDILSRNGLS FKGCCSQS DFGTTSLKYLDLS FNGVT TMS S1' FLGLEQLEHLDFQHSNLKQMSE FSVFLS LgNLIYL DI SHTHTRVAFNGI FNGLSS LEVL<MACNS FQEN FLP D IFTELRNLT FLDLSQCQLEQLS BTAFNSLS SLQVLNM

SIHNNFSLDTFPYKCLNSLQVLDYSLNHIMTSK{KQELQHFPSSLAFLNLTQNDEACTCEHQSFLQW!IKDQRQLLV

EVERMECATFSDKQGMPVLSLNITCQMNKTrIGVSVLSVLVVSVVAVLVYI<FYFNLMLLAGCLKYGRGENIYDAF VI YSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDF'r PGVAIAAN IIHEGFHKSRKVIVVVSQHFI QS RWC I FEYE TAQTWQFLS SRAG IIFI VLQKVEKTLLRQQVELYRLLS lNT YLEWE DSVLGRH I WRRLRKALLDGKSWN PEGTV

GTGCNWQEATSI

WO 0119011-1 PCT/USOI/16766 Table 5: Partial nucleotide and amino acid sequences (see SEQ ID NO: 9 and of a mammalian, primate, human, DNAX Tell like Receptor TOT TGG GAT OTT TTT GAG GGA CT TCT CAT CTT CAA OTT Cys

I

AAT

Asn

ACT

Thr

TCT

Ser

AAC

Asn

TTG

Leu

TTT

Phe

GCA

Ala

TTC

Phe

CTA

Leu 145

CTC

Leu

TGT

Cvs Oly Leu Ser TCC CTT CCA Set Leu Pro AGC CTC AAC Ser Leu Asn OCT AAT TTA Ala Asn Leu AAT CCT OAT Asn Pro Asp AAG TTC ATT Lys Phe lie CAC ACC AAT His Thr Asn 105 TAC CCT GAC Tyr Pro Asp 120 GGT TOT OAT Oly Cys Asp 135 ATT OTA TGC Ile Val Cys GTC ACA AAG Val The Lys AGA CTG GTG Arg Leu Val 135 CTO TAT Leu Tyr AGC CAT Ser His ACA OTT Thr Val ATA TCC Ile Ser CTT AGT Leu Ser CTT AGC Leu Ser CGG CCT Gly Pro 110 OTT TCC Val See TTA AAG Leu Lys ACT CTG Thr Leu TGT TTT Cys Phe 175 CCC CAG Pro Gin 190 ACA GAl CCT GAT ATG TAC AAA TAT OAT 0CC TAT TTG TGC TTC AOC AGO Thr Glu Pro Asp Met Tyr Lys 'Tyr Asp Ala Tyr Leu Cys Phe Set Ser wo 0119011-1 WO 0190151PCT/LISOI/16766 AAA GAC Lys Asp 210 TTC ACA TGG GTG CAG Phe Thr Trp Vai Gin 215 AAT GCT TTG CTC Asn Ala Leu Leu CAC CTG GAO ACT His Leu Asp Thr TAO AGT GAC CAA Tyr Ser Asp Gin AGA TTC AAC CTG Arg Phe.Asn Leu TTT GAA GAA AGA Phe Giu Glu Rrg TTT GTC CCA GGA Phe Val Pro Gly AAC CGC ATT c-CC Asn Arg Ile Ala ATC CAG GAT GC Ile Gin Asp Ala ATC TGG Ile Trp 255 AAC AGT AGA Asn Ser Arg GGC TGG TGC Gly Trp Cys 275 ATC GTT TGT CTT Ile Val Cys Leu AGO AGA CAC TTC Ser Arg His Phe CTT AGA GAT LeU Arg Asp 270 TGC TTA TCT Cys Leu Ser CTT GAA GCC TTC Leu Giu Ala Phe TAT CO CAG GGC Tyr Aia Gin Gly GAO OTT Asp Leu 290 AAG AGT GOT CTC Asn Ser Ale Leu ATG GTG GTG GTT Met Val Val Val.

TOO TTG TOO CAG Ser Leu Ser Gin CAG TTG ATG AAA Gin Leu Met Lys CAA TCC ATO AGA Gin Ser Ile Arg TTT GTA CAG AAA The Val (lln Lys GAG TAT TTG AGG Gin Tyr Leu Arg OAT AAA CTC TCT His Lys Leu Ser CCT GAG GAT OTO Pro Giu Asp Leu GAT GTT GGC TGG Asp Val Gly Trp TTT OTT ?he Leu 335 1008 1056 CAA GAG ATA CTA Gin Gin Ile Leu AAA GAA AAG Lys Giu Lys GAA AAG AAG AAA Giu Lys Lys Lys 350 GAC AAT AAC ATT Asp Asn Asn Ile 355 COG TTG CPA ACT GTA GCA ACC Pro Leu Gin Thr Val Ala Thr 360 ATO TOO TAATCAAAGG Ile Ser 365 AGCAATTTCC AACTTATCTC AAGCCACAAA TAACTCTTCA CTTTGTATTT GCACCAAGTT ATOATTTTGG GGTCCTCTCT GGAGGTTTTT TTTTTCTTTT TGCTACTATG AAAAOAACAT AAATCTCTCA ATTTTCGTAT CAAAAAAAAA AAAAAAAAAA TGGCGGCOGO 1165 1225 1275

CWDVFEGLSHLQVLYLNHNYLNSLPPGVFSHLTALRGLSLNSNRLTVLSHNDLPANLEILDISRNQLLAPNPDVF

VSLSVLDITHNKFICEOELST FINWLNHTNVTIAGPPADIYCVYPDSFSGVSLFSLSTEGCDSEEVLKSLKFSLF

IVCTVTLTLFLHTILTVTKFRGFCFICYKTAQRLVFKDHPQGTEPDMYKYDAYLCFSSKDFTWVQNALLKHLDTQ

YS OQNRFNLCFEERDFVPGENRIAN IQDAIWNSRKI VOLVSRHFLRDGWCLEAFSYAQGRCLS

DLNSALIMVVVG

SLSQYQLMI<MQS IRGFVQKQQYLPWPEDLQDVGWFLHKLSQQILKKEKEKKKDNNIPLQTVATIS wo 01/90151 WO 0190151PCT/USOI/16766 Table 6: Nucleotide and amnino acid or rodent DNAX Toll likJe Receptor 6 primate, human; SEQ ID NO: 13 sequences of mammalian, (DTLR6) SEQ ID NO: 11 and 14 are from rodent, primate and 12 arQ fron mouse.

primate: ATG TGG Met Trp -22 ATT TCC Ile Ser GAT GTC Asp Val GAC AAG Asp Lys 'aAC CTC Asn Leu TTT CAC Phe His GTA CCT Va]. Pro CAG ATT Gin Ile TAC CTG Tyr Leu AGC TTA Ser Leu AAA SAG Lys Gin.

140 CAA AAC Gin Asa 155 AAG AGA Lys Arg CTT GGG Len Gly GAT GTT Asp Val ACA GAA Thr Giu ACC ATT Thr Ile GAC CAT Asp His CTG GG Len Giy so AGA AGC Arg Ser AAC CAG Asn Gin CTC AGC Len Ser ACA GAA Thr Gin CTT TTT Leu Phe CCT AAA Pro Lys GTG ATC Val Ile ATT CCC Ile Pro GAC ATC Asp Ile GAT TTC Asp Phe ATG TGC Met Cys ACT TAT Thr Tyr CCG CAG Pro Gin AAC ATC Asn Ile GAA ATA Giu Ile 150 GTT TCA Val Ser 165 TGT TAT TAT Cys Tyr Tyr CGA AAT Arg Asn 160 OCT TGT TAT Pro Cys Tyr TAT TCA ATA Tyr Ser Ile WO 011901-1- PCT/US01/16766 AAA GAT GCC TTC CTA AAC TTG ACA AAG TTA AAA GTG CTC TCC CTC AAA 624 Lys Asp Ala Phe Len Asn Leu Thr Lys Leu Lys Val Leu Ser Leu Lys 175 180 185 GAT AAC AAT GTC ACA GCC GTC CCT ACT GTT TTGCCA TCT ACT TTA ACA 672 Asp Aen Asn Val Thr Ala Val Pro Thr Val Leu Pro Ser Thr Len Thr 190 195 200 GAA CTA TAT CTC TAC AAC AAC ATG ATT GCA AAA ATC CAA GAA GAT GAT 720 GLu Leu Tyr Len Tyr Asn Asn Met Ile Ala Lys Ile Gin Gin Asp Asp 205 210 215 TTT AAT AAC CTC AAC CAA TTA CAA ATT CIT GAC CTA AGT GCA AAT TGC 768 Phe Asn Asn Len Asn Gin Leu Gin Ile Leu Asp Len Ser Gly Asn Cys 220 225 230 CCT CGT TGT TAT AAT CCC CCA TTT CCT TCT GCG CCG TGT AAA AAT AAT 816 Pro Arg Cys Tyr Asn Ala Pro Phe Pro Cys Ala Pro Cys Lys Asn Asn 235 240 245 250 TCT CCC CTA CAC ATC CCT GTA AAT GCT ITT GAT GCG CTG ACA GAA TTA 864 Ser Pro Leu Gin Ile Pro Val Asn Ala Phe Asp Ala Len Thr Glu Leu 255 260 265 AAA GTT TTA CGT CTA CAC ACT AAC TCT CTT CAG CAT GIG CCC CCA AGA 912 Lys Val Leu Arg Leu His Ser Asn Ser Leu Gin His Val Pro Pro Arg 270 275 280 IGG TTT AAM AAC ATC AAC AAA CIC CAC GAA CIG GAT CTG TCC CAA AAC 960 Trp Phe Lys Asn Ile Asri Lys Len Gin Giu Len Asp Len Ser Gin Asn 285 290 295 TTC TTG GCC AAA GAA ATT GGG GAT GCT AAA TTT CTG CAT ITT CTC CCC 1008 Phe Leu Ala Lys Glu lIe Gly Asp Ala Lys Phe Leu His Phe Leu Pro 300 305 310 AGO CIC ATC CAA TTG GAT CTG TCT TIC AAT TTT CAA CTT CAG GTC TAT 1056 Ser Leu Ile Gin Len Asp Len Ser Phe Asn Phe Glu Leu Gin Val Tyr 315 320 325 330 CGT GCA TOT ATG AAT CIA TCA CAA CA TTT TCT TCA CTG AAA A.GC CTG 1104 Arg Ala Ser Met Asn Len Ser Gin Ala Phe Ser Ser Leu Lys Ser Len 335 340 345 AAA ATT CTG CGG AIC AGA GCA TAT GIC TTT AAA GAG TTG AAA AGC ITT 1152 Lys Ile Len Arg Ile Arg Gly Tyr Val Phe Lys Glu Len Lys Her Phe 350 355 360 AAC CIC TCG CC TTA CAT AAT CIT CAA AAT CIT GAA GTT CTT GAT CTI 1200 Aen Len Ser Pro Le His Asn Len Gin Asn Leu Gli Val Len Asp Len 365 370 375 GGC ACT AAC TTT ATA A.AA AT GCT AAC CTC AGO ATG TTT AAA CPA ITT 1248 Gly Thr Asn Phe Ile Lys Ile Ala Asn Len Ser Met Phe Lys Gin Phe 380 385 390 wo 011901-1-1 WO 0190151PCT/US01I16766

AAA

Lys 395

GGA

G.1y

GAA

Giu

AT

As p

ATG

Met

AGT

S er 475

TCT

Ser

OTT

Lau

TTC

Phe

OTT

Len

CAA

Gin 555

GTT

Val AGA CTG AAA Arg Leu Lys GAT TCA AGT Asp Ser Ser AGT TAT GAA Ser Tyr Giu 430 AAG TAT GCA Lys Tyr Ala 445 TCT GTT AAT Ser Val Asn 460 AAA AAT AGT Lys Asn Ser TIC CIG AAA Phe Leu Lys AAT GGC AGT Aen Gly Sex 52.0 TOG A-AC AAC Ser Asn Asn 525 CAC AAA CTG His Lys Leu 540 TGA GAA GGA Ser Giu Gly CIG CAG AAA Len Gin Lys GTC ATA GAT OTT TCA GIG AAT AA P9,TA TGA Val Ile Asp Leu Ser Vai Asn Lys Ile Ser 400 GAA GTT Gin Val 415 CCC CAG Pro Gin AGG AGT Arg Ser GAA AGO Gin Ser ATA TT Ile Phe 480 TGC CTG Cys Len 495 GAA TO Gin Phe CGG CT Arg Leu GAA GTT Glu Vai ATT ACT Ile Thr 560 CTG AIG Len Met 575 TGO TCA Cys Ser 420 GAA CAA Giu Gin 435 TTC AAA Phe Lys AAG TAT Lys Tyr AAG TCC Lye Ser TGA GGA Ser Gly 500 ITA GCA Leu Ala 5i5 CTC CAT Leu His ATA AGO Ile Ser OVA AAC Leu Asn GAC AAI Asp Asn 58C 405 AAT GOG AGA ACT Ast Ala Arq Thr TTA CAT TAT TTC Leu His Tyr Phe 440 AAC AAA GAG GOT Asn Lys Giu Ala 455 GGG GAG AGC TTG Giy Gin Thr Leu 470 TCT GAT TTT'CAG Ser Asp The Gin 485 AAT OTO ATT AGO Asn Len Ile Ser GAG CTG AGA TAT Giu Leu Arg Tyr TCA ACA GOA ITT Ser Thr Ala Phe 535 AGT A.AT AGO CAT Ser Asn Ser his 550 ITT ACG AAG A.AO The lhr Lye Asn 565 GAG ATC TCT TCO Asp Ile Ser Ser 1296 1344 1392 1440 1488 1.536 2584 1632 1680 1728 1'776 1824 1.872 AGO AGO ACC AVG GAG ACT GAG TOT OTT AGA ACT CIG GAA TIC AGA GGA Ser Arg Ihr Met Giu Ser Gin Ser Leu Arg Thr Len Gin Phe Arg Gly 590 .595 600 wo 011901-1-1 WO 0190151PCT/US01I16766 AAT CAC TTA GAT GTT TTA TGG AGA GAA GGT GAT PAC AGA TA C TTA CAA 1920 Asn His Len Asp Val Len Trp Arg Gin Gly Asp Asn Arg Tyr Leu Gin 605 610 615 TTA TTC AAG AAT CTG CTA AAA TTA GAG GAA TTA.GAC ATC TCT AAA AAT 1968 Len The Lys Asn Len Len Lys Leu Gin Gin Leu Asp lie Ser Lys Asn 620 625 630 TCC CTA AGT TTC TTG OCT TCT GGA GTT TTT GAT GGT ATG CCT CCA AAT 2016 Ser Leu Ser Phe Len Pro Ser Gly Val Phe Asp Gly Met Pro Pro Asn 635 640 645 650 CTA AAG AAT CTC TCT TTG GCC AAA AAT GGG CTC AAA TCT TTC ACT TGG 2064 Leu Lys Asn Len Ser Len Ala Lys Asn Ciy Leu Lys Ser Pne Ser Trp 655 660 665 AAC AAA CTC CAG TGT CTA AAC AAC CTG GAA ACT TTG GAC CTC ACC CAC 2112 Lys Lys Len Gin Cys Leu Lys Asn Len Gin Thr Len Asp Len Ser His 670 675 680 AAC CA.A CTG ACC ACT OTC CCT GAG AGA TTA TCC AAC TGT TCC AGA AGC 2160 Asn Gin Leu Thr Thr Val Pro Gin Arg Leu Ser Asn Cys Ser Arg Ser 685 690 695 CTC AAG AAT CTG ATT CTT AAG AAT AAT CAA ATC AGG AGT CTG P.CG AAG 2208 Lau Lys Asn Len Ile Len Lys Asn Asn Gin Ile Arg Ser Leu Thr Lys 700 705 710 TAT TTT CTA CAA GAT GCC TTC CAG TTG CGA TAT CTG GAT CTC AGC TCA 2256 Tyr The Len Gin Asp Ala Phe Gin Len Arg Tyr Leu Asp Leu Ser Ser 715 720 725 730 AAT AAA ATC CAC ATG ATC CAA AAG ACC AGC TTC CCA GAA AAT GTC CTC 2304 A.9n Lys Ile Gin Met Ile Gin Lys Thr Ser Phe Pro Gin Asn Val Len 735 740 745 AAC PAT CTG AAC ATG TTC CTT TTG CAT CAT AAT CGG TTT CTG TGC ACC 2352 Azn Asn Len Lys Met Leu Lau Len His His Asn Arg The Leu Cys Thr 750 755 76C TCT GAT CCT GTG TOG TTT CTC TGG TG GTT AAC CAT ACG GAG GTG ACT 2400 Cys Asp Ala Vai Trp Phe Val Trp Trp Val Asn His Thr Gin Val Thr 765 770 77 ATT CCT TAC CTG CCC ACA GAT CTG ACT TOT CTG GGG CCA GGA GCA CAC 2448 Ile Pro Tyr Len Ala Thr Asp Val Thtr Cys Val Gly Pro Gly Ala His 780 785 790 AAG CCC CAA ACT GTG ATC 17CC CTG GAT CTG TAC ACC TGT GAG T7A GAT 249E Lys Giy Gin Ser Val Ile Ser Len Asp Len Tyr Thr Cys Gin Len Asp 795 80C 805 810 CTG ACT AAC CTG ATT CTC TTC TCA CTT TCC ATA TCT GTA TCT CTC TTT 2544 Len Thr Asn Len Ile Len Phe Ser Len Ser Ile Ser Val Ser Leu Phe 815 820 825 wo 011901-1-1 WO 0190151PCT/US01I16766

CTC

Leu

TAT

Tyr

ATA

Ile

GAO

Asp 875

GAA

Glu

TGG

Trp

CTT

Leu

GAA

Glu

GAA

GIlu 955

AAG

Lys

CTT

Leu

CTA

Leu ATG GTG ATG Met Val. Met 830 ATT TAC CAT lie Tyr Fis 845 TCA CCA GAC Ser Pro Asp 860 CCA GCT GTG Pro Ala Val GAC CCA AGA Asp Pro Arg TTA CCA GGG Leu Pro Gly 910 AGC AAA AAG Ser Lys Lys 925 AAT TTT AAG Asn Phe Lys 940 AAA GTT GAT Lys Val Asp TCC AAG TTC Ser Lys Phe GAG TGG CCA Glu Trp Pro 990 AAG AJAC GCC Lys Asn Ala 1005 ATG ACA GCA AGT CAC CTC TAT TTC TGG GAT GTG TGG Met Thr Ala Ser His Leo Tyr Phe Trp 835 T TC Phe T GT Cys

ACC

T hr

GAG

Glu 895

CAG

Gin

ACA

Thr

ATA

I -e

GTG

Val

CTC

Leu 975

ACA

Thr

CTG

Leu

GTC

Asp Val Trp 840 GAG CGT CTA Gin Arg Leo GAC ACT AAA Asp Thr Lys GCC AAA CTG Ala Ly's Leu 890 GAA AGG GAC Glu Prg Asp 905 AGC ATA GAG Ser Ile Gin 920 GCA AAG ACT Ala Lys Thr CTC AIG GAT Leu Met Asp CCC TTT CAG Pro ?he Gin 970 AGT TCT GTC Ser Ser Val 985 TGC CAG TGT Trp Gin Cys 1000 2592 2640 2688 2736 27.84 2832 2880 2928 2976 3024 3072 3120 3138 GAO AAT CAT GTG GCC TAT AGT CAG GTG Asp Asn His Val Ala Tyr Ser Gin Val 1010 1015 TTC AAG GAA ACG The Lys Giu Thr Val 1020 MWTLKRLILILFNIILISKL-GARMFPKTLPCDVTLDVPKNEVIVDCT0KHLTEIPGGIPTNTTNLTLTINHI P DISPASFHRLfHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKPRSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQL WO 01/90151 PCT/USO1/16766 LSLEANUT FSTRRENLTELANTETLYLGQNCYYRZNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPST LTELYLYNNNIAKTQEDDENNLNQLQILDLSGN CFRCYNAPF'PCAPCNNNS PLOT PVNAFDALTELKVLRLHSN SLQHVPPRWFKNTNKLOELDLSQNFLAKEIGDAKFLHFLPSLIQLDLS FNFELQVYRASNINLSQAFSSLKSLKI LRIRCYVFKELKS FNLSPLHNLQNLEVLDLGTNFIK:ANLSMFKQFKRLKVTDLSVNKrSPSGDSSEVGPCSNA RTSVESYEPQVLEQLHYFRYDRYARSCRFKNKEASFMSVNESCYKYGQTLDLSKNSIFrVKSSDFQHLSFLKCL NLSGNLI SOTLNGSEFQPLAELRYLDFSNNRLDLLHSTAFELHKLEVLDSSNSHYFOSEGITINLNFTKNLK VLQKLMMNDNDISSSTSRTNESESLRTLERGN-LDVLWREGDNRYLQLFKNLLKLEELDI SKNSLSFLPSGvP

DGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLZTLDLSHNQLTTVFERLSNCSRSLKNLILKNNQIRSLTKYFLO

DAFQLRYLDLSSNKTOMIQK TSFPENVLNNLKMLLLHENRFLCTCDAVWFVWWVNHTEVTI FYLATDVTCVGPC

AHKGQSVISLDLYTCELDLTNLTLFSLSISVSLFLMVMMTASHLYFWDVWYIY-HFCKAKIKGYQRLTSFDCCYD

AFIVYDTKDPAVTEWVLAELVAKLEDPREKHFNLcLEERDWLPGQPVLENLSQS

IQLSKKTVFVNTDKYAKTEN

EIAFYLSHQRLMDEKVDVIILTFLEKPFQKSKFLQLRKRLCCSSVLEWPTNPQAR PYFWQCLKNALATDNHvA

YSQVFEETV

WO 01/90151 PCT/USOI/16766 rodent (SEQ ID NO: 13 and 14): CTT GGA AAA CCT CIT CAG AAG TCT AAG TTT CIT CAG CTC AGG AAG AGA 48 Len Gly Lys Pro Len Gin Lys Ser Lys Phe Leu Gin Len Arg Lys Ary 1 5 10 CTC TGC AGG AGC TCT GTC CTT GAG TGG CCT GCA AAT CCA CAG GCT CAC 96 Leu Cys Arg Ser Ser Val Len Glu Trp Pro Ala Asn Pro Gin Ala His 25 CCA TAC TTC TGG CAC TGC CTG AAA AAT GCC CTG ACC ACA GAC AAT CAT 144 Pro Tyr Phe Trp Gin Cys Len Lys Asn Ala Len Thr Ihr Asp Aso His 40 GIG GCT TAT AST CAA AIG TIC AAG CAA ACA GTC TAG 180 Val Ale Tyr Ser Gln Met Phe Lys Gln Thr Val LGKPLQKSKFLQLRKRLCRS SVLEWPANPQAHPYFHQCLKALTTDNHJAYSQMFKETV additional rodent, mouse sequences: upstream (SEQ ID NO: 27 and 28); nucleotides 186, 196, 217, 276, and 300 designated C, each may be A, C, G, or T: ICC TAT TCT AIG GAA AAA GAT GCT TTC CIA TTT ATC AGA AAT TTG AAG 48 Ser Tyr Ser Met Glu Lys Asp Ala Phe Len Phe Met Arg Asn Len Lys 1 5 10 GTT CTC ICA CIA AAA CAT AAC AAT GTC ACA GCT GTC CCC ACC ACT TTG 96 Val Len Ser Len Lys Asp Asn Asn Val Thr Ala Val Pro Ihr Ihr Leu 25 CCA CCI AAT TIA CIA GAG CTC TAT CTT TAT AAC AAT ATC ATT AAG AAA 144 Pro Pro Asn Len Leu Glu Leu Tyr Len Tyr Asn Asn Ile Ile Lys Lys 40 ATC CAA GAA AAT GAT TIC AAI AAC CIC AAT GAG TTG CAA GIC CIT GAC 192 Ile Gin Glu Asn Asp Phe Asn Asn Len Asn Glu Lou Cln Val Leu Asp 55 CTA CGT GGA AAT TGC CCI CGA TGT CAT AAT GTC CCA TAT CCG TGT ACA 240 Len Arg Sly Asn Cys Pro Arg Cys His Aso Val Pro Tyr Pro Cys Ihr 65 70 75 CCG TGT GAA AAT AAT ICC CCC ITA CAC AIC CAT CAC AAT GCT TTC AAT 288 Pro Cys Glu Asn Asn Ser Pro Len Gin Ile His Asp Asa Ala Phe Asn 90 ICA TCG ACA GAC 300 Ser Ser Thr Asp 100 WO 0119011-1 PCT/USOI/16766 SYSMEKDAFLFNRNLKVLSLKDNNVTAVPTTLPPNLLELYLYNNI I1CKIQENDFNNLNELQXLDLXGMCPRCXNV

PYPCTPCENNSPLQIHXNAFNSSTX

downstream (SEQ ID NO: 29 and 30); nucleotide 1643 designated A, may be A or G; nucleotide 1664 designated C, may be A, C, G, or T; nucleotides 1680 and 1735 designated G, may be G or T; nucle'otide 1719 designated C, may be C or T; and nucleotide 1727 designated A, may be A, G, or T: TCT OCA GAA ATT CCC TGG AAT TCC TTG CCT CCT GAG GTT TTT GAG GGT 48 Ser

I

ATG

Met

TCT

Ser

GAC

Asp

TGT

Cys

CAA

Gin

GAG

Asp

GAA

Glu

TTT

Phe

ACA

Thr 145 Ile Pro Trp Asn Ser Leu Pro Pro Glu Vai 5 PAT CT\ AAC Z\AT CTC TCC TTG GCC AAA AAT Asn Leu Lys Asn Leu Ser Len Ala Lys Asn TGG GAC AGA CTC GAG TTA CTG AAG CAT TTG Trp Asp Arg Len Gin Leu Leu Lys His Leu 40 CAT AAC CAG CTG ACA AAA GTA CCT GAG AGA His Asn Gin Leu Thr Lys Vai Pro Glu Arg AGT CTG ACA ACA CTG ATT CIT AAG CAT AAT Ser Len Thr Ihr Leu Ile Leu Lys His Asn 70 AAA TAT TTT CTA OAA OAT OCT TTG CAA TTG Lys Tyr Phe Leu Giu Asp Ala Leu Gin Leu TCA AAT AAA ATC CAG GIC ATT CAG AAG ACT Ser Asn Lys Ile Gin Val Ile Gin Lys Ihr 100 105 CTC AAC AAT CTG GAG ATO TTG GTT TTA CAT Len Asn Asri Leu Giu Met Len Val Len His 120 125 AAG TOT OAT GCT GTG TGS TTT GTC TGG TGG Asn Cys Asp Ala Val Trp Phe Val Trp Trp 135 14C ACT ATT GCA TAC CTG GCC ACT OAT GTG ACT Thr Ile Pro Tyr Len Ala Thr Asp Vai Thr 150 155 CCA GGA GCA CAC AAA GOT CAA ACT GTC ATA ICC CTT GAT CTG TAT ACG Pro Gly Ald His Lys Gly Gin Ser Val lie 165 170 Ser Leu Asp Leu Tyr Thr 175 WO 011901-1- PCT/US01/16766

TGT

Cys

TCA

Ser

TGG

Trp

TAT

Tyr 225

GTG

Val

CTG

Leu

OTA

Leu

TCC

Ser

AAA

Lys 305

CAG

Gin

GAA

Glu

TGC

Cys

TAC

Tyr

GCT

Ala TTA GAT CTC ACA AAC CTG ATT CTG TTC TCA OTT Leu Asp Lau Thr Asn Lau Ile Lau Phe Ser Val 180 1R5 ACA ACA AGT CAC Thr Thr Ser His 205 TGG AAA GCA AAG Trp Lys Ala Lys 220 CCT TGT TAT GAT Pro Cys Tyr Asp 235 ACA GAA TGG GTT Thr Glu Trp Val 250 GAA AAA CAC TTC Glu Lys His Phe CAG OCA OTT CTA Gin Pro Vol Lou 285 ACA OTG TTT GTG Thr Val Phe Val 300 ATG OCA TTT TAT Met Ala Phe Tyr 315 OTG ATT ATC G Val Ile Ile Lau 330 OTT CAG CTC AGO Leu Gin Leu Arg GCA AAT CCA CAC Ala Asn Pro Gin 365 OTG ACC AGA GAO Leu Thr Th: Asp TCC ATA Ser Ile 190 CTC TTT Leu Phe ATA AAG Ile Lys GCT TTT Ala Phe TTG CAG Leu Gin 255 AAT TTO Asn Lau 270 GAA AAC Glu Asn ATO ACA Met Thr TTO TCT Leu Ser ATA TTO lie Phe 335 AAO AGA Lys Arg 350 GCT CAC Ala His AAT OAT 576 624 672 720 769 816 864 912 960 1008 1104 1152 1202 Asn His Val TAT AGT CAA ATG TTG AAG GAA ACA GTC TAGCTGTCTO AAGAATGTCA Tyr Ser Gin Met Phe Lys*Olu Thr Val wo 0119011-1 WO 0190151PCT/t1S01/16766

CCACCTP.GGA

GAATTTITCC

GAAATTAAGA

AAATATTTCC

TTTATCTGCA

AGTAGTAGGG

TCAATGAAAT

is

ACCATGCAAT

CTC TTAT CAT

CATGCCTTGG

TAACAGTTGT

ACGGGAGACT

CCTAGCTCAA

TACA-ATT OAT

TAAAAATTAC

PLAAAGCCCAG

ATGCCACAAA

TTTCTTGGGG

TACCTGAAOT

CATGGCTCAG

CATAGAAGAT

ATCTOAAAAA

AAGAGCCACA

ACAAGCTTTC

AGAACTTCTC

ACCGCTACTG

CCCATGGAGG

TTTCATAAOG GTTTCCATAA ATTOOTOGGA AATCATCAAT AATTTCTTTC 'TTCATOTGCC CTGTOCCTAG OAGACAACAC CATCTCCCCT OAAOAAOTAC TCTCTCTCTG ATACTGAACT AO'rAAATOOT TTCATTATCA GTACAGGACA GCTOOTAGCT GOTTCTCTOG OAAAAOOGA

ATOAAOGTCT

ATATGGCTAA

ATOCTCAGTT

AAGGCTtTGA

TAGTACTTTT

GTACCAGACT

TOTAGTATCC

OCT TCAAGGC

AGGTTTTTTT

1262 1322 1382 1442 1502 L562 L622 1682 1742 TOGCCATCCA TGAA 1756

SPEIPWWTSLPPEVFEGMPPNLKNLSLAKNGLKSFFWDLQLL{HLEILDLSHNQLTKVPERLANCSKSLTTL:LK

HNQIRQLTKYFLEDALQLRYLDTS SNIQVIQKTSF PENVLNNLEMLVLHHNRFLCNCDAVWFVWWVNHTDVTIP YLATDVTCVGPGAHKGQSVISLDLYTCELjDLTNLILFSVS IS SVLFLWTVMTTSHLFFWDMYTYYWCAKIKGY

PASAT?WSPCYDAFIVYDTKNSAVTEWVLQELVAKLEDPREKFNLCLEERDWLPGQPVLENJSQSIQLSKKTVF

VITQKYAKPES FKMAFYLSHQRLLDEKVDVI ILTWFLERPLQKSBCFLQLRKRLCRS SVLEWPANPQAHPYFWQCLK

NALTTDNHVAYSQMFKETV

wo 011901-1-1 WO 0190151PCT/US01I16766 Table 7: Nucieotide and amino acid sequences of a mammalian, e.g., primate, human, DNAX Toll like Receptor 7 (DTLR7) upstream (SEQ ID NO: 15 and 16): G ?.AT TCC AGA CTT AT?. RAC TTG AAA? A?.T CTC TAT TTG GCC TGG AAC Asn Ser Arg Leu Ile Asn Leu Lys Asn Leu Tyr Len Ala Trp Asn 1 5 10 TGC TAT TTT ?.AC AAA? GTT TGC GAG AAA? ACT AAC AT?. GAA OAT GGA GTA Cys

TT

Phe

COTT

Leu

CTG

Leu

TG

Leu

TIC

Phe

AAT

Asn

AAC

Asn

AAT

Asn

GGA

Gly 160 Tyr

GA?.

TCA

Se r

AGO

Sec

AT?.

Ile

AAT

As n AzTA Ile

CTC

Leu

ATG

Met 145

GA.

Giu Phe

ACG

Thr

CAT

His so

?.AC

As n

AT

Asn

GCC

Al a

GAT

Asp

TCT

Ser 130

CT

Pro

AT?.

Ile As n

GG

Leu

GTG

ValI

ACO

Ihr

TTA

Leu

CC?.

Pro

CGT

Arg 115

AGC

S er

CAT

His

GCC

Ala Lys

ACA.

Thr

CC?.

Pro

CAG

Gin

ACA.

Thr

TTT

Pha 100

TTT

Phte

ACT

Thr

CIG

Leu

T

Ser ValI

PAT

Asn

CCC

Pro

ATC

Ile

T?.

Le I 85

CC?.

Pro

GCT

Ala

TCC

Ser

AAG

Lys

GG

Gly 165 Cys

TG

Leu

APA

Lys 70

CT?.

Leu

TGC

Cys

TTT

Phe

CTC

Leu

GTG

Val 150

GCA

Ala Gin

GAG

Gin

CTG

Leu 55

TAC

Tyr

GAT

Asp

GIG

Val

CAA.

Gin

AGO

Arg 135

CTG

Leu

III

Phe Lys

TIG

Leu 40

CC?.

Pro

AT

I le

II?.

Leu

CCI

Pro As n 120

AAG

Lys

GAT

Asp

T?.

Leu Thr 25

CIA

Leu

A.GC

Se r

AGT

Ser

AGC

Sec T GI C ys 105

TO

Leu

ATI

Ile

CTI

Leu

ACG

Thr Asn

IC?.

Sec

ICC

Sec

GA.

Olu

GG

Gly 90

GAT

Asp

ACC

Thr

AAI

Asn

GAR.

Giu

AIG

Met 170 Ile

CIA

Leu

CIA

Leu

GA?.

01 u

AAC

As n

GG

Gly

CAA?

Gin

GCT

Ala

TIC

Phe 155

CTG

Leu Giu

ICT

Ser

CGC

Arg

GAT

Asp

IT

Cys

GGT

Gly

OTT

Leu

GCC

Ala 140

AJFC

Asn

CCC

pr o Asp

TC

Phe

AAA?

Lys

TC

Phe

CCG

Pro

CI

Al a

CGA

Arg 125

TG

Trp

TAT

Tyr

CC

Arg Gly

AAT

?.sn

CII

Leu

AAG

Lys

AGG

Arg

IC?.

Se r 110

TAC

Tyr

III

Phe

T?.

Leu

TI?.

Le u

T

Sec Phe

GO?.

Gly 101- C ys ATl Ile

CIA

Leu

AAA

Lys

GIG

Val1

GA.

Glu 175 AT?. OTT GAC 110 TCT III AAC TAT AT?. A?.G GGG AGT TAT CC?. CAG CAT Ile Leu Asp Leu Sec Phe Asn Tlyr Ile Lys Gly Ser Tyr Pro Gin His wo 011901-1-1 WO 0190151PCTIUS01I16766 AlT AAT ATT TOG AGA AAG TWO TGT ARA CIT TTG TOT Ile As Ile Set Arg Asn The Set Lys Len Leu Sec CTA CCC SCA TIC Len Arg Ala Len 205 CAT TTA AGA His Len Arg 210 CCC CIG AIC Pro Len Met 225 GGT TAT GIG ITOC Cly Tyr Val Pile SPA CTC AGA CAA Gin Len Arg Gin CAT TTC GAG Asp Pile Gin GAG OTT OCA Gin Len Pro TIA TOG ACT Len Set lilt ATC AAC Ile Asn 235 CPA AAI Gin Asn 250 TIC GGT AlT AAI Len Gly Ile Pen

TIT

Pile 240 MTT APO CAA AIC CAT TO AAA OTT TIC Ile Lye Gin Ile Asp Pile Lys Len Pile 245 TO TOG AAT Pile Set Asn CAA ATT All TAG Gin Ile Ile Tyr TCA CAA AAC ACA Set Gin Asn Arg TOPA COG TTG CIA Set Pto Len Val AAA CAT Lye Asp 27)0 ACC CCC GAG Tilt Aty Gin TAT GGA AAI ACT Tyt Ala Asn Set TOT TTI CPA CT Set Pile Gin Atg CAT AIG CGG His Ile Atg 285 AAC ITT TAT Aso Pile Tyr AAA CA CC Lys Arg Arg 290 TOP AGA CAT ITT GAG III GAG OGA OAT Set Tilt Asp Pile Gin Pile Asp Pro His 295 9112 OAT TIO His Pile 305 AGO CT OCT TIP ATA AAGCR CAA ICT GOT GOT TAT CCA AAA lilt Arg Pto Leu ie Lys Pro Gin Cys Ala Aia Tyr Cly Lys CCC PTA CAT PTA AGO GIG AAG ACT ATI TIC TT set Ile Pile Ala Len 320 Asp Len Set Leu Asn 325 NSRLINLKNLYLAWNCYFNKVE TMIEDCVFETLINLELLSLSFMSLSHVPP

KLF:SSLRKLFLSNTQIKYISE

EDEKCLINLILLDLSGNGPROFNAPFPCVPCDGCASTNI DRFAFQNLTOLRYLNLS STSLRKTNAAM0FKMMPHL KVLDLEFNYLVCEIASGAFLIMLPRLE ILDLS FNYIKGSYPQFIINISRNFSKLLSLRALHLRCGYVFOELREDDF

OPLMOLPNLSIINLCTNFTKOTDFKLFQMFSNLEIIYLSENRTSPLVKDIRQSYANSSSFQRHIRKPRSTDFEF

DPHSNFYHFIRPLIKPCCAAYCKALDLSLNS

IF

downsttets (SEQ ID NO: 17 and 18) CAC TOT OTT ICC ACA TOG GAAL ACT TO TAT CAT GOT TAC AlT TOT TAT 48 Gin Set Leu Set Tilt Set Gin lilt Pile Tyr Asp Ala yt Ile Sec Tyt 1 5 1C GAG AGO AA CAT CO TOT CIT ACT GAO TCC GIG ATA AAT GAG GIG CCC 96 Asp Tilt Lys Asp Ala Set Val Tilt Asp Tsrp Vl Ile Asn Gin Len Arg 25 wo 011901-1-1 WO 0190151PCT/US01I16766 TAC CAC CT)? Tyr His Lau GAA GAG AGC CGA GAC AAA AAC GTT CTC OTT TCT OTA GAG Giu Giu Ser Arg Asp Lys Asn Val Leu Leu Cys Laeu Glu GAG AGG Giu Arg GAT TGG GAO CCG OGA TTG GCC P.TC Asp Trp Asp Pro Sly Leu Ala Ile

ATC.'GAO

Ile Asp AAC OTO ATG CAG Asn Leu Met Gin

AGO

Ser

GCA

Ala ATO AAC CAA AGC AAG AAA ACA GTA TTT Ile Aen Gin Ser Lys Lys Thr Val Phe OTT TTA ACC AAA AAA Val Lau Thr Lys Lys TAO TTG GO TTG CAG Tyr Lou Gly Leu Gin AAA AGC TGG Lys Ser Trp TTT AAA ACA GOT Phe Lys Thr Aia CTA ATG GOT Leu Met Gly OTG TTA CAG Vai Leu Gin 115 AAC ATG OAT GTG Asn Met Asp Val ATA TTT ATO OTG Ile Ph Ile Leu CAT TOT COG TAT His Ser Pro Tyr AGG OTA CG Arg Leu Arg AAC COG AAG Asn Pro Lys CAG CG Gin Arg OCA GAP.

Aia Giu 140 OTO GAG OCA Leu Oiu Pro 110 ATO TGT AAG Ile Cys Lys AGO TTS TTT Arg Leu Phe AGO TOO Ser Ser 130 TOG CAA Trp Gin 145 AAC AAT Aen Asn

ATO

Ile OTO CAG TG Leu Gin Trp ACT ZTG AGA Thr Leu Arg OTO TTG ACT Val Leu Thr AAT OAT TOA CGS Asn Asp Ser Arg ATO TAT Met Tyr TOO ATT AAG Ser Ile Lye CAA TAO Gin Tyr 170 TAACTOACST TAAGTCATGA

TTTCOCGCOA

GGT AAC CAAA

CCCACAGTTT

TOT OTCCAGA

TCOATGTOGT

TGTAAGCCAT

AGAGGTTGCT

CATOAOTTTG

GAGTGAOOAO

TCAGTTGGTC

TAATAAAGAT

TTACTCCCAA

TTGAGGGTOA

GGCTGCAATG

TGTTTTOTOG

GOGAGOCTAT

AGCAAGATGA

GOCATATTOT

CTOAGTCCAG

ATCA-ACTATT

GCAAAGGAAT

AAACCTTACG

TAOGTOTTOA

ATTCAATTCC

CCCACAACGG

AGTOACAATO

ATTTGTTAGA

GGAAAAOAGC

TTCCCTTGAC

GAOATTTOOO

TCGGTTTCAA

CCCAGCATAA

CCAGAGACAT

TOOTGGOTA

OAGCTTGCTT

TTTTGTAATC

AGTAAAOOAO

TGAAGACCAA

TGOTGTCCTG

TATTAGI TAT

AACAACCACA

OTGOTOTTO

AGGCATOACT

TTGGCCAAAG

CATOAGAGCT

GAATCAAAAA

AGTCCACO

GATOOTGAGC

GOAT 000000

CTATTGCTAC

TTCTGO

TGCTTCAGGO

GGGGTCACAC

OOTATAOTCA

AGCOkkAAAAG

AGTSATATCT

AGOTCCATOG

TCTGATTGCT

CTATCTI OAT 593 653 713 773 e33 893 953 1013 1073 1133 wo 0119011-1 WO 0190151PCT/LISOI/16766 GGATAGATTO TCAATATCAG GAGGCCAGGG P TCACTGTGG ACCATCTTAG CACT'DGACCT 1193 AACACATCTT CTTTTCAATA TC'PAAGAACT TTTGCCACTG TGACTAATGG TCCTAATATT .1253 AAGCTGTTGT TTATATTTAT CAPATATCTA TGGCTACATG GTTATATTAT GCTGI'GGTTG 1313 CGTTCGGTTT TATTTACAGT TGCTTTTACA AATATTTGCT GTAACATTTG ACTTCTAAGG 1373 TTT-ArAT-GCC ATTTAAGAAC TGAGATGGAT AGCTTTTAAA GCATCTTTTA CTTCTTACCA 1433 TTTTTTAAAA GTA"GCAGCT AAATTCGAAG CTTTTGC-TCT ATATTGTTAA. TTC-CCATTGC 1493 TGTAAATCTT AzAATGAATG AATAAAAATG TTTCATTTTA AAAAAAAAAA AAAAAAAAAA 1553 AAAA 1557 QSLSTSQTFYDAYISYDTXDASVTDWVINELRYHLEESRDKNVLLCLEERDWDPGLAII DNLMQSINQSKKTVFV

LTKEYAESWNFKTAFYLGLRLMGNMDVIIFILLEPVLQHSPYLRLRQRICKSSILQWPDNPKAERLFWQTLRN

2 0 VVLTENDSRYNNMYVDSIhQY Further 2S atg ntg Met Leu primate, e.g, human, DTLR7 seque ann tgc att ttc ctg nta ata tnt Cys Ile Phe Lau aat ttt tct aa Asn Phe Ser Arg Lau Ile Ser agc tat cnt Ser Tvr Pro nce (SEQ ID ggt tcn tgt Sly Sex Cys tgt gat gag Cys Asp Giu 36 and 37).

tta tgc 48 Leu Cys gcc Ala -1 eat As n 10 agc aat Cgt Ser Asai Arg aaa aag caa 96 Lys Lys Gin nag gaa gtt 144 Gin Giu Val tna gtt att Ser Val Ile ang gtg ggc Thr Val Gly gca gag tgc Ala Glu Cys Cga nta Arg Leu cnn caa Pro Gin aaa tat gtg ana Lys Tyr Vai Thr cta gan ntg Leu Asp Leu ttc atn aca Phe Ile Thr ata ang aat Ile Thr Asn gaa tca. ttt caa ggg Giu Sex Phe Gin Gly an cn aat gte nag Asn Pro Asn Val Gln tnt get eat Ser Asp Asn naa eat ctc Gin Asn Leu nag aan gga Gin Asn Gly ant aaa Thr Lys eat nnn Asn P)r o ctc eec Leu Asn ate Ile aat cta. eec Asn Lau Asn 192 240 288 336 384 ggt eta nee tca eat ggn ttg eat Gly Ile Gin Sex Asn Gly Leu Asn ace gen ggg gca Thr Asp Gly Ala nta sea eec Lau Lys Asn 100 nta egg gag tta Leu Arg Glu Leu pea gan sen Glu Asp Asn wo 01/90151 ccc caa ata ccc tct ggt ttg rca Pro Gin Ile Pro Ser Gly Len Pro 115 gag tct ttg aca gee Giu Sec Leu Thr Giu 120 act aaa gag ,ggc att Thr Lys Giu' Giy Ile 140 PCTIUSO1/16766 ott egt cta 4 Leu Sec Leu 125 caa Ile Gin Asn 130 eat ate tao aac Asn Ile Tyr Asn tca aga ott Sec Arg Lev eta aec Ile Asn 145 ttg aaa aatoctc Leu Lys Asn Leu ttg gcc tgg aec tgo tat ttt aac aaa Leu Ala Trp Asn Cys Tyr ?he Aen Lys 155 gtt Val 160 tgr gag aaa act Cys Gin Lys Thr ata gaa get gga Ile Gin Asp Gly ttt gee aeg otg Phe Giu Thr Leu eat ttg gag ttg Asn Leu Gin Len toe cta tot ttc Sec Len Ser The tot ott tca cat Ser Leu Ser His gtg cca Vel Pro 190 ccc aaa ctg Pro Lys Len ago tcc ota cgc Sar Ser Leu Arg ott ttt ctg agc Leu Phe Leu Sec aac acc rag Asn Thr Gin 205 aet tta aca Aen Len Thr etc aaa tac Ile Lys Tyr 210 tte ota get Leu Leu Asp 225 ett egt gee gee Ile Sec Ciii Cin tto aag gga ttg Phe Lys Giy Leu tte ego ggg Leu Ser Gly tgt cog egg tgc Cys Pro Arg Cys eec goc ore ttt Asn Ala Pro Phe rca Pro 240 tgo gtg rot tgt Cys Vai Pro Cys ggt ggt got toa Giy Giy Ala Sec aat ate get ogt Asn Ile Asp Arg got ttt rae ar Ala Phe Gin Asn ecc cee ott cae Thr Gin Len Arg ote eec oto tct Leu Asn Leu Ser ego act Ser Thr 270 too oto agg Sec Leu Arg att eat got gor Ile Asn Ala Ala ttt aee eat etg Phe Lys Asn Met rot cat otg Pro His Len 285 aag gtg otg Lys Val Lev 290 gat ott gee ttc Asp Len Gin Phe eec tat tta gtg gga gsa ate gco tot Asn Tyr Len Vai Gly Glu Ile Ala Sec 295 300 ggg gre Gly Ala 305 ttt tta erg etg Phe Leu Thr Met coo ogo. tta gee Pro Arg Len Gin ott gao ttg tot Len Asp Len Sec 1008 1056 ttt Phe 320 eec tat ate aeg Asn Tyr Ile Lye egt tat rca rag Sec Tyr Pro Gin att eat ett too Ile Asn Ile Sec wo 01/90151 WO 0190151PCTIUSO1/16766 aac ttc tct aaa.

Asn Phe Ser Lys ett ttg tet Lou. Lou Ser 340 cta egg gca ttg cat tta aga Lou Arg Ala Lou H-is Lou Arg 345 ggt tat Gly Tyr 350 gtg ttc cag Vai Phe Gin eca aac tta Pro Asn Lou 370 gat ttc as Asp Phe Lys 385 ctt aga gaa get Lou Arg Glu. Asp ttc cag ccc ctg Phe Gin' Pro Lau atg cag ett Met Gin Lau 365 sag caa atc Lys Gin Ile tcg act. atc aac Sor Thr Ile Asn ggt att aat ttt Gly Ile Asn Phe ctt ttc caa Lou Phe Gin ttc tcc aat ctg Pho Ser Asn Lou att att tao ttg Ile Ile Tyr Lou gsa aac aga ata tea ceg ttg gta. aaa C-lu Asn Arg Ile Ser Pro Lou Val Lys gat ace cgg cag Asp Thr Arg Gin 410 egg aaa ega egc Arg Lys Arg Arg agt tat See Tyr 415 tea aca Ser Thr 430 gee set agt. tcc Ala Asn Ser Ser ttt caa. cgt cat Phe Gin Arg His gat ttt gag Asp Phe Giu gac cca cat teg Asp Pro His Ser ttt tat cat tte Phe Tyr His Phe ace cgt cet Thr Arg Pro 445 gat tta ago Asp Lou Ser tta ats aag eca caa tgt get get tat gga aaa gee Lou Ile Lys Pro Gin Cys Aia Ala Tyr Gly Lys Ala 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 ctc aac Lau Asn 465 agt att ttc tte Ser Ile Phe Phe ggg eca aac cas Gly Pro Asn Gin gaa aat ctt cet Glu Asn Lou Pro att gee tgt tta Ile Ala Cys Lou ctg tot gee aat Lou Ser lia Asn eat got cee gtg Aen Ala Gin Val agt gga act gaa Ser Giv Thr Giu aca sac aat aga.

Th-r Asn Asn Arg 515 tea gee att oct Ser Ala Ile Pro gte aaa tat ttg Val Lys Tyr Leu get ttg Asp Lou 510 eta gao ttt gat sat get agt get ott Leu Asp Phe Asp Asn Ala Ser Ala Lell 520 act gas ttg Thr Glu Leu 525 tat tte aga Tyr The Arg tee gac ttg Ser Asp Leu 530 gae gtt eta gat Glu. VaI Lou Asp age tat aat tea Ser Tyr Asn Ser ate gee Ile Ala 545 gge gte ace. cat Gly Val Thr His eta gaa ttt att cae eat ttc sea aat Leu Giu. Phe Ile Gin Asn Phe The Asn 555 eta ass gtt tta. eac ttg age eac aec eec ett tat act tte ace get Leu Lys Val Lou Asn Lou Ser His Asn Asn Ile Tyr Thr Leu Thr Asp wo 01/90151 PCTIUSO1/16766 50565 570 575 aag tat aac ctg gaa agc aag tcc ctg gta gaa tta gtt ttc agt ggc 1824 Lys Tyr Asn Leu Glu Ser Lys Ser Leu Val Glu Leu Val Phe Ser Gly 580 585 590 aat cgc ctt gac att ttg tgg aat gat gat gac aac agg tat atc tcc 1872 Asn Arg Leu Asp Ile Leu Trp Asn Asp Asp Asp Asri Arg Tyr Ile Ser 595 600 605 wo 01/90151 WO 0190151PCTIUSO1/16766 ett ttc aa Ile Phe Lys 610 ggt etc aag aat Gly Leu Lys Asn aca c gt ctg gat Thr Arg Leu Asp toc ott eat Ser Leu Asn agg ctc Arg Leu 625 aag cac atec ca Lys His Ile Pro gaa gca tto Glu Ala Phe ott -aat Leu' Asn .635 ttg cca gog agt Leu Pro Ala Ser etc Leu 640 act gaa cta cat.

Thr Giu Leu His eat gat aat atg Asn Asp Asn Met aag ttt ttt aac Lys Phe Phe Asn aca tta etc cag Thr Leu Leu Gin ttt cot cgt ctc Phe Pro Arg Leu ttg ott gao tta Leu Leu Asp Leu cgt gga Arg Gly 670 eac aaa ota Asn Lye Leu ott cgg ace Leu Arg Thr 690 ttt tta act gat Phe Leu Thr Asp cta tot gao ttt Leu Ser Asp Phe ace tct too Thr 3cr Ser 685 cta ccc tot Leu Pro Ser ctg ctg ctg agt Leu Leu Leu Ser aec agg att tc Asn Arg Ile Ser ggc ttt Giy Phe 705 ott tot gaa gtc Leu 3cr Giu Val agt otg aag cac Ser Leu Lys His get tta agt too Asp Leu Ser Ser 1920 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2544 a at Asn 720 otg ota aaa aoa Leu Leu Lys Thr eac aaa too gee Asn Lys 3cr Ala gea act eag ae Gin Thr Lys Thr aco aaa tta tot Thr Lys Len Ser ttg gaa ote oao Leu Glu Leu His aac ccc ttt gaa Asn Pro Phe Gin tgo ace Cys Thr 750 tgt gao att Cys Asp Ile aaa att ccc Lye Ile Pro 770 gat tto oga age Asp Phe Arg Arg atg gat gaa oat Nat Asp Gin His otg eat gto Leu Asn Val 765 ggg gat cae Gly Asp Gin aqa ctq gte gat Arg Leu VJai Asp att tgt goe agt Ile Cys Ala 8cr aga ggg Arg Gly 785 aag aqt att gtg Lys Ser ile VJal ctg gag cta aca Leu Giu Leu Thr act tgt gtt toe get Thr Cys Val Ser Asp 795 ttt atc eoo aco atg Phe Ile Thr Thr Met gto act goa gtg ata Val Thr Ala Val Ile 800 ttt ttc ttc acg Phe Phe Phe Thr gtt atg ttg got Val Met Leu Ala ctg got cac cat Leu Ale His His ttt tao tgg get Phe Tyr Trp Asp gtt tgg Val Trp 830 wo 01/90151 WO 0190151PCTIUSOI/16766 ttt ata tat Phe Ile Tyr gtg tgt tta get Val Cys Leu Ala tta aaa gge tac agg tet ctt Leu Lys Gly Tyr Arg Ser Leu 845 tcc aca tcc Ser Thr Ser 850 get gcc tct Asp Ala Ser 865 caa act ttc tat Gin Thr Phe Tyr gct tae att ,tct Ala Tyr Iie'Ser gac ace aaa Asp Thr Lys gtt act. gac Val Thr Asp gtg ata eat gag Val Ile Ase Glu cgc tac eec ett Arg Tyr His Leu gaa Giu 880 gag agc ega gac Giu Ser Arg Asp nac gtt etc ctt Asn Val Leu Leu cta gag gag agg Leu Giu Glu Arg tgg gac ccg gga Trp Asp Pro Gly gee ate ate gee Ale Ile Ile Asp etc atg eag age Leu Met Gin Ser atc eac Ile Asn 910 caa agc aeg Gin Ser Lys aca gta ttt gtt Thr Vai Pbie Val eec aaa aea tat Thr Lys Lys Tyr gca aaa age Ala Lys Ser 925 eta atg ggt Leu Met Glv 2592 264C 2688 2736 2784 2832 2880 2928 2976 3024 3072 3099 tgg aae ttt Trp Asn Phe 930 gag eec etg Giu Asn Met 945 aaa aca get ttt Lys Thr Ala Phe ttg gee ttg cag Leu Ala Lau Gin get gtg att Asp Val Ie ttt ate ctg ctg Phe Ile Leu Leu eca gtg tta eag Pro Val Leu Gin cat His 960 tot ccg tat ttg Sec Pro Tyr Leu eta cgg cag egg Leu Arg Gin Arg atc tgt aag agc tcc atc Ile Cys Lys Ser Ser ile 970 975 etc eag tgg cet Leu Gin Trp Pro aac ccg aag gea Asn Pro Lys Ala ggc ttg ttt tgg Gly Leu Phe Trp caa act Gin Thr 990 etg aga aat Leu Arg Asn gte ttg act Val Leu Thr gaa aet gat Giu Asn Asp 1000 tca cgg tat Ser Arg Tyr eac aet etg Asn Asn Met 1005 tat gte gat tcc Tyr Vai Asp Ser 1010 att aag caa tac tae Ile Lys Gin Tyr 1015 WO 01/90151 PCT/USOI/16766 TNESFQGLQNLTKINLNHNNQHQNGNPGIQSNGLNITDGAFLNLKNLRELLLEDNQLFQI

PSGLPES

LTELSLIQNNIYNITKEGISRLINLKNLYLAWNCYFNKVCEKTNIEDGVFETLTNLELLSLS

FNSLSHV

PPKLPSSLRKLFLSNTQIKYISEEDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGAS

INIDRFAFQN

LTQLRYLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEILDLSFNYIKGSYP

QHINI SRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLGIMFIKQIDFKLFQNFSNLEII YLS ENRI SPLVKDTRQ SYANS SSFQRHIRKRRSTDFEFDPHSNFYHFTRPLIKFQCAAYGKALDLSLNS 1FF IGPNQFENLFDIACLNLSANSNAQVLSCTEFSAI

PHVKYLDLTNNRLDFDNASALTELSDLEVLDL

SYNSHYFRIAGVTHHLEFIQNFTNLKVLNLSHNNIYTLTDKYNLESKSLVELVFSGNRLDILWNDDDNR

YIS IFKGLKNLTRLDLSLNRLKHIPNEAFLNLPASLTELHINDNNLKFFNWTLLQQFPRLELLDLRGNK LLFLTDSLSDFTS SLRTLLLSHNRI SHI2SGFLSEVSSLKHLDLS

SNLLKTINKSALETKTTTKLSMLE

LHGNPFECTCDIGDFRRWMDEHLNVKI PRLVDVICASPGDQRGKS IVSLELTTCVSDVTAVI LFFFTFF ITTMVMLAALAHHLFYWDVWF

IYNVCLAKLKGYRSLSTSQTFYDAYISYDTKDASVTDWVINELRYNLE

ESRDKNVLLCLEERDWDPGLAI

IDNLMQSINQSKKTVFVLTKIYAKSWNFKTAFYLALQRLMGENMDVI

IFILLEPVLQHSFYLRLRQRICKSS

ILQWPDNPKAEGLFWQTLRINVVLTENDSRYNNMYIDSIKQY

WO 01/90151 PCTIUSOI/16766 Table 8: Partial nucleotidie and amino acid sequences (see SEQ ID NO: 19 and 20) of a mammalian, primate, human, DNAX Toll like Receptor 8 (DTLR8).

AAT GAA TTG ATC CCC AAT CTA GAG AAG GAA Asn 1

TGC

Cys

ATT

Ile

CCC

Pro

CAC

His

CTG

Leu

GAA

Glu

CGT

Arg

AAT

As n

TTA

Gin

CTT

Leu

GTA

Val1

AAC

As n

CAC

His

GAA

Gin s-CT Al a

AAA

Lys

GTA

Val 130

AAT

Leu

TAT

T yr

AGC

Ser 35

TT

Phe

AAT

Asn

CCC

Pro

CTC

Leu

TGT

Cys 115

TTA

Leu

GAA

Ile

GAA

Glu

TTC

Phe

GTC

Val

CTC

Leu

ATT

Ile

CTG

Len 100

GGG

Gly

CC

Ala

GAG

P ro 5

AGC

Ser

ATT

Ile

CAG

Gin

TTC

Phe

CCA

Pro

GAA

Gin

CTT

Leu

ACC

1 hr

TCT

Asn

TAO

Tyr

GAG

Gin

AAT

Asn

CAT

His 70 TT C Phe

AAA

Lys

TTC

EPhe

AGA

Arg

CGA

Leu

TTT

Phe

~AA

Lys

GAG

Gin 55

GAA

Glu

TAT

T yr

AAA

L ys

TG

Tip

GAA

Gin 135

GGT

Gin

GAC

Asp

AGC

Ser 40

TGG

Tip

AAT

As n

C

Cys

GCA

Al a

GCA

Ala 120

ATG

Met

TCI

Lys

CCT

Pro 25

TAT

Tyr

TGC

Cys

TCT

Se r

ATT

Ile

TAC

Tyr 105

AAC

Aso

TAT

Tyr

ACA

Glm 10

C

Gly

AAG

Lye

CAT

His

GAT

Asp

CCC

Pro 90

TT

Len

CTT

Leu

GAA

Giu

ATC

GAT'GGT TCT ATO Asp Gly Ser Ile AAA AGC ATT AGT Lys Ser Ile Ser TCC ATC TTT GTT Ser Ile Phe Val TAT GAA TTC TAC Tyr Gin Phe Tyr CAC ATA ATT CT is Ile Ile Leu 75 ACC AGG TAT CAT Thr Arg Tyr His GAA TGG CCC AAG Glu Trp Pro Lye 110 CGA GCT OCT GTT Arg Ala Ala Val 125 CTG CAG ACA TTC Len Gin Thr Phe 140 TCT CTG ATG AGI TTG ATT Len Ile GAA APT Gin Asn TTG TCC Len Ser ITT GCC Phe Ala ATC TTA Ile Len AAA CTG Lys Len CAT AGG Asp Arg AAT OTT Asn Val ACA GAG Thr Clu ACA GAG Thr Asp 160

,TTGGGAT

48 96 144 192 240 288 336 384 432 480 53E Len Asn Gin Gin Ser Arg Cly Ser Thr Ile Ser Len Met 145 150 155 TGT CTA TA.AAATCCCA CAGTCCTTGG GAAGTTGGGG ACCACATACA Cys Len Arg

CTC

GTACATTGAT ACAACCTTTA TCATGGCAAT TTGACAATAT TTATTAAAAT AAAAAATGGT 59 so TATTCCCTTC AAAAAAAAAA AAAAAAAAAA AAA 62S NEL I?NLEKEDGS ILI CLYESYFDPGXS ISEN IVS FI EKSYKS IFVLS PNFVQNEWCHYE FYFAHHNLFHEN 1 HIILILLE2IPFYCIPTRYHKLEALLEKKAYLEWPKDRRKCGLFWANLRAVVNVLATREMYELQTFTELNI

SRGSTISLMRTDCL

wo 01/90151 WO 0190151PCTIUSOI/16766 additional primate, human sequence (SEQ ID NO: 31 and 32) nucleotides 4 and 23 designated C, may be A, C, G, or T; nucleotide 845 designated C, may be C or T: C TCC GAT GCC AAG ATT COG CAC CAG GCA TA2 TCA GAS GTC ATG ATG Ser Asp Ala Lys Ile Arg His Gin Ala Tyr Ser Glu Val Met Met 1 5 10 GTT GGA TCG TCA GAT. TCA TAO ACC TGT GAA TAC CCT TTA AAC CTA AGO Val Gly Trp Ser Ser Tyr Thr Cys Giu Tyr Pro Leu 25 GGA ACT AGG Gly Thr Arq GCT CTG TTG Ala Leu Leu AAA GAO GTT CAT Lys Asp Val His CAC GAA TTA His Giu Leu TCT TGC AAC ACA Ser Cys Asn Thr ATT GTC ACC ATT Ile Val Thr Ile GTT ATT ATG CTA Val Ile Met Leu CTG GGG TTG Leu Gly Leu GCT GTG Ala Val GCC TTC TGC TOT Ala ?he Cys Cys CAC TTT GAT CTG His Phe Asp Leu TGG TAT CTO AGG Trp Tyr Leu Arg

ATG

Met so CTA OCT CAA TGC ACA CAA ACA TGG CAC Leu Sly Gin Cys Thr Gin Thr Trp His 85 OTT AGO AAA ACA Val Arg Lys Thr ACC 286 Thr CAA GAA CAA CTO Gin Giu Gin Leu AGA AAT CTC CGA Arg Asn Val Arg CAC OCA TTT ATT His Ala Phe Ile TCA TAC 334 Sec Tyr 110 ACT CAA CAT Ser Giu His GAG AAC GAA Glu Lys Clu 130 TCT CTG TGO OTO Ser Leu Trp Val AAT GAA TTG ATC Asn Giu Leu Ile CCC AAT CTA 382 Pro Asn Leu 125 CAT OCT TCT ATC Asp Cly Ser Ile ATT TGC CTT TAT GAR AGC TAC TTT 430 Ile Cys Leu Tyr Ciu Ser Tyr Phe 140 GAC CCT Asp Pro 145 OGC AAA ACC ATT Cly Lys Ser Ile GAA AAT ATT GTA Ciu Asn Ile Val TTC ATT GAG AAA Phe Ile Ciu Lys

AGO

Sec 160 TAT AAG TOO ATC Tyr Lys Ser Ile GTT TTG TCT CCC Val Leu Ser Pro TTT GTC CAC AAT Phe Val Gin Asn TGG TOO CAT TAT OAA TTC TAO ITT 000 CAC CAC AAT CTC TTC Trp Cys His Tyr Olu Phe Tyr Phe Ala His His Asn Leu Phe CAT GAA 57z His Giu 190 AAT TOT CAT CAC Asn Ser Asp His ATA ATT CTT ATC TTA OTO OAA CCC ATT CCA TTC TAT Ile Ile Leu Ile Leu Leu Glu Pro Ile Pro Phe Tyr 200 205 wo 01/90151 WO 0190151PCTIUSOI/16766 TGC ATT CCC Cys Ile Pro 210 GCA TAC TTG Ala Tyr Leu 225 ACC AGG TAT CAT Thr Arg Tyr His GA.A TOG CCC AAG Glu Trp Pro Lys AAA CTG Lys Leu 215 GA.A GCT CTC Giu Ala Leu GAA AAA AAA Glu Lys Lys GAT AGG CGT Asp Arg Arg

AAA~

Ly's GGG CTT TTC TG Gly Lou Phe Trp GCA AAC OTT OGA GCT Ala Asn Leu Arg Ala 240 ATG TAT GAA CTG CAG Met Tyr Glu Leu Gin GOT OTT AAT GTT Ala Val Asn Val 245 ACA TTC ACA GAG Thr Phe Thr Giu AAT GTA Asn Val 250 TTA GOC ACC AGA GAA Leu Ala Thr Arg Glu 255 TTA AAT GAA GAG TOT OGA GC-T Leu Asn Glu Giu Ser Arg Gly 265 270 TOT ACA ATC Ser Thr Ile OTO ATO AGA ACA Lou Met Arg Thr GAO TGT OTA TAAAATOOCA CAGTOOTTG Asp Cys Leu 280 GAAGTTGGGG ACOACATACA CTGTTGGGAT GTACATTGAT ACAAOCTTTA TGATGOCAAT 927 TTGAOAATAT TTATTAAAAT AAAAAATGGT TATTCOTTO AAA-AAAAAA AAAAAAAAAA 987 AAAAAAAAAA AA 999 SDAKIhHQAYSEVMMVGWSDSYTCEYPLNLRGTRLKDVHLHELSCNTALLIVT

IVVIMLVLGLAVAFCCLHFDI

WYLPMLGQCTQTWHRVRKTTQEQLKRNVRF{AFISYSEHDSLWVKNELT

PNLEKEDGSILIOLYESYFDPGKS:

ENIVSFIEKSYKSIFVLSFNFVQNEWCHYEFYFAHHNLFHENSDHTILILLEPIFFYCIPTRYHKLEALLEKKJ

LEWPKDRRKCGLFWANLRAAVNVNVLATREMYELQTFTELNEESRGST

ISLMRTDOL

Further primate, human, DTLR8 (SEQ ID NO: 38 and 39): gaatcatcca cgcacctgca gctctgctga yagagtgcaa gccgtggggg ttttgagctc atcttcatca ttcatatgag gaaataagtg gtaaaatcct tggaaataca atg aga Met Arg ctc atc aga aac att tac ata ttt Lou Ile Arg Asn Ile Tyr Ile Phe tgt agt att gtt atg aca gca gag Cys Ser Ile Val Met Thr Ala Giu ggt gat gct cca gag ctg cca gaa gaa agg gaa ctg atg.

Oly Asp Ala Pro Giu Lou Pro Giu Giu Arg Giu Leu Met -1 1 5 10 acc aac tgc Thr Asn Cys tcc aac atg tct cta aga aag gtt ccc Ser Asn Met Ser Lou Arg Lys Vai Pro gac ttg acc cca Asp Lou Thr Pro gcc aca Ala Thr acg aca ctg Thr Thr Leu tta tcc tat aac Lou Ser Tyr Asn ctt ttt caa ctc cag agt tca 308 Lou Phe Gin Lou Gin Ser Ser wo 01/90151 WO 0190151PCTIUSOI/16766 gat Asp age Arg aga Arg tta Leu acc Thr cta Leu got Ala cat His 160 att Ile ato Ile cae Gin aag Lys ott Leu 240 tt Phe att Ile tat Tyr ct g Leu at g Met ggt Gly cat His 145 tat T yr ott Val1 eag Lys ttt Phe ace Thr 225 tto Phe cat tct gtc too aaa ctg age gtt ttg at His Ser Val Ser Lys Leu Arq Val Leu Ile caa Gin tta Leu goa Al a ct Pro ttg Leu 130 oty Leu gas Glu tta Leu act Thr gte Vai 210 tog Ser ott Leu cag Gin gat Asp ggt Gi y ato Ile 115 agt Ser cat His ga Giu cca Pro toa S er 195 agt Ser gtt Val etc Ile ot g Leu t to.

L eu oto Leu 100 t gt Cys ggg Gi y ct a Leu ggt Gi y atg Met 180 aaa L ys tat Tyr ct a Leu tta Leu Asp tot Ser 85 egg Arg gag Giu gce Al a eat Asn ago Ser 165 gao Asp ata Ile gaa Glu tt g Leu ca Gin 245 Ct 0 Leu 70 at Asn tat Tyr gee Glu aa Lys act Thr 150 ctg Leu aca Thr t ta Leu atg Met ott Leu 230 tt t Phe aes Lys ac As n tt a Leu got Ala ata Ile 135 gtc Val 000 Pro eat As n gaa Giu cee Gin 215 eat As n gtt Val1 aco Thr age Arg get Asp ggc: Gly 120 cee Gin ttc Phe etc Ile tto Phe atg Met 200 oge Arg ese Lys tgg Trp ttt Phe ot g Leu ott Leu 105 eec Asn aaa Lys tte Leu tta Leu t gg T rp 185 ace T hr eat Asn gtt Val cat His gas ,rttc Glif Phe eag egt Lys Ser 90 tot ttt Ser Phe etg toe Met Ser toe get Ser Asp gge ttc Giy Phe 155 aec ace Asn Thr 170 gtt ctt Val Leu eat eta Asn Ile ott .agt Leu Her get tte Asp Leu 235 eca tca Thr Her 250 cta tgc cat eec Leu Cys His Asn sac sag gag toe Asa Lys Glu Leu gte act tgg tat Val Thr Trp Tyr eat gec ttt gac Asn Asp Phe Asp 110 cac ctg gee etc His Leu Gu Ile 125 ttc cag aae ett Phs Gin Lys Ile 140 ega act ctt oct Arg Thr Leu Pro ace aea otg ceo Thr Lys Leu His 1-75 ttg cgt get gga Leo Arg Asp Gly 190 get ggc aea egc Asp Gly Lys Ser 205 tta gee eat got Leu Glu Asn Ala 220 cto tgg gao gao Leo Trp Asp Asp gtg gsa ceo ttt Val Giu His Phe 255 356 404 452 500 548 596 644 692 740 788 836 884 932 cag etc oge eat gtg act ttt Gin le Arg Asn Val Thr Phe 260 ggt ggt eag got tat ott Gly Giy Ala Tyr Leu gac cac eat Asp His Asn 270 wo 01/90151 WO 0190151PCTIUSOI/16766 tca ttt gac Ser Phe Asp gta cat ttc Val His Phe 290 ttg acc aaa Leu Thr Lys 305 tca aat act gta Ser Asn Thr Val.

aga act ata aaa Arg Thr Ile Lys ttg gag cat Len Gin His 285 tat ttg ctt Tyr Len Leu aga gtg ttt tac Arg Vai ?he Tyr caa cag gat,aaa Gin Gin Asp Lys atg gao ata Met Asp Ile aac ctg aca ata Asn Len Thr Ile aat gca caa atg Asn Aia Gin Met LdL.

cca Pro 320 cac atg ctt ttc His Met Leu Phe aat tat cct acg Asn Tyr Pro Thr aaa ttc caad Lys Phe Gin 330 ttt aaa aga Phe Lys Arg T yr ta Len ttt gcc aat aat Phe Aia Asn Asn tta aca gac gag Leu Thr Asp Giu ctg cct cac Leu Pro His aaa act ctc att Lys Thr Len Ile aat ggc aat aaa Asn Giy Asn Lys act atc caa Thr Ile Gin 350 ctg gag aca Len Giu Thr 365 gaa cac ttg Gin His Leu 1028 1076 1124 1172 1220 12E8 1316 1364 1412 1460 1508 1556 1604 ctt tat tta Leu Ser Leu 370 gat ctg agt Asp Len Ser 385 gta agt tgc ttt Val Sec Cys Phe caa aat cta tta Gin Asn Leu Leu 390 aac aac aca ccc Asn Asn Thr Pro caa cat aaa aat Gin H-is Lys Asn gaa aat tgc tca Gin Asn Cys Ser t gg T rp 400 cca gaa act gtg Pro Gin Thr Val aat atg eat ctg Asn Met Asn Len tac aat aaa ttg Tyr Asn Lys Len gat tct gtc ttc Asp Ser Vai Phe agg tgc ttg ccc Arg Cys Len Pro 420 etc cee act gte Ile Gin Thr Val aaa agt att caa. eta Lys Ser Ile Gin Ile 425 act aaa gag act att Pro Lys Giu Thr Ile aat aat aac Asn Asn Asn ctt gec ca Len Asp Len 430 cat ctg atg His Len Met 445 get ata cct Asp Len Pro gcc tta cga Ala Len Arg 450 gge tgc agt Gly Cys Ser 465 gee cta eat att Gin Len Asn Ile ttt eat ttt ca Phe Asn Phe Len cat ttc egt His Phe Ser ctt tca gtt ctg Len Ser Val Leu att gaa atg eac Ile Gin Met Asn tt C Phe 480 ett atc agc cae Ile Len Ser Pro ctg gat ttt gtt Len Asp Phe Val egc tga cag gaa Ser Cys Gin Gin 1652 wo 01/90151 WO 0190151PCT/USOI/16766 aaa act cta aat gcg gga aga eat cca Lys Thr Leu Asn Ala Gly Arg Asn Pro 500 ttc cgg tgt acc tgt gaa tta Phe Arg Cys Thr Cys Glii Leu 505 510 aaa aat ttc Lys Asn Phe tgg tca gat Trp Ser Asp 530 agg tta aaa Arg Leo Lys 545 cag ctt gaa aca Gin Leu Glu Thr tca gag,.gtc atg Ser Giu Va]. Met atg qtt gga Met Val Gly 525 agg gga act Arg Gly Thr tca tao acc tgt Ser Tyr Thr Cys tac cct tta aac Tyr Pro Leu Asn gac gtt cat Asp Val His cac gaa tta tct His Giu Leu Ser aec ace gct ctg Asn Thr Ala Leu att gtc acc att Ile Val Thr Ile gtt att atg cta Val Ile Met Leu ctg ggg ttg gct Leu Gly Leu Ala gcc ttc tgc tgt Ala Phe Cys Cys cac ttt gat ctg His ?he Asp Leu ccc tgg tat ctc agg atg cta Pro Trp Tyr Leu Arg Met Leu 585 590 ggt caa tgc Gly Gin Cys caa ctc aag Gin Leu Lys 610 cat gat tct His Asp Ser 625 caa aca tgg ceo Gin Thr Trp His gtt agg aaa aca Val Arg Lys Thr acc cee ga Thr Gin Giu 605 tac agt gaa Tyr Ser Glu 1700 1748 1796 1844 1892 1940 1988 2036 2084 2132 2180 2228 2276 2324 aga aet gtc oga Arg Asn Val Arg cac gca ttt att His Ala Phe Ile ctg tgg gtg Leu Trp Val eat gaa ttg etc Asn Glu Leu Ile aat cta gag aeg Asn Leu Giu Lys gee get ggt tct atc ttg att tgc ctt tat gaa Giu Asp Giy Ser Ile Leu Ile Cys Leu Tyr Glu 640 645 650 agc tec ttt gac Ser Tyr Phe Asp ggc eaa agc att Gly Lys Ser Ile gee eat att gta Glu Asn Ile Val ttc att gag aaa Phe Ile Glu Lys agc tat Ser Tyr 670 aeg tcc atc Lys Ser Ile cat tat gaa His Tyr Giu 690 gtt ttg tct ccc Val Leu Ser Pro ttt gtc cag eat Phe VaIl Gin Asn gag tgg tgc Glu Trp Cys 685 gaa aat tct Giu Asn Ser ttc tac ttt gcc Phe Tyr ?he Ala cec aat ctc ttc His Asn Leu Phe gat cat Asp His 705 ate att ctt etc Ile Ile Leu Ile tta ctg ga Leu Leu Glu 710 ccc att. cca Pro Ile Pro 715 tto tat tgc att Phe Tyr Cys Ile wo 01/90151 PCT/USOI/16766 000 aco agg tat cat aaa otg aaa got ctc ctg gaa aaa aaa gca' tac 2372 Pro Thr Arg Tyr His Lys Leu Lys Ala Leu Leu Glu Lys Lys Ala Tyr 720 '725 730 735 ttg gaa tgg ccc a ag gat agg ogt aaa tgt ggg ctt ttc tgg gca aac 2420 Leu Glu Trp Pro Lys Asp Arg Arg Lys Cys Gly'Leu Phe Trp Ala Asn 740 745 750 ctt cga got gct att, aat gtt aat gta tta gcc acc aga gaa atg tat 2468 Leu Arg Ala Ala Ile Asn Val Asn Val Leu Ala Thr Arg Glu Met Tyr 755 760 765 gaa ctg cag aca tto aca gag tta aat gaa gag tot oga ggt tot aca 2516 Glu Leu Gin Thr Phe Thr Glu Leu Asn Glu Glu Ser Arg Gly Ser Thr 770 775 780 ato tot ctg atg aga aoa. gat tgt ota taaaatcoa cagtoottgg 2563 Ile Ser Leu Met Arg Thr Asp Cys Leu 785 790 gaagttgggg acoacataoa otgttgggat gtaoattgat acaaoottta tgatggoaat 2623 ttgaoaatat ttattaaaat aaaaaatggt tattcottc atatoagttt ctagaaggat 2683 ttotaagaat gtatcctata gaaacacott cacaagttta taagggotta tggaaaaagg 2743 tgttcatocc aggattgttt ataatoatga aaaatgtggo caggtgoagt ggctcactct 2803 tgtaatccca gcactatggg aggooaaggt gggtgarcca cgaggtCaag agatggagao 2863 catcctggco aaoatggtga aaccotgtot otaotaaaaa tacaaaaatt agotgggcgt 2923 gatggtgcac gcotgtagtc coagotactt gggaggctga ggoaggagaa tcgcttgaac 2983 oogggaggtg gcagttgoag tgagotgaga tcgagcoact goactccagc ctggtgaoag 3043 ago 3046 MRLIRNIYIFCS

IVTAEGDAPELPEERELMTNCSNMSLRKVPADLTPATTTLDLSYNLLFQLQSS

SVSKLRVLIZLCHNRIQQLDLKTFEFNKELRYLDLSNRLKSVTWYLALYDSNFTPC

GNMSHLEILGLSGAKIQKSDFQKIAHLHLNTVFLGFRTLPHYEEGSPLTKHVPDNWL

RDGIKTSKLEMTNIDGKSQFVSYEMQNLELENTSVLLNKDLLLFIQFVNHTSHFQI

RVTFGGKAYLDHNSFDYSNTVMRTIKLEHVHFRVFYIQQDKIYLTMINTSAPHFN

YPTKFQYLNFANN~ILTDELFKRTIQLPHLKTLILNGNKLETLSLVSCFNTLHDSNLHN

ENC SWPETVNNLSYNKLSDSVFRCLPKSIQILDLNNQIQTPKETIHLMLENANLDP C SHFSRLSVLNIENF

IIZSPSLDFVQSCQEVKTLNAGNPFRCTCELNFIQLETYSEMMGWSDY

KTTQEQLKRNVRFHAFISYSEHDSLWTKNELIPNLEKEDGSILCYEFDGSSNVIKY

KSIFVLSPNFVQNEWCHYEFYFAHHNLFHENSDHIILILLEPIPFYCPTRYHKLKALLEKKAYLEWPK

DRRKCGLFWANLRAAINVNVLATREMYELQTFTELNEESRGSTISLMRTDCL

wo 01/90151 WO 0190151PCT/USOI/16766 Table 9: Partial nucleotide and amino acid sequences (see SEQ ID NO: and 22) of a mammalian, primate, human, DNAX Toll like Receptor (DTLR9).

RAG AAC TCC AAA GAA AAC CTC CAG TTT CAT GCT -TTT ATT TCA TAT AGT Lys Asn Ser Lys Glu Asn Leu Gin 'Phe His Ala Phe Ile Ser Tyr Ser 10

GAA

Glu CAT GAT TCT GCC TGG GTG AAA ACT GAA TTG GTA CCT His Asp Ser Ala Irp Val Lys Ser Giu Leu Val Pro 25 CAT GAG AGA AAC TTT His Glu Arg Aso Phe TAC GTA GA.A Tyr Leu Giu GTC GCT GGC Val Pro Gly AAA GAA GAT Lys Glu Asp AlA CAG ATT TGT Ile Gin Ile Cys AAG AGG Lys Ser ATT GIG GAA AAT AIC RTC AAC TGC ATT Ilie Val Giu Asn Ile Ile Asn Gys Ile RAG AGT TAG RAG 192 Lys Ser Tyr Lys ATG TTT GTT tTG Ile Phe Val Leu CCC AAC ITT GTC Pro Asn Phe Val AGT GAG TGG TGC Ser Giu Trp Gys TAC GAR CTC TAT Tyr Giu Leu Tyr AAC TTA ATC GTC Asn Leu Ile Leu GCG CAT CAC AAT Ala His His Aso TTT CAT GAA GGA Phe His Giu Gly TGT AAT Ser Asn ATC TIA CTG GAR Ile Lau Leu Glu

CCC

Pro 105 ATT CGA CAG AAC Ile Pro Gin Asn AGC AlT CCC 336 Ser Ile Pro 110 AAC RAG TAG CAC AAG CTG RAG Asn Lys Tyr His Lys Leu Lys 115 GAG TGG CCC AAG GAG AAA AGC Gin Trp Pro Lys Glu Lys Ser 130 135 GIG ATG ACG GAG Leu Met Ihr Gin CGG ACT TAT ITG Arg Thr Tyr Leu 125 AAA CGI GGG GTG TTT TGG GCT Lys Arg Gly Leu Phe Trp Ala KNS KENLQFHAFI SYSEHDSAWVKSELVPYLEKEDIQIGLHERNFVPGKS IVEN I INC IEKSYKS I FVLS PNE SEWCHYELYFAHHNLFHEGSNNLILILLEPIPQNS

IPNKYHKLKALMTQRTYLQWPKEKSKRGLFWA

wo 01/90151 WO 0190151PCT/USOI/16766 Further primate, human DTLR9 (SEQ ID No: 40 and 41) aagaatttgg actcatatca agatgctctg aagaagaaoa acctttagg atagccactg oaaoatc atg aco Met Thr aaa gac aaa gaa Lys Asp Lys Giu cct att gtt ,aaa agc tto cat ttt Ile Val"Lys Ser gtt tgc ctt atg atc ata ata gtt gga aco aga atc cag ttc too gac Val Cys Leu Met Ile Ile Ile Val Gly Thr Arg Ile Gin Phe Ser Asp gga aat Gly Asfl -1 1 gaa ttt gca gta gac aag tca aaa Giu Phe Ala Val Asp Lys Ser Lys ggt ott att cat Gly Leu Ile His cca aaa gac cta Pro Lys Asp Leu ccg ctg aaa aco aaa gto tta gat atg tot cag aac Pro Leu Lys Thr Lys Val Leu Asp Met Ser Gin Asn 25 tao atc gct Tyr Ile Ala ott cag gtc tot Leu Gin Val Ser atg agc ttt ota Met Ser Phe Leu tca gag ttg Ser Glu Leu gat tta agt Asp Leu Ser 253 301 349 397 aca gtt ttg Thr Val Leu gtt ttc aag Val Phe Lys aga ott tcCcoat Arg Leu Ser His aga atc cag ota Arg Ile Gin Leu ttc aac cag Phe Asn Gin tta gaa tat ttg Leu Giu Tyr Leu tta tot oat aat Leu Ser His Asn oag Gin ttg oaa aag eta Leu Gin Lys Ile tgo oat oct att Cys His Pro Ile agt tto agg oat Ser Phe Arg His tta Leu 95 gat otO toa ttc Asp Leu Ser Phe gat tto aag goo Asp Phe Lys Ala 000 etc tgt aag Pro Ile Cys Lys gaa ttt Glu Phe 1.10 ggo- aeO tta Gly Asn Leu oae otg aat tto Gin Leu Asn Phe gga ttg. agt got Gly Leu Ser Ala atg aag ctg Met Lys Leu 125 oaa aaa tta gat ttg otg c0a att got oao ttg cat cta egt tat ato Gin Lys Leu Asp Leu Leu Pro Ile Ala His Leu His Leu Ser Tyr Ile 130 135 140 ott otg gat tte age aat tat tat eta aaa gee aat gag aoa gaa egt- Leu Leu Asp Leo Arg Asn Tyr Tyr Ile Lys Giu Asn Giu Thr Giu Ser ota cee att otg aat gca aa Leu Gin Ile Leu Asn Ala Lys 2.60 165 aco ott ceo ott Thr Leu His Leo 170 gtt ttt cac coa act Val Phe His Pro Thr 175 685 wo 01/90151 WO 0190151PCT/USOI/16766 agt tte ttc gct atc caa gtg aac ate tca gtt eat Ser Leu Phe Ala Ile Gin Val Asn Ile Ser Val Asn act tta ggg tgc Thr Leu GlY Cys 190 tte caa ctg Leu Gin Leu att aaa ttt Ile Lys Phe 210 act Thr 195 eat att aee ttg Asn Ile Lys Leu get gao eeac tgt Asp Asp' Asn Cys gtt tic Val Phe tta toe. gee ctc Leu Ser Giu Leu ega ggt cce acc Arg Gly Pro Thr ctg aat. ttt Leu Asn Phe aco ctc Thr Leu 225 eec ceo eta gaa Asn His Ile Giu act tgg eea tgc ctg gto age gtc ttt Thr Trp, Lys Cys Leu Val Arg Vel Phe 235 ttt ott tgg ccc Phe Leu Trp, Pro cot gtg gae tat Pro Vai Giu Tyr aat att tao aat Asn Ile Tyr Asn ace ate att gae Thr Ile Ile Giu ett ogt gaa gaa Ile Arg Giu Giu ttt aot tat tot Phe Thr Tyr Ser aee aog Lys Thr 270 ace ttg aee Thr Leu Lys ttt toa ceg Phe Ser Gin 290 etg tta aco Met Leu Thr 305 ttg ace eta gee Leu Thr Ile Giu etc eog eec cee Tie- Thr Asn Gin gtt ttt otg Val Phe Leu 285 eec ett etg Asn Ile Met ace got ttg tao Thr Ale Leu Tyr gtg ttt. tot gag Val Phe Ser Giu ett toe get Ile Ser Asp cot ttt ate ceo Pro Phe Ile His ctg tgt oct cat Leu Cys Pro His oca ego ace tto Pro Ser Thr Phe ttt ttg aao ttt ecc cag eec gtt tto Phe Leu Asn Phe Thr Gin Asn Val Phe 1021 1069 1117 1165 1213 1261 1309 1357 get agt ett ttt gee eee tgt too aog tta gtt aae ttg gag ace ott Asp Ser Ile Phe Giu Lys Cys Ser Thr Leu Val Lys Leu Glu Thr Leu 340 345 350 etc tte cee Ile Leu Gin eog eag get Thr Lys Asp 370 eat gge tte aee Asn Gly Leu Lys ott tto eaa gta Leu Phe Lys Val otg get gtt ego Leu Asp Val Her ggt oto atg Gly Leu Met 365 tgg eat tot Trp Asn Ser atg cot tot ttg gee eta Met Pro Ser Leu Giu Ile 375 ttg gee Leu Giu 385 tot ggt age oat Her Giy Arg His eea gee eec tgc act tgg gtt gag egt ate Lys Giu Asn Cys Thr Trp Val Giu Her Ile 390 395 wo 01/90151 WO 0190151PCT/USOI/16766 gtg tta aat ttg Val Len Asn Len tca sat atg ctt.

Ser Asn Met Len act gac Thr Asp 410 tot gtt ttc Ser Val Phe tgt tta oct ccc Cys Len Pro Pro aag agc gtt cot Lys Ser Vai Pro atc aag gta ctt Ile Lys Val Leu ctt cac ago aat Len His Ser Asn aaa ata Lys Ile 430 gaa ctc Gin Len aas caa gtc gta Lys Gin Val Val ctg gaa gct ttg Len Glu Ala Leu aat gtt got Asn Val Ala 450 ttc aat tot tta act gac ctt cct gga Phe Asn Ser Leu Thr Asp Len Pro Gly tgt ggc agc ttt Cys Gly Ser Phe 460 grit too cac oca Val Ser His Pro ago agc Ser Ser 465 ctt tot gta ttg Leu Ser Val Leu att gat oso aat Ile Asp His Asn gct gat ttc ttc Ala Asp Phe Phe agc tgc cag aag Ser Cys Gin Lys agg tos ata aaa Arg Ser Ile Lys 1405 1453 1501 1549 1597 1645 1693 1741 178 9 1837 1885 1933 ggg gac aat cca Gly Asp Asn Pro aat ata gac caa Asn Ile Asp Gin caa tgt acc tgt Gin Cys Thr Cys cta aga gaa ttt Leu Arg Giu Phe gtc aaa Val Lys 510 gta tca agt gas gtg tta gag ggc tgg cct gat tot Val Ser Ser Gin Val. Len Giu Gly Trp Pro Asp Ser tat aag tgt Tyr Lys Cys 530 gac tac cca gas agt tat aga gga ago Asp Tyr Pro Glu Ser Tyr Arg Gly Ser cta aag gac Leu Lys Asp ttt caO Phe His 545 atg tct gaa tta Met Ser Gin Len tcc tgo aac ata act otg ctg atc gtc acc Ser Cys Asn Ile Thr Leu Len Ile Val Thr 550 555 ggt goc acc atg otg gtg ttg got gtg act gtg acc tcc ctc tgc Gly Aia Thr Met Leu Val Len Ala Val Thr Val Thr Ser Leu Cys 565 570 575 ato tac ttg gat Ile Tyr Leu Asp cag act cgg cgc Gin Thr Arg Arg ccc tgg tat ctc Pro Trp Tyr Len atg gtg tgo cag Met Vai Cys Gin Top Thr 590 agq goc agg sac Arg Ala Arg Asn ccc tta ga Pro Leu Gin gaa ctc caa aga Gin Len Gin Arg 605 1981 2029 aac ctc cag ttt cat gct ttt Asn Len Gin Phe His Ala ?he att tca tat agt gas cat gat tot gcc Ile Ser Tyr Ser Giu His Asp Ser Ala 615 620 wo 01/90151 WO 0190151PCT/USOI/16766 tgg gtg aaa, agt gaa ttg gta cct tac ota gaa aaa gaa gat ata cag Trp Val Lys Ser Glu Leu Val Pro Tyr Leu Giu Lys Giu Asp Ile Gin att Ile 640 a at Asn tgt ctt cat Cys Leu His ato atc aac Ile Ile Asn gag agg Giu Arg 645 tgco att Cys Ile 660 gtc cag Val Gin aao ttt gto cot Asn Phe Val. Pro ggc.-aag agc att Gly'Lys Ser Ile 650 gtg gaa Val Giu gag aag agt Giu Lys Ser Lys Ser cat tac His

T

yr atc ttt gtt ttg Ile Phe Vai Leu 670 2 077 2125 2173 2221 2269 2317 tot ccc aac Ser pro Asn goc oat cao Ala His H-is 690 tta ctg gaa Leu Leu Giu 705 ttt Phe 675 agt gag Ser Giu tgg T rp 680 gga Gi y Giu aat oto ttt cat Asn Leu Phe His coo att cca cag pro Ile Pro Gin 710 tot aat aac Ser Asn Asn Leu Tyr Phe 685 ato ctc ato Ile Leu Ile tao cac aag Tyr His Lys ago att ccc aac Ser Ile Pro Asn 715 otg aag got oto atg aog cag Leu Lys Ala Lau Met Thr Gin '720 725 cgg act tat ttg cag tgg Rrg Thr Tyr Leu Gin Trp 730 coo aag gag Pro Lys Giu 735 got ttt aat Ala Phe Asn 750 aaa ago aaa. ogt ggg cto ttt tgg got aao att aga gc Lys Ser Lys Arg Giy Leu Phe Trp Ala Asn Ile Arg Ala 740 745 atg aaa tta aca ota gtc act gaa aao aat gat gtg aaa tot Mat Lys Leu Thr Leu Val Thr Giu Asn Asn Asp Vai Lys Sar 755 760 765 taaaaaaatt taggaaattO aaottaagaa acoattattt acttggatga tggtgaatag tacagtcgta agtnaotgtc tggaggtgoc tccattatoo tcatgOcttc aggaaagact taaoaaaaac aatgtttcat ctggggaact gagotaggog gtgaggttag cctgccagtt agagacagoc cagtctotto tggtttaatc attatgtttO aaattgaaac agtototttt.

gagtaaatgc toagtttttc agotoototo cactctgott toooaaatgg attctgttgg tgaag 2365 2413 2455 2515 2575 2635 2695 2755 2760 WO 01/90151 PCT/USOI/16766 MTKDKEF IVKSFHFVCLMII IVGTRIQFSDGNEFAVDKSKRGLIHVPKDLPLKTKVLDMSQNYIAELQV

SDMSFLSELTVLRLSHNRIQLLDLSVFKFNQDLEYLDLSHNQLQKISCHPIVSFRHLDLSFNDFKALPT

CKEFGNLSQLNFLGLSAMKLQKLDLLPIAHLHLSYILLDLNYYIKENETESLQILNAKTLHLVFHPTS

LFAIQVISNTLGCLQLTNIKLNDDNCQVFIKFLSELTRCPTLLNFTLNHIETTWKCLVVFQFLWPK

PVEYLNIYNLTI IES IREEDFTYSKTTLKALT.IEHITNQVFLFSQWALYTVFSEMIMLISDTPFIH MLC PHAPSTFKFLNFTQNVFTDS IFEKC STLVKLETLILQKNGLKDLFKVGLMTKDMPSLETLDVSIS LESCREKENCTWVESIVLNLSSNMLTDSVFRCLPPRIKVLDLHSNKIKSVFKQ1WKLEALQELNVAFN

SLDPCSSLVIDNVHSDFSCKRIADPQTERFKIQSEVL

EGWPDSYKCDYPESYRGSPLKCFHMSELSCNITLLITIGATMLVLAVPVTSLC

IYLDLFWYLRMVCQW

TQTRRREBNIPLiEELQRNLQFHAFISYSEHDSAWVXSELVPYLEKEDIQICLNERFVPGKS

IVENTIN

CIEKSYKSIFVIJSPNFVQSEWCHYELYFABNNLFEEGSNNLILILLEPIPQNSI

PNKYHKLKALMTQRT

YLWKKKGFAIAFMLLTNDK

wo 01/90151 WO 0190151PCT/U SOI/16766 Table 10: Nucleotide and amino acid sequences (see SEQ ID NO: 23 and 24) of a mnammalian, Primate, human, DNAX Toll like Receptor 10 (DTLR1O).

Nucleotides 54, 103, and 345 are designated A; each may be A or G; nucleotide 313 designated G, may be G or T; and nucleotides 316, 380, 407.

and 408 designated C;-each may be A, C, G, pr T.

OCT TCC ACC TGT GCC TOG CCT GGC TTC CCT GGC GGG GGC GGC AAA GTG 48 Ala Ser Thr Cys Ala.Trp Pro Gly Phe Pro Gly Gly Gly Gly Lys Val 1 5 10 GGC GAA ATG AGG ATO CCC TOO COT ACG ATG CCT TCG TOG TCT TCG ACA 96 Gly Gin Met Arg Met Pro Cys Pro Thr Met Pro er Trp Ser Ser Thr 25 AAA CGC AGA GCG CAG TGG CAG ACT GGG TGT ACA ACG AGO TTC G00 GGC 144 Lys Arg Arg Ala Gln Trp Gin Thr C-ly Cys Thr Thr Ser Phe Gly Gly 40 AGC TOO AGO ACT GCC GTG G00 OCT 000 CAC TOO GCC TGT 0CC TGG AGO 192 Ser Trp Arg Ser Ala Val Oly Ala Gly His Ser Ala Cys Ala Trp Arg 55 AAC GCG ACT GGC TGC OTO OCA AAA CCC TCT TTG AGA ACO TGT 000 OCT 240 Asn Ala Thr Gly Cys Leu Ala Lys Pro Ser Leu Arg Thr Cys Gly PrO 65 70 75 so COG TOT ATO- GCA 0CC GCA AGA CGC TC;T VT TOC TOG CCC ACA CGG ACC 288 Arg SEr Met Ala Ala Ala Arg Arg Cy3 Leu Cys Trp Pro Thr A--g ThE 90 000 TCA GTG GTC TCT TGC 000 COA OTT CTC CTG OTG GCC CAG CAG COO 336 Gly Ser Val Val er Cys Ala Pro Val Leu Leu Leu Ala Gin Gin Arg 100 105 110 OTG CTO GAA GAO OGC AAG GAO GTO OTO GTG CTO GTG ATC CTA ACG COT 384 Len Len Gin Asp Arg Lys Asp Val Val Val Lou Val Ile Leu Thr Pro 115 120 125 GAO 000 CAA G00 TCO OGA OTA' CCC OAT G00 OTO. ACC AGC GCC TOT CCC 432 Asp Oly Gln Ala Er Arg Leu Pro Asp Ala :Leu Thr See Ala Ser Ala 130 135 140 000 AGA GTG TOO TOO TOT GO CCC ACC AGO OCA GIG GTO 000 CAG OTT 480 Ale Arg Val er Ser Er Oly Pro Thr Ser Pro Val Val Ala Gin Leu 145 150 155 160 OTG AGG CCA GCA TOO ATO GCC OTG ACC AGO GAO AAC CAC CAC TIC TAT 528 Leu Arg Pro Ala Cys Met Ala Len Thr Arg Asp Asn His His Pha Tyr 165 170 17)5 AAO COG AAO TO TGC CAG OGA ACC CAC 000 OGA ATA 000 OTO AGO COG 57 6 Asn Arg Asn Phe Cys Gin Gly-Thr His Gly Aeg Ile Ala Val Ser Arg 180 185 190 WO 01/90151 PCT/USO1/16766 AAT COP GCA CGG TGC CAC OTC CAC ACA CAC CTA ACA TAT GCC TGC CTG 624 Asn Pro Ala Arg Cys His Leu His Thr His Leu Thr Tyr Ala Cys Leu 195 200 205 ATC TGACCAACAC ATGCTCGCCA CCCTCACCAC ACACC 662 Ile AS TCAW PG FPGGGGKVGEMRMPC PTMP SWSST KRRAQ WQTGCTTS FGGS WRSAVGAGHSAC AWRNAT GCLAK2S L RTCGPRSMAAARRCLCW2TRTGSVVSCAPVLLLAQQRLLEDRKDVVVLVILT PDGQASRLP DALTSASAARVSS S G PT S PWAQLLRPACMALT RDNHH FYN RN FCQGTHGRIAVS RN PARCHLHTHLTYACLI additional primate, human DTLR10 sequence (SEQ ID NO: 33 and 34); nucleotide 854 designated A, may be A or T; and nucleotides 1171 and 1172 designated C, each may be A, C, G, or T: CTG COT GCT GGC ACC CGG CTC CGG AGG CTG GAT GTC AGC TGC AAC AGC 48 Leu Pro Ala Gly Thr Arg Leu Azg Arg Leu Asp Val Ser Cys Asn Ser 1 5 10 ATC AGO TTC GTG GCC CCC GGC TTC TTT TCC AAG GCC AAG GAG CTGC GA 96 Ile Ser Phe Val Ala Pro Gly The Phe Ser Lys Ala Lys Glu Leu Arg 25 GAG CTC AAC OTT AGO GCC AAC GCC OTO AAG ACA GTG GAC CAC TCC TGG 144 Glu Leu Asn Leu Ser Ala Asn Ala Leu Lys Thr Val Asp His Ser Trp 40 TTI GGG CCC OTG GCG ACT GCC CTG CAA ATA CTA GAT GTA AC GCC AAC 192 Phe Gly Pro Leu Ala Ser Ala Leu Gin Ie Leu Asp Val Ser Ala Asn 55 OCT CTG CAC TGC GCC TGT GGG GOG CO TTT ATG GAO TIC CTG CTG GAG 240 Pro Leu His Cys Ala Cys Gly Ala Ala Phe Net Asp Phe Leu Leu Glu 65 70 75 GTG CAG GOT GCC GTG COO GGT CTG COO AGO CGG GTG AAG TGT GGO ACT 288 Val Gln Ala Ala Val Pro Gly Leu Pro Ser Arg Val Lys Cys Cly Ser 90 COG GGC CAG OTO CAG GGC CTO AGO ATO TTT GCA CAG GAO CTG CC CTO 336 Pro Gly Gin Leu Gin Gly Leu Ser Ile Phe Ala Gln Asp Leu Arg Leu 100 105 110 TGC OTG GAT GAG GOC OTO TOO TGG GAO TCT TTO GCC OTC TOG CTG CTG 384 Cys Leu Asp Giu Ala Leu Ser Trp Asp Cys Fhe Ala Leu Ser Leu Leu 115 120 125 GOT CTG CT OTG GGC CTG GGT GTG CCC ATG CTG CAT CAC OTO TGT GGC 432 Ala Val Ala Leu Gly Leu Gly Val Pro Met Leu His His Leu Cys Gly 130 135 140 TCC GAO CCC TGG TAO IGC TTC CAC OTG TGC OTG CC TGG OTT CCC TGG 480 Trp Asp Leu Trp Tyr Cys The His Leu Cys Leu Ala Trp Leu Pro Trp 145 150 155 160 wo 01/90151 WO 0190151PCT/USOI/16766

CGC

Arg

TTC

Phe

AAC

As n

CGC

Arg

GAG

Glu 225

CTG

Leu

CTG

Leu

GTG

Val1

CAG

Gin

GGT

Ci y 305

AAC

CAA ACT Gin Ser 165 TTC GAG Phe Asp 180 CGG GGG Arg Gly CTG GAG Leu Glu TGG GCC Trp Ala ACG GAC Tbr Asp 245 CAG CGC Gin Arg 260 AGC OCT Ser Pro TGC CGC Cys Arg AGC TTC Ser Phe TTC TAT GAT GAG Asp Gin GAG AGO Gin Ser 185 GAG GAG Gin Glu 200 GAO TGG Asp Trp TAT GGC Tyr Gly ACT GGT Ser Cly GAG GAO GI-a Asp 265 CGC CC Arg Arg 280 GTC CTC Val Leu CAG CTG Gin Leu AAC TTC GCC CTG Ala Leu GTG GCA Val Ala CGT GGG Arg Gly OCT GGC Pro Gly 220 CGC AAG Arg Lys 235 TTG CGC Leu Arg AAG GAO Lys Asp OGC TAO Arg Tyr TGG CCC Trp Pro 300 ATG GC Met Ala 315 CAG GGA GAT GCC Asp Ala 175 GTG TAO Val Tyr GCA OTO Ala Leu CTO TTT Len Phe TTT GTG Phe Val 240 TTO CTG Phe Leu 255 GTG OTG Val Leu CTG CGO Len Arg COO ACT Pro Ser AGG GAC Arg Asp 320 CCC GAA 528 576 624 672 720 768 816 864 912 960 1008 1068 1128 1173 Asn His His The Tyr Asn Arg Asn Phe Cys Gin Gly Pro Thr Ala Gin 330 335 TAGCOGTGAG CCGGAATOCT GOACGGTCC TGGTCTGAOC OTOOOOTCCT COOTOOOTO ATAAOPTGCTA COGAAGGOTA AAAAAAAAAA

ACCTCCACAC

ACCACACC

TOACOTOACO TOTGCOTGOO TGAOACAGAG CAGGOAOTOA

AACCA

wo 01/90151 WO 0190151PCTIUS01/16766 LPAG'PRLRRLDVSCINSI SFVAPGFFSKAKELPELNLSANALQPVDHSWIFGPLASALQILDVSA.NPLHCACGAAFM DFLLEVQAAVPGLPSRVKCGSPGQLQGLSI FAQDLRLCLDEALSDCFALSLLAVALGLGVPMLHLCGWDLWYC

FHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADMVYNELRGQLEECRGRWALRLCLEERDWLPOKTLFE

NLWASVYGSRKTLFVLAHT'DRVSGLLPJASFLLAQQRLLEDRKDVVVLVILSPDGRRSRY.

RLRQRLCRQSVLLWP

HgPSGQRSFWAQLG14ALTRDNHiHFYNEFCQ2GPTAE Further prim human, DTLR10 (SEQ ID NO: 42 and 43): atg ccc atg Met Pro Met ceo aca gct His Thr Ala tgg agt ggg tgg Trp Ser Gly Trp tgg agc tg qgg Trp Ser Trp Gly cog gcc act Pro Ala Thr gcc Ctg cac Ala Leu His ctc cca ccc cca cag ggt ttc tgc cgc Leu Pro Pro Pro Gin Gly Phe Cys Arg ccg ctg tot ttc ctg gtg Pro Leo Ser Leo Leu Val gcc ato atg Ctg Ala Ile Met Leo atg acc ctg Met Thr Leu ggt acc ttg Gly Thr Leu gcc ttc cta ccc Ala Phe Leu Pro gag otc tag ccc Glu Leu Gin Pro cat ggc His Gly ctg qtg aac Leu Val Rsn aac tgg ctg tt- Asn Tro Leo Ph.

aag tot gtg ccc Lys Set Val Pro cat tto too His Phe Ser too too aec Set Ser Asn atg gee qca ccc Cgt agc eat Met Ala Ala Pro Arg Gly Asn ogo etc cao cac etc cat gat Arg Ile His His !,eo His Asp etc ago ctt too Thr Ser Leo Set tot gao ttt gc Sec Asp Phe Ale ctg ccc ego ctg Leo Pro Set Leo C9g Arg 65 cat oto at cto His Leo Asn Leu aeg tgg at tgo cog cog gt- ggo ctc agc ccc Lys Trp Aen Cys Pro Pro Vel Gly Leo Set Pro 70 75 384 etg 000 ttc ccc Met His Phe Pro cac atg act etc His Met Thr Ile coo agt acc tto Pro Ser Thr Phe ttg got Leu Ala gtg ccc aco Vel Pro Thr gea gag ota eec ctg ego tao eec eec etc etg act Glu Glu Leo Asn Leo Ser Tyr Asn Asn Ile Met Thr gtg cot gog Vel Pro Ala 115 aac etc otg Asn Ile Leo 130 ctg ccc aaa too ctc ata too ctg tot Leo Pro Lys Ser Leo Ile Set Leo Ser 120 ago cat acc Ser -His Thr etg ote gac tot qcc ego ctc goc ggc ctg cat got otg Met Leo Asp Ser Ala Set Leo Ala Gly Leo His Ale Leo wo 01/90151 WO 0190151PCT/USOI/16766 ttc cta ttc atg Phe Leu Phe Met ggo aac tgt tat Gly Asn Cys, Tyr aag aao ccc tgc Lys Asn Pro Cys cag gca ctg gag Gin Ala Leu Giu gcc ccg ggt Ala Pro Gly gcc ctc ott gpo ctg ggc aac oto Ala Leu Leu 'Ply Leu Gly Asn Leu 170 1175 acc cac ctg Thr His Leu ctq occt tc Leu Pro Ser 195 ctc aag tac aac Leu'Lys Tyr Asn ctc act gtg gtg Leu Thr Val. Val ccc cgo aac Pro Arg Asn 190 cgc atc gtc Arg Ile Val agc ctg gag tat Ser Leu Giu Tyr ctp ttg tcc tac Leu Leu Ser Tyr aaa ctg Lys Leu 210 gcg cct gag gao Ala Pro Giu Asp gcc eat. ctg acc goc ctg cgt. gtg ctc Ala Asn Leu Thr Ala Leu Arg Val Leu gtg ggc gga aat Val Gly Gly Asn cgc cgc tgc gac Arg Arg Cys Asp got ccc aac CCC Ala Pro Asn Pro atg gag tgo cot Met Glu Cys Pro ceo ttc ccc cag His Phe Pro Gin cat coo gat ecc His Pro Asp Thr tto ago Phe Ser 255 cac otg agc His Leu Ser tgg otg aet Trp Leu Asn 275 ctt gee ggc ctg Leu Giu Gly Leu ttp aa gac agt Leu Lys Asp Ser tct oto tcc Ser Leu Ser 270 cpa ptg ctg Arg Val Leu gcc egt tgg ttc Ala Ser Trp Phe ggg otg gga aac Gly Leu Gly Asn gac otg Asp Leu 290 agt gag aac ttc Ser Giu Asn Phe tao aaa tgc etc Tyr Lys Cys Ile eea acc eag gcc Lys Thr Lys Ala cag gpc cta ace Gin Gly Leu Thr ctg ogo aeg ott Leu Arg Lys Leu ctg too ttc aat Leu Ser Phe Asn 960 1008 1056 1104 1152 1200 1248 cae aeg agp gtp Gin Lys Arg Val ttt gcc ceo otg Phe Ala His Leu ctg cC cot too Leu Ala Pro Sar ttc g Phe Gly 335 ago otg gto Ser Leu Val toe ctc pat Ser Leu Asp 355 ctg aag gag otg Leu Lys Glu Leu etg ceo ggc etc Met His Gly Ile ttc ttCcogo Phe Phe Arg 350 ccc etp ctc Pro Met Leu gag acO acg oto Giu Thr Thr Leu oca ctg p00 ogo Pro Leu Ala Arg wo 01/90151 WO 0190151PCT/USOI/16766 cag act ctg ogt ctg cag atg aac ttc etc Gin Thr Leu Arg Leu Gin Met Asn Phe Ile aac cag Asn Gin 380 gcc cag ctc ggc Ala Gin Leu Gly ctg tcg gac eec Leu Ser Asp Asn 400 ato Ile 385 ttc agg gcc ttc Phe Arg Ala Phe cot ggc Pro Giy 390 ctg ogo tac gtg gac Leu Arg Tyr ValiAsp 395 cgc atc ago gga Arg Ile Ser Gly tcg gag ctg ace Ser Giu Leu Thr acc atg ggg gag Thr Met Giy Glu gca gat Aia Asp 415 gga ggg gag Gly Giy Giu gtg gao act Vai Asp Thr 435 aag qto tgg ctg cag oct L~ys Val Trp Leu Gin Pro 420 425 ggg gao ott got Giy Asp Leu Aia ccg gcc cca Pro Aia Pro 430 agc acc ctc Ser Thr Leu ccc agc tct gaa Pro Ser Ser Giu tto agg ccc eac Phe Arg Pro Asn aac ttc Asn ?he 450 aco ttg gat ctg Thr Leu Asp Leu cgg aac aac ctg Arg Asn Asn Leu acc gtg cag ccg Thr Val Gin Pro gag Giu 465 atg ttt gcc cag Met Phe Ala Gin tcg oac ctg cag Ser His Leu Gin otg ogc ctg agg Leu Arg Leu Ser 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 177 6 1824 1872 aac tgc ato tog Asn Cys Ile Ser gca gtc eat ggc Ala Vai Asn Gly ceg tto ctg cog Gin Phe Leu Pro ctg acc Leu Thr 495 ggt ctg cag Gly Leu Gin gag cac tca Giu His Ser 515 cta gao ctg too cao aat aag ctg gao Leu Asp Leu Ser H-is Asn Lys Leu Asp 505 cto tao oao Leu Tyr His 510 gao otc ego Asp Leu Ser tto aog gag ota Phe Thr Giu Leu oga ctg gag gc Arg Leu Giu Ala tao eac Tyr Asn 530 ago oag ccc ttt Ser Gin Pro Phe ggo atg ceg ggc gtg ggo ceo eec tto ego Gly Met Gin Gly Vai-Gly His Asn Phe Ser 535 540 aco otg cgo ceo otc ego ctg goc ceo eec Thr Leu Arg His Leu Ser Leu Ale His Asn 555 560 tto gtg got ceo ctg Phe Vai Ala His Leu 545 eac atc ceo ago ca Asn Ile His Ser Gin gtg too ceg ceg Val Ser Gin Gin tgc egt acg tog Cys Ser Thr Ser otg cgg Lell Arg- 575 gcc ctg gao Ale Leu Asp ago ggc eat gca Ser Gly Asn Ala ctg ggc Leu Gly 585 cat atg tgg His Met Trp goc geg gge Ale Giu Gly 590 1920 wo 01/90151 WO 0190151PCT/USOI/16766 gac ctc tat Asp Leu Tyr 595 ctg car ttc ttc: Leu H-is Phe Phe ggc ctg age ggt G2ly Leu Ser Gly ate tgg etg Ile Trp Leu gac ttg Asp Leu 610 tee cag aac cgc Ser Gin Asn Arg eac acc etc is Thr Leu ctg ccc Leu Pro 620 caa aec etg cgc Gin Thr Leu Arg ac Asn 625 rtc ccc aag Leo Pro Lys agc cta Ser 'Leu 630 rag gtg ctg cgt Gin Val Leu Arg cgt gae aat tac Arg Asp Asn Tyr gcr ttc ttt aag Ala Phe Phe Lys tgg tgg agc etc cac ttc ctg ccc ace etg gaa gtc Trp Trp Ser Leu His Phe Leu Pro Lys Leu Giu Val 645 650 655 ctc gac ctg Leu Asp Leo ect gct ggc Pro Ala Gly 675 gga aac cag ctg Gly Asn Gin Leu gee ctg acc aat Ala Leo Thr Asn ggC age ctg Gly Ser Leo 670 eac agc atc: Asn Ser Ile arc cgg etc cgg Thr Arg Leo Arg ctg gat gtc agr JLeu Asp Va2l Ser agc ttc Ser Phe 690 gtg grr err ggc Val Ala Pro Gly tet tcC aag gee Phe Ser Lys Ala gag ctg cga gag Glu Leu Arg Glu 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2544 2592 etc Leu 705 aac ctt age gre Asn Leu Ser Ala grc etc aag ac Ala Leo Lys Thr gar car tre tgg Asp His Ser Trp ggg CCC etg gcg Gly Pro Leu Ala gee etg raa ata Ala Leu Gin Ile gat gta. agc gee Asp Val Ser Ala ac ect Asn Pro 735 ctg eac tgC Leu His Cys rag get gee Gin Ala Ala 755 tgt ggg geg gee Cyo Gly Ala Ale atg gac ttc rtg etg gag gtg Met Asp Phe Leu Leu Glu Val 750 gtg ccc ggt etg Val Pro Gly Leu age egg gtg aag Ser Arg Val Lys ggr agt erg Gly Ser Pro ggc rag Gly Gin 770 etc rag gge etc: Leu Gin Gly Leu ate ttt gca rag Ile Pha Ala Gin etg egC etc tge Leu Arg Leo Cys ctg Leo 785 gat gag gee etc Asp Glu Ala Lea tgg gae tgt ttc Trp Asp Cys Phe etc teg rtg etg Leu Ser Leu Leu gtg get ctg gge etg ggt gtg eec atg Val Ala Leo Gly Leu Giy Val Pro Met 805 eat ear etc tgt His His Leu Cys ggr tgg Gly Trp 815 wo 01/90151 WO 0190151PCT/USOI/16766 gac ctc tgg Asp Leu Trp tgc ttc cac ctg Cys Phe His Leu ctg gcc tgg ctt ccc tgg cgg Leu Ala Trp Leu Pro Trp Arg 830 ggg cgg caa Gly Arg Gin 835 gtg gtc ttc Val Val Phe 850 agt ggg cga gat Ser Gly Arg Asp gat gcc ctg ccc Asp Ala Leu,-Pro gat gcc ttc Asp Ala Phe gac aaa acg Asp Lys Thr agc gca gtg gca Ser Ala Val Ala tgg gtg tac aac Trp Val Tyr Asn gag Gi u 865 ctt cgg ggg cag Leu Arg Gly Gin gag gag tgc cgt Glu Glu Cys Arg cgc tgg gca ctc Arg Trp Ala Leu ctg tgc ctg gag Leu Cys Leu Glu cgc gac tgg ctg Arg Asp Trp Leu ggc aaa acc ctc Gly Lys Thr Leu ttt gag Phe Glu 895 aac ctg tgg Asn Leu Trp tcg gtc tat ggc Ser Val Tyr Gly cgc aag acg ctg Arg Lys Thr Leu ttt gtg ctg Phe Val Leu 910 ttc ctg ctg Phe Leu Leu 2640 2688 2736 2784 2832 2880 2928 2976 3024 3072 3120 31.68 gcc cac acgj Ala His Thr 915 gac cgg gtc agt Asp Arg Val Ser ctc ttg cgc gcc Leu Leu Arg Ala gcc cag Ala Gin 930 cag cgc ctg ctg Gin Arg Leu Leu gag gac cgc aag gac gtc gtg gtg ctg gtg Glu Asp Arg Lys Asp Val Val Val Leu Val 935 940 atc lie 945 ctg agc cct gac Leu Ser Pro Asp cgc cgc tcc cgc Arg Arg Ser Arg gtg cgg cty cgc Val Arg Leu Arg cgc ctc tgc cgc Arg Leu Cys Arg agt gtc ctc ctc Ser Val Leu Leu ccc cac cag ccc Pro His Gin Pro agt ggt Ser Gly 975 cag cgc agc Gin Arg Ser cac cac ttc His His Phe 995 tgg gcc cag ctg Trp Ala Gin Leu atg gcc ctg acc Met Ala Leu Thr agg-gac aac Arg Asp Asn 990 tat aac cgg aac ttc Tyr Asa Arg Asn Phe 1000 tgc cag gga ccc acg gcc gaa tag Cys Gin Gly Pro Thr Ala Glu 1005 wo 01/90151 PCT/US01/16766

MYPMKWSGWRSWGPATHTALPPPQGFCRSALHPLSLLVQAMLAMYTLALGTLPAFLE'CELQPHGLVNCN

WLFLKSVPHF SMAAPRGNVTSLSLS SNRIHHLHDSDFAHLPSkRHLNL(WNCPPVGLSPMHFPCHMTIE

PSTFLAVPTLEELNLSYNNIMTVPALPKSLISLSLSHTNLLMLDSASLAGLHALRFLFMDGNCYYKNPC

RQALEVAPGALLGLGNLTHLSLICYNNLTVWPRNLPSSLEYLLLSYNRIVKLAPEDLANLTALRVLDVGG

NCRRCDHAPNPCMECPRHFPQLHPDTFSHLSRLEGLVLKDSSLSWLNASWFRGLCNLRVLDLSENPLYK

CITKTKAFQGLTQLRKLNLSFNYQKRVSFAHLSLAPSFGSLVALKELDNHGIFFRSLDETTLRPLARLP

MLQTLRLQMNFINQAQLGIFRAFPGLRYVDLSDNRI SGASFLTATMGEADGGEKVWLQPGDLAPAPVD'2 PS SEDFRPNCSTLNFTLDLSRNNLVTVQPEI4FAQLSHLQCLRLSHNCI SQAVNGSQFLPLTOLQVLDLS

HNKLDLYHEHSFTELPRLEALDLSYNSQPFGMQGVGHNFSFVAHLRTLRHLSLAIINNIHSQVSQQLCST

SLRLDFSGALGMWAEDLYLHFFQGLSGLIWLDSQNRLhHTLLPQTLRNPKSLQVLRLRDNYLAF FKWWSLHiFLPKLEVLDLAGNQLKALTNGSLFAG PRLRRLDVSCNSI SFVAPGFFSKAKELRELNLSANA LKTVDHSWFGPLASALQILDVSANPLHCACGAAF.DFLLEVQAAVPGLPSRVKCOSPGQLQGLS

IFAQD

LRLCLDEALSWDCFALSLLAVAAMLGVPMLHHLCGWDLWYCFHLCLAtWLPWRGRQSGRDEDALPYDAFV

VFDKTQSAVADWVYNELRGQLEECRGRWALRLCLEERDWLPCKTLFENLWASVYGSRKTLFVLAHTDRV

SGLLRASFLLAQQRLLEDRKDVVVLVILS PDGRRSRYVRLRQLCRQSVLLWP-QFSGQRSFWAQLGMA

LTRDNHHFYNRNFCQGPTAE

partial rodent, mouse DTLR1O nucleotide sequence (SEQ ID NO: TGCCCCACAC GGACCGCGTC AGTGGCCTCC TGCGCACCAG CTTCCTGCTG GCTCAGCAGC GCCTGTTGGA AGACCGCAAG GACGTGGTGG TGTTGGTGAT CCTGCGTCCG GATGCCCCAC 1 CGTCCCGCTA TGTGCGACTG CGCCAGCGTC TCTGCCGCCA GAGTGTGCTC TTCTGGCCCC 1 AGCGACCCAA CGGGCAGGGG GGCTTCTGGG CCCAGCT'GAG TACAGCCCTG ACTAGGGACA 2 ACCGCGACTT CTATAACCAG AACTTCTGCC GGGGACCTAC AGCAGAATAG CTCAGAGCAA 3 CAGCTGGAAA CAGCTGCATC TTCATGTCTG GTTCCCGAGT TGCTCTGCCT GCCTTGCTCT 3 GTCTTACTAC ACCGCTATTT GGCAAGTGCG CAATATATGC TACCAAGCCA CCAGGCCCAC 4 GGAGCAAAGG TTGGCTGTAA AGGGTAGTTT TCTTCCCATG CATCTTTCAG GAGAGTGAAG 4 ATAGACACCA AACCCAC 4 Further rodent, mouse, DTLR10 (SEQ ID NO: 44 and aac ctg tcc ttc aat tac cgc aag Asn Leu Ser Phe Asn Tyr Arg Lys 1 5 aag gta tcc ttt Lys Val Ser Phe gcc cgc ctc cac Ala Arg Leu His gag ctg aac atg Giu Leu Asn Met ctg gca agt Leu Ala Ser ttt aag aac ctg Phe Lys Asn Leu tca ctg cay Ser Leu Gin aac ggc atc Asn Gly Ile ttc ttc cgc ttg Phe The Arg Leu aac aag tac acg ctc aga tgg ctg Asn Lys Tyr Thr Leu Arg Trp Leu gcc gat ctg ccc aaa ctc cac Ala Asp Leu Pro Lys Leu His act ctg cat ctt Thr Leu His Leu atg eec ttc atc Met Asn The Ile wo 01/90151 PCT/US01/16766 aac cag gca cag ctc ago ato ttt ggt acc ttc cga goc ctt ogc ttt 240 Asn Gin Ala Gin Leu Ser Ile Phe Gly Thr Phe Ar; Ala Leu Ar; Phe 70 75 gtg gac ttg tca gao aat cgc etc agt ggg cct tra aog otg tca gaa 288 Val Asp Leu Ser Asp Asn Arg Ile Ser Giy ProSer Thr Leu Ser Glu 90 gcc acc cot gaa gag gca gat gat gca gag cag gag gag ctg ttg tot 336 Ala Thr Pro Glu Giu Ala Asp Asp Ala Glu Gin Glu Glu Leu Leu Ser 100 105 110 gcg 'gat cot ceo oca got cog ctg ago eco cot got tot aag eac ttc 284 Ala Asp Pro His Pro Ala Pro Leu Ser Thr Pro Ala Ser Lys Asn Phe 115 120 125 atg gao ag; tgt aag aac ttc aag ttc aac atg gao otg tot cgg eec 432 Met Asp Arg Gys Lys Asn ?he Lys Phe Asn Met Asp Leu Ser Arg Asn 130 135 140 aac ctg gtg act atc aca gca gag at; ttt gta aet otc tca ogo oto 480 Asn Leu Val Thr Ile Thr Ala Giu Met Phe Val Aen Leu Ser Ar; Leo 145 150 155 160 cag tgt ott ago otg ago ceo aec toa att goa oag got gtc aat ggo 523 Gln Cys Leu Ser Leu Ser His Asn Ser Ile Ala Gin Ala Val Asn Gly 165 170 175 tot oag ttc otg oog otg aoc ggt c-g oag gtg cta gao otg too oao 576 Ser Gin Phe Leu Prc Leu Thr Gly Leo Gin Val Leo Asp Leu Ser His 180 185 190 aat aag ot; gao oto tao cao gag ceo tos ttc ac; gag ota coa oga 624 Asn Lys Leu Asp Leu Tyr His Glu His Ser Phe Thr Glu Leu Pro Ar; 195 200 205 ctg gag goc otg gao oto ago tao aao ago cag 000 ttt ago atg aag 67 2 Leu Glu Ala Leu Asp Leo Ser Tyr Asn Ser Gin Pro Phe Ser Met Lys 210 215 220 ggt ata ggc cac aat tto agt ttt gtg aco oat ctg too at; cte oag 720 Gly Ile Gly His Asn Phe Ser Phe Val Thr His Leo Ser Met Leo Gin 225 230 235 240 ago ctt ago ctg goa ceo aat gao att oat aoo cgt gtg too toe oat 768 Ser Leu Ser Leo Ala His Asn Asp Ile His Thr Ar; Val Ser Ser His 245 250 255 oto aso ago aao toa gtg agg ttt ott gao tto ego ggc aao ggt at; 816 Leo Asn Ser Asn Ser Val Ar; Phe Leo Asp Phe Ser Gly Asn Gly Mat 260 265 270 ggc ogo atg tgg gat gag ggg ggc ott tat oto oat tto tto caa ggo 864 Gly Ar; Met Trp Asp Glu Gly Gly Leo Tyr Leo His Phe Phe Gin Gly 275 280 285 wo 01/90151 WO 0190151PCT/USOI/16766 ctg agt Leu Ser 290 ggc gtg ctg aag Gly Val. Leu Lys gao ctg tct caa Asp Leu Ser Gin aac ctg cat ato Asn Leu His Ile ctc Leu 305 cgg ccc cag aac Arq Pro Gin Asn ott gao Leu Asp 310 sac ctc ccc Asn Leu Pro ago org aag ctg 'Ser Leu Lys Leu agc ctc oga gao Ser Leu Arg Asp sac tao Asn'Tyr 325 cta tot. ttc ttt sac tgg acc agt Leu Ser Phe Phe Asn Trp Thr Ser 330 ctg too Leu Ser 335 ttc ota ccc Phe Leu Pro gcc ctg ac Ala Leu Thr 355 ctg gaa gto 1Leu Glu Val cta -gao Leu Asp 345 ctg gca ggc adc Leu Ala Gly Asn cag cta sag Gin Leu Lys 350 cag aaa ctc Gin Lys Leu sat ggc soc ctg Asn Gly Thr Leu ast ggc soc ctc Asn Gly Thr Leu gat gto Asp Val 370 agt ago sac sgt Ser Ser Asn Ser gtc tot gtg gc Val Ser Val Ala ggc ttc tt-t too Gly Phe Phe Ser sag Lys 385 goc sag gag otg Ala Lys Glu Leu gag oto 550 ott Glu Leu Asn Leu gcc sac gc oto Ala Asn Ala Leu aca gtg gao cac Thr Val Asp His tgg ttt ggg coo Trp Phe Gly Pro gtg atg sac otg Val Met Asn Leu 505 gtt Thr Val 415 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 ota gao gtg Leu Asp Val gta gao tta Val Asp Leu 435 ago sac cot ctg Ser Asn Pro Leu tgt goc tyt ggg Cys Ala Cys Gly gos goc ttc Ala Ala Phe 430 ctg got aat Leu Ala Asn otg ttg gag gt g Leu Leu Glu Val 500 sag gtg oct Thr Lys Val Pro ggt gtg Gly Val 450 sag tgt ggc ago Lys Cys Gly Ser ggc cag ctg cag Gly Gin Leu Gin cgt ago sto ttc Arg Ser Ile Phe gog Ala 465 cag gao otg cgg Gin Asp Leu Arg otg tgo ctg gat gag gto oto tot tgg gao tgc Leu Cys Leu Asp Glu Val Leu Ser Trp Asp Cys 470 475 480 ttt ggo ott tos Phe Gly Leu Ser ttg got gtg gco Leu Ala Val Ala ggc atg gtg gtg Gly Met Val Val cot ata Pro Ile 495 otg cac cat oto tgc ggc tgg gao Leo His His Leo Cys Gly Trp Asp 500 tgg tao tgt ttt Trp Tyr Cys ?he cat otg tgc His Leo Cys 510 wo 01/90151 PCT/USOI/16766 ctg gca tgg cta cct ttg cta gcc cgc agc cga cgc agc gcc caa act Leu Ala Trp 515 ctc cct tat Leu Pro Tyr 530 gcc gac tgg Ala Asp Trp 545 ggc cgc tgg Giy Arg Trp ggc cag acg Gly Gin Thr aag act cta Lys Thr Leu 595 cgc acc agc Arg Thr Ser 610 gac gtg gtg Asp Val Val 625 tat gtg cga Tyr Val Arg ccc cag cag Pro Gin Gin gcc ctg act Ala Leu Thr 675 gga cct aca Gly Pro Thr 690 ctggttcccg a gcgcaatata t ttttcttccc a Arg tt c Phe cgg Arq ct g Leu tgg Trp 585 acg Th r cag Gin cgt Arg tgc Cys Gi y 665 tt c Arq Arg aag gca Lys,-Ala 540 cgg ctg Arg Leu 555 gac cga Asp Arg tcc ato Ser Ile cgc gtc Arg Vai ctg ttg Leu Leu 620 gat gcc Asp Ala 635 cag agt Gin Ser tgg gcc Trp Ala aac cag 1584 1632 1680 1728 1776 1824 1872 1920 1960 2016 2064 2119 2179 2239 2289 Ary Asp Asn Arg His Phe Tyr Asn Gin Aso Phe Cy5 Arg 680 685 gca gaa tagctcagag caacagctgg aaacagctgc atcttcatgt Ala Glu ~gttgctctg cctgccttgc tctgtcttac tacaccgcta tttggcaagt :gctaccaag ccaccaggcc cacggagcaa aggttggctg taaagggtag Ltgcatcttt caggagagtg aagatagaca ccaaacccac WO 01/90151 PCT/USOI/16766 NLSFNRKSALLSFNV ENNIFRLKTRLDPLTHQNIQQ S IFGTFRALRFVDILSDNRISGPSTLSEATPEEADDAEQEELLSADPHPAPLSTPASNFMDRCKNFKFN

MDLSRNNLVTITAEMFVNLSRLQCLSLSHNSIAQAVNGSQFLPLTGLQVLDLSHNKLDLYHEHSFTELP

RLADSNQFMGGNSVHSLSSANITVSLSSRLFGQGM

DEGGLYLHFFQGLSCVL!(LDLSQNNLHILRPQNLDNLPKSLKLLSLRDNYLSFFNTSLSFLPLEVJLD

LAGNQLKALTNGTLPNGTLLQKLDVSSNS IVSVAPGFFSKACELRELNLSANALKTVDHSWFGPIVMNL TVLDVRSNPLHCACGAAFVDLLLEVQTKVPGLNGVKCGS PGQLQGRSIFAQDLRLCLDEVLSWDCFGL SLLAVAVGMVVPILHHLCGWDVWYCFHLCLAWLPLLARSRRSAQTLPYDAFVVFDKAQSAVADWVYfNEL RVRLEERRGRWALRLCLEDRDWLPGQTLFENLWAS IYGSFRKTLFVLMliTDRVSGLLRTSFLLAQQRLLE

I)KVVVLPARRYRRRCQVFPQNQGW~SATDRFNNCG

TAE

wo 01/90151 PCT/USOI/16766 Table 11: Comparison of intracellular domains of human DTLRs. DTLR1 is SEQ ID NO: 2; DTLR2 is SEQ LID NO: 4; DTLR3 is SEQ ID NO: 6; DTLK4 is SEQ ID NO: 8; DTLRS is SEQ ID NO: 10; and DTLR6 is SEQ ID NO: 12.

Particularly important and conserved, characteristic, residues correspond, across the DTLRs, to SEQ ID NO: 18 residues tyrl0-tyrl3; trp26; cys46; trp52; pro54-gly55; ser69; lys71; trp134-proI35; and phel44-trpl4S.

DTLRI

DTLR9 DTLR8 DTLR2

DTLRG

DTLR-)

DTLRlO

DTLRZ

DTLRS

DTLRJ

DTLRI

DTLRS

DTLRS

DTLR2

DTLRG

DTLR7j DTLR1O DTLR4

OTLRS

DTLRJ

DTLR1

DTLRS

DTLRE

DTLR2

DTLRE

DTLR1O DTLR4 DTLR4

DTLRS

DTLR1 dS DTLRS DTLR8 DTLR2 DTLR6 0 DTLR7 DTLR4 DTLR3

QRNLQFHAFT.SYSGHD---SFWVRNELLPNLEKEG--MQICLRERNF

KENLQFEAFISYSEHD---SAWVKSELVPYLEKED--IQICLRERNF

NELIPNLEKEDGS ILICLYESYE SRNICYDAFVSYSERD AYWVENLMVQELENFNPP FKLCLHKRDF S DCDFVDKPATWLEVKEPRK-FLLEO PSQTFYDAYI SYDTKDASVTDWVINELRYHLEESRDK--NVLLCLEERDW S DALFYDAFVVFDKTXSAVADWVYNELRCQLEECRCRW-ALRLCLEERDW RGENTYDAFVIYSSQD EDWVRNELVKNLEEGVPP FQLCLRYRDE PIDMYKYDAYLCFSSND- FTWVQNALLI HLDTQYS DQNRFNLCFEERDF TEQFEYAAY: IH-AYKD--- -KDWVWEHFSSNEKEDQS LKFCLEERDF V2GKIVENITCIEKSKSIVLSPFVQEWCHYELFAHHL. H

VFGKSIVENIITC-IEKSYKSIFVLSPNFVQSEWCH-YELYFAHHNLFHE

VPGKSIVENIINC-IEKSYKSIE'VLSPNFVQSEWCH-YELYFAHHNLFHE

IPGKWIIDNI IDS-IEKSHKTVFVLSENEVKSEWCK-YELDFSHERLFEE LPCQPVLENLSQS-IQLSKKTVFVNTDKYAKTENFK- IAFYLSHQRLMDE DPOLAI IDNLMQS-INQSKK<TVFVLTKKYAKSWNFK-TAFYLXLQRLNCE LFGKTLFENLWAS-VYGS RKTLFVLAHTDRVSGLLR-AI FLLAQQRLLE- TPGVAIAANI IHEGFHKSRKVIVVVSQHFIQSRWCI-EEYEIAQTWQFLS

VPGENRIANIQDA-IWNSRKIVCLVSRNFLRDGWCL-EAFSYAQGRCLSD

EAGVFELEAIVNS-IKRSRKIIFVITHHLLKDPLCKREKVHHAVQQAIEQ

GSNSLILILLEPIFQYSIFSSYHKLKSLMARRTYLEWPKSKRCLFWAN

GSNNLILILLEPI FQNSIPNKYHKLKALMTQRTYLQWPKEKSKRGJFWA- NSDHIIL ILLEPI PFYCI PTRYHKLEALLEKKAYLEWPKDRRKCCLFWAN NNDAAILILLEPIEKKAI PQRFCKLRNTNNTKTYLEwPNDEAQREGFWVN KVDVIILI FLEKPEQK SKELQLRKRLCGSSVLEWPTNPQAHPYFWQC NMDVII FILLEPVLQH SPYLRLRQRICKSSILQWPDNPKAERLFWQT SRAGII FIVLQKVENT-LLRQQVELYRLLSRNTYLEWEDSvLGRHIFWRR LNSALIMVVVrGSLSQY-QLMKEQSIRGFVQKQQYLRWPEDLQDVGW'LHK NLDSIILVFLEEI PDYKLNHALCLRRGNFKSHCILNWPVQKERICAFRHK LRAAVNVNVLATRENYELQTFTELNEESRGST ISLMRT DCL

LNNALATDNHVAYSQVFKETV--------------------

LXNVVLTENDSRYNNMYVDSIKOY------------------

LRKALLDGKSWNPEGTVGTGCNWQEATSI LSQQILKKEKEKKKDNNI PLQTVATIS STransmembrane segments correspond approximately to S802-818 (791-823) of primate DTLR7 SEQ ID NO: 37; 559-575 S(550-586) of DTLR8 SEQ ID NO: 39; 553-569 (549-582) of DTLR9 SEQ ID NO: 41; 796-810 (790-814) of DTRL10 SEQ ID NO: 43; and 481-497 (475-503) of DTLR10 SEQ ID NO: As used herein, the term DNAX Toll like receptor 2 (DTLR2) shall be used to describe a protein comprising a 00 protein or peptide segment having or sharing the amino 00 acid sequence shown in Table 2, or a substantial fragment C 10 thereof. Similarly, with a DTLR3 and Table 3; DTLR4 and Table 4; DTLR5 and Table 5; DTLR 6 and Table 6; DTLR7 and 0 Table 7; DTLR8 and Table 8; DTLR9 and Table 9; and and Table 10. Rodent, mouse, DTLR11 sequence is provided, in EST AA739083; DTLR13 in ESTAI019567; DTLR14 in ESTs AT390330 and AA244663.

Also described herein are protein variations of the respective DTLR allele whose sequence is provided, a mutein agonist or antagonist. Typically, such agonists or antagonists will exhibit less than about 10% sequence differences, and thus will often have between 1- and 11fold substitutions, 7-fold, and others.

It also encompasses allelic and other variants, e.g., natural polymorphic, of the protein described. Typically, it will bind to its corresponding biological receptor with high affinity, at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM. The term shall also be used herein to refer to related naturally occurring forms, alleles, polymorphic variants, and metabolic variants of the mammalian protein.

Described herein are proteins or peptides having substantial amino acid sequence identity with the amino acid sequence in Table 2. It will include sequence variants with relatively few substitutions, e.g., preferably less than about 3-5. Similar features apply to WO 01/90151 PCT/US01/16766 the other DTLR sequences provided in Tables 3, 4, 5, 6, 7, 8, 9, or A substantial polypeptide "fragment", or "segment", is a stretch of amino acid residuesof at least about 8 amino acids, generally at least 10.amino acids, more generally at least 12 amino acids, often at least 14 amino acids, more often at least 16 amino acids, typically at least 18 amino acids, more typically at least 20 amino acids, usually at least 22 amino acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids. Sequences of segments of different proteins can be compared to one another over appropriate length stretches.

Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches, if necessary, by introducing gaps as required. See, Needleham, et al., (1970) J. Mol. Biol. 48:443-453; Sankoff, et al., (1983) chapter one in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; the University of Wisconsin Genetics Computer Group (GCG), Madison, WI; and the NCBI (NIH); each of which is incorporated herein by reference. This changes when considering conservative substitutions as matches.

Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Homologous amino acid sequences are intended to include natural allelic and interspecies variations in the cytokine sequence. Typical homologous proteins or peptides will have from 50-100% homology (if gaps can be introduced), to 60-100% homology WO 01/90151 PCT/US01/16766 (if conservative substitutions are included) with an amino acid sequence segment of Table 2, 3, 4, 5, 6, 7, 8, 9, or Homology measures will be at least about generally at least 76%, more generally at least 81%, often at least 85%, more often at least 88%, typically at least more typically at least 92%, usually at least 94%, more usually at least 95%, preferably at least 96%, and more preferably at least 97%, and in particularly preferred embodiments, at least 98% or more. The degree of homology will vary with the length of the compared segments. Homologous proteins or peptides, such as the allelic variants, will share most biological activities with the embodiments described in Table 2, 3, 4, 6, 7, 8, 9, or 10. Particularly interesting regions of comparison, at the amino acid or nucleotide levels, correspond to those within each of the blocks 1-10, or intrablock regions, corresponding to those indicated in Figures 2A- 2B.

As used herein, the term "biological activity" is used to describe, without limitation, effects on inflammatory responses, innate immunity, and/or morphogenic development by respective ligands. For example, these receptors should, like IL-1 receptors, mediate phosphatase or phosphorylase activities, which activities are easily measured by standard procedures.

See, Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al. (1991) Meth. Enzymol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant. Biol.

56:449-463; and Parker, et al. (1993) Nature 363:736-738.

The receptors exhibit biological activities much like regulatable enzymes, regulated by ligand binding.

However, the enzyme turnover number is more close to an enzyme than a receptor complex. Moreover, the numbers of occupied receptors necessary to induce such enzymatic WO 01/90151 PCT/US01/16766 activity is less than most receptor systems, and may number closer to dozens per cell, in contrast to most receptors which will trigger at numbers in the thousands per cell. The receptors, or portions thereof, may be useful as phosphate labeling enzymes to label general or specific substrates.

The terms ligand, agonist, antagonist, and analog of, a DTLR, include molecules that modulate the characteristic cellular responses to Toll ligand like proteins, as well as molecules possessing the more standard structural binding competition features of ligand-receptor interactions, where the receptor is a natural receptor or an antibody. The cellular responses likely are mediated through binding of various Toll ligands to cellular receptors related to, but possibly distinct from, the type I or type II IL-1 receptors. See, Belvin and Anderson (1996) Ann. Rev. Cell Dev. Biol.

12:393-416; Morisato and Anderson (1995) Ann. Rev.

Genetics 29:371-3991 and Hultmark (1994) Nature 367:116- 117.

Also, a ligand is a molecule which serves either as a natural ligand to which said receptor, or an analog thereof, binds, or a molecule which is a functional analog of the natural ligand. The functional analog may be a ligand with structural modifications, or may be a wholly unrelated molecule which has a molecular shape which interacts with the appropriate ligand binding determinants. The ligands may serve as agonists or antagonists, see, Goodman, et al. (eds. 1990) Goodman Gilman's: The Pharmacological Bases of Therapeutics, Pergamon Press, New York.

Rational drug design may also be based upon structural studies of the molecular shapes of a receptor or antibody and other effectors or ligands. Effectors may be other proteins which mediate other functions in response to ligand binding, or other proteins which WO 01/90151 PCT/US01/16766 normally interact with the receptor. One means for determining which sites interact with specific other proteins is a physical structure determination, xray crystallography or 2 dimensional NMR techniques.

These will provide guidance as to which amino acid residues form molecular contact regions. For a detailed description of protein structural determination, see, Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York, which is hereby incorporated herein by reference.

II. Activities The Toll like receptor proteins will have a number of different biological activities, in phosphate metabolism, being added to or removed from specific substrates, typically proteins. Such will generally result in modulation of an inflammatory function, other innate immunity response, or a morphological effect. The DTLR2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins are homologous to other Toll like receptor proteins, but each have structural differences. For example, a human DTLR2 gene coding sequence probably has about 70% identity with the nucleotide coding sequence of mouse DTLR2. At the amino acid level, there is also likely to be reasonable identity.

The biological activities of the DTLRs will be related to addition or removal of phosphate moieties to substrates, typically in a specific manner, but occasionally in a non specific manner. Substrates may be identified, or conditions for enzymatic activity may be assayed by standard methods, as described in Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al. (1991) Meth. Enzymol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al.

WO 01/90151 PCT/US01/16766 (1991) Cold Spring Harbor Symp. Quant. Biol. 56:449-463; and Parker, et al. (1993) Nature 363:736-738.

III. Nucleic Acids This invention contemplates use of isolated nucleic acid or fragments, which encode these or closely related proteins, or fragments thereof, to encode a corresponding polypeptide, preferably one which is biologically active. In addition, this invention covers isolated or recombinant DNA which encodes such proteins or polypeptides having characteristic sequences of the respective DTLRs, individually or as a group. Typically, the nucleic acid is capable of hybridizing, under appropriate conditions, with a nucleic acid sequence segment shown in Tables 2-10, but preferably not with a corresponding segment of Table 1. Said biologically active protein or polypeptide can be a full length protein, or fragment, and will typically have a segment of amino acid sequence highly homologous to one shown in Tables 2-10. Further, this invention covers the use of isolated or recombinant nucleic acid, or fragments thereof, which encode proteins having fragments which are equivalent to the DTLR2-10 proteins. The isolated nucleic acids can have the respective regulatory sequences in the 5' and 3' flanks, promoters, enhancers, poly-A addition signals, and others from the natural gene.

An "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially pure, separated from other components which naturally accompany a native sequence, such as ribosomes, polymerases, and flanking genomic sequences from the originating species. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, which are thereby distinguishable from naturally occurring compositions, and chemically WO 01/90151 PCT/US01/16766 synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule, either completely or substantially pure.

An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain heterogeneity, preferably minor.

This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.

A "recombinant" nucleic acid is typically defined either by its method of production or its structure. In reference to its method of production, a product made by a process, the process is use of recombinant nucleic acid techniques, involving human intervention in the nucleotide sequence. Typically this intervention involves in vitro manipulation, although under certain circumstances it may involve more classical animal breeding techniques. Alternatively, it can be a nucleic acid made by generating a sequencetcomprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, naturally occurring mutants as found in their natural state. Thus, for example, products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process. Such a process is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a restriction enzyme sequence recognition site.

Alternatively, the process is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms, encoding a fusion protein. Restriction WO 01/90151 PCT/US01/16766 enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design. A similar concept is intended for a recombinant, fusion, polypeptide. This will include a dimeric repeat. Specifically included are synthetic nucleic acids which, by genetic code redundancy, encode equivalent polypeptides to fragments of DTLR2-5 and fusions of sequences from various different related molecules, other IL-1 receptor family members.

A "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least 21 nucleotides, more generally at least 25 nucleotides, ordinarily at least 30 nucleotides, more ordinarily at least 35 nucleotides, often at least 39 nucleotides, more often at least 45 nucleotides, typically at least 50 nucleotides, more typically at least nucleotides, usually at least 60 nucleotides, more usually at least 66 nucleotides, preferably at least 72 nucleotides, more preferably at least 79 nucleotides, and in particularly preferred embodiments will be at least or more nucleotides. Typically, fragments of different genetic sequences can be compared to one another over appropriate length stretches, particularly defined segments such as the domains described below.

A nucleic acid which codes for a DTLR2-10 will be particularly useful to identify genes, mRNA, and cDNA species which code for itself or closely related proteins, as well as DNAs which code for polymorphic, allelic, or other genetic variants, from different individuals or related species. Preferred probes for such screens are those regions of the interleukin which are conserved between different polymorphic variants or which contain nucleotides which lack specificity, and will preferably be full length or nearly so. In other situations, WO 01/90151 PCT/US01/16766 polymorphic variant specific sequences will be more useful.

This invention further covers recombinant nucleic acid molecules and fragments havinga nucleic acid sequence identical to or highly homologous to the isolated DNA set forth herein. In particular, the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA replication. These additional segments typically assist in expression of the desired nucleic acid segment.

Homologous, or highly identical, nucleic acid sequences, when compared to one another or Table 2-10 sequences, exhibit significant similarity. The standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions. Comparative hybridization conditions are described in greater detail below.

Substantial identity in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 60% of the nucleotides, generally at least 66%, ordinarily at least 71%, often at least 76%, more often at least 80%, usually at least 84%, more usually at least 88%, typically at least 91%, more typically at least about 93%, preferably at least about 95%, more preferably at least about 96 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides, including, e.g., segments encoding structural domains such as the segments described below. Alternatively, substantial identity will exist when the segments will hybridize under selective hybridization conditions, to a strand or its complement, typically using a sequence derived from Tables 2-10.

Typically, selective hybridization will occur when there WO 01/90151 PCT/US01/16766 is at least about 55% homology over a stretch of at least about 14 nucleotides, more typically at least about preferably at least about 75%, and more preferably at least about 90%. See, Kanehisa (1984) Nucl. Acids Res.

12:203-213, which is incorporated herein by reference.

The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, generally at least about 20 nucleotides, ordinarily at least about 24 nucleotides, usually at least about 28 nucleotides, typically at least about 32 nucleotides, more typically at least about 40 nucleotides, preferably at least about nucleotides, and more preferably at least about 75 to 100 or more nucleotides.

Stringent conditions, in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions. Stringent temperature conditions will usually include temperatures in excess of about 30° C, more usually in excess of about 37" C, typically in excess of about 45° C, more typically in excess of about 55" C, preferably in excess of about 65° C, and more preferably in excess of about 70° C. Stringent salt conditions will ordinarily be less than about 500 mM, usually less than about 400 mM, more usually less than about 300 mM, typically less than about 200 mM, preferably less than about 100 mM, and'more preferably less than about 80 mM, even down to less than about 20 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370, which is hereby incorporated herein by reference.

Alternatively, for sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison WO 01/90151 PCT/US01/16766 algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparisop algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optical alignment of sequences for comparison can be conducted, by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The WO 01/90151 PCT/US01/16766 final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence/comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight and weighted end gaps.

Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described Altschul, et al. (1990) J. Mol. Biol. 215:403-410.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) WO 01/90151 PCT/US01/16766 Proc. Nat'l Acad. Sci. USA 89:10915) alignments of expectation of 10, M=5, N=4, and a comparison of both strands.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873- 5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.

Thus, a polypeptide is typically substantially identical to a second polypeptide, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

The isolated DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode this protein or its derivatives. These modified sequences can be used to produce mutant proteins (muteins) or to enhance the expression of variant species. Enhanced WO 01/90151 PCT/US01/16766 expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant DTLR-like derivatives include predetermined or site-specific mutations of the protein or its fragments, including silent mutations using genetic code degeneracy.. "Mutant DTLR" as used herein encompasses a polypeptide otherwise falling within the homology definition of the DTLR as set forth above, but having an amino acid sequence which differs from that of other DTLRlike proteins as found in nature, whether by way of deletion, substitution, or insertion. In particular, "site specific mutant DTLR" encompasses a protein having substantial homology with a protein of Tables 2-10, and typically shares most of the biological activities or effects of the forms disclosed herein.

Although site specific mutation sites are predetermined, mutants need not be site specific.

Mammalian DTLR mutagenesis can be achieved by making amino acid insertions or deletions in the gene, coupled with expression. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mammalian DTLR mutants can then be screened for the desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesis. See also Sambrook, et al.

(1989) and Ausubel, et al. (1987 and periodic Supplements).

The mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.

O The phosphoramidite method described by Beaucage and SCarruthers (1981) Tetra. Letts. 22:1859-1862, will produce Ssuitable synthetic DNA fragments. A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an 00 appropriate primer sequence.

00 0 Polymerase chain reaction (PCR) techniques can often be applied in mutagenesis. Alternatively, mutagenesis primers are commonly used methods for generating defined 0 mutations at predetermined sites. See, Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA; and Dieffenbach and Dveksler (eds. 1995) PCR Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY.

IV. Proteins, Peptides Described herein are primate DTLR2-10, whose sequences are disclosed in Tables 2-10, and described above. Allelic and other variants are also contemplated, including, fusion proteins combining portions of such sequences with others, including epitope tags and functional domains.

The present invention also provides recombinant proteins, heterologous fusion proteins using segments from these rodent proteins. A heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner.

Thus, the fusion product of a DTLR with an IL-1 receptor is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties, e.g., sequence or antigenicity, derived from each source peptide. A similar concept applies to heterologous nucleic acid sequences.

105 SIn addition, new constructs may be made from O combining similar functional or structural domains from Sother related proteins, IL-1 receptors or other DTLRs, including species variants. For example, ligandbinding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, Cunningham, et al. (1989) Science 243:1330-1336; and 00 O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992, each

OC

j00 of which is incorporated herein by reference. Thus, new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of Sreceptor-binding specificities. For example, the ligand binding domains from other related receptor molecules may be added or substituted for other domains of this or related proteins. The resulting protein will often have hybrid function and properties. For example, a fusion protein may include a targeting domain which may serve to provide sequestering of the fusion protein to a particular subcellular organelle.

Candidate fusion partners and sequences can be selected from various sequence data bases, GenBank, c/o IntelliGenetics, Mountain View, CA; and BCG, University of Wisconsin Biotechnology Computing Group, Madison, WI, which are each incorporated herein by reference.

Also described herein are muteins which bind Toll ligands, and/or which are affected in signal transduction.

Structural alignment of human DTLR1-10 with other members of the IL-1 family show conserved features/residues. See, Figure 3A. Alignment of the human DTLR sequences with other members of the IL-1 family indicates various structural and functionally shared features. See also, Bazan, et al. (1996) Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al. (1991) Protein Engineering 4:263-269.

106 WO 01/90151 PCT/US01/16766 The IL-la and IL-10 ligands bind an IL-1 receptor type I as the primary receptor and this complex then forms a high affinity receptor complex with the IL-1 receptor type III. Such receptor subunits are probably shared with the new IL-1 family members.

Similar variations in other species counterparts of DTLR2-10 sequences, in the corresponding regions, should provide similar interactions with ligand or substrate. Substitutions with either mouse sequences or human sequences are particularly preferred. Conversely, conservative substitutions away from the ligand binding interaction regions will probably preserve most signaling activities.

"Derivatives" of the primate DTLR2-10 include amino acid sequence mutants, glycosylation variants, metabolic derivatives and covalent or aggregative conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in the DTLR amino acid side chains or at the N- or C- termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, lysine or arginine.

Acyl groups are selected from the group of alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species.

In particular, glycosylation alterations are included, made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, mammalian WO 01/90151 PCT/US01/16766 glycosylation enzymes. Deglycosylation enzymes are also contemplated. Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, phosphotyrosine, phosphoserine, or phosphothreonine.

A major group of derivatives are covalent conjugates of the receptors or fragments thereof with other proteins of polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups.

Preferred derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.

Fusion polypeptides between the receptors and other homologous or heterologous proteins are also provided.

Homologous polypeptides may be fusions between different receptors, resulting in, for instance, a hybrid protein exhibiting binding specificity for multiple different Toll ligands, or a receptor which may have broadened or weakened specificity of substrate effect. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. Typical examples are fusions of a reporter polypeptide, luciferase, with a segment or domain of a receptor, a ligand-binding segment, so that the presence or location of a desired ligand may be easily determined. See, Dull, et al., U.S. Patent No. 4,859,609, which is hereby incorporated herein by reference. Other gene fusion partners include glutathione-S-transferase (GST), bacterial 8galactosidase, trpE, Protein A, Z-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor. See, Godowski, et al. (1988) Science 241:812-816.

r The phosphoramidite method described by Beaucage and SCurruthers (1981) Tetra. Letts. 22:1859-1862, will produce Ssuitable synthetic DNA fragments. A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an 00 appropriate primer sequence.

00 Such polypeptides may also have amino acid residues Ci 10 which have been chemically modified by phosphorylation, Ssulfonation, biotinylation, or the addition or removal of 0 other moieties, particularly those which have molecular shapes similar to phosphate groups. In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, affinity ligands.

Fusion proteins will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expressed are described generally, for example, in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al. (eds. 1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York, which are each incorporated herein by reference. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer.

Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232:341-347; and Atherton, et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford; each of which is incorporated herein by reference.

See also Dawson, et al. (1994) Science 266:776-779 for methods to make larger polypeptides.

Also described herein is the use of derivatives of a DTLR2-10 other than variations in amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical 109 WO 01/90151 PCT/US01/16766 moieties. These derivatives generally fall into three classes: salts, side chain and terminal residue covalent modifications, and adsorption complexes, for example with cell membranes. Such covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of a receptor or other binding molecule, an antibody. For example, a Toll ligand can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of a DTLR receptor, antibodies, or other similar molecules.

The ligand can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.

A DTLR of this invention can be used as an immunogen for the production of antisera or antibodies specific, capable of distinguishing between other IL-1 receptor family members, for the DTLR or various fragments thereof. The purified DTLR can be used to screen monoclonal antibodies or antigen-binding fragments prepared by immunization with various forms of impure preparations containing the protein. In particular, the term "antibodies" also encompasses antigen binding fragments of natural antibodies, Fab, Fab2, Fv, etc.

The purified DTLR can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of expression, or immunological disorders which lead to antibody production to the endogenous receptor. Additionally, DTLR fragments may also serve as immunogens to produce the antibodies of the present invention, as described immediately below. For example, this invention contemplates antibodies having binding WO 01/90151 PCT/US01/16766 affinity to or being raised against the amino acid sequences shown in Tables 2-10, fragments thereof, or various homologous peptides. In particular, this invention contemplates antibodies hiving binding affinity to, or having been raised against,.specific fragments which are predicted to be, or actually are, exposed at the exterior protein surface of the native DTLR.

The blocking of physiological response to the receptor ligands may result from the inhibition of binding of the ligand to the receptor, likely through competitive inhibition. Thus, in vitro assays of the present invention will often use antibodies or antigen binding segments of these antibodies, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either ligand binding region mutations and modifications, or other mutations and modifications, which affect signaling or enzymatic function.

This invention also contemplates the use of competitive drug screening assays, where neutralizing antibodies to the receptor or fragments compete with a test compound for binding to a ligand or other antibody. In this manner, the neutralizing antibodies or fragments can be used to detect the presence of a polypeptide which shares one or more binding sites to a receptor and can also be used to occupy binding sites on a receptor that might otherwise bind a ligand.

V. Making Nucleic Acids and Protein DNA which encodes the protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples. Natural sequences can be isolated using standard methods and the sequences provided herein, in Tables 2-10. Other species counterparts can be identified by hybridization WO 01/90151 PCT/US01/16766 techniques, or by various PCR techniques, combined with or by searching in sequence databases, GenBank.

This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length receptor or fragments which can in turn, for example, be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified ligand binding or kinase/phosphatase domains; and for structure/function studies. Variants or fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent. The protein, or portions thereof, may be expressed as fusions with other proteins.

Expression vectors are typically self-replicating DNA or RNA constructs containing the desired receptor gene or its fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation. Expression vectors also usually contain an origin of replication that WO 01/90151 PCT/US01/16766 allows the vector to replicate independently of the host cell.

The vectors of this invention include those which contain DNA which encodes a protein,' as described, or a fragment thereof encoding a biologically active equivalent polypeptide. The DNA can be under the control of a viral promoter and can encode a selection marker. This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic cDNA coding for such a protein in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNA coding for the receptor is inserted into the vector such that growth of the host containing the vector expresses the cDNA in question.

Usually, expression vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, it is possible to effect transient expression of the protein or its fragments in various hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of the protein encoding portion or its fragments into the host DNA by recombination.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host. Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, Pouwels, et al. (1985 and WO 01/90151 PCT/US01/16766 Supplements) Cloning Vectors: A Laboratory Manual, Elsevier, and Rodriquez, et al. (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, 1988, which are incorporated herein by reference.

Transformed cells are cells, preferably mammalian, that have been transformed or transfected with receptor vectors constructed using.recombinant DNA techniques.

Transformed host cells usually express the desired protein or its fragments, but for purposes of cloning, amplifying, and manipulating its DNA, do not need to express the subject protein. This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the receptor to accumulate in the cell membrane. The protein can be recovered, either from the culture or, in certain instances, from the culture medium.

For purposes of this invention, nucleic sequences are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.

Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotes include both gram negative and gram positive organisms, E.

coli and B. subtilis. Lower eukaryotes include yeasts, S. cerevisiae and Pichia, and species of the genus WO 01/90151 PCT/US01/16766 Dictyostelium. Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, insect cells, and birds, and of mammalian origin, human, primates, and rodents.

Prokaryotic host-vector systems include a wide variety of vectors for many different species. As used herein, E. coli and its vectors will be used generically to include equivalent vectors used in other prokaryotes.

A representative vector for amplifying DNA is pBR322 or many of its derivatives. Vectors that can be used to express the receptor or its fragments include, but are not limited to, such vectors as those containing the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (the. pN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540). See Brosius, et al. (1988) "Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in Vectors: A Survey of Molecular Cloning Vectors and Their Uses, (eds. Rodriguez and Denhardt), Buttersworth, Boston, Chapter 10, pp. 205-236, which is incorporated herein by reference.

Lower eukaryotes, yeasts and Dictyostelium, may be transformed with DTLR sequence containing vectors. For purposes of this invention, the most common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It will be used to generically represent lower eukaryotes although a number of other strains and species are also available. Yeast vectors typically consist of a replication origin (unless of the integrating type), a selection gene, a promoter, DNA encoding the receptor or its fragments, and sequences for translation termination, polyadenylation, and transcription termination. Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such inducible promoters as the alcohol dehydrogenase 2 WO 01/90151 PCT/US01/16766 promoter or metallothionine promoter. Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-series), self-replicating high copy number (such as the YEp-series); integrating types (such as the YIp-series), or mini-chromosomes (such as the YCp-series).

Higher eukaryotic tissue culture cells are normally the preferred host cells for expression of the functionally active interleukin protein. In principle, any higher eukaryotic tissue culture cell line is workable, insect baculovirus expression systems, whether from an invertebrate or vertebrate source.

However, mammalian cells are preferred. Transformation or transfection and propagation of such cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines. Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, from such sources as from adenovirus, parvoviruses, vaccinia virus, or cytomegalovirus.

Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo PolyA, see Thomas, et al.

(1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.

For secreted proteins, an open reading frame usually encodes a polypeptide that consists of a mature or secreted product covalently linked at its N-terminus to a signal peptide. The signal peptide is cleaved prior to WO 01/90151 PCT/US01/16766 secretion of the mature, or active, polypeptide. The cleavage site can be predicted with a high degree of accuracy from empirical rules, von-Heijne (1986) Nucleic Acids Research 14:4683-4690," and the precise amino acid composition of the signal peptide does not appear to be critical to its function, Randall, et al. (1989) Science 243:1156-1159; Kaiser, et al. (1987) Science 235:312-317.

It will often be desired to express these polypeptides in a system which provides a specific or defined glycosylation pattern. In this case, the usual pattern will be that provided naturally by the expression system. However, the pattern will be modifiable by exposing the polypeptide, an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system. For example, the receptor gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable in prokaryote or other cells.

The source of DTLR can be a eukaryotic or prokaryotic host expressing recombinant DTLR, such as is described above. The source can also be a cell line such as mouse Swiss 3T3 fibroblasts, but other mammalian cell lines are also contemplated by this invention, with the preferred cell line being from the human species.

Now that the sequences are known, the primate DTLRs, fragments, or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer-Verlag, New York; and Bodanszky (1984) The Principles of Peptide Synthesis, Springer-Verlag, New York; all of each which are incorporated herein by reference. For example, an WO 01/90151 PCT/US01/16766 azide process, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or-a dicyclohexylcarbodiimide (DCCD)/additive process can be used. Solid phase and solution phase syntheses are both applicable to the foregoing processes. Similar techniques can be used with partial DTLR sequences.

The DTLR proteins, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino groups that are not being used in the coupling reaction typically must be protected to prevent coupling at an incorrect location.

If a solid phase synthesis is adopted, the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group. Examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, and the like.

An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize the peptide step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide. This solid-phase approach is generally described by Merrifield, et al. (1963) in J. Am. Chem.

WO 01/90151 PCT/US01/16766 Soc. 85:2149-2156, which is incorporated herein by reference.

The prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis, various forms of chromatography, and the like. The receptors of this invention can be obtained in varying degrees of purity depending upon desired uses. Purification can be accomplished by use of the protein purification techniques disclosed herein, see below, or by the use of the antibodies herein described in methods of immunoabsorbant affinity chromatography. This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate cells, lysates of other cells expressing the receptor, or lysates or supernatants of cells producing the protein as a result of DNA techniques, see below.

Generally, the purified protein will be at least about 40% pure, ordinarily at least about 50% pure, usually at least about 60% pure, typically at least about pure, more typically at least about 80% pure, preferable at least about 90% pure and more preferably at least about 95% pure, and in particular embodiments, 97%- 99% or more. Purity will usually be on a weight basis, but can also be on a molar basis. Different assays will be applied as appropriate.

VI. Antibodies Antibodies can be raised to the various mammalian, primate DTLR proteins and fragments thereof, both in naturally occurring native forms and in their recombinant forms, the difference being that antibodies to the active receptor are more likely to recognize epitopes which are only present in the native conformations. Denatured WO 01/90151 PCT/US01/16766 antigen detection can also be useful in, Western analysis. Anti-idiotypic antibodies are also contemplated, which would be useful as agonists or antagonists of a natural receptor or" an antibody.

Preferred antibodies will exhibit properties of both affinity and selectivity. High affinity is generally preferred, while selectivity will allow distinction between various embodiment subsets. In particular, it will be desirable to possess antibody preparations characterized to bind, various specific combinations of related members while not binding others. Such various combinatorial subsets are specifically enabled, e.g., these reagents may be generated or selected using standard methods of immunoaffinity, selection, etc.

Antibodies, including binding fragments and single chain versions, against predetermined fragments of the protein can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins.

Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective protein, or screened for agonistic or antagonistic activity. These monoclonal antibodies will usually bind with at least a KD of about 1 mM, more usually at least about 300 pM, typically at least about 100pM, more typically at least about 30 uM, preferably at least about 10 pM, and more preferably at least about 3 pM or better.

The antibodies, including antigen binding fragments, of this invention can have significant diagnostic or therapeutic value. They can be potent antagonists that bind to the receptor and inhibit binding to ligand or inhibit the ability of the receptor to elicit a biological response, act on its substrate. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides to bind producing cells, or cells localized to the source of the interleukin.

WO 01/90151 PCT/US01/16766 Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker.

The antibodies of this invention can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they might bind to the receptor without inhibiting ligand or substrate binding.

As neutralizing antibodies,.they can be useful in competitive binding assays. They will also be useful in detecting or quantifying ligand. They may be used as reagents for Western blot analysis, or for immunoprecipitation or immunopurification of the respective protein.

Protein fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens. Mammalian DTLR and its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions, Dover Publications, New York; and Williams, et al. (1967) Methods in Immunology and Immunochemistry, Vol.

1, Academic Press, New York; each of which are incorporated herein by reference, for descriptions of methods of preparing polyclonal antisera. A typical method involves hyperimmunization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin is isolated.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, Stites, et al. (eds.) Basic and Clinical Immunology (4th Lange Medical Publications, Los WO 01/90151 PCT/US01/16766 Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and particularly in Kohler and Milstein (1975) in Nature 256: 495-497, which discusses one method of generating monoclonal antibodies.

Each of these references is incorporated herein by reference. Summarized briefly, this method involves injecting an animal with an immunogen. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See, Huse, et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544-546, each of which is hereby incorporated herein by reference.

The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, WO 01/90151 PCT/US01/16766 enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos. 3,817,837;.'3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant or chimeric immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567; or made in transgenic mice, see Mendez, et al. (1997) Nature Genetics 15:146-156. These references are incorporated herein by reference.

The antibodies of this invention can also be used for affinity chromatography in isolating the DTLRs. Columns can be prepared where the antibodies are linked to a solid support, particles, such as agarose, Sephadex, or the like, where a cell lysate may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby the purified protein will be released. The protein may be used to purify antibody.

The antibodies may also be used to screen expression libraries for particular expression products. Usually the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.

Antibodies raised against a DTLR will also be used to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the protein or cells which express the protein. They also will be useful as agonists or antagonists of the ligand, which may be competitive inhibitors or substitutes for naturally occurring ligands.

A DTLR protein that specifically binds to or that is specifically immunoreactive with an antibody generated against a defined immunogen, such as an immunogen consisting of the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, is typically WO 01/90151 PCT/US01/16766 determined in an immunoassay. The immunoassay typically uses a polyclonal antiserum which was raised, to a protein of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24. This antiserum is selected to have low crossreactivity against other IL-1R family members, e.g., DTLR1, preferably from the same species, and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the protein of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, or a combination thereof, is isolated as described herein. For example, recombinant protein may be produced in a mammalian cell line. An appropriate host, an inbred strain of mice such as Balb/c, is immunized with the selected protein, typically using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra). Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross reactivity against other IL-1R family members, mouse DTLRs or human DTLR1, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573. Preferably at least two DTLR family members are used in this determination in conjunction with either or some of the human DTLR2-10. These IL-1R family members can be produced as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques as described herein.

Immunoassays in the competitive binding format can be used for the crossreactivity determinations. For example, WO 01/90151 PCT/US01/16766 the proteins of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 22, and/or 24, or various fragments thereof, can be immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the protein of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and/or 24. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled. The crossreacting antibodies are then removed from the pooled antisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein the IL-1R like protein of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and/or 24). In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than twice the amount of the protein of the selected protein or proteins that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.

It is understood that these DTLR proteins are members of a family of homologous proteins that comprise at least so far identified genes. For a particular gene product, such as the DTLR2-10, the term refers not only to the amino acid sequences disclosed herein, but also to other proteins that are allelic, non-allelic or species variants. It also understood that the terms include nonnatural mutations introduced by deliberate mutation WO 01/90151 PCT/US01/16766 using conventional recombinant technology such as single site mutation, or by excising short sections of DNA encoding the respective proteins, or by substituting new amino acids, or adding new amino acids. Such minor alterations must substantially maintain the immunoidentity of the original .molecule and/or its biological activity.

Thus, these alterations include proteins that are specifically immunoreactive with a designated naturally occurring IL-1R related protein, for example, the DTLR proteins shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24. The biological properties of the altered proteins can be determined by expressing the protein in an appropriate cell line and measuring the appropriate effect upon lymphocytes. Particular protein modifications considered minor would include conservative substitution of amino acids with similar chemical properties, as described above for the IL-1R family as a whole. By aligning a protein optimally with the protein of DTLR2-10 and by using the conventional immunoassays described herein to determine immunoidentity, one can determine the protein compositions of the invention.

VII. Kits and quantitation Both naturally occurring and recombinant forms of the IL-1R like molecules of this invention are particularly useful in kits and assay methods. For example, these methods would also be applied to screening for binding activity, ligands for these proteins. Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds per year. See, a BIOMEK automated workstation, Beckman Instruments, Palo Alto, California, and Fodor, et al. (1991) Science 251:767-773, which is incorporated herein by reference. The latter describes means for testing binding by a plurality of defined polymers synthesized on a solid substrate. The development of suitable assays to screen for a ligand or Sagonist/antagonist homologous proteins can be greatly Sfacilitated by the availability of large amounts of purified, soluble DTLRs in an active state such as is provided by this invention.

Purified DTLR can be coated directly onto plates for use in the aforementioned ligand screening techniques.

00 However, non-neutralizing antibodies to these proteins can 00 ,be used as capture antibodies to immobilize the respective

NO

1 0 receptor on the solid phase, useful, in diagnostic uses.

Described herein is the use of DTLR2-10, fragments thereof, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of the protein or its ligand. Alternatively, or additionally, antibodies against the molecules may be incorporated into the kits and methods. Typically the kit will have a compartment containing either a defined DTLR peptide or gene segment or a reagent which recognizes one or the other. Typically, recognition reagents, in the case of peptide, would be a receptor or antibody, or in the case of a gene segment, would usually be a hybridization probe.

A preferred kit for determining the concentration of, DTLR4, a sample would typically comprise a labeled compound, ligand or antibody, having known binding affinity for DTLR4, a source of DTLR4 (naturally occurring or recombinant) as a positive control, and a means for separating the bound from free labeled compound, for example a solid phase for immobilizing the DTLR4 in the test sample. Compartments containing reagents, and instructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for mammalian DTLR or a peptide fragment, or receptor fragments are useful in diagnostic applications to detect the presence of elevated levels of ligand and/or 127 WO 01/90151 PCT/US01/16766 its fragments. Diagnostic assays may be homogeneous (without a separation step between free reagent and antibody-antigen complex) or heterogeneous (with a separation step). Various commercial assays exist, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA) and the like. For example, unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to DTLR4 or to a particular fragment thereof.

These assays have also been extensively discussed in the literature. See, Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH., and Coligan (ed. 1991 and periodic supplements) Current Protocols In Immunology Greene/Wiley, New York.

Anti-idiotypic antibodies may have similar use to serve as agonists or antagonists of DTLR4. These should be useful as therapeutic reagents under appropriate circumstances.

Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay. For the subject invention, depending upon the nature of the assay, the protocol, and the label, either labeled or unlabeled antibody, or labeled ligand is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like. Preferably, the kit will also contain instructions for proper use and disposal of the contents after use. Typically the kit has compartments for each useful reagent, and will contain instructions for proper use and disposal of reagents. Desirably, the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium WO 01/90151 PCT/US01/16766 having appropriate concentrations for performing the assay.

The aforementioned constituents of the diagnostic assays may be used without modification or may be modified in a variety of ways. For example, labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.

In any of these assays, a test compound, DTLR, or antibodies thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups: radiolabels such as 125I, enzymes Pat.

No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels Pat. No.

3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization. Both of the patents are incorporated herein by reference. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating the bound from the free ligand, or alternatively the. bound from the free test compound. The DTLR can be immobilized on various matrixes followed by washing. Suitable matrices include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the receptor to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling,, and biotin-avidin. The last step in this approach involves the precipitation of antibody/antigen complex by any of several methods including those utilizing, an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate. Other suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al.

(1984) Clin. Chem. 30(9):1457-1461, and the double antibody magnetic particle separation as described in U.S.

WO 01/90151 PCT/US01/16766 Pat. No. 4,659,678, each of which is incorporated herein by reference.

The methods for linking protein or fragments to various labels have been extensively reported in the literature and do not require detailed discussion here.

Many of the techniques involve the use of activated carboxyl groups either through the use of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like.

Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use of oligonucleotide or polynucleotide sequences taken from the sequence of a DTLR. These sequences can be used as probes for detecting levels of the respective DTLR in patients suspected of having an immunological disorder.

The preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature. Normally an oligonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several kilobases. Various labels may be employed, most commonly radionuclides, particularly 32 p. However, other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes. The antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound Sto the duplex can be detected. The use of probes to the CN1 Snovel anti-sense RNA may be carried out in any conventional techniques such as nucleic acid S 5 hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT), and hybrid arrested translation (HART). This also includes 00 amplification techniques such as polymerase chain reaction 00

(PCR)

\O

C- 10 Diagnostic kits which also test for the qualitative or quantitative presence of other markers are also Scontemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers.

Thus, kits may test for combination of markers. See, Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97.

VIII. Therapeutic Utility Described herein are reagents with significant therapeutic value. The DTLRs (naturally occurring or recombinant), fragments thereof, mutein receptors, and antibodies, along with compounds identified as having binding affinity to the receptors or antibodies, should be useful in the treatment of conditions exhibiting abnormal expression of the receptors of their ligands. Such abnormality will typically be manifested by immunological disorders. Additionally, this invention should provide therapeutic value in various diseases or disorders associated with abnormal expression or abnormal triggering of response to the ligand. The Toll ligands have been suggested to be involved in morphologic development, e.g., dorso-ventral polarity determination, and immune responses, particularly the primitive innate responses.

See, Sun, et al. (1991) Eur. J. Biochem. 196:247- 254; Hultmark (1994) Nature 367:116-117.

WO 01/90151 PCT/US01/16766 Recombinant DTLRs, muteins, agonist or antagonist antibodies thereto, or antibodies can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additionalactive ingredients, in conventional pharmaceutically acceptable carriers or diluents, along with physiologically innocuous stabilizers and excipients. These combinations can be sterile, filtered, and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies or binding fragments thereof which are not complement binding.

Ligand screening using DTLR or fragments thereof can be performed to identify molecules having binding affinity to the receptors. Subsequent biological assays can then be utilized to determine if a putative ligand can provide competitive binding, which can block intrinsic stimulating activity. Receptor fragments can be used as a blocker or antagonist in that it blocks the activity of ligand.

Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of ligand, inducing signaling. This invention further contemplates the therapeutic use of antibodies to DTLRs as antagonists.

The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage.

Various considerations.are described, in Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological WO 01/90151 PCT/US01/16766 Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, (current edition), Mack Publishing Co., Easton, Penn.; each of which is hereby incorporated herein by reference. Methods for administration are discussed therein and below, for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others.

Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, in the Merck Index, Merck Co., Rahway, New Jersey. Because of the likely high affinity binding, or turnover numbers, between a putative ligand and its receptors, low dosages of these reagents would be initially expected to be effective. And the signaling pathway suggests extremely low amounts of ligand may have effect. Thus, dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 uM concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier. Slow release formulations, or slow release apparatus will often be utilized for continuous administration.

DTLRs, fragments thereof, and antibodies or its fragments, antagonists, and agonists, may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration. Therapeutic formulations may be administered in any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof.

Each carrier must be both pharmaceutically and WO 01/90151 PCT/US01/16766 physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Penn.; Avis, et al.

(eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY. The therapy of this invention may be combined with or used in association with other therapeutic agents, particularly agonists or antagonists of other IL-1 family members.

IX. Ligands The description of the Toll receptors herein provide means to identify ligands, as described above. Such ligand should bind specifically to the respective receptor with reasonably high affinity. Various constructs are made available which allow either labeling of the receptor to detect its ligand. For example, directly labeling DTLR, fusing onto it markers for secondary labeling, e.g., FLAG or other epitope tags, etc., will allow detection of receptor. This can be histological, as an affinity method for biochemical purification, or labeling or selection in an expression cloning approach. A two-hybrid selection system may also be applied making appropriate constructs with the available DTLR sequences. See, Fields and Song (1989) Nature 340:245-246.

WO 01/90151 PCT/US01/16766 Generally, descriptions of DTLRs will be analogously applicable to individual specific embodiments directed to DTLR2, DTLR3, DTLR4, DTLR5, DTLR6, DTLR7, DTLR8, DTLR9, and/or DTLR10 reagents and compositions.

The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments.

WO 01/90151 PCT/US01/16766

EXAMPLES

I. General Methods Some of the standard methods are described or referenced, in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.

(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates, Brooklyn, NY; or Ausubel, et al. (1987 and Supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York. Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al. (1987 and periodic supplements); Coligan, et al. (ed. 1996) and periodic supplements, Current Protocols In Protein Science Greene/Wiley, New York; Deutscher (1990) "Guide to Protein Purification" in Methods in Enzymology, vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments, to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence. See, Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, and Crowe, et al. (1992) QIAexpress: The High Level Expression and Protein Purification System QUIAGEN, Inc., Chatsworth, CA.

Standard immunological techniques and assays are described, in Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current Protocols in Immunology WO 01/90151 PCT/US01/16766 Wiley/Greene, NY; and Methods in Enzymology volumes. 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.

Assays for vascular biological activities are well known in the art. They will cover angiogenic and angiostatic activities in tumor, or other tissues, e.g., arterial smooth muscle proliferation (see, Koyoma, et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascular epithelium (see McEvoy, et al. (1997) J. Exp.

Med. 185:2069-2077), etc. See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am. J. Pathol.

147:668-677; Thyberg, et al. (1990) Atherosclerosis 10:966-990; and Gumbiner (1996) Cell 84:345-357.

Assays for neural cell biological activities are described, in Wouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier; Methods in Neurosciences Academic Press; and Neuromethods Humana Press, Tptowa, NJ.

Methodology of developmental systems is described, e.g., in Meisami Handbook of Human Growth and Developmental Biology CRC Press; and Chrispeels (ed.) Molecular Techniques and Approaches in Developmental Biology Interscience.

Computer sequence analysis is performed, using available software programs, including those from the"GCG Wisconsin) and GenBank sources. Public sequence databases were also used, from GenBank, NCBI, EMBO, and others. Determination of transmembrane and other important motifs may be predicted using such bioinformatics tools.

Many techniques applicable to IL-10 receptors may be applied to DTLRs, as described, in USSN 08/110,683 receptor), which is incorporated herein by reference for all purposes.

II. Novel Family of Human Receptors WO 01/90151 PCT/US01/16766 Abbreviations: DTLR, DNAX Toll-like receptor; IL-1R, interleukin-1 receptor; TH, Toll homology; LRR, leucinerich repeat; EST, expressed sequence tag; STS, sequence tagged site; FISH, fluorescence in situ hybridization.

The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin-1 (IL-1) receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning" of a novel class of putative human receptors with a protein architecture that is closely similar to Drosophila Toll in both intra- and extra-cellular segments. Five human Toll-like receptors, designated DTLRs 1-5, are likely the direct homologs of the fly molecule., and as such could constitute an important and unrecognized component of innate immunity in humans; intriguingly, the evolutionary retention of DTLRs in vertebrates may indicate another role, akin to Toll in the dorsoventralization of the Drosophila embryo, as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human DTLRs. Using fluorescence in situ hybridization and Sequence-Tagged Site database analyses, we also show that the cognate DTLR genes reside on chromosomes 4 (DTLRs 1, 2, and 9 (DTLR4), and 1 (DTLR5). Structure prediction of the aligned Tollhomology (TH) domains from varied insect and human DTLRs, vertebrate IL-1 receptors, and MyD88 factors, and plant disease resistance proteins, recognizes a parallel p/a fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.

WO 01/90151 PCT/US01/16766 The seeds of the morphogenetic gulf that so dramatically separates flies from humans are planted in familiar embryonic shapes and patterns, but give rise to very different cell complexities. .DeRobertis and Sasai (1996) Nature 380:37-40; and Arendt and Nibler-Jung (1997) Mech. Develop. 61:7-21. This divergence of developmental plans between insects and vertebrates is choreographed by remarkably similar signaling pathways, underscoring a greater conservation of protein networks and biochemical mechanisms from unequal gene repertoires. Miklos and Rubin (1996) Cell 86:521-529; and Chothia (1994) Develop.

1994 Suppl., 27-33. A powerful way to chart the evolutionary design of these regulatory pathways is by inferring their likely molecular components (and biological functions) through interspecies comparisons of protein sequences and structures. Miklos and Rubin (1996) Cell 86:521-529; Chothia (1994) Develop. 1994 Suppl., 27- 33 and Banfi, et al. (1996) Nature Genet. 13:167- 174.

A universally critical step in embryonic development is the specification of body axes, either born from innate asymmetries or triggered by external cues. DeRobertis and Sasai (1996) Nature 380:37-40; and Arendt and Nubler-Jung (1997) Mech. Develop. 61:7-21. As a model system, particular attention has been focused on the phylogenetic basis and cellular mechanisms of dorsoventral polarization. DeRobertis and Sasai (1996) Nature 380:37and Arendt and Nbbler-Jung (1997) Mech. Develop. 61:7- 21. A prototype molecular strategy for this transformation has emerged from the Drosophila embryo, where the sequential action of a small number of genes results in a ventralizing gradient of the transcription factor Dorsal. St. Johnston and NUsslein-Volhard (1992) Cell 68:201-219; and Morisato-and Anderson (1995) Ann.

Rev. Genet. 29:371-399.

WO 01/90151 PCT/US01/16766 This signaling pathway centers on Toll, a transmembrane receptor that transduces the binding of a maternally-secreted ventral factor, Spatzle, into the cytoplasmic engagement of Tube, an accessory molecule, and the activation of Pelle, a Ser/Thr-kinase that catalyzes the dissociation of Dorsal from the inhibitor Cactus and allows migration of Dorsal to ventral nuclei (Morisato and Anderson (1995) Ann. Rev. Genet. 29:371-399; and Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol. 12:393- 416. The Toll pathway also controls the induction of potent antimicrobial factors in the adult fly (Lemaitre, et al. (1996) Cell 86:973-983); this role in Drosophila immune defense strengthens mechanistic parallels to IL-1 pathways that govern a host of immune and inflammatory responses in vertebrates. Belvin and Anderson (1996) Ann.

Rev. Cell Develop. Biol. 12:393-416; and Wasserman (1993) Molec. Biol. Cell 4:767-771. A Toll-related cytoplasmic domain in IL-1 receptors directs the binding of a Pellelike kinase, IRAK, and the activation of a latent NF-KB/I- KB complex that mirrors the embrace of Dorsal and Cactus.

Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol.

12:393-416; and Wasserman (1993) Molec. Biol. Cell 4:767- 771.

We describe the cloning and molecular characterization of four new Toll-like molecules in humans, designated DTLRs 2-5 (following Chiang and Beachy (1994) Mech. Develop. 47:225-239), that reveal a receptor family more closely tied to Drosophila Toll homologs than to vertebrate IL-1 receptors. The DTLR sequences are derived from human ESTs; these partial cDNAs were used to draw complete expression profiles in human tissues for the five DTLRs, map the chromosomal locations of cognate genes, and narrow the choice of cDNA libraries for fulllength cDNA retrievals. Spurred by other efforts (Banfi, et al. (1996) Nature Genet. 13:167-174; and Wang, et al.

(1996) J. Biol. Chem. 271:4468-4476), we are assembling, WO 01/90151 PCT/US01/16766 by structural conservation and molecular parsimony, a biological system in humans that is the counterpart of a compelling regulatory scheme in Drosophila. In addition, a biochemical mechanism driving Toll signaling is suggested by the proposed tertiary.fold of the Tollhomology (TH) domain, a core module shared by DTLRs, a broad family of IL-1 receptors, mammalian MyD88 factors and plant disease resistance proteins. Mitcham, et al.

(1996) J. Biol. Chem. 271:5777-5783; and Hardiman, et al.

(1996) Oncogene 13:2467-2475. We propose that a signaling route coupling morphogenesis and primitive immunity in insects, plants, and animals (Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol. 12:393-416; and Wilson, et al. (1997) Curr. Biol. 7:175-178) may have roots in bacterial two-component pathways.

Computational Analysis.

Human sequences related to insect DTLRs were identified from the EST database (dbEST) at the National Center for Biotechnology Information (NCBI) using the BLAST server (Altschul, et al. (1994) Nature Genet. 6:119- 129). More sensitive pattern- and profile-based methods (Bork and Gibson (1996) Meth. Enzymol. 266:162-184) were used to isolate the signaling domains of the DTLR family that are shared with vertebrate and plant proteins present in nonredundant databases. The progressive alignment of DTLR intra- or extracellular domain sequences was carried out by ClustalW (Thompson, et al. (1994) Nucleic Acids Res. 22:4673-4680); this program also calculated the branching order of aligned sequences by the Neighbor- Joining algorithm (5000 bootstrap replications provided confidence values for the tree groupings).

Conserved alignment patterns, discerned at several degrees of stringency, were drawn by the Consensus program (internet URL http://www.bork.emblheidelberg.de/Alignment/ consensus.html). The PRINTS WO 01/90151 PCT/US01/16766 library of protein fingerprints (http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/ PRINTS.html) (Attwood, et al. (1997) Nucleic Acids Res.

25:212-217) reliably identified the;myriad leucine-rich repeats (LRRs) present in the extracellular segments of DTLRs with a compound motif (PRINTS code Leurichrpt) that flexibly matches N- and C-terminal features of divergent LRRs. Two prediction algorithms whose three-state accuracy is above 72% were used to derive a consensus secondary structure for the intracellular domain alignment, as a bridge to fold recognition efforts (Fischer, et al. (1996) FASEB J. 10:126-136). Both the neural network program PHD (Rost and Sander (1994) Proteins 19:55-72) and the statistical prediction method DSC (King and Sternberg (1996) Protein Sci. 5:2298-2310) have internet servers (URLs http://www.emblheidelberg.de/predictprotein/phd pred.html and http://bonsai.lif.icnet.uk/bmm/dsc/dsc_read_align.html, respectively). The intracellular region encodes the THD region discussed, in Hardiman, et al. (1996) Oncogene 13:2467-2475; and Rock, et al. (1998) Proc. Nat'l Acad. Sci. USA 95:588-593, each of which is incorporated herein by reference. This domain is very important i7i the mechanism of signaling by the receptors, which transfers a phosphate group to a substrate.

Cloning of full-length human DTLR cDNAs.

PCR primers derived from the Toll-like Humrsc786 sequence (GenBank accession code D13637) (Nomura, et al.

(1994) DNA Res. 1:27-35) were used to probe a human erythroleukemic, TF-1 cell line-derived cDNA library (Kitamura, et al. (1989) Blood 73:375-380) to yield the DTLR1 cDNA sequence. The remaining DTLR sequences were flagged from dbEST, and the relevant EST clones obtained from the I.M.A.G.E. consortium (Lennon, et al. (1996) Genomics 33:151-152) via Research Genetics (Huntsville, WO 01/90151 PCT/US01/16766 AL): CloneID#'s 80633 and 117262 (DTLR2), 144675 (DTLR3), 202057 (DTLR4) and 277229 (DTLR5). Full length cDNAs for human DTLRs 2-4 were cloned by DNA hybridization screening of Xgtl0 phage, human adult lung, placenta, and fetal liver 5'-Stretch Plus cDNA libraries (Clontech), respectively; the DTLR5 sequence is derived from a human multiple-sclerosis plaque EST. All positive clones were sequenced and aligned to identify individual DTLR ORFs: DTLR1 (2366 bp clone, 786 aa ORF), DTLR2 (2600 bp, 784 aa), DTLR3 (3029 bp, 904 aa), DTLR4 (3811 bp, 879 aa) and (1275 bp, 370 aa). Similar methods are used for DTLRs 6-10. Probes for DTLR3 and DTLR4 hybridizations were generated by PCR using human placenta (Stratagene) and adult liver (Clontech) cDNA libraries as templates, respectively; primer pairs were derived from the respective EST sequences. PCR reactions were conducted using T. aquaticus Taqplus DNA polymerase (Stratagene) under the following conditions: 1 x (940 C, 2 min) 30 x (550 C, 20 sec; 720 C 30 sec; 940 C 20 sec), 1 x (720 C, 8 min). For DTLR2 full-length cDNA screening, a 900 bp fragment generated by EcoRI/XbaI digestion of the first EST clone (ID# 80633) was used as a probe.

mRNA blots and chromosomal localization.

Human multiple tissue (Cat# 1, 2) and cancer cell line blots (Cat# 7757-1), containing approximately 2 jg of poly(A) RNA per lane, were purchased from Clontech (Palo Alto, CA). For DTLRs 1-4, the isolated full-length cDNAs served as probes, for DTLR5 the EST clone (ID #277229) plasmid insert was used. Briefly, the probes were radiolabeled with a- 32 p] dATP using the Amersham Rediprime random primer labeling kit (RPN1633).

Prehybridization and hybridizations were performed at C in 0.5 M Na2HP04, 7% SDS, 0.5 M EDTA (pH All stringency washes were conducted at 650 C with two initial washes in 2 x SSC, 0.1% SDS for 40 min followed by a WO 01/90151 PCT/US01/16766 subsequent wash in 0.1 x SSC, 0.1% SDS for 20 min.

Membranes were then exposed at -700 C to X-Ray film (Kodak) in the presence of intensifying screens. More detailed studies by cDNA library Southerns (14) were performed with selected human DTLR.clones to examine their expression in hemopoietic cell subsets.

Human chromosomal mapping was conducted by the method of fluorescence in situ hybridization (FISH) as described in Heng and Tsui (1994) Meth. Molec. Biol. 33:109-122, using the various full-length (DTLRs 2-4) or partial cDNA clones as probes. These analyses were performed as a service by SeeDNA Biotech Inc. (Ontario, Canada). A search for human syndromes (or mouse defects in syntenic loci) associated with the mapped DTLR genes was conducted in the Dysmorphic Human-Mouse Homology Database by internet server (http://www.hgmp.mrc.ac.uk/DHMHD/ hum chromel.html).

Similar methods nare applicable to DTLRs 6-10.

Conserved architecture of insect and human DTLR ectodomains.

The Toll family in Drosophila comprises at least four distinct gene products: Toll, the prototype receptor involved in dorsoventral patterning of the fly embryo (Morisato and Anderson (1995) Ann. Rev.iGenet. 29:371-399) and a second named '18 Wheeler' (18w) that may also be involved in early embryonic development (Chiang and Beachy (1994) Mech. Develop. 47:225-239; Eldon, et al. (1994) Develop. 120:885-899); two additional receptors are predicted by incomplete, Toll-like ORFs downstream of the male-specific-transcript (Mst) locus (GenBank code X67703) or encoded by the 'sequence-tagged-site' (STS) Dm2245 (GenBank code G01378) (Mitcham, et al. (1996) J. Biol.

Chem. 271:5777-5783). The extracellular segments of Toll and 18w are distinctively composed of imperfect, -24 amino acid LRR motifs (Chiang and Beachy (1994) Mech. Develop.

WO 01/90151 PCT/US01/16766 47:225-239; and Eldon, et al. (1994) Develop. 120:885- 899). Similar tandem arrays of LRRs commonly form the adhesive antennae of varied cell surface molecules and their generic tertiary structure ispresumed to mimic the horseshoe-shaped cradle of a ribonuclease inhibitor fold, where seventeen LRRs show a repeating P/a-hairpin, 28 residue motif (Buchanan and Gay (1996) Prog. Biophys.

Molec. Biol. 65:1-44). The specific recognition of Spatzle by Toll may follow a model proposed for the binding of cystine-knot fold glycoprotein hormones by the multi-LRR ectodomains of serpentine receptors, using the concave side of the curved P-sheet (Kajava, et al. (1995) Structure 3:867-877); intriguingly, the pattern of cysteines in Spatzle, and an orphan Drosophila ligand, Trunk, predict a similar cystine-knot tertiary structure (Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol.

12:393-416; and Casanova, et al. (1995) Genes Develop.

9:2539-2544).

The 22 and 31 LRR ectodomains of Toll and 18w, respectively (the Mst ORF fragment displays 16 LRRs), are most closely related to the comparable 18, 19, 24, and 22 LRR arrays of DTLRs 1-4 (the incomplete DTLR5 chain presently includes four membrane-proximal LRRs) by sequence and pattern analysis (Altschul, et al. (1994) Nature Genet. 6:119-129; and Bork and Gibson (1996) Meth.

Enzymol. 266:162-184) (Fig. However, a striking difference in the human DTLR chains is the common loss of a -90 residue cysteine-rich region that is variably embedded in the ectodomains of Toll, 18w and the Mst ORF (distanced four, six and two LRRs, respectively, from the membrane boundary). These cysteine clusters are bipartite, with distinct 'top' (ending an LRR) and 'bottom' (stacked atop an LRR) halves (Chiang and Beachy (1994) Mech. Develop. 47:225-239; Eldon, et al. (1994) Develop. 120:885-899; and Buchanan and Gay (1996) Prog.

Biophys. Molec. Biol. 65:1-44); the 'top' module recurs in WO 01/90151 PCT/US01/16766 both Drosophila and human DTLRs as a conserved juxtamembrane spacer (Fig. We suggest that the flexibly located cysteine clusters in Drosophila receptors (and other LRR proteins), when mated 'top' to 'bottom', form a compact module with paired termini that can be inserted between any pair of LRRs without altering the overall fold of DTLR ectodomains; analogous 'extruded' domains decorate the structures of other proteins (Russell (1994) Protein Engin. 7:1407-1410).

Molecular design of the TH signaling domain.

Sequence comparison of Toll and IL-1 type-I (IL-R1) receptors has disclosed a distant resemblance of a -200 amino acid cytoplasmic domain that presumably mediates signaling by similar Rel-type transcription factors.

Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol.

12:393-416; and (Belvin and Anderson (1996) Ann. Rev.

Cell Develop. Biol. 12:393-416; and Wasserman (1993) Molec. Biol. Cell 4:767-771). More recent additions to this functional paradigm include a pair of plant disease resistance proteins from tobacco and flax that feature an N-terminal TH module followed by nucleotide-binding (NTPase) and LRR segments (Wilson, et al. (1997) Curr.

Biol. 7:175-178); by contrast, a 'death domain' precedes the TH chain of MyD88, an intracellular myeloid differentiation marker (Mitcham, et al. (1996) J. Biol.

Chem. 271:5777-5783; and Hardiman, et al. (1996) Oncogene 13:2467-2475) (Fig. New IL-1-type receptors include IL-1R3, an accessory signaling molecule, and orphan receptors IL-1R4 (also called ST2/Fit-1/Tl), IL-1R5 (IL- 1R-related protein), and IL-1R6 (IL-lR-related protein-2) (Mitcham, et al. (1996) J. Biol. Chem. 271:5777- 5783;Hardiman, et al. (1996) Oncogene 13:2467-2475). With the new human DTLR sequences, we have sought a structural definition of this evolutionary thread by analyzing the conformation of the common TH module: ten blocks of WO 01/90151 PCT/US01/16766 conserved sequence comprising 128 amino acids form the minimal TH domain fold; gaps in the alignment mark the likely location of sequence and length-variable loops (Fig. 2A-2B).

Two prediction algorithms that take advantage of the patterns of conservation and variation in multiply aligned sequences, PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (King and Sternberg (1996) Protein Sci. 5:2298- 2310), produced strong, concordant results for the TH signaling module (Fig. 2A-2B). Each block contains a discrete secondary structural element: the imprint of alternating -strands (labeled A-E) and a-helices (numbered 1-5) is diagnostic of.a P/a-class fold with ahelices on both faces of a parallel p-sheet. Hydrophobic P-strands A, C and D are predicted to form 'interior' staves in the P-sheet, while the shorter, amphipathic Pstrands B and E resemble typical 'edge' units (Fig. 2A- 2B). This assignment is consistent with a strand order of B-A-C-D-E in the core P-sheet (Fig. 2C); fold comparison ('mapping') and recognition ('threading') programs (Fischer, et al. (1996) FASEB J. 10:126-136) strongly return this doubly wound p/a topology. A surprising, functional prediction of this outline structure for tfe TH domain is that many of the conserved, charged residues in the multiple alignment map to the C-terminal end of the Psheet: residue Aspl6 (block numbering scheme Fig. 2A-2B) at the end of PA, Arg39 and Asp40 following OB, Glu75 in the first turn of a3, and the more loosely conserved Glu/Asp residues in the PD-a4 loop, or after PE (Fig. 2A- 2B). The location of four other conserved residues (Asp7, Glu28, and the Arg57-Arg/Lys58 pair) is compatible with a salt bridge network at the opposite, N-terminal end of the P-sheet (Fig. 2A-2B). Alignment of the other DTLR embodiments exhibit similar features, and peptide segments comprising these feataures, 20 amino acid segments containing them, are particularly important.

WO 01/90151 PCT/US01/16766 Signaling function depends on the structural integrity of the TH domain. Inactivating mutations or deletions within the module boundaries (Fig. 2A-2B) have been catalogued for IL-1R1 and Toll.. Heguy, et al. (1992) J. Biol. Chem. 267:2605-2609; Croston, et al. (1995) J.

Biol. Chem. 270;16514-16517; Schneider, et al. (1991) Genes Develop. 5:797-807; Norris and Manley. (1992) Genes Develop. 6:1654-1667; Norris and Manley (1995) Genes Develop. 9:358-369; and Norris and Manley (1996) Genes Develop. 10:862-872. The human DTLR1-5 chains extending past the minimal TH domain 0, 6, 22 and 18 residue lengths, respectively) are most closely similar to the stubby, 4 aa 'tail' of the Mst ORF. Toll and 18w display unrelated 102 and 207 residue tails (Fig. 2A-2B) that may negatively regulate the signaling of the fused TH domains.

Norris and Manley (1995) Genes Develop. 9:358-369; and Norris and Manley (1996) Genes Develop. 10:862-872.

The evolutionary relationship between the disparate proteins that carry the TH domain can best be discerned by a phylogenetic tree derived from the multiple alignment (Fig. Four principal branches segregate the plant proteins, the MyD88 factors, IL-1 receptors, and Toll-like molecules; the latter branch clusters the Drosophila and human DTLRs.

Chromosomal dispersal of human DTLR genes.

In order to investigate the genetic linkage of the nascent human DTLR gene family, we mapped the chromosomal loci of four of the five genes by FISH (Fig. The DTLR1 gene has previously been charted by the human genome project: an STS database locus (dbSTS accession number G06709, corresponding to STS WI-7804 or SHGC-12827) exists for the Humrsc786 cDNA (Nomura, et al. (1994) DNA Res.

1:27-35) and fixes the gene to chromosome 4 marker interval D4S1587-D42405 (50-56 cM) circa 4 pl 4 This assignment has recently been corroborated by FISH WO 01/90151 PCT/US01/16766 analysis. Taguchi, et al. (1996) Genomics 32:486-488. In the present work, we reliably assign the remaining DTLR genes to loci on chromosome 4q32 (DTLR2), 4q35 (DTLR3), 9q32-33 (DTLR4) and 1q33.3 (DTLR5). During the course of this work, an STS for the parent DTLR2 EST (cloneID 80633) has been generated (dbSTS accession number T57791 for STS SHGC-33147) and maps to the chromosome 4 marker interval D4S424-D4S1548 (14.3-153 cM) at 4q32 -in accord with our findings. There is a -50 cM gap between DTLR2 and DTLR3 genes on the long arm of chromosome 4.

DTLR genes are differentially expressed.

Both Toll and 18w have complex spatial and temporal patterns of expression in Drosophila that may point to functions beyond embryonic patterning. St. Johnston and Nusslein-Volhard (1992) Cell 68:201-219; Morisato and Anderson (1995) Ann. Rev. Genet. 29:371-399; Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol. 12:393-416; Lemaitre, et al. (1996) Cell 86:973-983; Chiang and Beachy (1994) Mech. Develop. 47:225-239; and Eldon, et al. (1994) Develop. 120:885-899. We have examined the spatial distribution of DTLR transcripts by mRNA blot analysis with varied human tissue and cancer cell lines using radiolabeled DTLR cDNAs (Fig. DTLR1 is found to be ubiquitously expressed, and at higher levels than the other receptors. Presumably reflecting alternative splicing, 'short' 3.0 kB and 'long' 8.0 kB DTLR1 transcript forms are present in ovary and spleen, respectively (Fig. 5, panels A and A cancer cell mRNA panel also shows the prominent overexpression of DTLR1 in a Burkitt's Lymphoma Raji cell line (Fig. 5, panel C).

DTLR2 mRNA is less widely expressed than DTLR1, with a kB species detected in lung and a 4.4 kB transcript evident in heart, brain and muscle. The tissue distribution pattern of DTLR3 echoes that of DTLR2 (Fig.

panel DTLR3 is also present as two major WO 01/90151 PCT/US01/16766 transcripts of approximately 4.0 and 6.0 kB in size, and the highest levels of expression are observed in placenta and pancreas. By contrast, DTLR4 and DTLR5 messages appear to be extremely tissue-specific. DTLR4 was detected only in placenta as a single transcript of kB in size. A faint 4.0 kB signal was observed for in ovary and peripheral blood monocytes.

Components of an evolutionarily ancient regulatory system.

The original molecular blueprints and divergent fates of signaling pathways can be reconstructed by comparative genomic approaches. Miklos and Rubin (1996) Cell 86:521- 529; Chothia (1994) Develop. 1994 Suppl., 27-33; Banfi, et al. (1996) Nature Genet. 13:167-174; and Wang, et al.

(1996) J. Biol. Chem. 271:4468-4476. We have used this logic to identify an emergent gene family in humans, encoding five receptor paralogs at present, DTLRs that are the direct evolutionary counterparts of a Drosophila gene family headed by Toll (Figs. The conserved architecture of human and fly DTLRs, conserved LRR ectodomains and intracellular TH modules (Fig. 1), intimates that the robust pathway coupled to Toll in Drosophila 7) survives in vertebrates. The best evidence borrows from a reiterated pathway: the manifold IL-1 system and its repertoire of receptor-fused TH domains, IRAK, NF-KB and I-KB homologs (Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol. 12:393-416; Wasserman (1993) Molec. Biol. Cell 4:767-771; Hardiman, et al. (1996) Oncogene 13:2467-2475; and Cao, et al. (1996) Science 271:1128-1131); a Tube-like factor has also been characterized. It is not known whether DTLRs can productively couple to the IL-1R signaling machinery, or instead, a parallel set of proteins is used. Differently from IL-1 receptors, the LRR cradle of human DTLRs is predicted to retain an affinity for Spatzle/Trunk-related WO 01/90151 PCT/US01/16766 cystine-knot factors; candidate DTLR ligands (called PENs) that fit this mold have been isolated.

Biochemical mechanisms of signal transduction can be gauged by the conservation of interacting protein folds in a pathway. Miklos and Rubin (1996) Cell 86:521-529; Chothia (1994) Develop. 1994 Suppl., 27-33. At present, the Toll signaling paradigm involves some molecules whose roles are narrowly defined by their structures, actions or fates: Pelle is a Ser/Thr kinase (phosphorylation), Dorsal is an NF-KB-like transcription factor (DNA-binding) and Cactus is an ankyrin-repeat inhibitor (Dorsal binding, degradation). Belvin and Anderson (1996) Ann. Rev. Cell Develop. Biol. 12:393-416. By contrast, the functions of the Toll TH domain and Tube remain enigmatic. Like other cytokine receptors (Heldin (1995) Cell 80:213-223), ligand-mediated dimerization of Toll appears to be the triggering event: free cysteines in the juxtamembrane region of Toll create constitutively active receptor pairs (Schneider, et al. (1991) Genes Develop. 5:797-807), and chimeric Torso-Toll receptors signal as dimers (Galindo, et al. (1995) Develop. 121:2209-2218); yet, severe truncations or wholesale loss of the Toll ectodomain results in promiscuous intracellular signaling (Norris and Manley (1995) Genes Develop. 9:358-369; and Winans and Hashimoto (1995) Molec. Biol. Cell 6:587-596), reminiscent of oncogenic receptors with catalytic domains (Heldin (1995) Cell 80:213-223). Tube is membrane-localized, engages the N-terminal (death) domain of Pelle and is phosphorylated, but neither Toll-Tube or Toll-Pelle interactions are registered by two-hybrid analysis (Galindo, et al. (1995) Develop. 121:2209-2218; and Grophans, et al. (1994) Nature 372:563-566); this latter result suggests that the conformational 'state' of the Toll TH domain somehow affects factor recruitment. Norris and Manley (1996) Genes Develop. 10:862-872; and Galindo, et al. (1995) Develop. 121:2209-2218.

WO 01/90151 PCT/US01/16766 At the heart of these vexing issues is the structural nature of the Toll TH module. To address this question, we have taken advantage of the evolutionary diversity of TH sequences from insects, plants and vertebrates, incorporating the human DTLR chains, and extracted the minimal, conserved protein core for structure prediction and fold recognition (Fig. The strongly predicted TH domain fold with its asymmetric cluster of acidic residues is topologically identical to the structures of response regulators in bacterial two-component signaling pathways (Volz (1993) Biochemistry 32:11741-11753; and Parkinson (1993) Cell 73:857-871) (Fig. 2A-2C). The prototype chemotaxis regulator CheY transiently binds a divalent cation in an 'aspartate pocket' at the C-end of the core P-sheet; this cation provides electrostatic stability and facilitates the activating phosphorylation of an invariant Asp. Volz (1993) Biochemistry 32:11741- 11753. Likewise, the TH domain may capture cations in its acidic nest, but activation, and downstream signaling, could depend on the specific binding of a negatively charged moiety: anionic ligands can overcome intensely negative binding-site potentials by locking into precise hydrogen-bond networks. Ledvina, et al. (1996) Proc.

Natl. Acad. Sci. USA 93:6786-6791. Intriguingly, the TH domain may not simply act as a passive scaffold for the assembly of a Tube/Pelle complex for Toll, or homologous systems in plants and vertebrates, but instead actively participate as a true conformational trigger in the signal transducing machinery. Perhaps explaining the conditional binding of a Tube/Pelle complex, Toll dimerization could promote unmasking, by regulatory receptor tails (Norris and Manley (1995) Genes Develop. 9:358-369; Norris and Manley (1996) Genes Develop. 10:862-872), or binding by small molecule activators of the TH pocket. However, 'free' TH modules inside the cell (Norris and Manley (1995) Genes Develop. 9:358-369; Winans and Hashimoto WO 01/90151 PCT/US01/16766 (1995) Molec. Biol. Cell 6:587-596) could act as catalytic, CheY-like triggers by activating and docking with errant Tube/Pelle complexes.

Morphogenetic receptors and immune.defense.

The evolutionary link between insect and vertebrate immune systems is stamped in DNA: genes encoding antimicrobial factors in insects display upstream motifs similar to acute phase response elements known to bind NF- KB transcription factors in mammals. Hultmark (1993) Trends Genet. 9:178-183. Dorsal, and two Dorsal-related factors, Dif and Relish, help induce these defense proteins after bacterial challenge (Reichhart, et al.

(1993) C. R. Acad. Sci. Paris 316:1218-1224; Ip, et al.

(1993) Cell 75:753-763; and Dushay, et al. (1996) Proc.

Natl. Acad. Sci. USA 93:10343-10347); Toll, or other DTLRs, likely modulate these rapid immune responses in adult Drosophila (Lemaitre, et al. (1996) Cell 86:973-983; and Rosetto, et al. (1995) Biochem. Biophys. Res. Commun.

209:111-116). These mechanistic parallels to the IL-1 inflammatory response in vertebrates are evidence of the functional versatility of the Toll signaling pathway, and suggest an ancient synergy between embryonic patterning and innate immunity (Belvin and Anderson (1996) Ann. Rev.

Cell Develop. Biol. 12:393-416; Lemaitre, et al. (1996) Cell 86:973-983; Wasserman (1993) Molec. Biol. Cell 4:767- 771; Wilson, et al. (1997) Curr. Biol. 7:175-178; Hultmark (1993) Trends Genet. 9:178-183; Reichhart, et al. (1993) C. R. Acad. Sci. Paris 316:1218-1224; Ip, et al. (1993) Cell 75:753-763; Dushay, et al. (1996) Proc. Natl. Acad.

Sci. USA 93:10343-10347; Rosetto, et al. (1995) Biochem.

Biophys. Res. Commun. 209:111-116; Medzhitov and Janeway (1997) Curr. Opin. Immunol. 9:4-9; and Medzhitov and Janeway (1997) Curr. Opin. Immunol. The closer homology of insect and human DTLR proteins invites an even stronger overlap of biological functions that supersedes WO 01/90151 PCT/US01/16766 the purely immune parallels to IL-1 systems, and lends potential molecular regulators to dorso-ventral and other transformations of vertebrate embryos. DeRobertis and Sasai (1996) Nature 380:37-40; and Arendt and NUbler-Jung (1997) Mech. Develop. 61:7-21.

The present description of an emergent, robust receptor family in humans mirrors the recent discovery of the vertebrate Frizzled receptors for Wnt patterning factors. Wang, et al. (1996) J. Biol. Chem. 271:4468- 4476. As numerous other cytokine-receptor systems have roles in early development (Lemaire and Kodjabachian (1996) Trends Genet. 12:525-531), perhaps the distinct cellular contexts of compact embryos and gangly adults simply result in familiar signaling pathways and their diffusible triggers having different biological outcomes at different times, morphogenesis versus immune defense for DTLRs. For insect, plant, and human Tollrelated systems (Hardiman, et al. (1996) Oncogene 13:2467- 2475; Wilson, et al. (1997) Curr. Biol. 7:175-178), these signals course through a regulatory TH domain that intriguingly resembles a bacterial transducing engine (Parkinson (1993) Cell 73:857-871).

In particular, the DTLR6 exhibits structural features which establish its membership in the family. Moreover, members of the family have been implicated in a number of significant developmental disease conditions and with function of the innate immune system. In particular, the DTLR6 has been mapped to the X chromosome to a location which is a hot spot for major developmental abnormalities.

See, The Sanger Center: human X chromosome website http://www.sanger.ac.uk/HGP/ChrX/index.shtml; and the Baylor College of Medicine Human Genome Sequencing website http://gc.bcm.tmc.edu:8088/cgi-bin/seq/home The accession number for the deposited PAC is AC003046. This accession number contains sequence from two PACs: RPC-164K3 and RPC-263P4. These two PAC WO 01/90151 PCT/US01/16766 sequences mapped on human chromosome Xp22 at the Baylor web site between STS markers DXS704 and DXS7166. This region is a "hot spot" for severe developmental abnormalities.

III. Amplification of DTLR fragment by PCR Two appropriate primer sequences are selected (see Tables 1 through 10). RT-PCR is used on an appropriate mRNA sample selected for the presence of message to produce a partial or full length cDNA, a sample which expresses the gene. See, Innis, et al. (eds.

1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA; and Dieffenbach and Dveksler (eds. 1995) PCR Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY. Such will allow determination of a useful sequence to probe for a full length gene in a cDNA library. The DTLR6 is a contiguous sequence in the genome, which may suggest that the other DTLRs are also. Thus, PCR on genomic DNA may yield full length contiguous sequence, and chromosome walking methodology would then be applicable. Alternatively, sequence databases will contain sequence corresponding to portions of the described embodiments, or closely reated forms, alternative splicing, etc. Expression cloning techniques also may be applied on cDNA libraries.

IV. Tissue distribution of DTLRs Message for each gene encoding these DTLRs has been detected. See Figures 5A-5F. Other cells and tissues will be assayed by appropriate technology, PCR, immunoassay, hybridization, or otherwise. Tissue and organ cDNA preparations are available, from Clontech, Mountain View, CA. Identification of sources of natural expression are useful, as described.

Southern Analysis: DNA (5 jg) from a primary amplifie cDNA library is digested with appropriate restriction WO 01/90151 PCT/US01/16766 enzymes to release the inserts, run on a 1% agarose gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH).

Samples for human mRNA isolation would typically include, peripheral blood mononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells), resting (T100); peripheral blood mononuclear cells, activated with anti-CD3 for 2, 6, 12 h pooled (T101); T cell, THO clone Mot 72, resting (T102); T cell, THO clone Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T103); T cell, THO clone Mot 72, anergic treated with specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 clone HY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06, anergic treated with specific peptide for 2, 6, 12 h pooled (T109); T cell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935, activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); T cells CD4+CD45RO- T cells polarized 27 days in anti-CD28, IL-4, and anti IFN-y, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h (T116); T cell tumor lines Jurkat and Hut78, resting (T117); T cell clones, pooled AD130.2, Tc783.12, Tc783.1 3 Tc783.58, Tc782.69, resting (T118); T cell random y5 T cell clones, resting (T119); Splenocytes, resting (B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); B cell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102); B cell line JY, activated with PMA and ionomycin for 1, 6 h pooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled, activated with PMA and ionomycin for 6 h (K101); NKL clone, derived from peripheral blood of LGL leukemia patient, IL-2 treated (K106); NK cytotoxic clone 640-A30-1, resting (K107); hematopoietic precursor line TF1, activated with PMA and ionomycin for 1, 6 h pooled (C100); U937 premonocytic line, resting (M100); U937 premonocytic line, activated WO 01/90151 PCT/US01/16766 with PMA and ionomycin for 1, 6 h pooled (M101); elutriated monocytes, activated with LPS, IFNy, for 1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated with LPS, IFNy, IL-10 for 2, 6, 12, 24 h pooled (M103); elutriated monocytes, activated with LPS, IFNy, anti-IL-10. for 4, 16 h pooled (M106); elutriated monocytes, activated with LPS, IFNy, IL-10 for 4, 16 h pooled (M107); elutriated monocytes, activated LPS for 1 h (M108); elutriated monocytes, activated LPS for 6 h (M109); DC 70% CDla+, from CD34+ GM-CSF, TNFa 12 days, resting (D101); DC 70% CDla+, from CD34+ GM-CSF, TNFa 12 days, activated with PMA and ionomycin for 1 hr (D102); DC CDla+, from CD34+ GM-CSF, TNFa 12 days, activated with PMA and ionomycin for 6 hr (D103); DC 95% CDla+, from CD34+ GM-CSF, TNFa 12 days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled (D104); DC 95% CD14+, ex CD34+ GM-CSF, TNF( 12 days FACS sorted, activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CDla+ CD86+, from CD34+ GM-CSF, TNFa 12 days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5 days, resting (D107); DC from monocytes GM- CSF, IL-4 5 days, resting (D108); DC from monocytes GM- CSF, IL-4 5 days, activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFa, monocyte supe for 4, 16 h pooled (D110); leiomyoma L11 benign tumor (X101); normal myometrium M5 (0115); malignant leiomyosarcoma GS1 (X103); lung fibroblast sarcoma line MRC5, activated with PMA and ionomycin for 1, 6 h pooled (C101); kidney epithelial carcinoma cell line CHA, activated with PMA and ionomycin for 1, 6 h pooled (C102); kidney fetal 28 wk male (0100); lung fetal 28 wk male (0101); liver fetal 28 wk male (0102); heart fetal 28 wk male (0103); brain fetal 28 wk male (0104); gallbladder fetal 28 wk male (0106); small intestine fetal 28 wk male (0107); adipose tissue fetal 28 wk male (0108); ovary fetal 25 wk female (0109); uterus fetal 25 wk female WO 01/90151 PCT/US01/16766 (0110); testes fetal 28 wk male (0111); spleen fetal 28 wk male (0112); adult placenta 28 wk (0113); and tonsil inflamed, from 12 year old (X100).

Samples for mouse mRNA isolation can include, e.g.: resting mouse fibroblastic L cell line (C200); Braf:ER (Braf fusion to.estrogen receptor) transfected cells, control (C201); T cells, TH1 polarized (Mell4 bright, CD4+ cells from spleen, polarized for.7 days with IFN-y and anti IL-4; T200); T cells, TH2 polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IL-4 and anti-IFN-y; T201); T cells, highly TH1 polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T202); T cells, highly TH2 polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T203); CD44- CD25+ pre T cells, sorted from thymus (T204); TH1 T cell clone D1.1, resting for 3 weeks after last stimulation with antigen (T205); TH1 T cell clone D1.1, 10 jg/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, resting for 3 weeks after last stimulation with antigen (T207); TH2 T cell clone g/ml ConA stimulated 15 h (T208); Mell4+ naive T cells from spleen, resting (T209); Mell4+ T cells, polarized to Thl with IFN-y/IL-12/anti-IL-4 for 6, 12, 24 h pooled (T210); Mell4+ T cells, polarized to Th2 with IL-4/anti- IFN-y for 6, 13, 24 h pooled (T211); unstimulated mature B cell leukemia cell line A20 (B200); unstimulated B cell line CH12 (B201); unstimulated large B cells from spleen (B202); B cells from total spleen, LPS activated (B203); metrizamide enriched dendritic cells from spleen, resting (D200); dendritic cells from bone marrow, resting (D201); monocyte cell line RAW 264.7 activated with LPS 4 h (M200); bone-marrow macrophages derived with GM and M-CSF (M201); macrophage cell line J774, resting (M202); macrophage cell line J774 LPS anti-IL-lO at 0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line J774 LPS WO 01/90151 PCT/US01/16766 at 0.5, 1, 3, 5, 12 h pooled(M204); aerosol challenged mouse lung tissue, Th2 primers, aerosol OVA challenge 7, 14, 23 h pooled (see Garlisi, et al. (1995) Clinical Immunology and Immunopathology 75:75-83; X206); Nippostrongulus-infected lung tissue (see Coffman, et al.

(1989) Science 2.45:308-310; X200); total adult lung, normal (0200); total lung, rag-1 (see Schwarz, et al.

(1993) Immunodeficiency 4:249-252; 0205); IL-10 K.O.

spleen (see Kuhn, et al. (1991) Cell 75:263-274; X201); total adult spleen, normal (0201); total spleen, rag-i (0207); IL-10 K.O. Peyer's patches (0202); total Peyer's patches, normal (0210); IL-10 K.O. mesenteric lymph nodes (X203); total mesenteric lymph nodes, normal (0211); IL- K.O. colon (X203); total colon, normal (0212); NOD mouse pancreas (see Makino, et al. (1980) Jikken Dobutsu 29:1-13; X205); total thymus, rag-1 (0208); total kidney, rag-l (0209); total heart, rag-i (0202); total brain, rag- 1 (0203); total testes, rag-1 (0204); total liver, rag-i (0206); rat normal joint tissue (0300); and rat arthritic joint tissue (X300).

The DTLR10 has been found to be highly expressed in precursor dendritic cell type 2 (pDC2). See, e.g., Rissoan, et al. (1999) Science 283:1183-1186; and Siegal, et al. (1999) Science 284:1835-1837. However, it is not expressed on monocytes. The restricted expression of reinforces the suggestions of a role for the receptor in host immune defense. The pDC2 cells are natural interferon producing cells (NIPC), which produce large amounts of IFNc in response to Herpes simplex virus infection.

V. Cloning of species counterparts of DTLRs Various strategies are used to obtain species counterparts of these DTLRs, preferably from other primates. One method is by cross hybridization using closely related species DNA probes. It may be useful to go into evolutionarily similar species as intermediate WO 01/90151 PCT/US01/16766 steps. Another method is by using specific PCR primers based on the identification of blocks of similarity or difference between particular species, human, genes, areas of highly conserved or nonconserved polypeptide or nucleotide sequence. Alternatively, antibodies may be used for expression cloning.

VI. Production of mammalian DTLR protein An appropriate, GST, fusion construct is engineered for expression, in E. coli. For example, a mouse IGIF pGex plasmid is constructed and transformed into E. coli. Freshly transformed cells are grown in LB medium containing 50 gg/ml ampicillin and induced with IPTG (Sigma, St. Louis, MO). After overnight induction, the bacteria are harvested and the pellets containing the DTLR protein are isolated. The pellets are homogenized in TE buffer (50 mM Tris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters. This material is passed through a microfluidizer (Microfluidics, Newton, MA) three times.

The fluidized supernatant is spun down on a Sorvall GS-3 rotor for 1 h at 13,000 rpm. The resulting supernatant containing the DTLR protein' is filtered and passed over a glutathione-SEPHAROSE column equilibrated in 50 mM Trisbase pH 8.0. The fractions containing the DTLR-GST fusion protein are pooled and cleaved with thrombin (Enzyme Research Laboratories, Inc., South Bend, IN). The cleaved pool is then passed over a Q-SEPHAROSE column equilibrated in 50 mM Tris-base. Fractions containing DTLR are pooled and diluted in cold distilled H20, to lower the conductivity, and passed back over a fresh Q-Sepharose column, alone or in succession with an immunoaffinity antibody column.. Fractions containing the DTLR protein are pooled, aliquoted, and stored in the -70° C freezer.

Comparison of the CD spectrum with DTLR1 protein may suggest that the protein is correctly folded. See Hazuda, et al. (1969) J. Biol. Chem. 264:1689-1693.

WO 01/90151 PCT/US01/16766 VII. Biological Assays with DTLRs Biological assays will generally be directed to the ligand binding feature of the prote.in or to the kinase/phosphatase activity of the-receptor. The activity will typically be reversible, as are many other enzyme actions, and will mediate phosphatase or phosphorylase activities, which activities are easily measured by standard procedures. See, Hardie, et al. (eds.

1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al. (1991) Meth. Enzymol.

200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. .Quant. Biol. 56:449-463; and Parker, et al.

(1993) Nature 363:736-738.

The family of interleukin Is contains molecules, each of which is an important mediator of inflammatory disease.

For a comprehensive review, see Dinarello (1996) "Biologic basis for interleukin-1 in disease" Blood 87:2095-2147.

There are suggestions that the various Toll ligands may play important roles in the initiation of disease, particularly inflammatory responses. The finding of novel proteins related to the IL-1 family furthers the identification of molecules that provide the molecular basis for initiation of disease and allow for the development of therapeutic strategies of increased range and efficacy.

VIII. Preparation of antibodies specific for, DTLR4 Inbred Balb/c mice are immunized intraperitoneally with recombinant forms of the protein, purified DTLR4 or stable transfected NIH-3T3 cells. Animals are boosted at appropriate time points with protein, with or without additional adjuvant, to further stimulate antibody production. Serum is collected, or hybridomas produced with harvested spleens.

WO 01/90151 PCT/US01/16766 Alternatively, Balb/c mice are immunized with cells transformed with the gene or fragments thereof, either endogenous or exogenous cells, or with isolated membranes enriched for expression of the antigen. Serum is collected at the appropriate time,.typically after numerous further administrations. Various gene therapy techniques may be useful, in producing protein in situ, for generating an immune response.

Monoclonal antibodies may be made. For example, splenocytes are fused with an appropriate fusion partner and hybridomas are selected in growth medium by standard procedures. Hybridoma supernatants are screened for the presence of antibodies which bind to the desired DTLR, by ELISA or other assay. Antibodies which specifically recognize specific DTLR embodiments may also be selected or prepared.

In another method, synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press. In appropriate situations, the binding reagent is either labeled as described above, fluorescence or otherwise, or immobilized to a substrate for panning methods. Nucleic acids may also be introduced into cells in an animal to produce the antigen, which serves to elicit an immune response. See, e.g., Wang, et al. (.1993) Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniques 16:616-619; and Xiang, et al. (1995) Immunity 2: 129-135.

IX. Production of fusion proteins with, Various fusion constructs are made with DTLR5. This portion of the gene is fused to an epitope tag, a FLAG tag, or to a two hybrid system construct. See, e.g., Fields and Song (1989) Nature 340:245-246.

WO 01/90151 PCT/US01/16766 The epitope tag may be used in an expression cloning procedure with detection with anti-FLAG antibodies to detect a binding partner, ligand for the respective The two hybrid system may also be used to isolate proteins which specifically bind to X. Chromosomal mapping of DTLRs Chromosome spreads are prepared. In situ hybridization is performed on chromosome preparations obtained from phytohemagglutinin-stimulated lymphocytes cultured for 72 h. 5-bromodeoxyuridine is added for the final seven hours of culture (60 jg/ml of medium), to ensure a posthybridization chromosomal banding of good quality.

An appropriate fragment, a PCR fragment, amplified with the help of primers on total B cell cDNA template, is cloned into an appropriate vector. The vector is labeled by nick-translation with 3 H. The radiolabeled probe is hybridized to metaphase spreads as described in Mattei, et al. (1985) Hum. Genet. 69:327-331.

After coating with nuclear track emulsion (KODAK NTB2), slides are exposed, for 18 days at 40 C. To avoid any slipping of silver grains during the banding procedure, chromosome spreads are first stained with buffered Giemsa solution and metaphase photographed. Rbanding is then performed by the fluorochrome-photolysis- Giemsa (FPG) method and metaphases rephotographed before analysis.

Alternatively, FISH can be performed, as described above. The DTLR genes are located on different chromosomes. DTLR2 and DTLR3 are localized to human chromosome 4; DTLR4 is localized to human chromosome 9, and DTLR5 is localized to human chromosome 1. See Figures 4A-4D.

WO 01/90151 PCT/US01/16766 XI. Structure activity relationship Information on the criticality of particular residues is determined using standard procedures and analysis.

Standard mutagenesis analysis is performed, by generating many different variants.at determined positions, at the positions identified above, and evaluating biological activities of the variants. This may be performed to the extent of determining positions which modify activity, or to focus on specific positions to determine the residues which can be substituted to either retain, block, or modulate biological activity.

Alternatively, analysis of natural variants can indicate what positions tolerate natural mutations. This may result from populational analysis of variation among individuals, or across strains or species. Samples from selected individuals are analyzed, by PCR analysis and sequencing. This allows evaluation of population polymorphisms.

XI. Isolation of a ligand for a DTLR A DTLR can be used as a specific binding reagent to identify its binding partner, by taking advantage of its specificity of binding, much like an antibody would be used. A binding reagent is either labeled as described above, fluorescence or otherwise, or immobilized to a substrate for panning methods.

The binding composition is used to screen an expression library made from a cell line which expresses a binding partner, ligand, preferably membrane associated. Standard staining techniques are used to detect or sort surface expressed ligand, or surface expressing transformed cells are screened by panning.

Screening of intracellular expression is performed by various staining or immunofluorescence procedures. See also McMahan, et al. (1991) EMBO J. 10:2821-2832.

WO 01/90151 PCT/US01/16766 For example, on day 0, precoat 2-chamber permanox slides with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min at room temperature. Rinse once with PBS.

Then plate COS cells at 2-3 x 105 cells per chamber in ml of growth media. Incubate overnight at 37' C.

On day 1 for each sample, prepare 0.5 ml of a solution of 66 Lg/ml DEAE-dextran, 66 pM chloroquine, and 4 pg DNA in serum free DME. For each set, a positive control is prepared, of DTLR-FLAG cDNA at 1 and 1/200 dilution, and a negative mock. Rinse cells with serum free DME. Add the DNA solution and incubate 5 hr at 37" C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth medium and incubate overnight.

On day 2, change the medium. On days 3 or 4, the cells are fixed and stained. Rinse the cells twice with Hank's Buffered Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3X with HBSS. The slides may be stored at -80" C after all liquid is removed. For each chamber, 0.5 ml incubations are performed as follows. Add HBSS/saponin with 32 pl/ml of 1 M NaN 3 for 20 min. Cells are then washed with HBSS/saponin 1X. Add appropriate DTLR or DTLR/antiboy complex to cells and incubate for 30 min. Wash cells twice with HBSS/saponin. If appropriate, add first antibody for 30 min. Add second antibody, Vector anti-mouse antibody, at 1/200 dilution, and incubate for min. Prepare ELISA solution, Vector Elite ABC horseradish peroxidase solution, and preincubate for min. Use, 1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash cells twice with HBSS, second wash for 2 min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of H 2 0 2 per 5 ml of glass distilled water. Carefully remove chamber and rinse slide in water. Air dry for a few minutes, then add 1 Sdrop of Crystal Mount and a cover slip. Bake for 5 min at Z 85-90 0

C.

Evaluate positive staining of pools and progressively subclone to isolation of single genes responsible for the binding.

00 Alternatively, DTLR reagents are used to affinity 00 ,purify or sort out cells expressing a putative ligand.

\O

IND 10 See, Sambrook, et al. or Ausubel, et al.

Another strategy is to screen for a membrane bound Sreceptor by panning. The receptor cDNA is constructed as described above. The ligand can be immobilized and used to immobilize expressing cells. Immobilization may be achieved by use of appropriate antibodies which recognize, a FLAG sequence of a DTLR fusion construct, or by use of antibodies raised against the first antibodies.

Recursive cycles of selection and amplification lead to enrichment of appropriate clones and eventual isolation of receptor expressing clones.

Phage expression libraries can be screened by mammalian DTLRs. Appropriate label techniques, e.g., anti-FLAG antibodies, will allow specific labeling of appropriate clones.

All citations herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

166 v It is to be understood that a reference herein to a prior art document does not constitute an admission that Sthe document forms part of the common general knowledge in the art in Australia or any other country.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The 00 specific embodiments described herein are offered by way 00 ,I of example only, and the invention is to be limited by the

\O

1 0 terms of the appended claims, along with the full scope of equivalents to which such claims are entitled; and the 166a WO 01/90151 PCT/US01/16766 invention is not to be limited by the specific embodiments that have been presented herein by way of example.

Humans have two distinct types of dendritic cell (DC) precursors. Peripheral blood monocytes (pDCl) give rise to immature myeloid DCs after culturing with GMCSF and IL- 4. These immature cells become mature myeloid DCs (DC1) after stimulation with CD40 ligand (CD40L). The CD4+CD3- CDllc- plasmacytoid cells (pDC2) from blood or tonsils give rise to a distinct type of immature DC after culture with IL-3, and differentiate into mature DCs (DC2) after stimulation. Rissoan, et al. (1999) Science 283:1183-1186.

Siegal, et al. (1999) Science 284:1835-1837, show that pDC2 is the "Natural Interferon Producing Cell" (IPC). Interferons (IFNs) are the most important cytokines in antiviral immune responses. "Natural IFNproducing cells" (NIPCs) in human blood express CD4 and major histocompatibility complex class II proteins, but have not been isolated and further characterized because of their rarity, rapid apoptosis, and lack of lineage markers. Purified NIPCs are here shown to be the CD4(+)CDllc- type 2 dendritic cell precursors (pDC2s), which produce 200 to 1000 times more IFN than other blood cells after microbial challenge. pDC2s are thus an effector cell type of the immune system, critical for antiviral and antitumor immune responses. They are implicated as important cells in HIV infected patients Toll-like receptor (TLR) molecules belong to the IL- 1/Toll receptor family. Ligands for TLR2 and TLR4 have been identified, and their functions are related to the host immune response to microbial antigen or injury.

Takeuchi, et al. (1999) Immunity 11:443-451; and Noshino, et al. (1999) J. Immunol. 162:3749-3752. The pattern of expression of TLRs seem to be restricted. Muzio, et al.

(2000) J. Immunol. 164:5998-6004. With these findings that: i) TLR10 is highly expressed and restricted in pDC2s, and ii) pDC2 is the NIPC, it is likely that will play an important role in the host's innate immune response.

Claims (10)

1. An isolated antigenic polypeptide comprising the amino Sacid sequence of SEQ ID NO: 12. S

2. A fusion protein comprising the polypeptide of claim 1. 00 00

3. An isolated antibody or antibody fragment which ~1O 10 specifically binds to the polypeptide of claim 1.

4. An isolated nucleic acid encoding the polypeptide of claim 1.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the vector of claim

7. A process for recombinantly producing a polypeptide comprising culturing the host cell of claim 6 under conditions in which the polypeptide is expressed.

8. An isolated antigenic polypeptide according to claim 1, substantially as herein described with reference to any one of the Examples or Figures.

9. An isolated antibody or antibody fragment according to claim 3, substantially as herein described with reference to any one of the Examples or Figures. 168 Is0

10. An isolated nucleic acid according to claim 4, substantially as herein described with reference to any one of the Examples or Figures. Dated this 1st day of June 2006 SCHERING CORPORATION By its Patent Attorneys GRIFFITH HACK 00 O 0 0I 169