AU741504B2 - Receptor activator of NF-kappa B, receptor is member of TNF receptor superfamily - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Immunex Corporation Actual Inventor(s): DIRK M ANDERSON, LAURENT J GALIBERT, EUGENE MARASKOVSKY Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: RECEPTOR ACTIVATOR OF NF-KAPPA B, RECEPTOR IS MEMBER OF TNF RECEPTOR

SUPERFAMILY

S Our Ref: 610431 POF Code: 44735/44735 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 60o6q Ti'-

TITLE

RECEPTOR ACTIVATOR OF NF-KAPPA B, RECEPTOR IS MEMBER OF TNF RECEPTOR SUPERFAMILY The present application is a divisional from Australian patent application number 57184/98 the entire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION The present invention relates generally to the field of cytokine receptors, and more specifically to cytokine receptor/ligand pairs having immunoregulatory activity.

BACKGROUND OF THE INVENTION Efficient functioning of the immune system requires a fine balance between cell proliferation and differentiation and cell death, to ensure that the immune system is capable of reacting to foreign, but not self antigens. Integral to the process of regulating the immune and inflammatory response are various members of the Tumor Necrosis Factor (TNF) Receptor/Nerve Growth Factor Receptor superfamily (Smith et al., Science 248:1019; 1990). This family of receptors includes two different TNF receptors (Type I and Type II; Smith et al., supra; and Schall et al., Cell 61:361, 1990), nerve growth factor receptor (Johnson et al., Cell 47:545, 1986), B cell antigen CD40 (Stamenkovic et al., EMBO J. 8:1403, 1989), CD27 (Camerini et al., J. Immunol. 147:3165, 1991), (Durkop et al., Cell 68:421, 1992), T cell antigen OX40 (Mallett et al., EMBO J. 9:1063, 1990). human Fas antigen (Itoh et al., Cell 66:233, 1991), murine 4-1BB receptor (Kwon 20 et al., Proc. Natl. Acad. Sci. USA 86:1963, 1989) and a receptor referred to as Apoptosis- *Inducing Receptor (AIR; USSN 08/720,864, filed October 4, 1996).

CD40 is a receptor present on B lymphocytes, epithelial cells and some carcinoma S cell lines that interacts with a ligand found on activated T cells, CD40L (USSN S 08/249,189, filed May 24, 1994). The interaction of this ligand/receptor pair is essential 25 for both the cellular and humoral immune response. Signal transduction via CD40 is mediated through the association of the cytoplasmic domain of this molecule with members of the TNF receptor-associated factors (TRAFs; Baker and Reddy, Oncogene 12:1, 1996).

It has recently been found that mice that are defective in TRAF3 expression due to a targeted disruption in the gene encoding TRAF3 appear normal at birth but develop *30 progressive hypoglycemia and depletion of peripheral white cells, and die by about ten days of age (Xu et al., Immunity 5:407, 1996). The immune responses of chimeric mice reconstituted with TRAF3- 7 fetal liver cells resemble those of CD40-deficient mice, although TRAF3- 7 B cells appear to be functionally normal.

The critical role of TRAF3 in signal transduction may be in its interaction with one of the other members of the TNF receptor superfamily, for example, CD30 or CD27, which are present on T cells. Alternatively, there may be other, as yet unidentified 2 members of this family of receptor that interact with TRAF3 and play an important role in postnatal development as well as in the development of a competent immune system. Identifying additional members of the TNF receptor superfamily would provide an additional means of regulating the immune and inflammatory response, as well as potentially providing further insight into post-natal development in mammals.

SUMMARY OF THE INVENTION The present invention provides a method of screening a molecule for its capacity to antagonize or agonize RANK, said method comprising an assay selected from the group consisting of: a) i) contacting a RANK polypeptide with a RANKL polypeptide in the presence of the molecule under conditions that permit binding of the RANK and RANKL polypeptides; ii) detecting the binding of the RANK and RANKL polypeptides; iii) determining that the molecule is a RANK antagonist if the amount of RANK-RANKL binding detected is decreased when the molecule is present; and :oo: 20 iv) determining that the molecule is a RANK agonist if the amount of RANK biological activity detected is increased when the molecule is present; b) i) triggering RANK in a culture of cells that is transfected with a plasmid 25 in which an NF-KB-dependent promoter is operably linked to a reporter gene, wherein during said triggering the cultured cells are exposed to the molecule; ii) detecting a product of the reporter gene in said exposed cells; iii) determining that the molecule is a RANK antagonist if the amount of the reporter gene product detected in said exposed cells is decreased; and iv) determining that the molecule is a RANK agonist if the amount of the _reporter gene product detected in said exposed cells is increased; IC W.WOMVS1WmnWArdLM19M)d= 2a c) i) incubating the molecule and a RANKL polypeptide with isolated CD1a' dendritic cells; ii) monitoring the formation of dendritic cell clusters; iii) determining that the molecule is a RANK antagonist if dendritic cell clustering is decreased when the molecule is present; and iv) determining that the molecule is a rank agonist if dendritic cell clustering is increased when the molecule is present; d) i) incubating the molecule and a RANKL polypeptide with isolated CDla* dendritic cells in a medium comprising GM-CSF, IL-4, TNF-a and Flt3L; ii) irradiating the incubated dendritic cells; iii) monitoring the allostimulatory capacity of the irradiated dendritic cells S 15 in a mixed lymphocyte reaction; iv) determining that the molecule is a RANK antagonist if the allostimulatory capacity of the irradiated dendritic cells is decreased when the molecule is present; and v) determining that the molecule is a RANK agonist if the allostimulatory capacity of the irradiated dendritic cells is increased when the molecule is present; e) i) triggering RANK in the presence of the molecule in cells capable of expressing RANK; ii) measuring TNF-a production or NF-KB activation in the cells; iii) determining that the molecule is a RANK antagonist if TNF-a production or NF-cB activation in said cells is decreased when the molecule is present; and iv) determining that the molecule is a RANK agonist if TNF-a production or NF-KB activation in said cells is increased when the molecule is present; IC W:lon SharonSJJspecisp20619(2).doc 2b f) i) activating peripheral blood T cells in culture in the presence of the molecule and one or more cytokines capable of upregulating RANK, said cytokine(s) being selected from the group consisting of TGF-p and 1IL-4; ii) incubating said T cells for at least 4 days; iii) detecting viable T cells in said culture; iv) determining that the molecule is a RANK antagonist if the number of viable T cells detected is reduced when the molecule is present; and v) determining that the molecule is a RANK agonist if the number of viable T cells detected is increased when the molecule is present; g) i) incubating the molecule with cells expressing RANK; ii) detecting TNF-a or activated NF-KB in the incubated cells; iii) determining that the molecule is a RANK antagonist if the amount of TNF-oa or activated NF-KB detected is decreased when themolecule is *-iv present; and *iv) determining that the molecule is a RANK antagonist if the amount of TNF-a or activated NF-KB detected is increased when the molecule is 20 present; and h) i) incubating the molecule with cells expressing RANK, said cells being 9* transfected with a plasmid in which an NF-KB-dependent promoter is operably linked to a reporter gene; ii) detecting a product of the reporter gene in the incubated cells; iii) determining that the molecule is a RANK antagonist if the amount of the reporter gene product detected is decreased when the molecule is present; and iv) determining that the molecule is a RANK agonist if the amount of the reporter gene product detected in said incubated cells is increased when the molecule is present.

[C W:%o-aSh.,Sjjspe."lp206 l 9 2 ),d.c 2c Another aspect of the invention is a method of therapeutically inhibiting RANK activity in a subject having a tumor or neoplastic disease, septic shock, an inflammatory condition or a graft-versus-host reaction comprising administering to said subject a RANK polypeptide selected from the group consisting of: a) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, of SEQ ID NO:6, and amino acids 3-232 of SEQ ID NO:8; b) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID wherein x is amino acid 1 or 31 of SEQ ID NO:15, and amino acids 3-232 of SEQ ID NO:8; 15 c) amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any S. one of amino acids 24-33, inclusive, or fragments thereof; and d) a RANK/GST fusion protein.

A further aspect of the present invention is a use of a composition comprising 20 a RANK antagonist for the manufacture of a medicament for therapeutically inhibiting o: RANK activity in a mammal.

a a IC W.Vion SharonlSJJspeCisp2619(3).doc 2d The present invention also provides a novel receptor, referred to as RANK (for receptor activator of NF-KB), that is a member of the TNF receptor superfamily. RANK is a Type I transmembrane protein having 616 amino acid residues that interacts with TRAF3.

Triggering of RANK by over-expression, co-expression of RANK and membrane bound RANK ligand (RANKL), and with addition of soluble RANKL or agonistic antibodies to RANK results in the upregulation of the transcription factor NF-KB, a ubiquitous transcription factor that is most extensively utilized in cells, of the immune system.

Soluble forms of the receptor can be prepared and used to interfere with signal transduction through membrane-bound RANK, and hence upregulation of NF-KB; accordingly, pharmaceutical compositions comprising soluble forms of the novel receptor are also provided. Inhibition of NF-KB by RANK antagonists may be useful in ameliorating negative effects of an inflammatory response that result from triggering of RANK, for example in treating toxic shock or sepsis, graft-versus-host reactions, or acute inflammatory reactions. Soluble forms of the receptor will also be useful in vitro to screen for agonists or antagonists of RANK activity.

The cytoplasmic domain of RANK will be useful in developing assays for inhibitors of signal transduction, for example, for screening for molecules that inhibit S interaction of RANK with TRAF2 or TRAF3. Deleted forms and fusion proteins 20 comprising the novel receptor are also disclosed.

The present invention also identifies a counterstructure, or ligand, for RANK, referred to as RANKL. RANKL is a Type 2 transmembrane protein with an intracellular domain of less than about 50 amino acids, a transmembrane domain and an extracellular domain of from about 240 to 250 amino acids. Similar to other members of the TNF family to which it belongs, RANKL has a 'spacer' region between the transmembrane 25 domain and the receptor binding domain that is not necessary for receptor binding.

Accordingly, soluble forms of RANKL can comprise the entire extracellular domain or fragments thereof that include the receptor binding region.

These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention.

IC W:\ona\SnaronSJJsp e c isp2061 9 (2).aoc BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 demonstrates the influence of RANK.Fc and hRANKL on activated T cell growth. Human peripheral blood T cells were cultured as described in Example 12; viable T cell recovery was determined by triplicate trypan blue countings.

Figure 2 illustrates the ability of RANKL to induce human DC cluster formation.

Functionally mature dendritic cells (DC) were generated in vitro from CD34+ bone marrow (BM) progenitors and cultured as described in Example 13. CD a+ DC were cultured in a cytokine cocktail alone (Figure 2A), in cocktail plus CD40L (Figure 2B), RANKL (Figure 2C), or heat inactivated (AH) RANKL (Figure 2D), and then photographed using an inversion microscope.

Figure 3 demonstrates that RANKL enhances DC allo-stimulatory capacity.

Allogeneic T cells were incubated with varying numbers of irradiated DC cultured as described in Example 13. The cultures were pulsed with 3 H]-thymidine and the cells harvested onto glass fiber sheets for counting. Values represent the mean standard deviation (SD) of triplicate cultures.

Figure 4 presents an alignment of human RANK with other TNFR family members in the region of structurally conserved extracellular cysteine-rich pseudorepeats. Predicted disulfide linkages (DS1-DS3) are indicated. RANK and CD40 contain identical amino acid substitutions C^G) eliminating DS2 in the second pseudorepeat.

Figure 5 presents an alignment of human RANKL with other TNF family members.

DETAILED DESCRIPTION OF THE INVENTION o. A novel partial cDNA insert with a predicted open reading frame having some similarity to CD40 was identified in a database containing sequence information from 25 cDNAs generated from human bone marrow-derived dendritic cells The insert was used to hybridize to colony blots generated from a DC cDNA library containing full-length cDNAs. Several colony hybridizations were performed, and two clones (SEQ ID NOs:l and 3) were isolated. SEQ ID NO:5 shows the nucleotide and amino acid sequence of a predicted full-length protein based on alignment of the overlapping sequences of SEQ ID 30 NOs:l and 3.

RANK is a member of the TNF receptor superfamily; it most closely resembles in the extracellular region. Similar to CD40, RANK associates with TRAF2 and TRAF3 (as determined by co-immunoprecipitation assays substantially as described by Rothe et al., Cell 83:1243, 1995). TRAFs are critically important in the regulation of the immune and inflammatory response. Through their association with various members of the TNF receptor superfamily, a signal is transduced to a cell. That signal results in the proliferation, differentiation or apoptosis of the cell, depending on which receptor(s) is/are triggered and which TRAF(s) associate with the receptor(s); different signals can be 1' W transduced to a cell via coordination of various signaling events. Thus, a signal transduced through one member of this family may be proliferative, differentiative or apoptotic, depending on other signals being transduced to the cell, and/or the state of differentiation of the cell. Such exquisite regulation of this proliferative/apoptotic pathway is necessary to develop and maintain protection against pathogens; imbalances can result in autoimmune disease.

RANK is expressed on epithelial cells, some B cell lines, and on activated T cells.

However, its expression on activated T cells is late, about four days after activation. This time course of expression coincides with the expression of Fas, a known agent of apoptosis. RANK may act as an anti-apoptotic signal, rescuing cells that express

RANK

from apoptosis as CD40 is known to do. Alternatively, RANK may confirm an apoptotic signal under the appropriate circumstances, again similar to CD40. RANK and its ligand are likely to play an integral role in regulation of the immune and inflammatory response.

Moreover, the post-natal lethality of mice having a targeted disruption of the TRAF3 gene demonstrates the importance of this molecule not only in the immune response but in development. The isolation of RANK, as a protein that associates with TRAF3, and its ligand will allow further definition of this signaling pathway, and development of diagnostic and therapeutic modalities for use in the area of autoimmune and/or inflammatory disease.

DNAs, Proteins and Analogs The present invention provides isolated RANK polypeptides and analogs (or muteins) thereof having an activity exhibited by the native molecule RANK muteins that bind specifically to a RANK ligand expressed on cells or immobilized on a surface or 25 to RANK-specific antibodies; soluble forms thereof that inhibit RANK ligand-induced signaling through RANK). Such proteins are substantially free of contaminating endogenous materials and, optionally, without associated native-pattern glycosylation.

Derivatives of RANK within the scope of the invention also include various structural forms of the primary proteins which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, a RANK protein may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction. The primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants. Covalent derivatives are prepared by linking particular functional groups to amino acid side chains or at the N- or C-termini.

Derivatives of RANK may also be obtained by the action of cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and IV lysine residues. The inventive proteins may also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide-activated, bisoxiraneactivated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking). Once bound to a substrate, the proteins may be used to selectively bind (for purposes of assay or purification) antibodies raised against the proteins or against other proteins which are similar to RANK or RANKL, as well as other proteins that bind RANK or RANKL or homologs thereof.

Soluble forms of RANK are also within the scope of the invention. The nucleotide and predicted amino acid sequence of the RANK is shown in SEQ ID NOs: 1 through 6.

Computer analysis indicated that the protein has an N-terminal signal peptide; the predicted cleavage site follows residue 24. Those skilled in the art will recognize that the actual cleavage site may be different than that predicted by computer analysis. Thus, the Nterminal amino acid of the cleaved peptide is expected to be within about five amino acids on either side of the predicted, preferred cleavage site following residue 24. Moreover a soluble form beginning with amino acid 33 was prepared; this soluble form bound RANKL. The signal peptide is predicted to be followed by a 188 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 383 amino acid cytoplasmic tail.

Soluble RANK comprises the signal peptide and the extracellular domain (residues 1 to 213 of SEQ ID NO:6) or a fragment thereof. Alternatively, a different signal peptide can be substituted for the native leader, beginning with residue 1 and continuing through a residue selected from the group consisting of amino acids 24 through 33 (inclusive) of SEQ ID NO:6. Moreover, fragments of the extracellular domain will also provide soluble forms of RANK. Fragments can be prepared using known techniques to isolate a desired portion 25 of the extracellular region, and can be prepared, for example, by comparing the extracellular region with those of other members of the TNFR family and selecting forms similar to those prepared for other family members. Alternatively, unique restriction sites or PCR techniques that are known in the art can be used to prepare numerous truncated forms which can be expressed and analyzed for activity.

30 Fragments can be prepared using known techniques to isolate a desired portion of the extracellular region, and can be prepared, for example, by comparing the extracellular region with those of other members of the TNFR family (of which RANK is a member) and selecting forms similar to those prepared for other family members. Alternatively, unique restriction sites or PCR techniques that are known in the art can be used to prepare numerous truncated forms which can be expressed and analyzed for activity.

Other derivatives of the RANK proteins within the scope of this invention include covalent or aggregative conjugates of the proteins or their fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or posttranslationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall the yeast a-factor leader).

Protein fusions can comprise peptides added to facilitate purification or identification of RANK proteins and homologs poly-His). The amino acid sequence of the inventive proteins can also be linked to an identification peptide such as that described by Hopp et al., Bio/Technology 6:1204 (1988). Such a highly antigenic peptide provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. The sequence of Hopp et al. is also specifically cleaved by bovine mucosal enterokinase, allowing removal of the peptide from the purified protein. Fusion proteins capped with such peptides may also be resistant to intracellular degradation in E. coli.

Fusion proteins further comprise the amino acid sequence of a RANK linked to an immunoglobulin Fc region. An exemplary Fc region is a human IgG, having a nucleotide an amino acid sequence set forth in SEQ ID NO:8. Fragments of an Fc region may also be used, as can Fc muteins. For example, certain residues within the hinge region of an Fc region are critical for high affinity binding to FcyRI. Canfield and Morrison Exp. Med.

173:1483; 1991) reported that Leu(23 4 and Leu(235)were critical to high affinity binding of IgG3 to FcyRI present on U937 cells. Similar results were obtained by Lund et al. (J.

Immunol. 147:2657, 1991; Molecular hnmunol. 29:53, 1991). Such mutations, alone or in combination, can be made in an IgG, Fc region to decrease the affinity of IgG, for FcR.

Depending on the portion of the Fc region used, a fusion protein may be expressed as a dimer, through formation of interchain disulfide bonds. If the fusion proteins are made 25 with both heavy and light chains of an antibody, it is possible to form a protein oligomer with as many as four RANK regions.

In another embodiment, RANK proteins further comprise an oligomerizing peptide such as a leucine zipper domain. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988). Leucine zipper domain is a term used to refer to a conserved peptide domain present in these (and other) proteins, which is responsible for dimerization of the proteins. The leucine zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptad repeat, with four or five leucine residues interspersed with other amino acids. Examples of leucine zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Two nuclear transforming proteins, fos and jun, also exhibit leucine zipper domains, as does the gene product of the murine proto-oncogene, c-mnyc (Landschulz et al., Science 240:1759, 1988). The products of the nuclear oncogenesfos andjun comprise leucine zipper domains preferentially form a heterodimer (O'Shea et al., Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989). The leucine zipper domain is necessary for biological activity (DNA binding) in these proteins.

The fusogenic proteins of several different viruses, including paramyxovirus, coronavirus, measles virus and many retroviruses, also possess leucine zipper domains (Buckland and Wild, Nature 338:547,1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and Human Retroviruses 6:703, 1990). The leucine zipper domains in these fusogenic viral proteins are near the transmembrane region of the proteins; it has been suggested that the leucine zipper domains could contribute to the oligomeric structure of the fusogenic proteins. Oligomerization of fusogenic viral proteins is involved in fusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523, 1991).

Leucine zipper domains have also been recently reported to play a role in oligomerization of heat-shock transcription factors (Rabindran et al., Science 259:230, 1993).

Leucine zipper domains fold as short, parallel coiled coils. (O'Shea et al., Science 254:539; 1991) The general architecture of the parallel coiled coil has been well characterized, with a "knobs-into-holes" packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). The dimer formed by a leucine zipper domain is stabilized by the heptad repeat, designated (abcdefg)n according to the notation of McLachlan and Stewart Mol. Biol. 98:293; 1975), in which residues a and d are generally hydrophobic residues, with d being a leucine, which line up on the same face of a helix. Oppositelycharged residues coraimonly occur at positions g and e. Thus, in a parallel coiled coil S formed from two helical leucine zipper domains, the "knobs" formed by the hydrophobic side chains of the first helix are packed into the "holes" formed between the side chains of the second helix.

The leucine residues at position d contribute large hydrophobic stabilization energies, and are important for dimer formation (Krystek et al., Int. J. Peptide Res.

38:229, 1991). Lovejoy et al. recently reported the synthesis of a triple-stranded a-helical S bundle in which the helices run up-up-down (Science 259:1288, 1993). Their studies confirmed that hydrophobic stabilization energy provides the main driving force for the formation of coiled coils from helical monomers. These studies also indicate that electrostatic interactions contribute to the stoichiometry and geometry of coiled coils.

Several studies have indicated that conservative amino acids may be substituted for individual leucine residues with minimal decrease in the ability to dimerize; multiple changes, however, usually result in loss of this ability (Landschulz et al., Science 243:1681, 1989; Turner and Tjian, Science 243:1689, 1989; Hu et al., Science 250:1400, 1990). van Heekeren et al. reported that a number of different amino residues can be substituted for the leucine residues in the leucine zipper domain of GCN4, and further found that some GCN4 proteins containing two leucine substitutions were weakly active (Nucl. Acids Res. 20:3721, 1992). Mutation of the first and second heptadic leucines of the leucine zipper domain of the measles virus fusion protein (MVF) did not affect syncytium formation (a measure of virally-induced cell fusion); however, mutation of all four leucine residues prevented fusion completely (Buckland et al., J. Gen. Virol. 73:1703, 1992). None of the mutations affected the ability of MVF to form a tetramer.

Amino acid substitutions in the a and d residues of a synthetic peptide representing the GCN4 leucine zipper domain have been found to change the oligomerization properties of the leucine zipper domain (Alber, Sixth Symposium of the Protein Society, San Diego, CA). When all residues at position a are changed to isoleucine, the leucine zipper still forms a parallel dimer. When, in addition to this change, all leucine residues at position d are also changed to isoleucine, the resultant peptide spontaneously forms a trimeric parallel' coiled coil in solution. Substituting all amino acids at position d with isoleucine and at position a with leucine results in a peptide that tetramerizes. Peptides containing these substitutions are still referred to as leucine zipper domains.

Also included within the scope of the invention are fragments or derivatives of the intracellular domain of RANK. Such fragments are prepared by any of the hereinmentioned techniques, and include peptides that are identical to the cytoplasmic domain of RANK as shown in SEQ ID NO:5, or of murine RANK as shown in SEQ ID NO: 15, and those that comprise a portion of the cytoplasmic region. All techniques used in preparing soluble forms may also be used in preparing fragments or analogs of the cytoplasmic domain RT-PCR techniques or use of selected restriction enzymes to prepare truncations). DNAs encoding all or a fragment of the intracytoplasmic domain will be o. useful in identifying other proteins that are associated with RANK signalling, for example :o using the immunoprecipitation techniques described herein, or another technique such as a 25 yeast two-hybrid system (Rothe et al., supra).

The present invention also includes RANK with or without associated native-pattern glycosylation. Proteins expressed in yeast or mammalian expression systems, COS-7 cells, may be similar or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of DNAs encoding the inventive proteins in bacteria such as E. coli provides non-glycosylated molecules. Functional mutant analogs of RANK protein having inactivated

N-

glycosylation sites can be produced by oligonucleotide synthesis and ligation or by sitespecific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems.

N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asnwhere A, is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate.

Such a site can be eliminated by substituting another amino acid for Asn or for residue

Z,

(V I deleting Asn or Z, or inserting a non-Z amino acid between Al and Z, or an amino acid other than Asn between Asn and A.

RANK protein derivatives may also be obtained by mutations of the native RANK or subunits thereof. A RANK mutated protein, as referred to herein, is a polypeptide homologous to a native RANK protein, respectively, but which has an amino acid sequence different from the native protein because of one or a plurality of deletions, insertions or substitutions. The effect of any mutation made in a DNA encoding a mutated peptide may be easily determined by analyzing the ability of the mutated peptide to bind its counterstructure in a specific manner. Moreover, activity of RANK analogs, muteins or derivatives can be determined by any of the assays described herein (for example, inhibition of the ability of RANK to activate transcription).

Analogs of the inventive proteins may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation. Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present.

When a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be considered. Subunits of the inventive proteins may be constructed by deleting terminal or internal residues or sequences. Soluble forms of RANK can be readily prepared and tested for their ability to inhibit RANK-induced NF- :r rB activation. Polypeptides corresponding to the cytoplasmic regions, and fragments thereof (for example, a death domain) can be prepared by similar techniques. Additional 25 guidance as to the types of mutations that can be made is provided by a comparison of the sequence of RANK to proteins that have similar structures, as well as by performing structural analysis of the inventive RANK proteins.

Generally, substitutions should be made conservatively; the most preferred substitute amino acids are those which do not affect the biological activity of RANK ability of the inventive proteins to bind antibodies to the corresponding native protein in 0 substantially equivalent a manner, the ability to bind the counterstructure in substantially the 0600 same manner as the native protein, the ability to transduce a RANK signal, or ability to induce NF-icB activation upon overexpression in transient transfection systems, for o example). Examples of conservative substitutions include substitution of amino acids outside of the binding domain(s) (either ligand/receptor or antibody binding areas for the extracellular domain, or regions that interact with other, intracellular proteins for the cytoplasmic domain), and substitution of amino acids that do not alter the secondary and/or tertiary structure of the native protein. Additional examples include substituting one aliphatic residue for another, such as fle, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.

Mutations in nucleotide sequences constructed for expression of analog proteins or fragments thereof must, of course, preserve the reading frame phase of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins which would adversely affect translation of the mRNA.

Not all mutations in the nucleotide sequence which encodes a RANK protein or fragments thereof will be expressed in the final product, for example, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see EPA 75,444A, incorporated herein by reference), or to provide codons that are more readily translated by the selected host, the well-known E. coli preference codons for E. coli expression.

Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants, random mutagenesis may be conducted and the expressed mutated proteins screened for the desired activity. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic *30 Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent NOs.

4,518,584 and 4,737,462 disclose suitable techniques, and are incorporated by reference herein.

Other embodiments of the inventive proteins include RANK polypeptides encoded by DNAs capable of hybridizing to the DNA of SEQ ID NO:6 under moderately stringent .35 conditions (prewashing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of 50C, 5 X SSC, overnight) to the DNA sequences encoding RANK, or more preferably under stringent conditions (for example, hybridization in 6 X SSC at 63"C overnight; washing in 3 X SSC at 55C), and other sequences which are degenerate to those which encode the RANK. In one embodiment, RANK polypeptides are at least about 70% identical in amino acid sequenze to the amino acid sequence of native RANK protein as set forth in SEQ ID NO:5. In a preferred embodiment, RANK polypeptides are at least about 80% identical in amino acid sequence to the native form of RANK; most preferred polypeptides are those that are at least about 90% identical to native

RANK.

Percent identity may be determined using a computer program, for example, the GAP computer program described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). For fragments derived from the RANK protein, the identity is calculated based on that portion of the RANK protein that is present in the fragment The biological activity of RANK analogs or muteins can be determined by testing the ability of the analogs or muteins to inhibit activation of transcription, for example as described in the Examples herein. Alternatively, suitable assays, for example, an enzyme immunoassay or a dot blot, employing an antibody that binds native RANK, or a soluble form of RANKL, can be used to assess the activity of RANK analogs or muteins, as can assays that employ cells expressing RANKL. Suitable assays also include, for example, signal transduction assays and methods that evaluate the ability of the cytoplasmic region of RANK to associate with other intracellular proteins TRAFs 2 and 3) involved in signal transduction will also be useful to assess the activity of RANK analogs or muteins.

Such methods are well known in the art.

Fragments of the RANK nucleotide sequences are also useful. In one embodiment, such fragments comprise at least about 17 consecutive nucleotides, preferably at least about 25 nucleotides, more preferably at least 30 consecutive nucleotides, of the RANK DNA 25 disclosed herein. DNA and RNA complements of such fragments are provided herein, S along with both single-stranded and double-stranded forms of the RANK DNA of SEQ ID and those encoding the aforementioned polypeptides. A fragment of RANK DNA generally comprises at least about 17 nucleotides, preferably from about 17 to about :nucleotides. Such nucleic acid fragments (for example, a probe corresponding to the extracellular domain of RANK) are used as a probe or as primers in a polymerase chain reaction (PCR).

The probes also find use in detecting the presence of RANK nucleic acids in in vitro assays and in such procedures as Northern and Southern blots. Cell types S: expressing RANK can be identified as well. Such procedures are well known, and the skilled artisan can choose a probe of suitable length, depending on the particular intended application. For PCR, 5' and 3' primers corresponding to the termini of a desired RANK DNA sequence are employed to amplify that sequence, using conventional techniques.

0 If Other useful fragments of the RANK nucleic acids are antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target RANK mRNA (sense) or RANK DNA (antisense) sequences.

The ability to create an antisense or a sense oligonucleotide, based upon a cDNA sequence for a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

Uses of DNAs, Proteins and Analogs The RANK DNAs, proteins and analogs described herein will have numerous uses, including the preparation of pharmaceutical compositions. For example, soluble forms of RANK will be useful as antagonists of RANK-mediated NF-KB activation, as well as to inhibit transduction of a signal via RANK. RANK compositions (both protein and DNAs) will also be useful in development of both agonistic and antagonistic antibodies to RANK.

The inventive DNAs are useful for the expression of recombinant proteins, and as probes for analysis (either quantitative or qualitative) of the presence or distribution of RANK transcripts.

The inventive proteins will also be useful in preparing kits that are used to detect soluble RANK or RANKL, or monitor RANK-related activity, for example, in patient specimens. RANK proteins will also find uses in monitoring RANK-related activity in other samples or compositions, as is necessary when screening for antagonists or mimetics of this activity (for example, peptides or small molecules that inhibit or mimic, respectively, the interaction). A variety of assay formats are useful in such kits, including (but not limited to) ELISA, dot blot, solid phase binding assays (such as those using a biosensor), rapid format assays and bioassays.

The purified RANK according to the invention will facilitate the discovery of inhibitors of RANK, and thus, inhibitors of an inflammatory response (via inhibition of NF-KB activation). The use of a purified RANK polypeptide in the screening for potential inhibitors is important and can virtually eliminate the possibility of interfering reactions with *contaminants. Such a screening assay can utilize either the extracellular domain of RANK, 30 the intracellular domain, or a fragment of either of these polypeptides. Detecting the inhibiting activity of a molecule would typically involve use of a soluble form of RANK derived from the extracellular domain in a screening assay to detect molecules capable of binding RANK and inhibiting binding of, for example, an agonistic antibody or RANKL, or using a polypeptide derived from the intracellular domain in an assay to detect inhibition of the interaction of RANK and other, intracellular proteins involved in signal transduction.

Moreover, in vitro systems can be used to ascertain the ability of molecules to antagonize or agonize RANK activity. Included in such methods are uses of RANK chimeras, for example, a chimera of the RANK intracellular domain and an extracellular domain derived from a protein having a known ligand. The effects on signal transduction of various molecule can then be monitored by utilizing the known ligand to transduce a signal.

In addition, RANK polypeptides can also be used for structure-based design of RANK-inhibitors. Such structure-based design is also known as "rational drug design." The RANK polypeptides can be three-dimensionally analyzed by, for example, X-ray crystallography, nuclear magnetic resonance or homology modeling, all of which are wellknown methods. The use of RANK structural information in molecular modeling software systems to assist in inhibitor design is also encompassed by the invention. Such computerassisted modeling and drug design may utilize information such as chemical conformational analysis, electrostatic potential of the molecules, protein folding, etc. A particular method of the invention comprises analyzing the three dimensional structure of RANK for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described above.

Expression of Recombinant RANK The proteins of the present invention are preferably produced by recombinant DNA methods by inserting a DNA sequence encoding RANK protein or an analog thereof into a recombinant expression vector and expressing the DNA sequence in a recombinant expression system under conditions promoting expression. DNA sequences encoding the proteins provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being inserted in a recombinant expression vector and expressed in a recombinant transcriptional unit.

Recombinant expression vectors include synthetic or cDNA-derived DNA fragments encoding RANK, or homologs, muteins or bioequivalent analogs thereof, operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation, as described in detail below. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated.

DNA regions are operably linked when they are functionally related to each other.

For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame.

DNA

sequences encoding RANK, or homologs or analogs thereof which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA.

Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMI (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectors include the P-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.

412, 1982). A particularly useful bacterial expression system employs the phage k PL promoter and cI857ts thermolabile repressor. Plasmid vectors available'from the American Type Culture Collection which incorporate derivatives of the k PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).

Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3 -phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate *30 dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322 for 35 selection and replication in E. coli (Ampr gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and a-factor secretion leader.

The ADH2 promoter has been described by Russell et al. Biol. Chem. 258:2674. 1982) and Beier et al. (Nature 300:724, 1982). The yeast a-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. See, Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc. Natl. Acad.

Sci. USA 81:5330, 1984. The leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the BglI site located in the viral origin of replication is included. Further, viral genomic promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen.

Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol.

3:280, 1983).

A useful system for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A preferred eukaryotic vector for expression of RANK DNA is referred to as pDC406 (McMahan et al., EMBO J. 10:2821, 1991), and includes regulatory sequences derived from SV40, human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV). Other preferred vectors include pDC409 and pDC410, which are derived from pDC406. pDC410 was derived from pDC406 by substituting the EBV origin of replication with sequences encoding the SV40 large T antigen. pDC409 differs from pDC406 in that a Bgl II restriction site outside of the multiple cloning site has been deleted, making the Bgl II site within the multiple cloning site unique.

A useful cell line that allows for episomal replication of expression vectors, such as pDC406 and pDC409, which contain the EBV origin of replication, is CV-1/EBNA (ATCC CRL 10478). The CV-1/EBNA cell line was derived by transfection of the CV-1 cell line with a gene encoding Epstein-Barr virus nuclear antigen-I (EBNA-1) and constitutively express EBNA-1 driven from human CMV immediate-early enhancer/promoter.

Host Cells Transformed host cells are cells which have been transformed or transfected with expression vectors constructed using recombinant DNA techniques and which contain sequences encoding the proteins of the pres-nt invention. Transformed host cells may express the desired protein (RANK, or homologs or analogs, thereof), but host cells transformed for purposes of cloning or amplifying the inventive DNA do not need to express the protein. Expressed proteins will preferably be secreted into the culture supematant, depending on the DNA selected, but may be deposited in the cell membrane.

Suitable host cells for expression of proteins include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacillus spp. Higher eukaryotic cells include established cell lines of mammalian origin as described below.

Cell-free translation systems could also be employed to produce proteins using RNAs derived from the DNA constructs disclosed herein. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant disclosure of which is hereby incorporated by reference.

Prokaryotic expression hosts may be used for expression of RANK, or homologs or analogs thereof that do not require extensive proteolytic and disulfide processing.

Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host. Suitable prokaryotic hosts for transformation include E. coli S Bacillus subtilis, Salmonella typhimurium, and various species within the genera :.25 Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

Recombinant RANK may also be expressed in yeast hosts, preferably from the Saccharomyces species, such as S. cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of **30 replication from the 2g yeast plasmid or an autonomously replicating sequence

(ARS),

promoter, DNA encoding the protein, sequences for polyadenylation and transcription termination and a selection gene. Preferably, yeast vectors will include an origin of replication and selectable marker permitting transformation of both yeast and E. coli, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae trpI gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and a promoter derived from a highly expressed yeast gene to induce transcription of a structural sequence downstream. The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable yeast transformation protocols are known to those of skill in the art; an exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp+ transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 gg/ml adenine and 20 Rtg/ml uracil.

Host strains transformed by vectors comprising the ADH2 promoter may be grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 gg/ml adenine and 80 gg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4°C prior to further purification.

Various mammalian or insect cell culture systems can be employed to express recombinant protein. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, CV-I/EBNA (ATCC CRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

'25 Purification of Recombinant RANK Purified RANK, and homologs or analogs thereof are prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts. For example, supernatants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.

Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix can comprise a counter structure protein or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Gel filtration chromatography also provides a means of purifying the inventive proteins.

Affinity chromatography is a particularly preferred method of purifying RANK and homologs thereof. For example, a RANK expressed as a fusion protein comprising an immunoglobulin Fc region can be purified using Protein A or Protein G affinity chromatography. Moreover, a RANK protein comprising an oligomerizing zipper domain may be purified on a resin comprising an antibody specific to the oligomerizing zipper domain. Monoclonal antibodies against the RANK protein may also be useful in affinity chromatography purification, by utilizing methods that are well-known in the art. A ligand may also be used to prepare an affinity matrix for affinity purification of RANK.

Finally, one or more reversed-phase high performance liquid chromatography

(RP-

HPLC) steps employing hydrophobic RP-HPLC media, silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a RANK composition.

Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express the inventive protein as a secreted protein .25 greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J: Chromnatog. 296:171, 1984). This reference describes two sequential, reversed-phase HPLC steps for purification of recombinant human GM-CSF on a preparative

HPLC

column.

Protein synthesized in recombinant culture is characterized by the presence of cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover the inventive protein from the culture. These components ordinarily will be of yeast, prokaryotic or non-human higher eukaryotic origin and preferably are present in innocuous contaminant quantities, on the order of less than :35 about 1 percent by weight. Further, recombinant cell culture enables the production of the inventive proteins free of other proteins which may be normally associated with the proteins as they are found in nature in the species of origin.

Uses and Administration of RANK Compositions The present invention provides methods of using therapeutic compositions comprising an effective amount of a protein and a suitable diluent and carrier, and methods for regulating an immune or inflammatory response. The use of RANK in conjunction with soluble cytokine receptors or cytokines, or other immunoregulatory molecules is also contemplated.

For therapeutic use, purified protein is administered to a patient, preferably a human, for treatment in a manner appropriate to the indication. Thus, for example, RANK protein compositions administered to regulate immune function can be given by bolus injection, continuous infusion, sustained release from implants, or other suitable technique.

Typically, a therapeutic agent will be administered in the form of a composition comprising purified RANK, in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed.

Ordinarily, the preparation of such protein compositions entails combining the inventive protein with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with conspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions sucrose) as diluents. Appropriate dosages can be determined in trials. The amount and frequency of administration will S depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.

Soluble forms of RANK and other RANK antagonists such as antagonistic monoclonal antibodies can be administered for the purpose of inhibiting RANK-induced induction of NF-KB activity. NF-KB is a transcription factor that is utilized extensively by cells of the immune system, and plays a role in the inflammatory response. Thus, inhibitors of RANK signalling will be useful in treating conditions in which signalling through RANK has given rise to negative consequences, for example, toxic or septic shock, or graft-versus-host reactions. They may also be useful in interfering with the role S of NF-KB in cellular transformation. Tumor cells are more responsive to radiation when their NF-KB is blocked; thus, soluble RANK (or other antagonists of RANK signalling) will be useful as an adjunct therapy for disease characterized by neoplastic cells that express

RANK.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and comprises", is not intended to exclude other additives, components, integers or steps".

The following examples arc offercd by way of illustration, and noi by way of limitation. Those skilled in the art will recognize that variations of the inventionl embodied in the examples can be miade, es)pecially in light of the teachings of lthe various references cited herein, the disclosures of which are incorporated by referencc.

EXAMPLE 1 The example describes the identification and isolation of a DNA encoding a novel member of the TNF receptor supcrfamily. A partial cDNA insert with a predicted open reading frame having some similarity to CD40 (a cell-surface antigen present on the surface of both normal and neoplastic human B cells that has been shown to play an important role in B-cell proliferation and differentiation; Stamenkovic et al., EMBO J. 8:1403, 1989), was identified in a database containing sequence information from cDNAs generated from human bone marrow-derived dendritic cells The insert was excised from the vector by restriction endonuclease digestion, gel purified, labeled with 2 p, and used to hybridize to colony blots generated from a DC cDNA library containing larger cDNA inserts using high stringency hybridization and washing techniques (hybridization in 5xSSC, formamide at 42 0 C overnight, washing in 0.5xSSC at 63°C); other suitable high stringency conditions are disclosed in Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1989), 9.52-9.55.

Initial experiments yielded a clone referred to as 9D-8A (SEQ ID NO:1); subsequent analysis indicated that this clone contained all but the extreme 5' end of a novel cDNA, with predicted intron sequence at the extreme 5' end (nucleotides 1-92 of SEQ ID NO:1).

SAdditional colony hybridizations were performed, and a second clone was isolated. The second clone, referred to as 9D-15C (SEQ ID NO:3), contained the 5' end without intron interruption but not the full 3end. SEQ ID NO:5 shows the nucleotide and amino acid sequence of a predicted full-length protein based on alignment of the overlapping sequences of SEQ ID NOs:1 and 3.

The encoded protein was designated RANK, for receptor activator of NF-KB. The cDNA encodes a predicted Type 1 transmembrane protein having 616 amino acid residues, with a predicted 24 amino acid signal sequence (the computer predicted cleavage site is after Leu24), a 188 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 383 amino acid cytoplasmic tail. The extracellular region of RANK displayed significant amino acid homology (38.5% identity, 52.3% similarity) to CD40. A cloning vector (pBluescriptSK-) containing human RANK sequence, designated pBluescript:huRANK (in E. coli DHIOB), was deposited with the American Type Culture Collection, Rockville, MD (ATCC) on December 20, 1996, under terms of the Budapest Treaty, and given accession number 98285.

EXAMPLE 2 This example describes construction of a RANK DNA construct to express a RANK/Fc fusion protein. A soluble form of RANK fused to the Fc region of human IgG, was constructed in the mammalian expression vector pDC409 (USSN 08/571,579). This expression vector encodes the leader sequence of the Cytomegalovirus (CMV) open reading frame R27080 (SEQ ID NO:9), followed by amino acids 33-213 of RANK, followed by a mutated form of the constant domain of human IgG, that exhibits reduced affinity for Fc receptors (SEQ ID NO:8; for the fusion protein, the Fc portion of the construct consisted of Arg3 through Lys232). An alternative expression vector encompassing amino acids 1-213 of RANK (using the native leader sequence) followed by the IgG, mutein was also prepared. Both expression vectors were found to induce high levels of expression of the RANK/Fc fusion protein in transfected cells.

To obtain RANK/Fc protein, a RANK/Fc expression plasmid is transfected into CV-1/EBNA cells, and supernatants are collected for about one week. The RANK/Fc fusion protein is purified by means well-known in the art for purification of Fc fusion proteins, for example, by protein A sepharose column chromatography according to manufacturer's recommendations Pharmacia, Uppsala, Sweden). SDSpolyacrylamide gel electrophoresis analysis indicted that the purified RANK/Fc protein migrated with a molecular weight of -55kDa in the presence of a reducing agent, and at a molecular weight of 10lkDa in the absence of a reducing agent.

N-terminal amino acid sequencing of the purified protein made using the CMV S R27080 leader showed 60% cleavage after Ala20,. 20% cleavage after Pro22 and cleavage after Arg28 (which is the Furin cleavage site; amino acid residues are relative to SEQ ID NO:9); N-terminal amino acid analysis of the fusion protein expressed with the 25 native leader showed cleavage predominantly after Gln25 (80% after Gln25 and 20% after Arg23; amino acid residues are relative to SEQ ID NO:6, full-length RANK). Both fusion proteins were able to bind a ligand for RANK is a specific manner they bound to the surface of various cell lines such as a murine thymoma cell line, EL4), indicating that the presence of additional amino acids at the N-terminus of RANK does not interfere with its ability to bind RANKL. Moreover, the construct comprising the CMV leader encoded RANK beginning at amino acid 33; thus, a RANK peptide having an N-terminus at an amino acid between Arg23 and Pro33, inclusive, is expected to be able to bind a ligand for RANK in a specific manner.

Other members of the TNF receptor superfamily have a region of amino acids between the transmembrane domain and the ligand binding domain that is referred to as a 'spacer' region, which is not necessary for ligand binding. In RANK, the amino acids between 196 and 213 are predicted to form such a spacer region. Accordingly, a soluble form of RANK that terminates with an amino acid in this region is expected to retain the ability to bind a ligand for RANK in a specific manner. Preferred C-terminal amino acids for soluble RANK peptides are selected from the group consisting of amino acids 213 and 196 of SEQ ID NO:6, although other amino acids in the spacer region may be utilized as a C-terminus.

EXAMPLE 3 This example illustrates the preparation of monoclonal antibodies against

RANK.

Preparations of purified recombinant RANK, for example, or transfected cells expressing high levels of RANK, are employed to generate monoclonal antibodies against

RANK

using conventional techniques, such as those disclosed in U.S. Patent 4,411,993.

DNA

encoding RANK can also be used as an immunogen, for example, as reviewed by Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies are likely to be useful in interfering with RANK-induced signaling (antagonistic or blocking antibodies) or in inducing a signal by cross-linking RANK (agonistic antibodies), as components of diagnostic or research assays for RANK or RANK activity, or in affinity purification of

RANK.

To immunize rodents, RANK immunogen is emulsified in an adjuvant (such as complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, MT), and injected in amounts ranging from 10-100 .tg subcutaneously into a selected rodent, for example, BALB/c mice or Lewis rats. DNA may be given intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has been found to be a suitable diluent for DNA-based antigens. Ten days to three weeks days later, the immunized animals are boosted with additional immunogen and periodically boosted thereafter on a weekly, biweekly or every third week immunization schedule.

5 Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay (antibody sandwich), ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other suitable assays, including FACS analysis.

Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are 30 sacrificed, splenocytes harvested, and fused to a murine myeloma cell line NS 1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a selective medium (for example, one containing hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit proliferation of non-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocyte hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivity with RANK, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Patent 4,703,004. A preferred screening technique is the antibody capture technique described by Beckman et al., J. Immunol.

144:4212 (1990). Positive clones are then injected into the peritoneal cavities of syngeneic rodents to produce ascites containing high concentrations mg/ml) of anti-RANK monoclonal antibody. The resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can also be used, as can affinity chromatography based upon binding to RANK protein.

Monoclonal antibodies were generated using RANK/Fc fusion protein as the immunogen. These reagents were screened to confirm reactivity against the RANK protein. Using the methods described herein to monitor the activity of the mAbs, both blocking antibodies that bind RANK and inhibit binding of a ligand to RANK) and non-blocking antibodies that bind RANK and do not inhibit ligand binding) were isolated.

EXAMPLE 4 This example illustrates the induction of NF-KB activity by RANK in 293/EBNA cells (cell line was derived by transfection of the 293 cell line with a gene encoding Epstein- Barr virus nuclear antigen-1 (EBNA-1) that constitutively express EBNA-1 driven from human CMV immediate-early enhancer/promoter). Activation of NF-KB activity was measured in 293/EBNA cells essentially as described by Yao et al. (Immunity 3:811, 1995). Nuclear extracts were prepared and analyzed for NF-KB activity by a gel retardation assay using a 25 base pair oligonucleotide spanning the NF-KB binding sites. Two million cells were seeded into 10 cm dishes two days prior to DNA transfection and cultured in DMEM-F12 media containing 2.5% FBS (fetal bovine serum). DNA transfections were performed as described herein for the IL-8 promoter/reporter assays.

*25 Nuclear extracts were prepared by solubilization of isolated nuclei with 400 mM NaCI (Yao et al., supra). Oligonucleotides containing an NF-KB binding site were annealed and endlabeled with 32 P using T4 DNA polynucleotide kinase. Mobility shift reactions contained 10 gLg of nuclear extract, 4 gg of poly(dI-dC) and 15,000 cpm labeled double-stranded oligonucleotide and incubated at room temperature for 20 minutes.

Resulting protein-DNA complexes were resolved on a 6% native polyacrylamide gel in 0.0 0.25 X Tris-borate-EDTA buffer.

Overexpression of RANK resulted in induction of NF-KB activity as shown by an appropriate shift in the mobility of the radioactive probe on the gel. Similar results were observed when RANK was triggered by a ligand that binds RANK and transduces a signal to cells expressing the receptor by co-transfecting cells with human RANK and murine RANKL DNA; see Example 7 below), and would be expected to occur when triggering is done with agonistic antibodies.

EXAMPLE This example describes a gene promoter/reporter system based on the human Interleukin-8 (IL-8 promoter used to analyze the activation of gene transcription in vivo.

The induction of human IL-8 gene transcription by the cytokines Interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-a) is known to be dependent upon intact NF-KB and NF-IL-6 transcription factor binding sites. Fusion of the cytokine-responsive IL-8 promoter with a cDNA encoding the murine IL-4 receptor (mIL-4R) allows measurement of promoter activation by detection of the heterologous reporter protein (mlL-4R) on the cell surface of transfected cells.

Human kidney epithelial cells (293/EBNA) are transfected (via the DEAE/DEXTRAN method) with plasmids encoding: the reporter/promoter construct (referred to as pIL-8rep), and the cDNA(s) of interest. DNA concentrations are always kept constant by the addition of empty vector DNA. The 293/EBNA cells are plated at a density of 2.5 x 104 cells/ml (3 ml/ well) in a 6 well plate and incubated for two days prior to transfection. Two days after transfection, the mIL-4 receptor is detected by a radioimmunoassay (RIA) described below.

In one such experiment, the 293/EBNA cells were co-transfected with DNA encoding RANK and with DNA encoding RANKL (see Example 7 below). Co-expression of this receptor and its counterstructure by cells results in activation of the signaling process of RANK. For such co-transfection studies, the DNA concentration/well for the DEAE transfection were as follows: 40 ng of pIL-8rep [pBluescriptSK- vector (Stratagene)]; 0.4 ng CD40 (DNA encoding CD40, a control receptor; pCDM8 vector); 0.4 ng RANK

(DNA

encoding RANK; pDC409 vector), and either 1-50 ng CD40L (DNA encoding the ligand for CD40, which acts as a positive control when co-transfected with CD40 and as a negative control when co-transfected with RANK; in pDC304) or RANKL (DNA encoding a ligand for RANK; in pDC406). Similar experiments can be done using soluble

RANKL

or agonistic antibodies to RANK to trigger cells transfected with RANK.

For the mIL-4R-specific RIA, a monoclonal antibody reactive with mlL-4R is labeled with 1251 via a Chloramine T conjugation method; the resulting specific activity is typically 1.5 x 1016 cpm/nmol. After 48 hours, transfected cells are washed once with media (DMEM/F12 5% FBS). Non-specific binding sites are blocked by the addition of pre-warmed binding media containing 5% non-fat dry milk and incubation at 37°C/5%

CO

2 in a tissue culture incubator for one hour. The blocking media is decanted and binding buffer containing '2I anti-mlL-4R (clone M rat IgG 1) is added to the cells and incubated with rocking at room temperature for 1 hour. After incubation of the cells with the radiolabeled antibody, cells are washed extensively with binding buffer (2X) and twice with phosphate-buffered saline (PBS). Cells are lysed in 1 ml of 0.5M NaOH, and total radioactivity is measured with a gamma counter.

Using this assay, 293/EBNA co-transfected with DNAs encoding RANK demonstrated transcriptional activation, as shown by detection of mulL-4R on the cell surface. Overexpression of RANK resulted in transcription of muIL-4R, as did triggering of the RANK by RANKL. Similar results are observed when RANK is triggered by agonistic antibodies.

EXAMPLE 6 This example illustrates the association of RANK with TRAF proteins. Interaction of RANK with cytoplasmic TRAF proteins was demonstrated by co-immunoprecipitation assays essentially as described by Hsu et al. (Cell 84:299; 1996). Briefly, 293/EBNA cells were co-transfected with plasmids that direct the synthesis of RANK and epitope-tagged (FLAG®; SEQ ID NO:7) TRAF2 or TRAF3. Two days after transfection, surface proteins were labeled with biotin-ester, and cells were lysed in a buffer containing 0.5% RANK and proteins associated with this receptor were immunoprecipitated with anti- RANK, washed extensively, resolved by electrophoretic separation on a 6-10% SDS polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane for Western blotting. The association of TRAF2 and TRAF3 proteins with RANK was visualized by probing the membrane with an antibody that specifically recognizes the FLAG® epitope. TRAFs 2 and 3 did not immunopreciptitate with anti-RANK in the absence of RANK expression.

EXAMPLE 7 This example describes isolation of a ligand for RANK, referred to as RANKL, by direct expression cloning. The ligand was cloned essentially as described in USSN 08/249,189, filed May 24, 1994 (the relevant disclosure of which is incorporated by reference herein), for CD40L. Briefly, a library was prepared from a clone of a mouse thymoma cell line EL-4 (ATCC TIB 39), called EL-40.5, derived by sorting five times with biotinylated CD40/Fc fusion protein in a FACS (fluorescence activated cell sorter). The cDNA library was made using standard methodology; the plasmid DNA was isolated and transfected into sub-confluent CV1-EBNA cells using a DEAE-dextran method.

Transfectants were screened by slide autoradiography for expression of RANKL using a two-step binding method with RANK/Fc fusion protein as prepared in Example 2 followed by radioiodinated goat anti-human IgG antibody.

A clone encoding a protein that specifically bound RANK was isolated and sequenced; the clone was referred to as 11H. An expression vector containing murine RANKL sequence, designated pDC406:muRANK-L (in E. coli DHIOB), was deposited with the American Type Culture Collection, Rockville, MD (ATCC) on December 1996, under terms of the Budapest Treaty, and given accession number 98284. The nucleotide sequence and predicted amino acid sequence of this clone are illustrated in SEQ ID NO: 10. This clone did not contain an initiator methionine; additional, full-length clones were obtained from a 7B9 library (prepared substantially as described in US patent 5,599,905, issued February 4, 1997); the 5' region was found to be identical to that of human RANKL as shown in SEQ ID NO: 12, amino acids 1 through 22, except for substitution of a Gly for a Thr at residue 9.

This ligand is useful for assessing the ability of RANK to bind RANKL by a number of different assays. For example, transfected cells expressing RANKL can be used in a FACS assay (or similar assay) to evaluate the ability of soluble RANK to bind RANKL. Moreover, soluble forms of RANKL can be prepared and used in assays that are known in the art ELISA or BIAcore assays essentially as described in USSN 08/249,189, filed May 24, 1994). RANKL is also useful in affinity purification of RANK, and as a reagent in methods to measure the levels of RANK in a sample. Soluble RANKL is also useful in inducing NF-KB activation and thus protecting cells that express RANK from apoptosis.

EXAMPLE 8 IThis example describes the isolation of a human RANK ligand (RANKL) using a PCR-based technique. Murine RANK ligand-specific oligonucleotide primers were used in S PCR reactions using human cell line-derived first strand cDNAs as templates. Primers e corresponded to nucleotides 478-497 and to the complement of nucleotides 858-878 of murine RANK ligand (SEQ ID NO:10). An amplified band approximately 400 bp in length from one reaction using the human epidermoid cell line KB (ATCC CCL-17) was gel purified, and its nucleotide sequence determined; the sequence was 85% identical to the corresponding region of murine RANK ligand, confirming that the fragment was from human RANKL.

To obtain full-length human RANKL cDNAs, two human RANKL-specific oligonucleotides derived from the KB PCR product nucleotide sequence were radiolabeled and used as hybridization probes to screen a human PBL cDNA library prepared in lambda gtlO (Stratagene, La Jolla, CA), substantially as described in US patent 5,599,905, issued February 4, 1997 Several positive hybridizing plaques were identified and purified, their inserts subcloned into pBluescript SK- (Stratagene, La Jolla, CA), and their nucleotide sequence determined. One isolate, PBL3, was found to encode most of the predicted human RANKL, but appeared to be missing approximately 200 bp of 5' coding region. A second isolate, PBL5 was found to encode much of the predicted human RANKL, including the entire 5' end and an additional 200 bp of 5' untranslated sequence.

The 5' end of PBL5 and the 3' end of PBL3 were ligated together to form a full length cDNA encoding human RANKL. The nucleotide and predicted amino acid sequence of the full-length human RANK ligand is shown in SEQ ID NO:12. Human RANK ligand shares 83% nucleotide and 84% amino acid identity with murine RANK ligand. A plasmid vector containing human RANKL sequence, designated pBluescript:huRANK-L (in E. coli DHIOB), was deposited with the American Type Culture Collection, Rockville, MD (ATCC) on March 11, 1997 under terms of the Budapest Treaty, and given accession number 98354.

Murine and human RANKL are Type 2 transmembrane proteins. Murine RANKL contains a predicted 48 amino acid intracellular domain, 21 amino acid transmembrane domain and 247 amino acid extracellular domain. Human RANKL contains a predicted 47 amino acid intracellular domain, 21 amino acid transmembrane domain and 249 amino acid extracellular domain.

EXAMPLE 9 This example describes the chromosomal mapping of human RANK using PCRbased mapping strategies. Initial human chromosomal assignments were made using RANK and RANKL-specific PCR primers and a BIOS Somatic Cell Hybrid PCRable DNA kit from BIOS Laboratories (New Haven, CT), following the manufacturer's instructions. RANK mapped to human chromosome 18; RANK ligand mapped to human chromosome 13. More detailed mapping was performed using a radiation hybrid mapping °o panel Genebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville, AL; described in Walter, MA et al., Nature Genetics 7:22-28, 1994). Data from this analysis was then submitted electronically to the MIT Radiation Hybrid Mapper (URL: http://wwwgenome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) following the instructions contained therein. This analysis yielded specific genetic marker names which, when submitted electronically to the NCBI Entrez browser (URL: http://www3.ncbi.nlm.nih.gov/htbinpost/Entrez/query?db=c&form=0), yielded the specific map locations. RANK mapped to chromosome 18q22.1, and RANKL mapped to chromosome 13q 14.

EXAMPLE This example illustrates the preparation of monoclonal antibodies against RANKL.

Preparations of purified recombinant RANKL, for example, or transfixed cells expressing high levels of RANKL, are employed to generate monoclonal antibodies against RANKL using conventional techniques, such as those disclosed in US Patent 4,411,993. DNA encoding RANKL can also be used as an immunogen, for example, as reviewed by Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies are likely to be useful in interfering with RANKL signaling (antagonistic or blocking antibodies), as components of diagnostic or research assays for RANKL or RANKL activity, or in affinity purification of

RANKL.

To immunize rodents, RANKL immunogen is emulsified in an adjuvant (such as complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, MT), and injected in amounts ranging from 10-100 gg subcutaneously into a selected rodent, for example, BALB/c mice or Lewis rats. DNA may be given intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has been found to be a suitable diluent for DNA-based antigens. Ten days to three weeks days later, the immunized animals are boosted with additional immunogen and periodically boosted thereafter on a weekly, biweekly or every third week immunization schedule.

Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay (antibody sandwich), ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other suitable assays, including FACS analysis.

Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to a murine myeloma cell line NS I or preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a selective medium (for example, one containing hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit proliferation of non-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocyte hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivity with RANKL, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem. 8:871 (1971) and in US Patent 4,703,004. A preferred screening technique 25 is the antibody capture technique described by Beckman et al., J. Immunol. 144:4212 (1990). Positive clones are then injected into the peritoneal cavities of syngeneic rodents to produce ascites containing high concentrations mg/ml) of anti-RANK monoclonal S antibody. The resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can also be S used, as can affinity chromatography based upon binding to RANKL protein. Using the methods described herein to monitor the activity of the mAbs, both blocking antibodies that bind RANKL and inhibit binding to RANK) and non-blocking antibodies that bind RANKL and do not inhibit binding) are isolated.

EXAMPLE 11 This example demonstrates that RANK expression can be up-regulated. Human peripheral blood T cells were purified by flow cytometry sorting or by negative selection using antibody coated beads, and activated with anti-CD3 (OKT3, Dako) coated plates or phytohemagglutinin in the presence or absence of various cytokines, including Interleukin- 4 (IL-4),Transforming Growth Factor-1 (TGF-3) and other commercially available cytokines ILl-a, IL-2, IL-3, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-y, TNF- a).

Expression of RANK was evaluated by FACS in a time course experiment for day 2 to day 8, using a mouse monoclonal antibody mAbl44 (prepared as described in Example as shown in the table below. Results are expressed as to referring to the relative increase in intensity of staining with anti-RANK. Double labeling experiments using both anti-RANK and anti-CD8 or anti-CD4 antibodies were also performed.

Table 1: Upregulation of RANK by Cytokines r r r r Cytokine (concentration) Results: IL-4 (50 ng/ml) TGF-B (5 ng/ml) to IL-4 (50 ng/ml) +TGF-B (5 ng/ml) ILl-a IL-2 IL-3 IL-7 IL-8 (lOng/ml) IL-10 IL-12 IFN-y (100U/ml) TNF-a Of the cytokines tested, IL-4 and TGF-B increased the level of RANK expression on both CD8+ cytotoxic and CD4+ helper T cells from day 4 to day 8. The combination of IL-4 and TGF-B acted synergistically to upregulate expression of this receptor on activated T cells. This particular combination of cytokines is secreted by suppresser T cells, and is believed to be important in the generation of tolerance (reviewed in Mitchison and Sieper, Z. Rheumatol. 54:141, 1995), implicating the interaction of RANK in regulation of an immune response towards either tolerance or induction of an active immune response.

EXAMPLE 12 This example illustrates the influence of RANK.Fc and hRANKL on activated

T

cell growth. The addition of TGFB to anti-CD3 activated human peripheral blood T lymphocytes induces proliferation arrest and ultimately death of most lymphocytes within the first few days of culture. We tested the effect of RANK:RANKL interactions on TGF3treated T cells by adding RANK.Fc or soluble human RANKL to T cell cultures.

Human peripheral blood T cells (7x 10 S PBT) were cultured for six days on anti- CD3 (OKT3, 5gg/ml) and anti-Flag (M l, 5g/ml) coated 24 well plates in the presence of TGF (Ing/ml) and IL-4 (10ng/ml), with or without recombinant FLAG-tagged soluble hRANKL (1g/ml) or RANK.Fc (10pg/ml). Viable T cell recovery was determined by triplicate trypan blue countings.

The addition of RANK.Fc significantly reduced the number of viable T cells recovered after six days, whereas soluble RANKL greatly increased the recovery of viable T cells (Figure Thus, endogenous or exogenous RANKL enhances the number of viable T cells generated in the presence of TGFB. TGFB, along with IL-4, has been implicated in immune response regulation when secreted by the TH 3 /regulatory T cell subset. These T cells are believed to mediate bystander suppression of effector T cells.

Accordingly, RANK and its ligand may act in an auto/paracrine fashion to influence T cell tolerance. Moreover, TGFB is known to play a role in the evasion of the immune system effected by certain pathogenic or opportunistic organisms. In addition to playing a role in the development of tolerance, RANK may also play a role in immune system evasion by S pathogens.

25 EXAMPLE 13 This example illustrates the influence of the interaction of RANK on CDla+ dendritic cells Functionally mature dendritic cells (DC) were generated in vitro from S CD34+ bone marrow (BM) progenitors. Briefly, human BM cells from normal healthy volunteers were density fractionated using Ficoll medium and CD34+ cells immunoaffinity isolated using an anti-CD34 matrix column (Ceprate, CellPro). The CD34+ BM cells were then cultured in human GM-CSF (20 ng/ml), human IL-4 (20 ng/ml), human TNF-a ng/ml), human CHO-derived Flt3L (FL; 100 ng/ml) in Super McCoy's medium supplemented with 10% fetal calf serum in a fully humidified 37°C incubator CO2) for 14 days. CDla+, HLA-DR+ DC were then sorted using a FACStar PlusTM, and used for biological evaluation of RANK On human CDla+ DC derived from CD34+ bone marrow cells, only a subset of CDla+ DC expressed RANK at the cell surface as assessed by flow cytometric analysis. However, addition of CD40L to the DC cultures resulted in RANK surface expression on the majority of CDla DC. CD40L has been shown to activate DC by enhancing in vitro cluster formation, inducing DC morphological changes and upregulating HLA-DR, CD54, CD58, CD80 and CD86 expression Addition of RANKL to DC cultures significantly increased the degree of DC aggregation and cluster formation above control cultures, similar to the effects seen with (Figure Sorted human CDla+ DC were cultured in a cytokine cocktail (GM- CSF, IL-4, TNF-a and FL) (upper left panel), in cocktail plus CD40L (lug/ml) (upper right), in cocktail plus RANKL (lg/ml) (lower left), or in cocktail plus heat inactivated RANKL (ltg/ml) (lower right) in 24-well flat bottomed culture plates in 1 ml culture media for 48-72 hours and then photographed using an inversion microscope. An increase in DC aggregation and cluster formation above control cultures was not evident when heat inactivated RANKL was used, indicating that this effect was dependent on biologically active protein. However, initial phenotypic analysis of adhesion molecule expression indicated that RANKL-induced clustering was not due to increased levels of CD2, CDI la, CD54 or CD58.

The addition of RANKL to CD a+ DC enhanced their allo-stimulatory capacity in a o mixed lymphocyte reaction (MLR) by at least 3- to 10-fold, comparable to S DC (Figure Allogeneic T cells (lx105) were incubated with varying numbers of irradiated (2000 rad) DC cultured as indicated above for Figure 2 in 96-well round bottomed culture plates in 0.2 ml culture medium for four days. The cultures were pulsed with 0.5 mCi 3 H]-thymidine for eight hours and the cells harvested onto glass fiber sheets for counting on a gas phase P counter. The background counts for either T cells or DC cultured alone were <100 cpm. Values represent the mean SD of triplicate cultures. Heat inactivated RANKL had no effect. DC allo-stimulatory activity was not further enhanced when RANKL and CD40L were used in combination, possibly due to DC functional capacity having reached a maximal level with either cytokine alone. Neither RANKL nor enhanced the in vitro growth of DC over the three day culture period. Unlike CD40L, RANKL did not significantly increase the levels of HLA-DR expression nor the expression of CD80 or CD86.

RANKL can enhance DC cluster formation and functional capacity without modulating known molecules involved in cell adhesion (CD 18, CD54), antigen presentation (HLA-DR) or costimulation (CD86), all of which are regulated by CD40/CD40L signaling. The lack of an effect on the expression of these molecules suggests that RANKL may regulate DC function via an alternate pathway(s) distinct from CD40/CD40L. Given that CD40L regulates RANK surface expression on in vitrogenerated DC and that CD40L is upregulated on activated T cells during DC-T cell interactions, RANK and its ligand may form an important part of the activation cascade that is induced during DC-mediated T cell expansion. Furthermore, culture of DC in RANKL results in decreased levels of CDlb/c expression, and increased levels of CD83. Both of these molecules are similarly modulated during DC maturation by CD40L (Caux et al. J.

Exp. Med. 180:1263; 1994), indicating that RANKL induces DC maturation.

Dendritic cells are referred to as "professional" antigen presenting cells, and have a high capacity for sensitizing MHC-restricted T cells. There is growing interest in using dendritic cells ex vivo as tumor or infectious disease vaccine adjuvants (see, for example, Romani, et al., J. Exp. Med., 180:83, 1994). Therefore, an agent such as RANKL that induces DC maturation and enhances the ability of dendritic cells to stimulate an immune response is likely to be useful in immunotherapy of various diseases.

EXAMPLE 14 This example describes the isolation of the murine homolog of RANK, referred to as muRANK. MuRANK was isolated by a combination of cross-species PCR and colony hybridization. The conservation of Cys residues in the Cys-rich pseudorepeats of the extracellular domains of TNFR superfamily member proteins was exploited to design human RANK-based PCR primers to be used on murine first strand cDNAs from various sources. Both the sense upstream primer and the antisense downstream primer were 20 designed to have their 3' ends terminate within Cys residues.

The upstream sense primer encoded nucleotides 272-295 of SEQ ID NO:5 (region encoding amino acids 79-86); the downstream antisense primer encoded the complement of nucleotides 409-427 (region encoding amino acids 124-130). Standard PCR reactions were set up and run, using these primers and first strand cDNAs from various murine cell line or tissue sources. Thirty reaction cycles of 94°C for 30 seconds, 50 0 C for 30 seconds, and 72°C for 20 seconds were run. PCR products were anlyzed by electrophoresis, and .specific bands were seen in several samples. The band from one sample was gel purified and DNA sequencing revealed that the sequence between the primers was approximately identical to the corresponding human RANK nucleotide sequence.

A plasmid based cDNA library prepared from the murine fetal liver epithelium line FLE18 (one of the cell lines identified as positive in the PCR screen) was screened for fulllength RANK cDNAs using murine RANK-specific oligonucleotide probes derived from the murine RANK sequence determined from sequencing the PCR product. Two cDNAs, one encoding the 5' end and one encoding the 3' end of full-length murine RANK (based on sequence comparison with the full-length human RANK) were recombined to generate a full-length murine RANK cDNA. The nucleotide and amino acid seqeunce of muRANK are shown in SEQ ID Nos:14 and The cDNA encodes a predicted Type 1 transmembrane protein having 625 amino acid residues, with a predicted 30 amino acid signal sequence, a 184 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 390 amino acid cytoplasmic tail. The extracellular region of muRANK displayed significant amino acid homology (69.7% identity, 80.8% similarity) to huRANK. Those of skill in the art will recognize that the actual cleavage site can be different from that predicted by computer; accordingly, the N-terminal of RANK may be from amino acid 25 to amino acid Other members of the TNF receptor superfamily have a region of amino acids between the transmembrane domain and the ligand binding domain that is referred to as a 'spacer' region, which is not necessary for ligand binding. In muRANK, the amino acids between 197 and 214 are predicted to form such a spacer region. Accordingly, a soluble form of RANK that terminates with an amino acid in this region is expected to retain the ability to bind a ligand for RANK in a specific manner. Preferred C-terminal amino acids for soluble RANK peptides are selected from the group consisting of amino acids 214, and 197 of SEQ ID NO: 14, although other amino acids in the spacer region may be utilized as a C-terminus.

EXAMPLE This example illustrates the preparation of several different soluble forms of RANK *20 and RANKL. Standard techniques of restriction enzyme cutting and ligation, in S combination with PCR-based isolation of fragments for which no convenient restriction sites existed, were used. When PCR was utilized, PCR products were sequenced to ascertain whether any mutations had been introduced; no such mutations were found.

In addition to the huRANK/Fc described in Example 2, another RANK/Fc fusion protein was prepared by ligating DNA encoding amino acids 1-213 of SEQ ID NO:6, to DNA encoding amino acids 3-232 of the Fc mutein described previously (SEQ ID NO:8).

A similar construct was prepared for murine RANK, ligating DNA encoding amino acids 1- S 213 of full-length murine RANK (SEQ ID NO:15) to DNA encoding amino acids 3-232 of the Fc mutein (SEQ ID NO:8).

*.jo0 A soluble, tagged, poly-His version of huRANKL was prepared by ligating DNA encoding the leader peptide from the immunoglobulin kappa chain (SEQ ID NO:16) to DNA encoding a short version of the FLAGTM tag (SEQ ID NO: 17), followed by codons encoding Gly Ser, then a poly-His tag (SEQ ID NO: 18), followed by codons encoding Gly Thr Ser, and DNA encoding amino acids 138-317 of SEQ ID NO: 13. A soluble, poly-His tagged version of murine RANKL was prepared by ligating DNA encoding the CMV leader (SEQ ID NO:9) to codons encoding Arg Thr Ser, followed by DNA encoding poly-His (SEQ ID NO: 18) followed by DNA encoding amino acids 119-294 of SEQ ID NO: 11.

A soluble, oligomeric form of huRANKL was prepared by ligating DNA encoding the CMV leader (SEQ ID NO:9) to a codon encoding Asp followed by DNA ending a trimer-former "leucine" zipper (SEQ ID NO:19), then by codons encoding Thr Arg Ser followed by amino acids 138-317 of SEQ ID NO:13.

These and other constructs are prepared by routine experimentation. The various DNAs are then inserted into a suitable expression vector, and expressed. Particularly preferred expression vectors are those which can be used in mammalian cells. For example, pDC409 and pDC304, described herein, are useful for transient expression. For stable transfection, the use of CHO cells is preferred; several useful vectors are described in USSN 08/785,150, now allowed, for example, one of the 2A5-3 X-derived expression vectors discussed therein.

EXAMPLE 16 This example demonstrates that RANKL expression can be up-regulated on muriie T cells. Cells were obtained from mesenteric lymph nodes of C57BJL6 mice, and activated with anti-CD3 coated plates, Concanavalin A (ConA) or phorbol myristate acetate in combination with ionomycin (anti-CD3: 500A2; Immunex Corporation, Seattle WA; ConA, PMA, ionomycin, Sigma, St. Louis, MO) substantially as described herein, and cultured from about 2 to 5 days. Expression of RANKL was evaluated in a three color analysis by FACS, using antibodies to the T cell markers CD4, CD8 and CD45RB, and RANK/Fc, prepared as described herein.

SRANKL was not expressed on unstimulated murine T cells. T cells stimulated with either anti-CD3, ConA, or PMA/ionomycin, showed differential expression of RANKL: see CD4 /CD45RBLo and CD4+/CD45RBHi cells were positive for RANKL, but CD8+ cells were not. RANKL was not observed on B cells, similar to results observed with human cells.

EXAMPLE 17 This example illustrates the effects of murine RANKL on cell proliferation and 30 activation. Various cells or cell lines representative of cells that play a role in an immune response (murine spleen, thymus and lymphnode) were evaluated by culturing them under conditions promoting their viability, in the presence or absence of RANKL. RANKL did not stimulate any of the tested cells to proliferate. One cell line, a macrophage cell line referred to as RAW 264.7 (ATCC accession number TIB 71) exhibited some signs of activation.

RAW cells constitutively produce small amounts of TNF-a. Incubation with either human or murine RANKL enhanced production of TNF-a by these cells in a dose dependent manner. The results were not due to contamination of RANKL preparations with endotoxin, since boiling RANKL for 10 minutes abrogated TNF-a production, whereas a similar treatment of purified endotoxin (LPS) did not affect the ability of the LPS to stimulate TNF-o production. Despite the fact that RANKL activated the macrophage cell line RAW T64.7 for TNF-a production, neither human RANKL nor murine RANKL stimulated nitric oxide production by these cells.

EXAMPLE 18 This example illustrates the effects of murine RANKL on growth and development of the thymus in fetal mice. Pregnant mice were injected with 1 mg of RANK/Fc or vehicle control protein (murine serum albumin; MSA) on days 13, 16 and 19 of gestation. After birth, the neonates continued to be injected with RANK/Fe intraperitoneally (IP) on a daily basis, beginning at a dose of 1 gg, and doubling the dose about every four days, for a final dosage of 4 gg. Neonates were taken at days 1, 8 and 15 post birth, their thymuses and spleens harvested and examined for size, cellularity and phenotypic composition.

A slight reduction in thymic size at day 1 was observed in the neonates born to the female injected with RANK/Fc; a similar decrease in size was not observed in the control i neonates. At day 8, thymic size and cellularity were reduced by about 50% in the RANK/Fc-treated animals as compared to MSA treated mice. Phenotypic analysis demonstrated that the relative proportions of different T cell populations in the thymus were S the same in the RANK/Fc mice as the control mice, indicating that the decreased cellularity was due to a global depression in the number of thymic T cells as opposed to a decrease in a specific population(s). The RANK/Fc-treated neonates were not significantly different from the control neonates at day 15 with respect to either size, cellularity or phenotype of thymic cells. No significant differences were observed in spleen size, cellularity or composition at any of the time points evaluated. The difference in cellularity on day 8 and not on day 15 may suggest that RANK/Fc may assert its effect early in thymic development.

EXAMPLE 19 This example demonstrates that the C-terminal region of the cytoplasmic domain of RANK is important for binding of several different TRAF proteins. RANK contains at least two recognizable PXQX(X)T motifs that are likely TRAF docking sites. Accordingly, the importance of various regions of the cytoplasmic domain of RANK for TRAF binding was evaluated. A RANK/GST fusion protein was prepared substantially as described in Smith and Johnson, Gene 67:31 (1988), and used in the preparation of various truncations as described below.

Comparison of the nucleotide sequence of murine and human RANK indicated that there were several conserved regions that could be important for TRAF binding.

Accordingly, a PCR-based technique was developed to facilitate preparation of various Cterminal truncations that would retain the conserved regions. PCR primers were designed to introduce a stop codon and restriction enzyme site at selected points, yielding the truncations described in Table I below. Sequencing confirmed that no undesired mutations had been introduced in the constructs.

Radio-labeled (35S-Met, Cys) TRAF proteins were prepared by in vitro translation using a commercially available reticulocyte lysate kit according to manufacturer's instructions (Promega). Truncated GST fusion proteins were purified substantially as described in Smith and Johnson (supra). Briefly, E. coli were transfected with an expression vector encoding a fusion protein, and induced to express the protein. The bacteria were lysed, insoluble material removed, and the fusion protein isolated by precipitation with glutathione-coated beads (Sepahrose 4B, Pharmacia, Uppsala Sweden) The beads were washed, and incubated with various radiolabeled TRAF proteins.

After incubation and wash steps, the fusion protein/TRAF complexes were removed from the beads by boiling in 0.1% SDS 1-mercaptoethanol, and loaded onto 12% SDS gels (Novex). The gels were subjected to autoradiography, and the presence or absence of radiolabeled material recorded. The results are shown in Table 2 below.

Table 2: Binding of Various TRAF Proteins to the Cytoplasmic Domain of RANK C terminal Truncations: E206-S339 E206-Y421 E206-M476 E206-G544 Full length TRAF1 TRAF2 TRAF3 TRAF4 TRAF6 These results indicate that TRAFI, TRAF2, TRAF3, TRAF 5 and TRAF6 bind to the most distal portion of the RANK cytoplasmic domain (between amino-acid G544 and A616). TRAF6 also has a binding site between S339 and Y421. In this experiment, also bound the cytoplasmic domain of RANK.

SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Immunex Corporation (ii) TITLE OF INVENTION: Receptor Activator of NF-kappaB (iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Immunex Corporation, Law Department STREET: 51 University Street CITY: Seattle STATE: WA COUNTRY: USA ZIP: 98101 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: Apple Power Macintosh OPERATING SYSTEM: Apple Operating System 7.5.5 SOFTWARE: Microsoft Word for Power Macintosh 6.0.1 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 22 DECEMBER 1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: USSN 60/064,671 FILING DATE: 14 OCTOBER 1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: USSN 08/813,509 FILING DATE: 07 MARCH 1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: USSN 08/772,330 (60/064,671) FILING DATE: 23 DECEMBER 1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Perkins, Patricia Anne REGISTRATION NUMBER: 34,693 REFERENCE/DOCKET NUMBER: 2851-WO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (206)587-0430 TELEFAX: (206)233-0644 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3115 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (vii) IMMEDIATE SOURCE: LIBRARY: BONE-MARROW DERIVED DENDRITIC CELLS CLONE: 9D-8A (ix) FEATURE: NAME/KEY: CDS LOCATION: 93..1868 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GCTGCTGCTG CTCTGCGCGC TGCTCGCCCG GCTGCAGTTT TATCCAGAAA

GAGCTGTGTG

GACTCTCTGC CTGACCTCAG TGTTCTTTTC

AG

GTG GCT TTG CAG ATC GCT CCT Val Ala Leu Gln Ile Ala Pro 113

S

S

S.

S

S

S

CCA TGT ACC Pro Cys Thr 10 AGT GAG AAG CAT Ser Glu Lys His

TAT

Tyr 15 GAG CAT CTG GGA Glu His Leu Gly

CGG

Arg TGC TGT AAC Cys Cys Asn AAA TGT Lys Cys 25 GAA CCA GGA AAG Glu Pro Gly Lys

TAC

Tyr 30 ATG TCT TCT AAA Met Ser Ser Lys

TGC

Cys ACT ACT ACC TCT Thr Thr Thr Ser

GAC

Asp 40 AGT GTA TGT CTG Ser Val Cys Leu TGT GGC CCG GAT Cys Gly Pro Asp TAC TTG GAT AGC Tyr Leu Asp Ser AAT GAA GAA .GAT Asn Glu Glu Asp TGC TTG CTG CAT Cys Leu Leu His

AAA

Lys GTT TGT GAT ACA Val Cys Asp Thr GGC AAG Gly Lys GCC CTG GTG Ala Leu Val GCG TGC ACG Ala Cys Thr GTG GTC GCC GGC Val Val Ala Gly AGC ACG ACC CCC Ser Thr Thr Pro CGG CGC TGC Arg Arg Cys TGC TGC CGC Cys Cys Arg GCT GGG TAC CAC Ala Gly Tyr His

TGG

Trp 95 AGC CAG GAC TGC Ser Gln Asp Cys CGC AAC Arg Asn 105 ACC GAG TGC GCG Thr Glu Cys Ala

CCG

Pro 110 GGC CTG GGC GCC CAG CAC CCG TTG CAG Gly Leu Gly Ala Gin His Pro Leu Gln 115 449 497

CTC

Leu 120 AAC AAG GAC ACA Asn Lys Asp Thr

GTG

Val 125 TGC AAA CCT TGC Cys Lys Pro Cys

CTT

Leu 130 GCA GGC TAC TTC Ala Gly Tyr Phe

TCT

Ser 135 GAT GCC TTT TCC Asp Ala Phe Ser

TCC

Ser 140 ACG GAC AAA TGC Thr Asp Lys Cys

AGA

Arg 145 CCC TGG ACC AAC Pro Trp Thr Asn TGT ACC Cys Thr 150 TTC CTT OGA Phe Leu Gly OTT TGC AGT Val Cys Ser 170

AAG

Lys 155 AGA GTA GAA CAT Arg Val 01u His

CAT

His 160 GGG ACA GAG AAA Gly Thr Glu Lys TCC GAT GCG Ser Asp Ala 165 GAA CCC CAT Glu Pro His TCT TCT CTG CCA Ser Ser Leu Pro AGA AAA CCA CCA Arg Lys Pro Pro

AAT

Asn 180 GTT TAC Val Tyr 185 TTG CCC GGT TTA Leu Pro Gly Leu ATT CTG CTT CTC Ile Leu Leu Leu

TTC

Phe 195 GCG TCT GTG 0CC Ala Ser Val Ala

CTG

Leu 200 GTO OCT GCC ATC Val Ala Ala Ile TTT GOC OTT TOC Phe Gly Val Cys AGO AAA AAA 000 Arg Lys Lys Oly 689 737 785 OCA CTC ACA OCT Ala Leu Thr Ala

AAT

Asn 220 TTG TOG CAC TG Leu Trp His Trp

ATC

Ile 225 AAT GAG OCT TOT Asn Glu Ala Cys GOC COC Gly Arg 230 CTA AGT OGA Leu Ser Oly ACO OCA AAC Thr Ala Asn 250

OAT

Asp 235 AAG GAG TCC TCA Lys Giu Ser Ser

GGT

Gly 240 GAC AGT TOT OTC Asp Ser Cys Val AOT ACA CAC Ser Thr His 245 TTA CTG CTG Leu Leu Leu 06 S@ S

S

*.OOS@

0 @0 0 @005 0 *5 0 0* 0@ TTT GOT CAG CAG Phe Gly Gin Gin

OGA

Gly 255 OCA TOT GAA GOT Ala Cys Glu Oly ACT CTG Thr Leu 265 GAG GAO AAG ACA Giu Glu Lys Thr

TTT

Phe 270 CCA GAA OAT ATO Pro Glu Asp Met

TOC

Cys 275 TAC CCA OAT CA-A Tyr Pro Asp Gin

GOT

Gly 280 GOT OTC TOT CAG Gly Val Cys Gin ACO TOT OTA OGA Thr Cys Val Gly GOT CCC TAC GCA Oly Pro Tyr Ala

CAA

Gin 295 GOC OAA OAT 0CC Gly Oiu Asp Ala

AGO

Arg 300 ATO CTC TCA TTO Met Leu Ser Leu

GTC

Val 305 AOC AAG ACC GAO Ser Lys Thr Oiu ATA GAO Ile Giu 310 833 881 929 977 1025 1073 1121 1169 1217 V. 5* oe o 0000 OAA GAC AOC Olu Asp Ser CCC TCC CAG Pro Ser Gin 330

TTC

Phe 315 AGA CAG ATO CCC Arg Gin Met Pro

ACA

Thr 320 GA.A OAT GAA TAC Olu Asp Glu Tyr ATO GAC AGO Met Asp Arg 325 CCT OGA AGC Pro Gly Ser CCC ACA GAC CAG Pro Thr Asp Gin CTG TTC CTC ACT Leu Phe Leu Thr AAA TCC Lys Ser 345 ACA CCT CCT TTC Thr Pro Pro Phe TCT GAA CCC CTG GAO OTO Ser Giu Pro Leu Olu Val 350 *355 000 GAO AAT GAC Gly Olu Asn Asp TTA AGC CAG TOC TTC ACO 000 ACA CAG Leu Ser Gin Cys Phe Thr Gly Thr Gin 365

AGC

Ser 370 ACA OTO GOT TCA Thr Val Gly Ser

GAA

Oiu 375

AGC

Ser TGC AAC TGC ACT GAG CCC CTG Cys Asn Cys Glu Pro Leu TGC AGG Cys Arg 385 ACT GAT TGG ACT CCC ATG Thr Asp Trp Thr Pro Met 390 1265 1313 TCC TCT GAA Ser Ser Glu CAC TGG GCA His Trp Ala 410

AAC

Asn 395 TAC TTG CAA AAA Tyr Leu Gin Lys

GAG

Glu 400 GTG GAC AGT CCC CAT TGC CCG Val Asp Ser Cly His Cys Pro 405 GCC AGC CCC AGC Ala Ser Pro Ser AAC TGG CCA GAT Asn Trp Ala Asp TGC ACA GGC Cys Thr Gly 1361 TCC CGG Cys Arg 425 AAC CCT CCT GGG Asn Pro Pro Gly

GAG

Clu 430 GAC TCT GAA CCC Asp Cys Glu Pro

CTC

Leu 435 GTG GOT TCC CCA Val Cly Ser Pro

AAA

Lys 440 CGT GGA CCC TTG Arg Gly Pro Leu GAG TGC GCC TAT Gin Gys Ala Tyr

GGC

Cly 450 ATG GGC CTT CCC Met Gly Leu Pro

CCT

Pro GAA GAA GAA GCC Glu Glu Glu Ala AGG ACG GAG GCC Arg Thr Glu Ala

AGA

Arg 465 GAC GAG CCC GAG Asp Gin Pro Glu GAT GG Asp Gly OCT GAT GGG Ala Asp Cly AGC TCC CCT Ser Ser Pro 490

AGO

Arg 475 CTC CCA AGC TCA Leu Pro Ser Ser

GCG

Ala 480 AGG GCA GOT GCC Arg Ala Gly Ala GGG TCT GGA Cly Ser Gly 485 ACT GGA AAC Thr Gly Asn GGT CCC GAG TCC Gly Gly Gin Ser

CCT

Pro 495 GCA TCT GGA AAT Ala Ser Cly Asn

GTG

Val1 500 1409 1457 1505 1553 1601 1649 1697 1745 1793 1841 AGT AAC Ser Asn 505 TCC ACG TTC ATC Ser Thr Phe Ile TCC AGC GOG GAG Ser Ser Gly Gin 510 GTG ATG Val Met 515 AAC TTC AAG GGC Asn Phe Lys Gly

GAG

Asp 520 ATC ATC GTG GTC Ile Ile Val Val TAG GTC Tyr Val 525 AGC GAG ACC Ser Gin Thr GAG GAG GGC C Gin Giu Cly Ala GCG GCT GCG GAG Ala Ala Ala.Giu ATG GGC CGC CCG Met Gly Arg Pro GAG GAG GAG ACC Gin Glu Glu Thr CTG CC Leu Ala CCC CCA GAG Arg Arg Asp CCC CCC CCC Gly Cly Pro 570

TCC

Ser 555 TTC CC CCC AAC Phe Ala Cly Asn CCC CCC TTC CCC Pro Arg Phe Pro GAG CCC TC Asp Pro Cys 565 ACC CCC GTG Arg Pro Val GAG GO CTC CCC Glu Gly Leu Arg

GAG

Giu 575 CCC GAG AAG CC Pro Clu Lys Ala

TCG

Ser GAG GAG Gin Ciu 585 CAA GOC CCC Gin Gly Cly CCC AAG OCT TCA CCGCCCCCCA

TCCCTGOCAC

Ala Lys Ala 590 1888 CCCCAACCTC CCACCCACGG CTCCCGAGCG CAGCACCCCA GCCTCTCCCC CAGCCCCCCC 1948 CACCCAGCGA TCGATCGGTA CAGTCCAGGA ACACCACCCG GCATTCTCTG CCCACTTTc 2008 CTTCCAGGAA ATGCCTTTT CAGOAACTCA ATTCATGACG ACTGTCCCCA TGCCCACGGA 2068

TGCTCAGCAG

AGTAATTTGT

CTATGTTTTC

TCTTTTTTAA

CTCTTTTCCT

TGGTGCGATT

TCCCACCTCA

CCCCGACTCC

CCCCAGCTAA

CCCCCACGCT

ATGGCTTTCC

CCTGGAGATA

GTTCTATATT

ATCTTTTTAA

CTAGGTGGTT

AAAACTCCAA

TGGAGAAAAT

GAATAAAGTT

CCCGCCGCAC TGGGGCAC GGCACTATGA CAGCTAT1 CCCCCATATT TGTATTCC TGTAAAGGTT TTCTCAA.

TTTTTTTTTC TTTTTTTC ATAGCCCGGT GCAGCCTC ACCTTCGGAG TAGCTGGC CCCCCCCCAG AGACACGC AGCAGTCCTC CAGCCTCC GGCCTGCTTT ACGTATT9) CAGTGTGTGT TCATTGTI GTTGCTAAGT TGCTAGG CTCATTTTTC TAAAAGA GTTTGTGTCG TTCCTTA2 AATTTATCCA TGCTGGC~ GTTGCTGCAG CTTGGCA GAACAGGACA TGGGGCT( GAAATTTTAA AAAAAAA

AT

'TT

TT

;GC

~TA

;AT

;TC

;GC

'TC

kAA

AC

k.AG

~GC

%GA

rTC

GTCTCCCCTG

TATGACTATC

TTCATAACTT

TTCTCCTAAA

AACCTGGCTC

ACTCCTGGGC

CACAGCTGCA

CCACCATGTT

CTCCCAAAGT

TTTTGTGCCC

CACTTTTGGG

ATGTGGTGGG

AAAAAAGGAA

AGAACTAAGC

GGCACTCAGG

TTCTTATTCT

CCACTCCTCA

CTGTTCTGTG

TTCTTGATAT

GGTGAGGGTC

TGGCCCAGGC

TCAAGCAATC

GGCCACGCCC

ACCCAGCCTG

ACTGGGATTA

CTGCTCACAG

AAAGGGCTAA

ACTTTCATAT

ACCCGATTTA

TCAGTATGTG

TACTTGGTAA

AGAGGTCTCT

ALACTCGCAGC

GGGGGGGGGT

CTTTCCTCCC

TCTTTCTTTT

TAGAGTGCAG

CAAGTGATCC

AGCTTCCTCC

GTCTCAAACT

CAGGCGTGAG

TGTTTTAGAG

ACATGTGAGG

TCTGAAAAAT

TTTCTCCTGA

ACCTTACCCG

GCAAATTTCT

CTGGAAAAGA

2128 2188 2248 2308 2368 2428 2488 2548 2608 2668 2728 2788 2848 2908 2968 3028 .0.

CCT GGAAAGAAAG GGCCCGGGAA GTTCAAGGAA 3088 3115 INFORM4ATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 591 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Val Ala Leu Gin Ile Ala Pro Pro Cys Thr Ser Glu 1 5 10 His Leu Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly 25 Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu 40 Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys 55 Lys His Tyr Glu Lys Pro Cys Tyr Met Ser Cys Gly Pro Leu Leu His Lys Ser Gin Val1 Thr Asp Cys Th r Cys Asp Thr Pro Arg Glu Cys Gly 70 Arg Cys Lys Cys Arg Ala Ala Arg Leu Cys Asn 105 Ala 75 Ala Glu 100 Gly Ala Gin His Pro Leu Gin Leu Asi'I Cys Arg 145 Gly Lys Leu 130 Pro Thr Pro 115 Ala Trp Glu Pro Gly Thr Lys Asn 180 Tyr Asn Ser 165 Glu Ph CyE 150 Asr Pro Leu Leu Phe Ala Ser Val 195 Cys Ile 225 Asp Cy s Asp Gly Val1 305 Glu Phe Leu Tyr 210 Asn Ser Giu Met Gly 290 Ser Asp Leu Glu Arg Giu Cys Gly Cys 275 Gly Lys Glu Thr Val 355 Lys Ala Val1 Val1 260 Tyr Pro Thr Tyr G1u 340 Gly Lys Cys Ser 245 Leu Pro Tyr Giu Met 325 Pro Giu Gly Gly 230 Thr Leu Asp Ala Ile 310 Asp Gly Asn 2Se: 13' Th~ Ale IHis Ala Lys 215 Arg His Leu Gin Gin 295 Giu Arg Ser Asp 120 r Asp r Phe Val Val Leu 200 Ala Leu Thr Thr Gly C 280 Gly C Glu A Pro S Lys .S 3 Ser L 360 Ala Leu Cys Tyr 185 Val1 Leu Ser Al1a eu ~65 fly flu2 ~sp er C er TI 45 eu S Ly Ph Gi1 Ser 170C Leu Ala Thr Gly Asn 250 Glu Val1 %sp ~er ;in 30 ~hr er sAsp Ser 'Lys 155 -Ser *Pro Ala Ala Asp 235 Phe Glu Cys Ala Phe I 315 Pro IJ Pro P Gin c Th: Se~ 14( Arc Sei Gl) Ile Asn 220 Lys Gly Lys Gln %rg 300 \rg 'hr ~ro .ys 1 Val y Tyr Ala Val 125 r Thr 0 j Val Leu Leu Ilie 205 Leu Gu Gin Thr Gly 'i 285 Met L Gin M~ Asp G Phe S 3 Phe T Al Hi Pr Cy As] Gil Prc Ilc 190 Phe r Ser fin 3he ~hr ~eu let in er hr a Gly S Trp o Gly 0 s; Lys p Lys 'His )Ala 175 Sle Gly His Ser C Gly 255 Pro G Cys V Ser L Pro T 3 Leu L 335 Giu p Gly TI Asn Ser Leu Pro Cys His 160 Arg Leu la 1 Prp fly 1 a 1iu 'a 1 eu hr eu ro Gin Ser 370 Thr Val Gly Ser Giu 375 Ser Cys Asn Cys Thr 380 Giu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr Leu Gin Lys Glu 385 390 395 400 Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser Pro Ser Pro Asn 405 410 415 Trp Ala Asp Val Cys Thr Gly Cys Arg Asn Pro Pro Gly Glu Asp Cys 420 425 430 Glu Pro Leu Val Gly Ser Pro Lys Arg Gly Pro Leu Pro Gin Cys Ala 435 440 445 Tyr Gly Met Gly Leu Pro Pro Glu Glu Glu Ala Ser Arg Thr Glu Ala 450 455 460 Arg Asp Gin Pro Glu Asp Gly Ala Asp Gly Arg Leu Pro Ser Ser Ala 465 470 475 480 Arg Ala Gly Ala Gly Ser Gly Ser Ser Pro Gly Gly Gin Ser Pro Ala 485 490 495 Ser Gly Asn Val Thr Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly 500 505 510 Gin Val Met Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gin 515 520 525 Thr Ser Gin Glu Gly Ala Ala Ala Ala Ala Glu Pro Met Gly Arg Pro 530 535 540 Val Gin Glu Glu Thr Leu Ala Arg Arg Asp Ser Phe Ala Gly Asn Gly 545 550 555 560 Pro Arg Phe Pro Asp Pro Cys Gly Gly Pro Glu Gly Leu Arg Glu Pro 565 570 575 Glu Lys Ala Ser Arg Pro Val Gin Glu Gin Gly Gly Ala Lys Ala 580 585 590 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1391 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (vii) IMMEDIATE SOURCE: LIBRARY: BONE-MARROW DERIVED DENDRITIC CELLS CLONE: 9D-15C (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 39..1391 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCGCTGAGGC CCCGGCGCCC CCCAGCCTGT CCCGCCCC ATG CCC CCG CCC GCC Met Ala Pro Arg Ala 1 q CGG CGG CGC CGC Arg Arg Arg Arg

CCG

Pro CTG TTC GCG CTC Leu Phe Ala Leu

CTC

Leu 15 CTG CTC TCC 6CG Leu Leu Cys Ala CTC CTC Leu Leu CCC CGG CTC Ala Arg Leu AAG CAT TAT Lys His Tyr GTG GCT TTC CAC Val Ala Leu Cmn

ATC

Ile GCT CCT CCA TGT Ala Pro Pro Cys ACC ACT GAG Thr Ser Clu CAA CCA GGA Clu Pro Gly GAC CAT CTG GGA Clu His Leu Cly

CGG

Arg 45 TGC TGT AAC AAA Cys Cys Asn Lys

TGT

Cys 50 AAC TAC Lys Tyr ATC TCT TCT AAA Met Ser Ser Lys

TGC

Cys 60 ACT ACT ACC TCT Thr Thr Thr Ser

GAC

Asp AGT CTA TCT CTC Ser Val Cys Leu

CCC

Pro TGT GGC CCG GAT Cys Cly Pro Asp TAC TTG GAT AGC Tyr Leu Asp Ser

TGG

Trp 80 AAT CAA CAA CAT Asn Clu Clu Asp

AAA

Lys TGC TTC CTC CAT Cys Leu Leu His

AAA

Lys CTT TCT CAT ACA Val Cys Asp Thr

GGC

Cly 95 AAC GCC CTG GTG Lys Ala Leu Val CCC GTG Ala Val 100" CTC CCC GGC Val Ala Cly TAC CAC TGG Tyr His Trp 120

AAC

Asn 105 AGC ACC ACC CCC Ser Thr Thr Pro

CGG

Arg 110 CGC TCC GCG TGC Arg Cys Ala Cys ACG GCT GGG Thr Ala Cly 115 ACC GAG TGC Thr Clu Cys 149 197 245 293 341 389 437 485 533 581 629 AGC CAG GAC TGC Ser Cmn Asp Cys TGC TCC CCC CGC Cys Cys Arg Arg

AAC

Asn 130 GCG CCC Ala Pro 135 GGC CTG GGC GCC Cly Leu Cly Ala

CAC

140 140 CAC CCC TTG CAC His Pro Leu Cmn

CTC

Leu 145 AAC AAG GAC ACA Asn Lys Asp Thr

GTG

Va1 150 TGC AAA CCT TGC Cys Lys Pro Cys

CTT

Leu 155 CCA GGC TAC TTC Ala Cly Tyr Phe

TCT

Ser 160 CAT CCC TTT TCC Asp Ala Phe Ser

TCC

Ser 165 ACG GAC AAA TGC Thr Asp Lys Cys

AGA

Arg 170 CCC TCG ACC AAC Pro Trp Thr Asn

TGT

Cys 175 ACC TTC CTT GGA Thr Phe Leu Cly AAC AGA Lys Arg 180 GTA CAA CAT Val Clu His

CAT

His 185 GGG ACA GAG AAA Cly Thr Clu Lys

TCC

Ser 190 CAT GCG GTT TGC Asp Ala Val Cys AGT TCT TCT Ser Ser Ser 195 CTG CCA GCT Leu Pro Ala 200 AGA AAA CCA CCA Arg Lys Pro Pro AAT GAA Asn Glu 205 CCC CAT GTT TAC TTG CCC GGT Pro His Val Tyr 210 Leu Pro Gly TTA ATA Leu Ile 215 ATT CTG CTT Ile Leu Leu CTC TTC Leu 'Phe 220 GCG TCT GTG GCC Ala Ser Val Ala

CTG

Leu 225 GTG GCT GCC ATC Val Ala Ala Ile TTT GGC GTT TGC Phe Gly Val Cys AGG AAA AAA GGG Arg Lys Lys Gly

AAA

Lys 240 GCA CTC ACA GCT Ala Leu Thr Ala TTG TGG CAC TGG Leu Trp His Trp

ATC

Ile 250 AAT GAG GCT TGT Asn Glu Ala Cys

GGC

Gly 255 CGC CTA AGT GGA Arg Leu Ser Gly GAT AAG Asp Lys 260 GAG TCC TCA Glu Ser Ser CAG CAG GGA Gin Gin Gly 280 GAC AGT TGT GTC Asp Ser Cys Val ACA CAC ACG GCA Thr His Thr Ala AAC TTT GGT Asn Phe Gly 275 GAG GAG AAG Glu Glu Lys GCA TGT GAA GGT Ala Cys Giu Gly

GTC

Val 285 TTA CTG CTG ACT Leu Leu Leu Thr

CTG

Leu 290 ACA TTT Thr Phe 295 CCA GAA GAT ATG Pro Giu Asp Met

TGC

Cys 300 TAC CCA GAT CAA Tyr Pro Asp Gin

GGT

Gly 305 GGT GTC TGT CAG Gly Val Cys Gin a

GGC

Gly 310 ACG TGT GTA GGA Thr Cys Val Gly

GGT

Gly 315 GGT CCC TAC GCA Gly Pro Tyr Ala

CAA

Gin 320 GGC GAA GAT GCC Gly Glu Asp Ala

AGG

Arg 325 ATG CTC TCA TTG Met Leu Ser Leu AGC AAG ACC GAG Ser Lys Thr Glu GAG GAA GAC AGC Glu Giu Asp Ser TTC AGA Phe Arg 340 CAG ATG CCC Gin Met Pro GAC CAG TTA Asp Gin Leu 360

ACA

Thr 345 GAA GAT GAA TAC Glu Asp Giu Tyr

ATG

Met 350 GAC AGG CCC TCC Asp Arg Pro Ser CAG CCC ACA Gin Pro Thr 355 ACA CCT CCT Thr Pro Pro CTG TTC CTC ACT Leu Phe Leu Thr

GAG

Glu 365 CCT GGA AGC AAA Pro Gly Ser Lys

TCC

Ser 370 965 1013 1061 1109 1157 1205 1253 1301 1349 TTC TCT Phe Ser 375 GAA CCC CTG GAG Glu Pro Leu Glu

GTG

Val 380 GGG GAG AAT GAC Gly Giu Asn Asp

AGT

Ser 385 TTA AGC CAG TGC Leu Ser Gin Cys

TTC

Phe 390 ACG GOG ACA CAG Thr Gly Thr Gin

AGC

Ser 395 ACA GTG GGT TCA Thr Val Gly Ser AGC TGC AAC TGC Ser Cys Asn Cys

ACT

Thr 405 GAG CCC CTG TGC Glu Pro Leu Cys ACT GAT TGG ACT Thr Asp Trp Thr ATG TCC TCT GAA Met Ser Ser Glu AAC TAC Asn Tyr 420 TTG CAA AAA Leu Gin Lys

GAG

Glu 425 GTG GAC AGT GGC CAT TGC CCG CAC TGG GCA GCC AGC Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser CCC AGC CCC AAC TOG GCA GAT GTC TGC ACA GGC TGC COG AAC Pro Ser Pro Asn Trp Ala Asp Val *Cys Thr Gly Cys Arg Asn 440 445 450 INFORMATION FOR SEQ ID NO:4: SEQUENCE

CHARACTERISTICS:

LENGTH: 451 amino acids 1391 0* 00.

Mel Let Prc Lys Asp Asn Ala Ala Arg Leu 145 Asp Phe Val1 Val (ii) (xi) tAla Pro .Cys Ala Cys Thr Cys Glu Ser Val Glu Glu Leu Val Cys Thr 115 Asn Thr 130 Asn Lys Ala Phe Leu Gly Cys Ser 195 Tyr Leu TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein Arc Le.

Ser Pro Cys Asp Ala 100 Ala Olu Asp Ser Lys 180 Ser Pro Ala Leu *Glu Gly Leu Lys Val Oly Cys Thr Ser 165 Arg Ser Gly Arg Arg Arg Arg Pro Leu Phe Ala Let.

Ala Lys Lys Pro 70 Cys Val1 Tyr Ala Val1 150 Thr Vl Leu Leu Arg His Tyr 55 Cys Leu Ala His Pro 135 Cys Asp Glu Pro Ile 215 Leu Tyr 40 Met Gly Leu Gly Trp 120 Gly Lys Lys His Ala 200 Ile Gln 25 Glu Ser Pro His Asn 105 Ser Leu Pro Cys His 185 Arg Leu 10 Val1 His Ser Asp Lys 90 Ser Gln Gly Cys Arg 170 Gly Lys Leu Ala Let.

Lys Glu 75 Val1 Thr Asp Ala Leu 155 Pro rhr Pro Lieu Leu IGly Cys Tyr Cys Thr Cys Gln 140 Ala Trp Glu Pro Phe 220 Gin Arg Thr Leu Asp Pro Glu 125 His Gly Thr Lys Asn 205 Ala I1e Cys Thr Asp Thr Arg 110 cys Pro Tyr lksn Ser 190 G1u e r Leu Ala Cys Thr Ser Oly Arg Cys Leu Phe Cys 175 Asp Pro Val Leu Pro Asn Ser Trp Lys Cys Arg Gin Ser 160 Thr Ala H[is Ala SEQUENCE DESCRIPTION: SEQ ID NO:4: 210 Val Ala Ala Ile Ile 230 Phe Gly Val Cys Tyr 235 Arg Lys Lys Gly Ala Leu Thr Ala Asn Leu Trp His 245 Leu Thr Thr Gly 305 Gly Giu Pro Lys Ser 385 Ser Ser His Cys Ser Al a Leu 290 Gly Giu Asp Ser Ser 370 Leu Cys Ser Trp Arg Gly As n 275 Glu Val1 Asp Ser Gin 355 Thr Ser Asn Glu Ala 435 As n Asp 260 Phe Glu Cys Ala Phe 340 Pro Pro Gin Cys Asn 420 Ala Lys Gly Lys Gin Arg 325 Arg Thr Pro Cys Thr 405 Tyr Ser Glu Gin Thr Gly 310 Met Gin Asp Phe Phe 390 Giu Leu Pro Ser Gin Phe 295 Thr Leu Met Gin Ser 375 Thr Pro Gin Ser Ser Gly 280 Pro Cys Ser Pro Leu 360 Giu Gly Leu Lys Pro 440 Trp Gly 265 Ala Glu Val Leu Thr 345 Leu Pro Thr Cys Giu 425 Asn Ile 250 Asp Cys Asp Gly Val1 330 Giu Phe Leu Gin Arg 410 Val1 Trp Asn Ser Giu Met Gly 315 Ser Asp Leu Giu Ser 395 Thr Asp Ala Glu Cys Gly Cys 300 Gly Lys Giu Thr Val 380 Thr Asp Ser Asp Ala Val1 Val1 285 Tyr Pro Thr Tyr Glu 365 Gly Val Trp Gly Val1 445 Cys Ser 270 Leu Pro Tyr Glu Met 350 Pro Glu Gly Thr His 430 Cys Gly 255 Thr Leu Asp Ala Ile 335 Asp Gly Asn Ser Pro 415 Cys Thr Arg His Leu Gin Gin 320 Glu Arg Ser Asp Giu 400 Met Pro Gly

S

*5*S

*SS*

S. S S INFORMATION FOR SEQ ID ii) SEQUENCE CHARACTERISTICS: LENGTH: 3136 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (vii) IMMEDIATE SOURCE: LIBRARY: BONE-MARROW DERIVED DENDRITIC

CELLS

CLONE: FULL LENGTH

RANK

(ix) FEATURE: NAME/KEY:

CDS

LOCATION: 39..1886 (xi) SEQUENCE DESCRIPTION: SEQ ID CCCCTGAGGC CGCGGCGCCC GCCAGCCTGT CCCGCCCC ATG GCC CCG CGC GCC Met Ala Pro Arg Ala CGG CGG CCC CGC CCG Arg Arg Arg Arg Pro CTG TTC GCG CTC CTG Leu Phe Ala Leu Leu 15 CTG CTC TGC GCG Leu Leu Cys Ala CTG CTC Leu Leu GCC CCG CTC Ala Arg Leu AAG CAT TAT Lys His Tyr CAG GTG Gin Val GCT TTG CAG Ala Leu Gin GCT CCT CCA TGT ACC ACT GAG Ala Pro Pro Cys Thr Ser Clu GAG CAT CTG CGA Glu His Leu Gly

CGG

Arg 45 TGC TGT AAC AAA Cys Cys Asn Lys

TGT

Cys CAA CCA GGA Clu Pro Gly AAG TAC Lys Tyr ATC TCT TCT AAA Met Ser Ser Lys

TGC

Cys 60 ACT ACT ACC TCT Thr Thr Thr Ser

GAC

Asp AGT GTA TCT CTC Ser Val Cys Leu .5

S

*SS*

*SSS

*5SS TGT GGC CCG GAT Cys Cly Pro Asp

CAA

Glu 75 TAC TTG CAT AGC Tyr Leu Asp Ser

TGC

Trp 80 AAT GAA CAA CAT Asn Glu Clu Asp

AAA

Lys TGC TTC CTC CAT Cys Leu Leu His

AAA

Lys 90 GTT TGT CAT ACA Val Cys Asp Thr AAG CCC CTG CTG Lys Ala Leu Val CCC CTG Ala Val 100 GTC CCC GGC Val Ala Gly TAC CAC TGG Tyr His Trp 120

AAC

Asn 105 AGC ACC ACC CCC Ser Thr Thr Pro

CGG

Arg 110 CGC TCC GCG TGC Arg Cys Ala Cys ACG GCT GGG Thr Ala Cly 115 ACC GAG TGC Thr Glu Cys AGC CAG CAC TGC Ser Gin Asp Cys

GAG

Clu 125 TGC TCC CCC CGC Cys Cys Arg Arg

AAC

Asn 130 245 293 341 389 437 485 533 581 629 GCG CCC Ala Pro 135 GGC CTG GGC GCC Cly Leu Cly Ala

CAC

Gin 140 CAC CCC TTC CAG His Pro Leu Gin

CTC

Leu 145 AAC AAG GAC ACA Asn Lys Asp Thr

GTC

Va1 150 TGC AAA CCT TGC Cys Lys Pro Cys

CTT

Leu 155 CCA GGC TAC TTC Ala Gly Tyr Phe

TCT

Ser 160 CAT CCC TTT TCC Asp Ala Phe Ser

TCC

Ser 165 ACC GAC AAA TGC Thr Asp Lys Cys

AGA

Arg 170 CCC TGG ACC AAC Pro Trp Thr Asn

TGT

Cys 175 ACC TTC CTT CGA Thr Phe Leu Cly AAC AGA Lys Arg 180 GTA CAA CAT Val Clu His

CAT

His 185 GGG ACA GAG AAA Gly Thr Glu Lys CAT GCG GTT TGC Asp Ala Val Cys AGT TCT TCT Ser Ser Ser 195 CTG CCA.GCT Leu Pro Ala 200 AGA AAA CCA CCA Arg Lys Pro Pro

AAT

Asn 205 GAA CCC CAT GTT Glu Pro His Val

TAC

Tyr 210 TTG CCC GGT Leu Pro Gly TTA ATA Leu Ile 215 ATT CTG CTT CTC Ile Leu Leu Leu GCG TCT GTG GCC Ala Ser Val Ala

CTG

Leu 225 GTG GCT GCC ATC Val Ala Ala Ile

ATC

Ile 230 TTT GGC GTT TGC Phe Gly Val Cys

TAT

Tyr 235 AGG AAA AAA GGG Arg Lys Lys Gly GCA CTC ACA GCT Ala Leu Thr Ala

AAT

Asn 245 725 773 821 TTG TGG CAC TGG Leu Trp His Trp AAT GAG OCT TGT Asn Glu Ala Cys

GGC

Gly 255 CGC CTA AGT GGA Arg Leu Ser Oly GAT AAG Asp Lys 260 GAG TCC TCA Glu Ser Ser CAG CAG GOA Gln Gln Gly 280

GOT

Gly 265 GAG AGT TGT GTC Asp Ser Gys Val

AGT

Ser 270 ACA GAG AG GA Thr His Thr Ala AAC TTT GOT Asn Phe Gly 275 GAG GAG AAG Olu Olu Lys 869 917 GA TGT GAA GOT Ala Gys Glu Gly

GTG

Val1 285 TTA GTG CTO ACT Leu Leu Leu Thr

CTG

Leu 290 AGA TTT Thr Phe 295 GGA OAA GAT ATG Pro Glu Asp Met

TG

Gys 300 TAG GGA OAT CAA Tyr Pro Asp Glri

GT

Oly 305 GOT OTC TOT CG Oly Val Gys Gin

S

S.

S *5*t S S *5 0

SO

Os

GC

Oly 310 ACG TOT OTA OGA Thr Gys Val Oly GGT CCC TACGOCA Gly Pro Tyr Ala

CAA

Gln 320 GOC OAA OAT 0CC Oly Oiu Asp Ala

AGO

Arg 325 965 1013 1061 ATG CTC TCA TTO Met Leu Ser Leu

OTC

Val1 330 AOC AAO ACC GAG Ser Lys Thr Glu GAG GAA GAG AOC Olu Olu Asp Ser TTG AGA Phe Arg 340 GAG ATO CCC Gln Met Pro GAG CG TTA Asp Gin Leu 360 GAA OAT GAA TAG Glu Asp Giu Tyr

ATO

Met 350 GAG AGO CCC TCC Asp Arg Pro Ser CG CCC ACA Gin Pro Thr 355 ACA GGT GGT Thr Pro Pro 1109 1157 .c S C

C

CTG TTC GTC ACT Leu Phe Leu Thr

GAG

Giu 365 CCT OGA AOC AAA Pro Gly Ser Lys

TCC

Ser 370 TTG TCT Phe Ser 375 GAA CCC GTG GAO Glu Pro Leu Glu GGG GAO AAT GAG Oly Oiu Asn Asp

AOT

Ser 385 TTA AOC CG TG Leu Ser Gin Gys

TTG

Phe 390 AG 000 AGA GAG Thr Gly Thr Gin

AG

Ser 395 ACA OTO GOT TCA Thr Val Oly Ser AGG TG AAC TG Ser Gys Asn Cys

ACT

Thr 405 1205 1253 1301 GAG CCC GTG TG Olu Pro Leu Gys

AGO

Arg 410 ACT OAT TOG ACT Thr Asp Trp Thr ATO TCG TGT GAA Met Ser Ser Oiu AAG TAG Asn Tyr 420 TTO CAA AAA Leu Gin Lys OTO GAG AGT GOC Val Asp Ser Oly

CAT

His 430 TG CCO GAG TG Cys Pro His Trp OCA 0CC AOC Ala Ala Ser 435 1349 CCC AGC CCC AAC TOG GCA GAT GTC TGC ACA GGC TOC CGG AAC CCT CCT Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly Cys Arg Asn Pro Pro

,AA%

GGG GAG Gly Glu 455 CCC CAG Pro Gin 470 GAC TOT OAA CCC Asp Cys Olu Pro

CTC

Leu 460

ATO

Met 445J

OTO

Val1 GOT TCC CCA Oly Ser Pro

AAA

Lys 465

GAA

Glu COT OGA CCC TTG Arg Oly Pro Leu TOC 0CC TAT Cys Ala Tyr

GOC

Gly 475

GAC

Asp GOC CTT CCC Gly Leu Pro

CCT

Pro 480 000 Gly GAA GAA 0CC Glu Glu Ala

AOC

Ser 485 AGO ACO GAG 0CC Arg Thr Giu Ala

AGA

Arg 490

AGG

Arg CAG CCC GAG Gin Pro Giu

OAT

Asp 495

TCT

Ser OCT OAT GG Ala Asp Gly AGO CTC Arg Leu 500 CCA AGC TCA Pro Ser Ser CAG TCC CCT Gin Ser Pro 520 ATC TCC AGC Ile Ser Ser

OCO

Ala 505

GCA

Ala OCA GOT 0CC Ala Oly Ala 000 Gly 510

ACT

Thr OGA AGC TCC Gly Ser Ser TCT GOA AAT Ser Gly Asn

GTO

Val1 525

AAC

Asn GGA AAC AGT Gly Asn Ser

AAC

Asn 530

ATC

Ile CCT GOT GOC Pro Gly Oly 515 TCC ACG TTC Ser Thr Phe ATC GTO GTC Ile Val Val 000 CAG GTG Gly Gin Val 535 TAC GTC Tyr Val

ATG

Met 540

CAG

Gin TTC AAG GOC Phe Lys Gly

GAC

Asp 545

GCG

Ala AGC CAG ACC Ser Gin Thr 550

ATG

Met

TCG

Ser 555

CAG

Gin GAG GOC OCO Giu Oly Ala

OCG

Ala 560

OCG

Ala OCT GCG GAG Ala Ala Oiu

CCC

Pro 565 1397 1445 1493 1541 1589 1637 1685 1733 1781 1829 1877 1926 1986 2046 2106 2166 2226 2286 2346 GOC CGC CCG Gly Arg Pro

OTG

Val1 570 GAG GAG ACC Glu Giu Thr

CTG

Leu 575

CCG

Pro COC CGA GAC Arg Arg Asp TCC TTC Ser Phe 580 GCO 000 AA Ala Gly As CTG COG GA Leu Arg 01 0CC AAG GC Ala Lys Al 615

GGOCTCOCGA

GTACAOTCOA

TTTCAGGAAG

CACTGGCA

TGACAOCTAT

ATTTOTATTC

GTTTTCTCAA

GOC CCO Gly Pro 585 CCG GAG Pro Glu CGC TTC CCG GAC Arg Phe Pro Asp 590 AAG 0CC TCO AGO Lys Ala Ser Arg TOC GOC GOC Cys Gly Gly CCC GAG 000 Pro Glu Oly 595 CAA GOC 000 Gin Oly Gly CCG GTO CAG GAG Pro Val Gin Glu T' TGAGCGCCCC CCATGGCTGG GAGCCCGAAG

CTCGGAGCCA

GOGCAGCACC

OGAAGACCAC

TGAATTGATG

GATGTCTCCC

TTTTATGACT

CTTTTCATA.A

AAATTCTCCT

GCAGCCTCTG

CCGGCATTCT

AGGACTGTCC

CTGCCACTCC

ATCCTGTTCT

CTTTTCTTGA

AAAGGTGAGG

CCCCAOCCCC

CTGCCCACTT

CCATGCCCAC

TCAAACTCGC

OTGOGGG

TATCTTTCCT

GTCTCTTTCT

GGCCACCCAG

TGCCTTCCAG

GGATGCTCAG

AGCAGTA\TT

GGTCTATGTT

CCCTCTTTTT

TTTCTCTTTT

GOATCGATCG

OAAATGGGCT

CAGCCCGCCG

TGTGGCACTA

TTCCCCCCAT

TAATGTAAAG

CCTT'TTTTT

TTCTTTTTTT

GGTGCAGCCT

GAGTAGCTGG

CAGAGACACG

CTCCAGCCTC

TTTACGTATT

TGTTCATTGT

AGTTGCTAGG

TTCTAAAAGA

TCGTTCCTTA

CCATGCTGGC

CAGCTTGGCA

ACATGGGGCT

TAAAAAAAAA

GGCAACCTGG

CTAACTCCTG

GATCACAGCT

GTCCCACCAT

GGCCTCCCAA

TTCTTTTGTG

AAACACTTTT

AACATGTGGT

AAGAAAAAAG

AGCAGAACTA

AGAGGCACTC

TTCTTCTTAT

CCTGGAAAGA

CTCTGGCCCA

GGCTCAAGCA

GCAGGCCACG

GTTACCCAGC

AGTACTGGGA

CCCCTGCTCA

GGGAAAGGGC

GGGACTTTCA

GAAACCCGAT

AGCTCAGTAT

AGGTACTTGG

TCTAGAGGTC

AAGGGCCCGG

GGCTAGAGTG

ATCCAAGTGA

CCCAGCTTCC

CTGGTCTCAA

TTACAGGCGT

CAGTGTTTTA

TAAACATGTG

TATTCTGAAA

TTATTTCTCC

GTGACCTTAC

TAAGCAAATT

TCTCTGGAAA

GAAGTTCAAG

CAGTGGTGCG

TCCTCCCACC

TCCCCCCGAC

ACTCCCCAGC

GAGCCCCCAC

GAGATGGCTT

AGGCCTGGAG

AATGTTCTAT

TGAATCTTTT

CCGCTAGGTG

TCTAAAACTC

AGATGGAGAA

GAAGAATAAA

ATTATAGCCC

TCAACCTTCG

TCCCCCCCCC

TAAAGCAGTC

GCTGGCCTGC

TCCCAGTGTG

ATAGTTGCTA

ATTCTCATTT

TAAGTTTGTG,

GTTAATTTAT

CAAGTTGCTG

AATGAACAGG

GTTGAAATTT

2406 2466 2526 2586 2646 2706 2766 2826 2886 2946 3006 3066 3126 3136 INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: LENGTH: 616 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:6: (ii) (xi) Met Ala Pro Arg Ala Arg Arg Arg Arg Pro Leu Phe Ala Leu 1 Leu Cys Ala Leu Leu 10 Ala Arg Leu Gln Val 25 Lys His Tyr Glu His 40 Lys Tyr Met Ser Ser Pro Cys Thr Lys Cys Glu Asp Ser Val Ser Glu Pro Gly Leu Leu Ala Leu Gln Leu Gly Arg Lys Cys Thr Ile Ala Pro Cys Cys Agn Thr Thr Ser Cys Leu Pro 70 55 Cys Gly Pro Asp Asn Ala Glu 75 Val Tyr Leu Asp Ser Trp Lys Glu Glu Asp Lys Leu Val Ala Val 100 Cys Leu Leu His Lys 90 Val Ala Gly Asn Ser 105 Cys Asp Thr Gly 935C Thr Thr Pro Arg Arg Cys 110 Ala Cys Thr 115 Ala Gly Tyr His Trp Ser Gin Asp Cys Glu Cys Cys Arg 120 125 Arg Leu 145 Asp Phe Val1 Val1 Leu 225 Ala Leu Thr Thr Gly 305 Gly Giu Pro Lys Ser 385 Ser As 13( Asr Ala Leu Cys Tyr 210 Val1 Leu Ser Ala Leu 290 Gly Giu A~sp Ser Ser 370 Leu -i Thr 0 *Lys Phe Gly Ser 195 Leu A].a Thr Gly Asn 275 Glu Val1 Asp Set Gin 355 Thr Ser Asn Gil Asp Ser Lys 180 Ser Pro Ala Ala Asp 260 Phe Giu Cys Ala Phe 340 Pro Pro Gln :ys ICys Thr Ser 165 Arg Ser Gly Ile Asn 245 Lys Gly Lys Gin Arg 325 Arg Thr Pro Cys ThrC 405 Ala Val1 150 Thr Val1 Leu Leu Ile 230 Leu Giu Gin Thr Sly 310 Me t Gln %.sp The ?he 390 flu Pro 135 Cys Asp Glu Pro Ile 215 Phe Trp Ser Gin Phe 295 Thr Leu Met Gin SerC 375 ThrC Pro G 1) Lys Lys His Ala 200 Ile Gly His Ser Sly 280 Pro :ys Ser Pro eu 360 'lu fly ,eu Lei- Pro Cys His 185 Arg Leu Val Trp Gly 265 Ala Giu Val1 Leu Thr 345 Leu Pro Thr Cys Giu 425 Gly Cys Arg 170 Gly Lys Leu Cys Ile 250 Asp Cys Asp Gly Val1 330 Giu Phe Leu Gin Arg 410 Val1 AL1 Let, 155 Pro Thr Pro Leu Tyr 235 Asn Ser Glu Met Gly 315 Ser Asp Leu Glu Ser 395 Thr Asp IGin 140 IAla Trp Giu Pro Phe 220 Arg Glu Cys Gly Cys 300 Gly Lys Giu Thr ValC 380 Thr N Asp 9] Ser C His Gly Thr Lys Asn 205 Ala Lys Ala Val Val1 285 tyr Pro Thr ryr flu 365 fly al1 rrp ;ly Prc Tyr Asn Ser 190 Giu Ser Lys Cys Ser 270 Leu Pro Tyr Giu Met 350 Pro Glu Gly Thr His 430 Leu -Phe Cys 175 *Asp Pro Val1 Gly Gly 255 Thr Leu Asp Ala Ile 335 Asp Gly Asn Ser Pro 415 Cys Gin Ser 160 Thr Ala His Ala Lys 240 Arg His Leu Gin Sin 320 Glu Arg Ser %.sp flu 100 d1et ?ro Ser Ser Giu Asn Tyr Leu Gin Lys 420 His Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly 435 440 Cys Lys 465 Glu Ala Ser Ser Asp 545 Ala Arg Gly Gin Arg 450 Arg Glu Asp Ser Asn 530 Ile Ala Arg Gly Glu 610 Asn Pro Gly Pro Glu Ala Gly Arg 500 Pro Gly 515 Ser Thr Ile Val Ala Glu Asp Ser 580 Pro Glu 595.

Gin Gly Pro Leu Ser 485 Leu Gly Phe Val Pro 565 Phe Gly Gly Gly Pro 470 Arg Pro Gin Ile Tyr 550 Met Ala Leu Ala Glu 455 Gin Thr Ser Ser Ser 535 Val Gly Gly Arg Lys 615 Asp Cys Glu Ser Pro 520 Ser Ser Arg Asn Glu 600 Ala Cys Glu Ala Tyr Ala Arg 490 Ala Arg 505 Ala Ser Gly Gin Gin Thr Pro Val 570 Gly Pro 585 Pro Glu Pro Gly 475 Asp Ala Gly Val Ser 555 Gin Arg Lys Leu 460 Met Gin Gly Asn Met 540 Gin Glu Phe Ala 445 Val Gly Pro Ala Val 525 Asn Glu Glu Pro Ser 605 Gly Leu Glu Gly 510 Thr Phe Gly Thr Asp 590 Arg Ser Pro Asp 495 Ser Gly Lys Ala Leu 575 Pro Pro Pro Pro 480 Gly Gly Asn Gly Ala 560 Ala Cys Val INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vii) IMMEDIATE SOURCE: CLONE: FLAG® peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asp Tyr Lys Asp Asp Asp Asp Lys 1 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 232 amino acids TYPE: amino acid (i i) (vi) (vii) (xi) STRANDEDNESS: not relevant TOPOLOGY: linear MOLECULE TYPE: protein ORIGINAL

SOURCE:

ORGANISM: Human IMIMEDIATE

SOURCE:

CLONE: IgGl Fc mutein SEQUENCE DESCRIPTION: SEQ ID NO:8: Glu Pro Arg Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Prc 1 r Pro Lys Giu Asp Ala Thr Val Asp Tyr Asp Leu Arg Lys 145 His Lys Ser Ser Ser 225 -Asp Gly *Asn Trp Pro Giu 130 Asn Ile Thr Lys Cys 210 Val Val Ser Leu Ala 115 Pro Gin Ala Thr Leu 195 Ser Giu Leu Ser Giu Thr Asn 100 Pro Gin Val Val Pro 180 Thr N Val b.

His Val2 Tyr Gly Met Val1 Ser 31u 165 Pro al1 lE Glu *His *Arg *Lys Gin Tyr Leu 150 Trp Val Asp Pro Ser Val 25 Ser Arg Thr 40 Asp Pro Giu 55 Asn Ala Lys Val Val Ser Asp Tyr Lys 105 Lys Thr Ile 120 Thr Leu Pro 135 Thr Cys Leu Glu Ser Asn Leu ASP Ser 185 Lys Ser Arg 200 Phe Let Pro Glu Val1 Thr Val1 90 Cys Ser Pro Val1 Gly 170 Asp rrp Lys Lys 75 Leu Lys Lys Ser Lys 155 Gin Gly Gin Phe Pro Pro Lys Val Thr Cys Val Phe Asn Trp Tyr 60 Pro Arg Giu Giu Thr Val Leu His Val Ser Asn Lys 110 Ala Lys Gly Gin.

125 Arg Asp Giu Leu 140 Gly Phe Tyr Pro Pro Giu Asn Asn 175 Ser Phe Phe Leu 190 Gin Gly Asn Val 205 Ala Pro Val1 Val1 Gin Gin Ala Pro Thr Arg 160 T'yr Tyr Phe iet His Giu Ala Leu His Asn 215 His 220 Tyr Thr Gin Lys Leu Ser Leu Ser Pro Gly Lys 230 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 31 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: CMV (R2780 Leader) (ix) FEATURE: OTHER INFORMATION: Metl-Arg28 is the actual leader peptide; Arg29 strengthens the furin cleavage site; nucleotides encoding Thr30 and Ser31 add a Spel site.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr 1 5 10 Val Ala Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser 25 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1630 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: A) ORGANISM: Mus musculus (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE: RANKL (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..884 (xi) SEQUENCE DESCRIPTION: SEQ ID CC GGC GTC CCA CAC GAG GGT CCG CTG CAC CCC GCG CCT TCT GCA CCG 47 Gly Val Pro His Glu Gly Pro Leu His Pro Ala Pro Ser Ala Pro 1 5 10 GCT CCG GCG Ala Pro Ala CCG CCA CCC GCC Pro Pro Pro Ala CCC TCC CGC TCC ATG TTC CTG Ala Ser Arg Ser Met Phe Leu 25 GCC CTC Ala Leu CTG GGG CTC GGA Leu Gly Leu TAC TTT CGA Tyr Phe Arg so Gly CTC GGC CAC GTG GTC TGC Leu Cly Gin Val. Val Cys 40 AGC ATC GCT Ser Ile Ala CTG TTC CTG Leu Phe LLu GAC ACC ACT Asp Ser Thr 143 191 GCG CAG ATG GAT Ala Gin Met Asp

CCT

Pro 55 AAC AGA ATA TCA Asn Arg Ile Ser

GAA

Ciu CAC TGC His Cys TTT TAT AGA ATC Phe Tyr Arg Ile ACA CTC CAT GAA Arg Leu His Giu

AAC

Asn GCA GAT TTG CAG Ala Asp Leu Gin

GAC

Asp TCC ACT CTG GAG Ser Thr Leu.Glu

ACT

Ser 85 CAA GAC ACA CTA Giu Asp Thr Leu

CCT

Pro 90 GAC TCC TGC AGG Asp Ser Cys Arg

AGC

Arg ATO AAA CAA CC Met Lys Gin Ala

TTT

Phe 100 CAG CGG GCC GTG Gin Cly Ala Val

CAG

Gin 105 AAG CAA CTG CAA Lys Glu Leu Gin CAC ATT His Ile GTG CCC CCA Val Gly Pro TCG TTG CAT Trp Leu Asp 130

CAG

Gin 115 CCC TTC TCA GGA Arg Phe Ser Gly

GCT

Ala 120 CCA GCT ATC ATC Pro Ala Met Met CAA CGC TCA Ciu Gly Ser 125 CCA TTT CCA Pro Phe Ala GTG CCC CAC CCA Val Ala Gin Arg

GCC

Gly 135 AAC CCT GAG CC Lys Pro Clu Ala

CAC

Gin 140 239 287 335 383 431 479 527 575 623 671 CAC CTC His Leu 145 ACC ATC AAT GCT Thr Ile Asn Ala

CC

Ala 150 ACC ATC CCA TCG Ser Ile Pro Ser

GGT

Gly 155 TCC CAT AAA GTC Ser His Lys Val

ACT

Thy 160 CTG TCC TCT TGG Leu Ser Ser Trp

TAC

Tyr 165 CAC CAT CGA GGC His Asp Arg Gly

TGC

Trp 170 CCC AAC ATC TCT Ala Lys Ile Ser ATG ACC TTA AGC Met Thr Leu Ser GGA AAA CTA AG Gly Lys Leu Arg

CTT

Val1 185 AAC CAA CAT GC Asn Gin .Asp Gly TTC TAT Phe Tyr 9 TAC CTG TAC Tyr Leu Tyr GTA CCT ACA Val Pro Thy 210

CC

Ala 195 AAC ATT TGC TTT Asn Ile Cys Phe

CGC

Arg 200 CAT CAT GAA ACA His His Giu Thy TCG GCA AGC Ser Gly Ser 205 AAA ACC AGC Lys Thr Ser GAC TAT CTT CAG Asp Tyr Leu Gin

CTG

Leu 215 ATG GTG TAT GTC Met Vai Tyr Val

GTT

Val1 220 ATC AAA Ile Lys 225 ATC CCA ACT TCT Ile Pro Ser Ser AAC CTC ATG AAA Asn Leu Met Lys

GGA

Cly 235 CCC ACC ACG AAA Gly Ser Thr Lys

AAC

Asn 240 TGC TCG CCC AAT Trp Ser Gly Asn

TCT

Ser 245 GAA TTC CAC TTT Giu Phe His Phe

TAT

Tyr 250 TCC ATA AAT GTT Ser Ile Asn Val GGA TTT TTC AAG Gly Phe Phe Lys CTC CGA GCT GGT GAA GAA ATT AGC ATT CAG GTG TCC Leu Arg Ala Gly Glu Glu Ile Ser Ile Gin Val Ser 260 265 270 CTG GAT CCG GAT CAA GAT GCG ACG TAC TTT GGG GCT Leu Asp Pro Asp Gin Asp Ala Thr Tyr Phe Gly Ala 280 285 GAC ATA GAC TGAGACTCAT TTCGTGGAAC ATTAGCATGG AAC CCT TCC Asn Pro Ser TTC AAA GTT

CTG

Leu 275

CAG

815 863 914 Phe Lys Val Gin Asp Ile Asp 290 ATGTCCTAGA TGTTTGGAAA

ACTAAGAGAC

GTTGTGTATA

ACAATTTTGT

GAAAAACTTA

TGTGCCACTG

TGAAGGGTTA

TTTCTAATGA

GTGTAATGTT

AATATTTAAA

ACTGGTGCAC

CTGGTGACCA

ATGGCCCACG

TGTAAAGTCC

AATGATTTCC

CACGTGAGCT

AGAACCTTGA

AGTTCTTTTG

GGAGAGAAAA

TTCTGTGCAA

AATGTCTCAC

TTTGTAATTC

CATGTAGTTT

CTTCTTAAA.A

GTGTATGAAA

ATAGGTGATG

TAGAATTGAA

ATGGAAGGGG

AATTAAGAGG

AATTGTTACA

ATATATGTAT

AGTTTTGTA.A

TGTTGACATA

CCCTGAAGGT

ATTTCTTTAT

AATGGATGAT

CTCACAGCCC

TTAGATTCAT

CCAGATTGGG

GTCACAGTCT

ATGCCATGTC

TTGCGC!TGGG

TTTTATATAA

ATTATATTTG

TTTA.ATGTTT

ACTCGTAGCT

TCTTTTTAAC

GTCTATACAT

TCTCTCTTGA

GGTGATTACA

AGAGGTATTC

CTGGGTCTAA

ATTGCAAAGA

ACCTGCAAAT

TGTCTAAAGT

TGCTATAGTA

TAAATGTACA

A.AGGGGGCAG

TTAATAGAGT

GTGTAAGACT

GCCTGTACAG

CAACGGTTTT

CGATGCTTAT

CCCCTGGACA

AATGATAGTG

AAGTTCTTTT

TATATTTCAG

TTTGATTCAA

GATGTATTTA

AATACTGTTT

CTTCAG

974 1034 1094 1154 1214 1274 1334 1394 1454 1514 1574 1630 INFORMATION FOR SEQ ID NO:l1: SEQUENCE CHARACTERISTICS: LENGTH: 294 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Gly Val Pro His Glu Gly Pro Leu His Pro Ala Pro Ser Ala Pro Ala 1 5 10 Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser Met Phe Leu Ala Leu Leu 25 Gly Leu Gly Leu Gly Gin Val Val Cys Ser Ile Ala Leu Phe Leu Tyr 40 Phe Arg Ala Gin Met Asp Pro Asn Arg Ile Ser Glu Asp Ser Thr His 55 Cys Phe Tyr Ser Thr Leu Lys Gin Ala Gly Pro Gin 115 Arg Glu Phe 100 Arg Ile Leu 70 Ser Glu Gin Gly Phe Ser Arg Asp Aia Leu Thr Val1 Hi o Leu Gin 105 Pro Glu Pro 90 Lys Ala Asn Ala 75 Asp Ser Giu Leu Met Met Asp Leu Gin Cys Arg Arg Gin His Ile 110 Asp Met Val1 120 Gi1 Gy5 e Leu Leu 145 Leu Thr Leu Pro Lys 225 Trp Phe Asp 130 Thr Ser Leu Tyr Thr 210 Ile Ser Phe Val Ala Gin Arg Gly Lys Pro Giu Sle Asn Ser Trp Ser Asn 180 Ala Asn 195 Asp Tyr Pro Ser Giy Asn Lys Leu 260 Ala Tyr 165 Gly Il.e Leu Ser Ser 245 Ala 150 His Lys Cys Gin His 230 Giu 135 Ser Asp Leu Phe Leu 215 Asn Phe Ile Arg A rg Arg 200 Met Leu His Pro Gly Val1 185 His Val1 Met Phe Ser Trp 170 Asn His Tyr Lys Tyr 250 *Ala Gly 155 Ala Gin Glu Val1 Gly 235 Ser Gir 140 Ser Lys Asp Thr Val1 220 Gly Ile Pro *His Ile Gly Ser 205 Lys Ser Asn Phe Lys Ser Phe 190 Giy Thr Thr Val1 Ala Val1 Asn 175 Tyr Ser Ser Lys Giy His Thr 160 Met Tyr Val1 Ile Asn 240 Gly Arg Ala Gly Glu Glu 265 Ile Ser Ile Gin Val Ser Asn 270 Pro Ser Leu Leu Asp Pro Asp Gin Asp Ala Thr Tyr 275 280 Lys Val Gin Asp Ile Asp 290 INFORMATION FOR SEQ ID NO:i2: SEQUENCE

CHARACTERISTICS:

LENGTH: 954 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: No (vi) ORIGINAL

SOURCE:

Phe 285 Gly Ala Phe ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE: huRANKL (full length) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..951 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

ATG

Met 1 CGC CGC GCC Arg Arg Ala

AGC

Ser 5 AGA GAC TAC ACC Arg Asp Tyr Thr AAG TAC Lys Tyr 10 CTG CGT GGC TCG GAG Leu Arg Gly Ser Glu GAG ATG GGC Glu Met Gly CCG CCG CCG Pro Pro Pro GGC CCC GGA GCC Gly Pro Gly Ala CAC GAG GGC CCC His Glu Gly Pro CTG CAC GCC Leu His Ala CGC TCC ATG Arg Ser Met CCT GCG CCG CAC Pro Ala Pro His

CAG

Gin 40 CCC CCC GCC GCC Pro Pro Ala Ala

TCC

Ser TTC GTG Phe Val GCC CTC CTG GGG Ala Leu Leu Gly

CTG

Leu 55 GGG CTG GGC CAG Gly Leu Gly Gin

GTT

Val GTC TGC AGC GTC Val Cys Ser Val

GCC

Ala CTG TTC TTC TAT Leu Phe Phe Tyr AGA GCG CAG ATG Arg Ala Gin Met

GAT

Asp 75 CCT AAT AGA ATA Pro Asn Arg Ile

TCA

Ser GAA GAT GGC ACT Glu Asp Gly Thr TGC ATT TAT AGA Cys Ile Tyr Arg TTG AGA CTC CAT Leu Arg Leu His GAA AAT Glu Asn 192 240 288 336 384 GCA GAT TTT Ala Asp Phe CCT GAT TCA Pro Asp Ser 115

CAA

Gin 100 GAC ACA ACT CTG Asp Thr Thr Leu

GAG

Glu 105 AGT CAA GAT ACA Ser Gin Asp Thr AAA TTA ATA Lys Leu Ile 110 GCT GTG CAA Ala Val Gin TGT AGG AGA ATT Cys Arg Arg Ile

AAA

Lys 120 CAG GCC TTT CAA Gin Ala Phe Gin

GGA

Gly 125 too.

.000 AAG GAA Lys Glu 130 TTA CAA CAT ATC Leu Gln His Ile GGA TCA CAG CAC Gly Ser Gin His

ATC

Ile 140 AGA GCA GAG AAA Arg Ala Glu Lys

GCG

Ala 145 ATG GTG GAT GGC Met Val Asp Gly

TCA

Ser 150 TGG TTA GAT CTG Trp Leu Asp Leu AAG AGG AGC AAG Lys Arg Ser Lys 432 480 528 GAA GCT CAG CCT Glu Ala Gin Pro

TTT

Phe 165 GCT CAT CTC ACT Ala His Leu Thr

ATT

Ile 170 AAT GCC ACC GAC Asn Ala Thr Asp ATC CCA Ile Pro 175 TCT GGT TCC Ser Gly Ser CAT AAA His Lys 180 GTG AGT CTG Val Ser Leu TCT TGG TAC CAT Ser Trp Tyr His GAT CGG GGT Asp Arg Gly 190 576

TG(

Tr~ AA1 Asr

CAT

His 225

TAC

TPyr

AAA

Lys

TAT

Tyr

ATC

Ile

GCA

Ala 305 (2) 3CCC AAG ATC TCC AAC ATG ACT TTT AGC )Ala Lys Ile Ser Asn Met Thr Phe Sex 195 200 CAG CAT GGC TTT TAT TAC CTG TAT CC Gin Asp Gly Phe Tyr Tyr Leu Tyr Ala 210 215 GAA ACT TCA GGA GAC CTA GCT ACA GAG Glu Thr Ser Gly Asp Leu Ala Thr Glu 230 GTC ACT AAA ACC AGC ATC AAA ATC CCA Val Thr Lys Thr Ser Ile Lys Ile Pro 245 250 GGA GGA AGC ACC AAG TAT TGG TCA CCC .Gly Gly Ser Thr Lys-Tyr Trp Ser Gly 260 265 TCC ATA AAC GTT GGT GGA TTT TTT AAG Ser Ile Asn Val Gly Gly Phe Phe Lys 275 280 ACC ATC GAG GTC TCC AAC CCC TCC TTA Ser Ile Giu Val Ser Asn Pro Ser Leu 290 295 ACA TAC TTT CCC GCT TTT AAA GTT CGA Thr Tyr Phe Gly Ala Phe Lys Vai Arg 310 INFORMATION FOR SEQ ID NO:13: SEQUENCE

CHARACTERISTICS:

LENGTH: 317 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ IDI Arg Arg Ala Ser Arg Asp Tyr Thr Lys 10 4et Giy Gly Gly Pro Gly Ala Pro His C 20 25 ?ro Pro Pro Ala Pro His Gin Pro Pro l 40 'al.Ala Leu Leu Gly Leu Gly Leu Gly C 55 ~eu Phe Phe Tyr Phe Arg Ala Gin Met A 70 .sp Gly Thr His Cys Ile Tyr Arg Ile L 90 Asr

AAC

As n

TAT

Tyr 235

ACT

Ser

AAT

As n

TTA

Leu

CTG

Leu

CAT

Asp 315 CGA AAA Gly Lys 205 ATT TC Ile Cys 220 CTT CAA Leu Gin TCT CAT Ser His TCT CAA Ser Giu CCC TCT Arg Ser 285 CAT CCG Asp Pro 300 ATA CAT TI Ile Asp

CT)

LeL TT1 Phe

CTA

Leu

ACC

Thr

TTC

Phe 270

'GA

i1Y

AT

~sp

~GA

\ATA

CGA

Arg

ATC

Met

CTG

Leu 255

CAT

His

GAG

Clu CAG C Gin

GTT

Val1

CAT

His

GTG

Val1 240

ATG

Met

~TTT

Phe 3AA 1lu

;AT

~sp 624 672 720 768 816 864 912 Met Glu Pro Phe Ala Glu

L

A

'JO: 13: Pyr Leu l1u Cly la Ala ;ln Vai S5p Pro eu Arg Arg Gly Pro Leu Ser. Arg Val Cys Asn Arg Leu His Ser His Ser Ser Ile Clu Giu Ala Met Val1 Ser As n Ala Asp Phe Gin Asp Thr Thr Leu Glu Ser Gin Asp.Thr Lys Leu Ile 100 Pro Lys Ala 145 Glu Ser Trp Asn His 225 Tyr Lys Tyr Ile Ala 305 Asp Glu 130 Met Ala Gly Ala Gin 210 Glu Val Gly Ser Ser 290 Thr Ser 115 Leu Val Gin Ser Lys 195 Asp Thr Thr Gly Ile 275 Ile Tyr Cys Gin Asp Pro His 180 Ile Gly Ser Lys Ser 260 Asn Glu Phe Arg His Gly Phe 165 Lys Ser Phe Gly Thr 245 Thr Val Val Gly Arg Ile Ser 150 Ala Val Asn Tyr Asp 230 Ser Lys Gly Ser Ala 310 Ile Val 135 Trp His Ser Met Tyr 215 Leu Ile Tyr Gly Asn 295 Phe Lys 120 Gly Leu Leu Leu Thr 200 Leu Ala Lys Trp Phe 280 Pro Lys 105 Gin Ser Asp Thr Ser 185 Phe Tyr Thr Ile Ser 265 Phe Ser Val 110 Ala Gin Leu Ile 170 Ser Ser Ala Glu Pro 250 Gly Lys Leu Arg Phe His Ala 155 Asn Trp Asn Asn Tyr 235 Ser Asn Leu Leu Asp 315 Gin Ile 140 Lys Ala Tyr Gly Ile 220 Leu Ser Ser Arg Asp 300 Ile Gly 125 Arg Arg Thr His Lys 205 Cys Gin His Glu Ser 285 Pro Asp Ala Ala Ser Asp Asp 190 Leu Phe Leu Thr Phe 270 Gly Asp Val Glu Lys Ile 175 Arg Ile Arg Met Leu 255 His Glu Gin Gin Lys Leu 160 Pro Gly Val His Val 240 Met Phe Glu Asp 9* 9* *t INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 1878 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Murine (vii) IMMEDIATE

SOURCE:

LIBRARY: Murine Fetal Liver Epithelium CLONE: muRANK (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 1875 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AT CC CCC CGC Met Ala Pro Arg GCC CGG Ala Arg CGC CGC CGC Arg Arg Arg

CAG

Gin 10 CTG CCC GCG CCG Leu Pro Ala Pro CTG CTC Leu Leu GCG CTC TGC Ala Leu Cys CCT OCA TGO Pro Pro Cys

GTG

Val1 CTG CTC GTT CCA Leu Leu Val Pro CAG GTG ACT CTC Gin Val Thr Leu.

CAC GTC ACT Gin Val Thr CGG TGT TC Arg Cys Cys 48 96 144 ACC CAG GAG AGG Thr Gin Clu Arg

CAT

His 40 TAT GAG CAT CTC Tyr Clu His Leu.

GGA

Gly AGC AGA Ser Arg TGC GAA CCA GCA Cys Glu. Pro Gly

AAG

Lys 55 TAC CTG TCC TCT Tyr Leu. Ser Ser TCC ACT COT ACC Cys Thr Pro Thr GAO AGT CTG TGT Asp Ser Val Cys 000 TGT GGC CCC Pro Cys Gly Pro

CAT

Asp GAG TAO TTC GAO Glu Tyr Leu Asp

ACC

Thr 0 *000 S S 0 TCC A.AT GAA GAA Trp Asn Clu Clu

CAT

Asp AAA TCC TTC CTC Lys Cys Leu Leu AAA GTC TCT CAT Lys Val Cys Asp GOA GCC Ala Cly AAC CCC CTC Lys Ala Leu.

TCT CT TC Cys Ala Cys 115

CTC

Val1 100 CC CTC CAT COT Ala Val. Asp, Pro AAC CAC ACC CC Asn His Thr Ala CCC CT CC Pro Arg Arg 110 GAG TCC TC Clu Cys Cys 192 240 288 336 384 432 480 528 ACC GOT CCC TAO Thr Ala Cly Tyr TGC AAC TCA GAO Trp Asn Ser Asp

TC

Cys 125 CCC ACC Arg Arg 130 AAC AOG GAC TCT Asn Thr Clu Cys

CCA

Ala 135 COT CCC TTC GCA Pro Gly Phe Cly CAGCOAT CCC TTG Gin His Pro Leu.

CAC

Gin 145 OTO AAC AAG CAT Leu Asn Lys Asp

AC

Thr 150 CTG TC ACA COO Val Cys Thr Pro

TC

Cys 155 OTO OTC CCC TTC Leu Leu. Cly Phe

TTO

Phe TCA CAT CTC TTT Ser Asp Val Phe

TOG

Ser 165 TOO ACA GAO AAA Ser Thr Asp Lys

TC

Cys 170 AAA OCT TGG ACC Lys Pro Trp Thr AAO TC Asn Cys ACC OTO OTT Thr Leu Leu

CCA

Cly 180 AAGCOTA GAA CCA Lys Leu Clu. Ala CAC CCC ACA ACG Gin Gly Thr Thr GAA TOA CAT Clu Ser Asp 190 576 GTG GTC TGC Val Val Cys 195 AGC TCT TCC ATG Ser Ser Ser Met

ACA

Thr 200 CTG AGG AGA CCA Leu Arg Arg Pro

CCC

Pro 205 AAG GAG GCC Lys Giu Ala CAG GCT Gin Ala 210 TAC CTG CCC AGT Tyr Leu Pro Ser

CTC

Leu 215 ATC GTT CTG CTC Ile Val Leu Leu

CTC

Leu 220 TTC ATC TCT GTG Phe Ile Ser Val 672 720

GTA

Val1 225 GTA GTG GCT GCC Vai Vai Ala Ala ATC TTC GGC GTT Ile Phe Gly Val TAC AGG AAG GGA Tyr Arg Lys Giy AAA GCG CTG ACA GCT AAT TTG TGG AAT Lys Ala Leu Thr Ala Asn Leu Trp Asn 245

TGG

Trp 250 GTC AAT GAT GCT Val Asn Asp Ala TGC AGT Cys Ser 255 AGT CTA AGT Ser Leu Ser CAC TCG GCA His Ser Ala 275

GGA

Gly 260 AAT AAG GAG TCC Asn Lys Glu Ser

TCA

Ser 265 GGG GAC CGT TGT Giy Asp Arg Cys GCT GGT TCC Ala Gly Ser 270 ATC TTA CTA Ile Leu Leu ACC TCC AGT GAG Thr Ser Ser Gin

CAA

Gin 280 GAA GTG TGT GAA Giu Val Cys Giu

GGT

Gly 285 ATG ACT Met Thr 290 CGG GAG GAG AAG Arg Giu Glu Lys

ATG

Met 295 GTT CCA GAA GAC Val Pro Giu Asp

GGT

Gly 300 GCT GGA GTC TGT Ala Gly Vai Cys GGG CCT GTG TGT GCG GCA Gly Pro Vai Cys Ala Ala 305 310 GGT GGG CCC TGG Gly Gly Pro Trp

GCA

Ala 315 GAA GTC AGA GAT Giu Val Arg Asp

TCT

Ser 320 .0000 so 0 0:0 AGG ACG TTC ACA Arg Thr Phe Thr

CTG

Leu 325 GTC AGC GAG GTT Val Ser Glu Val

GAG

Giu 330 ACG CAA GGA GAC Thr Gin Gly Asp CTC TCG Leu Ser 335 912 960 1008 1056 1104 AGG, AAG ATT Arg Lys Ile TCG ACT GGT Ser Thr Gly 355 ACA GAG GAT GAG Thr Giu Asp Giu ACG GAC CGG CCC Thr Asp Arg Pro TCG CAG CCT Ser Gin Pro 350 TCT ATA CCC Ser Ile Pro TCA CTG CTC CTA Ser Leu Leu Leu

ATC

Ile 360 CAG CAG GGA AGC Gin Gin Gly Ser

AAA

Lys 365 0* @4 0

S

S

0000 CCA TTC Pro Phe 370 CAG GAG CCC CTG Gin Glu Pro Leu

GAA

Giu 375 GTG GGG GAG AAC Val Gly Giu Asn AGT TTA AGC CAG Ser Leu Ser Gin

TGT

Cys 385 TTC ACC GGG ACT Phe Thr Gly Thr AGC ACG GTG GAT Ser Thr Val Asp

TCT

Ser 395 GAG GGC TGT GAC Giu Gly Cys Asp

TTC

Phe 400 1152 1200 1248 1296 ACT GAG CCT CCG Thr Glu Pro Pro CAC CTG ACA AAA His Leu Thr Lys 420

AGC

Ser 405 AGA ACT GAC TCT Arg Thr Asp Ser

ATG

Met 410 CCC GTG TCC CCT Pro Val Ser Pro GAA AAG Giu Lys 415 GAA ATA GAA GGT Giu Ile Giu Gly AGT TGC CTC CCC TGG GTG GTC Ser Cys Leu Pro Trp Val Val 430 AGC TCC AAC Ser Ser Asn 435 TCA ACA GAT GGC Ser Thr Asp Gly

TAC

Tyr 440 ACA GGC AGT GGG Thr Gly Ser Gly

AAC

Asn 445 ACT CCT GGG Thr Pro Gly 1344 GAG GAC CAT GAA CCC TTT CCA Ulu Asp 450 His Glu Pro Phe GGG TCC CTG AAA TGT Gly Ser Leu Lys Cys 460 GGA CCA TTG CCC Gly Pro Leu Pro

CAG

Gin 465 TGT GCC TAC AGC Cys Ala Tyr Ser

ATG

Met 470 GGC TTT CCC AGT Gly Phe Pro Ser

GAA

Glu 475 GCA GCA GCC AGC Ala Ala Ala Ser

ATG

Met 480 GCA GAG GCG GGA Ala Glu Ala Gly

GTA

Val 485 CGG CCC CAG GAC Arg Pro Gin Asp

AGG

Arg 490 GCT GAT GAG AGG Ala Asp Glu Arg GGA GCC Gly Ala 495 TCA GGG TCC Ser Gly Ser GTG ACT GGA Val Thr Gly 515

GGG

Gly 500 AGC TCC CCC AGT Ser Ser Pro Ser

GAC

Asp 505 CAG CCA CCT GCC Gin Pro Pro Ala TCT GGG AAC Ser Gly Asn 510 CAG GTG ATG Gin Val Met AAC AGT AAC TCC Asn Ser Asn Ser

ACG

Thr 520 TTC ATC TCT AGC Phe Ile Ser Ser

GGG

Gly 525 AAC TTC Asn Phe 530 AAG GGT GAC ATC Lys Gly Asp Ile

ATC

Ile 535 GTG GTG TAT GTC Val Val Tyr Val CAG ACC TCG. CAG Gin Thr Ser Gin 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1878

GAG

Glu 545 GGC CCG GGT TCC Gly Pro Gly Ser

GCA

Ala 550 GAG CCC GAG TCG Glu Pro Glu Ser

GAG

Glu 555 CCC GTG GGC CGC Pro Val Gly Arg

CCT

Pro 560 GTG CAG GAG GAG Val Gin Glu Glu CTG GCA CAC AGA Leu Ala His Arg

GAC

Asp 570 TCC TTT GCG GGC Ser Phe Ala Gly ACC GCG Thr Ala 575 CCG CGC TTC Pro Arg Phe GGG GCA CCC Gly Ala Pro 595 GGT GGG GCG Gly Gly Ala 610 GAC GTC TGT GCC Asp Val Cys Ala

ACC

Thr 585 GGG GCT GGG CTG Gly Ala Gly Leu CAG GAG CAG Gin Glu Gin 590 CAG GAG CAG Gin Glu Gin CGG CAG AAG GAC Arg Gin Lys Asp

GGG

Gly 600 ACA TCG CGG CCG Thr Ser Arg Pro CAG ACT TCA Gin Thr Ser

CTC

Leu 615 CAT ACC CAG GGG His Thr Gin Gly GGA CAA TGT GCA Gly Gin Cys Ala GAA TGA Glu 625 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 625 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Pro Arg Ala Arg Arg Arg Arg Gin Leu Pro Ala Pro Leu Leu 1 5 10 Ala Pro Ser Ser Trp Lys Cys Arg Gin 145 Ser Thr Val1 Gin Val1 225 Lys Ser His Leu Pro Arg Asp Asn Ala Ala Arg 130 Leu Asp Leu Val1 Ala 210 Val Ala Leu Ser Cys Cys Cys Ser Glu Leu Cys 115 Asn Asn Val Leu Cys 195 Tyr Val1 Leu Ser Ala 275 Val1 Thr Glu Val1 Glu Val1 100 Thr Thr Lys Phe Gly 180 S er Leu Ala Thr Gly 260 Thr Leu Gin Pro Cys Asp Ala Ala Glu Asp Ser 165 Lys Ser Pro Ala Ala 245 Asn Ser Leu Giu Gly Leu 70 Lys Val1 Gly Cys Thr 150 Ser Leu Ser Ser Ile 230 Asn Lys Ser Val1 Arg Lys 55 Pro Cys Asp Tyr Ala 135 Val1 Thr Glu Met Leu 215 Ile Leu Glu Gin Pro His 40 Tyr Cys Leu Pro His 120 Pro Cys Asp Ala Thr 200 Ile Phe Trp Ser Gin 280 Leu 25 Tyr Leu Gly Leu Gly 105 Trp Gly Thr Lys His 185 Leu Val1 Gly Asn Ser 265 Giu Gin Val Glu His Ser Ser Pro Asp 75 His Lys 90 Asn His Asn Ser Phe Gly Pro Cys 155 Cys Lys 170 Gin Gly Arg Arg Leu Leu Val Tyr 235 Trp Val 250 Gly Asp Val Cys Thr Leu Lys Glu Val1 Thr Asp Ala 140 Leu Pro Thr Pro Leu 220 Tyr Asn Arg Glu Gly 300 Leu Gly Cys Tyr Cys Ala Cys 125 Gin Leu Trp Thr Pro 205 Phe Arg Asp Cys Gly 285 Gin Arg Thr Leu Asp Pro 110 Giu His Gly Thr Glu 190 Lys Ile Lys Ala Ala 270 Ile Val1 Cys Pro Asp Ala Arg Cys Pro Phe Asn 175 Ser Glu Ser Gly Cys 255 Gly Leu Thr Cys Thr Thr Gly Arg Cys Leu Phe 160 Cys Asp Ala Val1 Gly 240 Ser Ser Leu Met Thr Arg Glu Glu Lys Met Val Pro Glu Asp 290 295 Ala Gly Val Cys WO 98/284 Gly Pro 305 124 Ii Val Cys Ala Ala Gly Gly Pro Trp Ala Glu Val Arg Asp 'CTIUS97/2386 6 Ser Arg Thr Phe Thr Leu Val Ser Glu Va 325 Arg Ser Pro Cys 385 Thr His Ser Giu Gin 465 Ala Ser( Val 91 Asn 1 Glu G 545 Val G Pro A Gly A Ly, Th PhE 37( PhE Gil Leu Ser Asp 450 Mlu Mly rhr ~he in .rg la s Il r GI G1 Th~ iPrc Thit Asn 435 His Ala Ala Ser Gly 515 Lys Pro Glu Phe Pro e Pro Thr Glu 340 Y Ser Leu Leu 5 n Giu Pro Leu r Gly Thr Giu 390 Pro Ser Arg 405 Lys Giu Ile 420 Ser Thr Asp Giu Pro Phe Tyr Ser Met 470 Gly Val Arg 485 Gly Ser Ser 500 Asn Ser Asn Gly Asp Ile Gly Ser Ala C 550 Giu Thr Leu 1 565 Pro Asp Val c 580 Arg Gin Lys A As] Lei Gli 375 Sei Thr Gil Gly Pro 455 Gly Pro Pro Ser Ilie 335 iu lia :ys ~sp eu 15 p Glu uIle 360 .1 Val Thr -Asp Gly Tyr 440 Gly Phe Gin Ser I Thr 520 Val Pro G His A Ala Tr 5 Gly T 600 Ty 34 Gl Gl Va Sezi Asp 425 Thr Ser Pro ksp sp 305 ~he ral iu .rg hr 85 hr 315 1 Giu Thr Gin 330 r Thr Asp Arg 5 n Gin Gly Ser v{ Giu Asn Asp 380 LAsp Ser Giu 395 Met Pro Val 4i0 Ser Cys Leu Gly Ser Gly Leu Lys Cys 460 Ser Giu Ala 475 Arg Aia Asp 490 Gin Pro Pro Ile Ser SerC Tyr Val SerC 540 Ser Giu Pro 555 Asp Ser PheP 570 Gly Ala Gly L Ser Arg Pro V 6 Gly Pro Asp Leu 335 350Gn r Lys Ser Il( 36! Se~ Gil Sei Pro Asn 445 Giy Ala Glu !\la 525 in al1 la e u 'al.

rLeu ICys Pro Trp 430 *Thr *Pro Ala Arg Ser 510 din Thr Gly Gly Gin 590 dinC Se] Asr Gil 415 Val1 Pro Leu Ser Gly 495 Gly Val1 Ser Airg rhr 575 Mlu flu e Pro -Gin Phe 400 Lys Val1 Gly Pro Met 480 Ala As n Met Gin Pro 560 Ala Gin Gin Ser 595 Gly Gly 610

L

6 Ala Gin Thr Ser His Thr Gin Gly Ser 620 Gly Gin Cys Ala Glu 625 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 Gly Ser Thr Gly INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: *o Asp Tyr Lys Asp Glu INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: His His His His His His INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 33 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Arg Met Lys Gin Ile Giu Asp Lys Ile Glu Giu Ile Leu Ser Lys Ile 1 5 10 Tyr His Ile Glu Asn Giu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu 25 Arg 68

Claims (26)

1. A method of screening a molecule for its capacity to antagonize or agonize RANK, said method comprising an assay selected from the group consisting of: a) i) contacting a RANK polypeptide with a RANKL polypeptide in the presence of the molecule under conditions that permit binding of the RANK and RANKL polypeptides; ii) detecting the binding of the RANK and RANKL polypeptides; iii) determining that the molecule is a RANK antagonist if the amount of RANK-RANKL binding detected is decreased when the molecule is present; and iv) determining that the molecule is a RANK agonist if the amount of 15 RANK biological activity detected is increased when the molecule is present; b) i) triggering RANK in a culture of cells that is transfected with a plasmid in which an NF-KB-dependent promoter is operably linked to a reporter gene, wherein during said triggering the cultured cells are exposed to oe the molecule; ii) detecting a product of the reporter gene in said exposed cells; iii) determining that the molecule is a RANK antagonist if the amount of the reporter gene product detected in said exposed cells is decreased; S 25 and iv) determining that the molecule is a RANK agonist if the amount of the reporter gene product detected in said exposed cells is increased; c) i) incubating the molecule and a RANKL polypeptide with isolated CD1 a dendritic cells; ii) monitoring the formation of dendritic cell clusters; iii) determining that the molecule is a RANK antagonist if dendritic cell clustering is decreased when the molecule is present; and tC WAc.1a .ShsvfSJrsped1sI713473.d0C iv) determining that the molecule is a rank agonist if dendritic cell clustering is increased when the molecule is present; d) i) incubating the molecule and a RANKL polypeptide with isolated CDla' dendritic cells in a medium comprising GM-CSF, IL-4, TNF-a and FIt3L; ii) irradiating the incubated dendritic cells; iii) monitoring the allostimulatory capacity of the irradiated dendritic cells in a mixed lymphocyte reaction; iv)determining that the molecule is a RANK antagonist if the allostimulatory capacity of the irradiated dendritic cells is decreased when the molecule is present; and v) determining that the molecule is a RANK agonist if the allostimulatory capacity of the irradiated dendritic cells is increased when the molecule is present; e) i) triggering RANK in the presence of the molecule in cells capable of expressing RANK; 20 ii) measuring TNF-a production or NF-KB activation in the cells; iii) determining that the molecule is a RANK antagonist if TNF-a production or NF-KB activation in said cells is decreased when the molecule is present; and *O iv) determining that the molecule is a RANK agonist if TNF-a production or NF-KB activation in said cells is increased when the molecule is present; f) i) activating peripheral blood T cells in culture in the presence of the molecule and one or more cytokines capable of upregulating RANK, said cytokine(s) being selected from the group consisting of TGF-3 and IL-4; IC W:\ilona\Sharon\SJJspec\SP713473.doc 71 ii) incubating said T cells for at least 4 days; iii) detecting viable T cells in said culture; iv) determining that the molecule is a RANK antagonist if the number of viable T cells detected is reduced when the molecule is present; and v) determining that the molecule is a RANK agonist if the number of viable T cells detected is increased when the molecule is present; g) i) incubating the molecule with cells expressing RANK; ii) detecting TNF-a or activated NF-KB in the incubated cells; iii) determining that the molecule is a RANK antagonist if the amount of TNF-a or activated NF-KB detected is decreased when the molecule is present; and iv) determining that the molecule is a RANK antagonist if the amount of TNF-a or activated NF-KB detected is increased when the molecule is present; and h) I) incubating the molecule with cells expressing RANK, said cells being transfected with a plasmid in which an NF-KB-dependent promoter is operably linked to a reporter gene; ii) detecting a product of the reporter gene in the incubated cells; iii) determining that the molecule is a RANK antagonist if the amount of the reporter gene product detected is decreased when the molecule is present; and iv) determining that the molecule is a RANK agonist if the amount of the reporter gene product detected in said incubated cells is increased when the molecule is present.

2. A method according to claim 1 wherein the cells capable of expressing RANK are selected from the group consisting of macrophages, epithelial cells, B- cells, activated T cells and cells transfected with recombinant RANK. IC W:ilona\Shamn\SJJspecilSP713473.doc 72

3. The method of claim 1 or 2, wherein the molecule is a soluble RANK polypeptide selected from the group consisting of: a) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID NO: 6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, of SEQ ID NO:6, and amino acids 3-232 of SEQ ID NO:8; b) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID wherein x is amino acid 1 or 31 SEQ ID NO: 15, and amino acids 3-232 of SEQ ID NO:8; and c) amino acids x to 213 of SEQ ID NO:6, Wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, or fragments thereof.

4. A method according to any one of claims 1 to 3, wherein the assay employs a RANKL polypeptide and the RANKL polypeptide is selected from the group consisting of membrane-associated RANKL and soluble RANKL.

5. A method according to any one of claims 1,2 or 4, wherein the molecule is an 20 antibody against RANK or RANKL.

6. A method according to any one of claims 1 to 5, wherein the detecting step is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), dot blot employing an antibody that binds native RANK, fluorescence activated cell 25 sorter assay (FACS assay) and a solid phase binding assay.

7. A method according to claim 1 wherein said NF-KB-dependent promoter is IL-8 promoter.

8. A method according to claim 1, wherein the assay employs RANK triggering and the RANK triggering step is selected from the group consisting of RANK over- expression, expression of membrane-bound RANKL in a cell that is expressing RANK, addition of a soluble RANKL polypeptide to cells expressing RANK, incubation of IC W:\ilona\Sharon\SJJspeci\SP713473.doc cells expressing RANK with cells expressing membrane-bound RANKL and addition of an agonistic antibody against RANK to cells expressing RANK.

9. A method according to claim 4, wherein the RANKL polypeptide is selected from the group consisting of: a) a polypeptide having the amino acid sequence of SEQ ID NO:11; b) a polypeptide having the amino acid sequence of SEQ ID NO:13; c) a soluble poly-His RANKL comprising a kappa chain leader having the amino acid sequence of SEQ ID NO: 16, a FLAGTM tag having the amino acid sequence of SEQ ID NO: 17, Gly Ser, a poly-His tag having the amino acid sequence of SEQ ID NO:18, Gly Thr Ser and amino acids 138-317 of SEQ ID NO:13; d) a soluble poly-His RANKL comprising amino acids x to 31 of SEQ ID NO:9, wherein x is amino acid 21, 32 or 29 of SEQ ID NO:9, Arg Thr 20 Ser, a poly-His tag having the amino acid sequence of SEQ ID NO:18, and amino acids 119-294 of SEQ ID NO:11; and e) a soluble oligomeric form of RANKL comprising amino acids x to 31 of SEQ ID NO:9, wherein x is amino acid 21, 32 OR 29 OF SEQ ID NO:9, 25 Asp, a leucine zipper having the amino acid sequence of SEQ ID NO:19, Thr Arg Ser and amino acids 138-317 of SEQ ID NO:13. A method of screening a molecule for its capacity to antagonize or agonize the binding of a TRAF-binding RANK polypeptide to a TRAF polypeptide comprising the steps of: JD W:jeannie\species20619-OO.doc 74 a) incubating a mixture comprising the molecule, a TRAF polypeptide and a TRAF-binding RANK polyeptide, wherein said mixture is incubated under conditions that allow the RANK polypeptide and the TRAF polypeptide to form RANK/TRAF complexes; b) detecting the RANK/TRAF complexes; c) determining that the molecule antagonizes binding of the rank polypeptide to the TRAF polypeptide if the amount of the RANK/TRAF complexes detected is decreased when the molecule is present; and d) determining that the molecule agonizes binding of the RANK polyeptide to the TRAF polypeptide if the amount of the RANK/TRAF complexes detected is increased when the molecule is present.

11. A method according to claim 10, wherein the TRAF polypeptide is selected from the group consisting of TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6. 20 12. A method according to claim 10, wherein the TRAF-binding RANK polypeptide is selected from the group consisting of: a) a RANK/GST fusion protein; and 25 b) a RANK/Fc fusion protein c) a polypeptide comprising amino acids 234-616 of SEQ ID NO:6; d) a polypeptide having an amino terminus selected from the group consisting of an amino acid between amino acid 1 and amino acid 33, inclusive, of SEQ ID NO:6, and a carboxy terminus selected from the group consisting of an amino acid between amino acid 234 and amino acid 616, inclusive; IC W:lona\Sharon\SJJspeci\SP713473.doc e) a polypeptide having an amino terminus selected from the group consisting of an amino acid between amino acid 1 and amino acid inclusive, of SEQ ID NO:15, and a carboxy terminus selected from the group consisting of an amino acid between amino acid 246 and amino acid 625, inclusive; f) a polypeptide comprising amino acids 544-616 of SEQ ID NO:6; g) a polypeptide comprising amino acids 339-421 of SEQ ID NO:6; h) a polypeptide comprising amino acids 236-625 of SEQ ID i) a fragment of a polypeptide of or j) a polypeptide having an amino acid sequence e at least about identical to a polypeptide of or

13. A method for therapeutically inhibiting RANK activity in a subject in need 20 thereof, said method comprising the step of administering to said subject a therapeutically sufficient amount of a composition comprising a RANK antagonist.

14. A method according to claim 13, wherein the RANK antagonist is selected from the group consisting of an antagonistic antibody against RANK, an 25 antagonistic antibody against RANKL and a soluble RANK polypeptide.

15. A method according to claim 14, wherein the RANK antagonist is a soluble RANK polypeptide that is selected from the group consisting of: a) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, of SEQ ID NO;6, and amino acids 3-232 of SEQ ID NO:8; JD W:jeannie\species20619-00,doc 76 b) a RANK;Fc polypeptide comprising amino acids x to 213 of SEQ ID wherein x is amino acid 1 or 31 of SEQ ID NO:15, and amino acids 3-232 of SEQ ID NO:8: c) amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1 or any one of amino acids 24-33, inclusive, or fragments thereof; and d) a RANK/GST fusion protein.

16. A method according to any one of claims 13 to 15, wherein said composition further comprises a physiologically acceptable carrier, excipient or diluent.

17. A method according to any one of claims 13 to 16, wherein said composition is administered by bolus injection, continuous infusion or sustained release from an implant.

18. A method according to claims 13 to 17, wherein the subject is selected from the group consisting of a subject having a tumor or neoplastic disease, a subject 20 having an inflammatory condition, a subject having septic shock and a subject having graft-versus-host reaction.

19. A method according to any one of claims 13 to 18, wherein the subject has a tumor or neoplastic condition and is undergoing radiation therapy. A method of therapeutically inhibiting RANK activity in a subject having a tumor or neoplastic disease, septic shock, an inflammatory condition or a graft- versus-host reaction comprising administering to said subject a RANK polypeptide selected from the group consisting of: a) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, of SEQ ID NO:6, and amino acids 3-232 of SEQ ID NO:8; IC W\Iona\Shamn\SJJspea\SP713473.doc b) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID wherein x is amino acid 1 or 31 of SEQ ID NO:15, and amino acids 3-232 of SEQ ID NO:8; c) amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, or fragments thereof; and d) a RANK/GST fusion protein.

21. Use of a composition comprising a RANK antagonist for the manufacture of a medicament for therapeutically inhibiting RANK activity in a mammal.

22. A use according to claim 21, wherein the RANK antagonist is selected from 15 the group consisting of an antagonistic antibody against RANK, an antagonistic antibody against RANKL and a soluble RANK polypeptide.

23. A use according to claim 21, wherein the RANK antagonist is a soluble RANK polypeptide that is selected from the group consisting of: a) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, of SEQ ID NO:6, and amino acids 3-232 of SEQ ID NO:8; S 25 b) a RANK;Fc polypeptide comprising amino acids x to 213 of SEQ ID wherein x is amino acid 1 or 31 of SEQ ID NO:15, and amino acids 3-232 of SEQ ID NO:8; c) amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1 or any one of amino acids 24-33, inclusive, or fragments thereof; and d) a RANK/GST fusion protein. IC W:JlonXShamnSJJspecSP2619(3).dmc 78

24. A use according to any one of claims 21 to 23, wherein said composition further comprises a physiologically acceptable carrier, excipient or diluent. 25 A use according to any one of claims 21 to 24, wherein said composition is administered by bolus injection, continuous infusion or sustained release from an implant.

26. A use according to any one of claims 21 to 25, wherein said mammal is selected from the group consisting of a mammal having a tumor or neoplastic disease, a mammal having an inflammatory condition, a mammal having septic shock and a mammal having graft-virus host reaction.

27. A use according to any one of claims 21 to 26, wherein the mammal has a 15 tumor or neoplastic condition and is undergoing reaction therapy.

28. A use of a RANK polypeptide for the manufacture of a medicament for therapeutically inhibiting RANK activity in a mammal having a tumor or neoplastic disease, septic shock, an inflammatory condition or a graft-versus-host reaction, wherein said RANK polypeptide is selected from the group consisting of: a) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, of SEQ ID NO:6, and amino acids 3-232 of SEQ ID NO:8; b) a RANK:Fc polypeptide comprising amino acids x to 213 of SEQ ID wherein x is amino acid 1 or 31 of SEQ ID NO:15, and amino acids 3-232 of SEQ ID NO:8; c) amino acids x to 213 of SEQ ID NO:6, wherein x is amino acid 1, or any one of amino acids 24-33, inclusive, or fragments thereof; and d) a RANK/GST fusion protein. IC W:ilonaSharon\SJJspeciSp20619(3).doc 79

29. A method according to claim 1, substantially as hereinbefore described.

30. A use according to claim 21, substantially as hereinbefore described. DATED: 8 June, 2001 PHILLIPS ORMONDE FITZPATRICK Attorneys for: IMMUNEX CORPORATION IC W:%iJlnavSharon'.SJJspeisp2O61 9(3).doc