ES2359297T3 - NF-KAPPA B RECEIVER ACTIVATOR, THE RECEIVER IS A MEMBER OF THE TNF RECEPTOR SUPERFAMILY. - Google Patents

Abstract

ISOLATED RECEIVERS, ADNS THAT CODIFY THESE RECIPIENTS AND PHARMACEUTICAL COMPOSITIONS PREPARED FROM THESE RECEPTORS. THESE ISOLATED RECEIVERS CAN BE USED TO REGULATE AN IMMUNE REACTION. THESE RECEIVERS ARE ALSO TO LOOK FOR THEIR INHIBITORS THROUGH THE SCREEN.

Description

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates, in general, to the field of cytokine receptors and, more specifically, to cytokine / ligand receptor pairs that have immunoregulatory activity.

BACKGROUND OF THE INVENTION

[0002] The effective functioning of the immune system requires a good balance between cell proliferation and differentiation and cell death, to ensure that the immune system is capable of reacting with foreign antigens but not with autoantigens. Various members of the Superfamily of the Tumor Necrosis Factor (TNF) / Nerve Growth Factor Receptor (Smith et al., Science 248: 1019; 1990) are essential for the immune and inflammatory response regulation procedure. This family of receptors includes two different TNF receptors (Type I and Type II; Smith et al., Mentioned above; and Schall et al., Cell 61: 361, 1990), nerve growth factor receptor (Johnson et al. , Cell 47: 545, 1986), CD40 B cell antigen (Stamenkovic et al., EMBO J. 8: 1403, 1989), CD27 (Camerini et al., J. Immunol. 147: 3165, 1991), CD30 ( Durkop et al., Cell 68: 421, 1992), OX40 T cell antigen (Mallett et al., EMBO J. 9: 1063, 1990), human Fas antigen (Itoh et al., Cell 66: 233, 1991) , murine 4-1BB receptor (Kwon et al., Proc. Natl. Acad. Sci. USA 86: 1963, 1989) and a receptor called Apoptosis Induction Receptor (AIR; USSN 08 / 720,864, filed October 4 of 1996).

[0003] CD40 is a receptor present in B lymphocytes, epithelial cells and some carcinoma cell lines, which interacts with a ligand found in activated T cells, CD40L (USSN 08 / 249,189, filed May 24, 1994 ). The interaction of this ligand / receptor pair is essential for both the cellular and humoral immune response. Signal transduction through CD40 is mediated by 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). Recently it has been discovered that mice that are defective in TRAF3 expression due to a targeted alteration in the gene encoding TRAF3, appear normal at birth, but develop progressive hypoglycemia and reduction of peripheral white blood cells, and die approximately at ten days of age (Xu et al., Immunity 5: 407, 1996). The immune responses of chimeric mice reconstituted with TRAF3 - / - fetal liver cells resemble those of CD40-deficient mice, although TRAF3 - / - B cells appear to be functionally normal.

[0004] 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 in T cells. Alternatively, it may There are other members, although not yet identified, of this family of receptors that interact with TRAF3 and play an important role in postnatal development as well as in the development of a competent immune system. The identification of additional members of the TNF receptor superfamily would provide an additional means to regulate the immune and inflammatory response, and could also provide an additional perspective of postnatal development in mammals.

SUMMARY DESCRIPTION OF THE INVENTION

[0005] The present invention provides a new receptor, called RANK (NF-B receptor activator), which is a member of the TNF receptor superfamily. RANK is a Type I transmembrane protein that has 616 amino acid residues, which interacts with TRAF3. The induction of RANK by overexpression, co-expression of RANK and membrane bound RANK ligand (RANKL), and by the addition of soluble RANKL or agonist antibodies to RANK, results in positive regulation of the NF transcription factor -B, a ubiquitous transcription factor that is widely used in cells of the immune system.

[0006] Soluble forms of the receptor can be prepared and used to interfere with the transduction of signals through membrane-bound RANK, and therefore the positive regulation of NF-B; consequently, pharmaceutical compositions comprising soluble forms of the new receptor are also provided. Inhibition of NF-porB by RANK antagonists may be useful for improving the negative effects of an inflammatory response resulting from the induction of RANK, for example in the treatment of toxic shock or septicemia, graft versus host reactions or inflammatory reactions. acute Soluble forms of the receptor will also be useful in vitro to select agonists or antagonists of RANK activity.

[0007] The cytoplasmic domain of RANK will be useful for developing assays for signal transduction inhibitors, for example, to select molecules that inhibit the interaction of RANK with TRAF2 or TRAF3. Deleted forms and fusion proteins comprising the new receptor are also described.

[0008] The present invention also identifies a counter-structure, or ligand, for RANK, called 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 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 domain and the receptor binding domain that is not necessary for receptor binding. Accordingly, soluble forms of RANKL may comprise the entire extracellular domain or fragments thereof that include the receptor binding region.

[0009] These and other aspects of the present invention will become apparent upon reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 demonstrates the influence of RANK.Fc and hRANKL on the growth of activated T cells. Human peripheral blood T cells were cultured as described in Example 12; The recovery of viable T cells was determined by triplicate counts with trypan blue.

Figure 2 illustrates the ability of RANKL to induce the formation of human DC clusters. Functionally mature dendritic (DC) cells were generated in vitro from bone marrow (BM) CD34 + progenitors and cultured as described in Example 13. DC CD1a + were cultured in a cytokine cocktail alone (Figure 2A), in cocktail plus CD40L (Figure 2B), RANKL (Figure 2C) or RANKL heat inactivated (H) (Figure 2D), and then photographed using an inversion microscope.

Figure 3 demonstrates that RANKL enhances the allo-stimulatory capacity of DC. Allogeneic T cells with varying numbers of irradiated cultured DC were incubated as described in Example 13. Cultures were pulsed with [3 H] -thymidine and the cells were collected in fiberglass sheets for counting. The values represent the mean típica standard deviation (SD) of triplicate cultures.

Figure 4 shows an alignment of human RANK with other members of the TNFR family in the region of structurally preserved extracellular cysteine-rich pseudo-repeats. The expected disulfide bonds (DS1-DS3) are indicated. RANK and CD40 contain identical amino acid substitutions (C ^ H, C ^ G) that eliminate DS2 in the second pseudo-repeat.

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

DETAILED DESCRIPTION OF THE INVENTION

[0011] A new partial cDNA insert was identified with an expected open reading phase that had some similarity with CD40 in a database containing cDNA sequence information generated from dendritic cells (DC) derived from human bone marrow . The insert was used to hybridize with colony transfer spots generated from a DC cDNA library containing full length cDNA. Several colony hybridizations were performed and two clones were isolated (SEQ ID NO: 1 and 3). SEQ ID NO: 5 shows the nucleotide and amino acid sequence of a predicted full length protein based on the alignment of the overlapping sequences of SEQ ID NO: 1 and 3.

[0012] RANK is a member of the TNF receptor superfamily; It looks a lot like CD40 in the extracellular region. Similar to CD40, RANK is associated 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 its association with various members of the TNF receptor superfamily, a signal is transduced into a cell. This signal results in cell proliferation, differentiation or apoptosis, depending on the receptor that is induced and the TRAF that is associated with the receptor or receptors; Different signals can be transduced to a cell by coordinating various signaling events. In this way, a signal transduced through a member of this family can be proliferative, differentiated or apoptotic, depending on other signals that are transduced to the cell, and / or the state of differentiation of the cell. This exquisite regulation of this proliferative / apoptotic pathway is necessary to develop and maintain protection against pathogens; imbalances can result in an autoimmune disease.

[0013] RANK is expressed in epithelial cells, some B cell lines and in activated T cells. However, its expression in activated T cells is late, approximately four days after activation. This time course of the expression coincides with the expression of Fas, a known apoptosis agent. RANK can act as an anti-apoptotic signal, rescuing cells that express RANK from apoptosis as CD40 is known to do. Alternatively, RANK can confirm an apoptotic signal in the appropriate circumstances, again similar to CD40. RANK and its ligand probably play an essential role in regulating the immune and inflammatory response.

[0014] In addition, the postnatal lethality of mice that have a targeted alteration of the TRAF3 gene demonstrates the importance of this molecule not only in the immune response, but also in development. The isolation of RANK, as a protein that is associated with TRAF3, and its ligand will allow an additional definition of this signaling pathway and the development of diagnostic and therapeutic modalities for use in the area of autoimmune and / or inflammatory diseases.

DNA, Proteins and Analogs

[0015] The present invention provides isolated human RANK polypeptides and analogs (or muteins) thereof that have an activity shown by the native molecule (ie, RANK muteins that specifically bind to a RANK ligand expressed in cells or immobilized on a surface or to antibodies specific for RANK; soluble forms thereof that inhibit the signaling induced by the RANK ligand through RANK). Such proteins substantially lack contaminating endogenous materials and, optionally, have no associated native pattern glycosylation. RANK derivatives within the scope of the invention also include various structural forms of primary proteins that 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 it may be in neutral form. Individual amino acid residues can also be modified by oxidation or reduction. The primary structure of the amino acid can be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or creating amino acid sequence mutants. Covalent derivatives are prepared by attaching particular functional groups to amino acid side chains or to the N or C end.

[0016] RANK derivatives can also be obtained by the action of crosslinking agents such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, in the cysteine and lysine residues. The proteins of the invention can also be covalently linked through reactive side groups to various insoluble substrates, such as cyanogen bromide activated, bisoxyran activated, carbonyldiimidazole activated or tosyl activated agarose structures, or by adsorption on polyolefin surfaces (with or without crosslinking with glutaraldehyde). Once bound to a substrate, proteins can be used to selectively bind (for testing or purification purposes) to antibodies induced against proteins or against other proteins that are similar to RANK or RANKL, as well as other proteins that bind RANK or RANKL or counterparts thereof.

[0017] Soluble forms of RANK are also within the scope of the invention. The nucleotide sequence and the predicted amino acid sequence of RANK are shown in SEQ ID NOS 1 to 6. Computer analysis indicated that the protein has an N-terminal signal peptide; The planned cleavage site follows the rest 24. Those skilled in the art will recognize that the actual cleavage site may be different from that provided by computer analysis. Thus, it is expected that the N-terminal amino acid of the cleaved peptide is within approximately five amino acids on either side of the preferred expected cleavage site after the remainder 24. In addition, a soluble form was prepared starting with amino acid 33 ; This soluble form bound RANKL. The signal peptide, as predicted, is followed by an extracellular domain of 188 amino acids, a transmembrane domain of 21 amino acids and a cytoplasmic tail of 383 amino acids.

[0018] Soluble RANK comprises the signal peptide and the extracellular domain (residues 1 to 213 of SEQ ID NO: 6) or a fragment thereof. Alternatively, the native leader may be substituted for a different signal peptide, beginning with residue 1 and continuing to a residue selected from the group consisting of amino acids 24 to 33 (inclusive) of SEQ ID NO: 6. In addition, the fragments Extracellular domain will also provide soluble forms of RANK. Fragments can be prepared using known techniques to isolate a desired part 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 shapes similar to those prepared for other family members. . Alternatively, unique restriction sites or PCR techniques that are known in this field can be used to prepare numerous truncated forms that can be expressed and analyzed with respect to activity.

[0019] Fragments can be prepared using known techniques to isolate a desired part 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 this field can be used to prepare numerous truncated forms that can be expressed and analyzed with respect to activity.

[0020] Other derivatives of 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 those obtained 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 in the N-terminal region of the protein, which directs co-translational or post-translational transfer of the protein from its synthesis site to its function site within or outside the membrane or cell wall (for example, the leader of the  yeast factor).

[0021] Protein fusions may comprise peptides added to facilitate purification or identification of RANK proteins and homologs (eg poly-His). The amino acid sequence of the proteins of the invention can also be linked to an identification peptide such as that described by Hopp et al., Bio / Technology 6: 1204 (1988). Said highly antigenic peptide provides an epitope reversibly linked by a specific monoclonal antibody, which allows rapid assay and easy purification of the expressed recombinant protein. The sequence of Hopp et al. It is also specifically cleaved by the bovine mucosa enterokinase, allowing the separation of the peptide from the purified protein. Fusion proteins protected with said peptides can also be resistant to intracellular degradation in E. coli.

[0022] Fusion proteins further comprise the amino acid sequence of a RANK bound to an Fc region of immunoglobulin. An exemplary Fc region is a human IgG1 having a nucleotide and amino acid sequence set forth in SEQ ID NO: 8. Fc region fragments may also be used, as well as Fc muteins. For example, certain residues within the hinge region of an Fc region are critical for high affinity binding to FcRI. Canfield and Morrison (J. Exp. Med. 173: 1483; 1991) reported that Leu (234) and Leu (235) were critical for the high affinity binding of IgG3 to FcRI present in U937 cells. Similar results were obtained by Lund et al. (J. Immunol. 147: 2657, 1991; Molecular Immunol. 29:53, 1991). These mutations, alone or in combination, can be made in an Fc region of IgG1 to reduce the affinity of IgG1 for FcR. Depending on the part of the Fc region used, a fusion protein can be expressed as a dimer, by forming intercatenary disulfide bonds. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a protein oligomer with up to four RANK regions.

[0023] In another embodiment, the RANK proteins further comprise an oligomerization peptide such as a leucine zipper domain. Leucine zippers were originally identified in several DNA binding proteins (Landschulz et al., Science 240: 1759, 1988). The leucine zipper domain is a term used to refer to a conserved peptide domain present in these (and other) proteins, which is responsible for protein dimerization. The leucine zipper domain (also referred to herein as an oligomerization or oligomer formation domain) comprises a repetitive heptad, with four or five leucine residues interspersed with other amino acids. Examples of leucine zipper domains are found in the GCN4 yeast transcription factor and in a thermostable DNA binding protein found in rat liver (C / EBP; Landschulz et al., Science 243: 1681, 1989). Two nuclear transformation proteins, fos and jun, also have leucine zipper domains, as well as the c-myc murine proto-oncogene gene product (Landschulz et al., Science 240: 1759, 1988). The products of fos and jun nuclear oncogenes comprise leucine zipper domains preferably of a heterodimer (O’Shea et al., Science 245: 646, 1989; Turner and Tjian, Science 243: 1689, 1989). The leucine zipper domain is necessary for the biological activity (DNA binding) of these proteins.

[0024] Fusonogenic 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 leucine zipper domains could contribute to the oligomeric structure of fusogenic proteins. Fusomeric viral protein oligomerization is involved in the formation of fusion pores (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88: 3523, 1991). Recently it has also been reported that leucine zipper domains play a role in the oligomerization of heat stroke transcription factors (Rabindran et al., Science 259: 230, 1993).

[0025] Leucine zipper domains fold as short parallel alpha helices packages (O'Shea et al., Science 254: 539; 1991). The general architecture of parallel alpha helic packing is well characterized, with a "buttonhole" ("knobs-into-holes") packing as proposed by Crick in 1953 (Crystallogr Act 6: 689). by a leucine zipper domain it is stabilized by repeated heptad, called (abcdefg) n according to the notation of McLachlan and Stewart (J. Mol. Biol. 98: 293; 1975), in which the remains a and d are generally hydrophobic moieties, being a leucine, which are aligned on the same face of a propeller. The oppositely charged moieties commonly appear in positions g and E. Thus, in a parallel alpha helic packing formed from two domains With helical leucine zipper, the "buttons" formed by the hydrophobic side chains of the first propeller are packaged in the "eyelets" formed between the side chains of the second helix.

[0026] Leucine residues in position d provide great hydrophobic stabilization energies and are important for the formation of dimers (Krystek et al., Int. J. Peptide Res. 38: 229, 1991). Lovejoy et al. recently reported the synthesis of a triple-chain -helical beam in which the propellers go up-up-down (Science 259: 1288, 1993). Their studies confirmed that hydrophobic stabilization energy provides the main driving force for the formation of alpha helic packings from helical monomers. These studies also indicate that electrostatic interactions contribute to the stoichiometry and geometry of alpha helic packing.

[0027] Several studies have indicated that individual leucine residues can be replaced by conservative amino acids with a minimal reduction in the ability to dimerize; however, multiple changes normally result in a loss of this capacity (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 several different amino acids can replace leucine residues in the GCN4 leucine zipper domain, and also discovered that some GCN4 proteins that contained two leucine substitutions were weakly active (Nucl. Acids Res. 20: 3721, 1992) . Mutation of the first and second hepatic leucines of the leucine zipper domain of measles virus fusion protein (MVF) did not affect syncytium formation (a measure of virally induced cell fusion); however, the mutation of the four leucine residues totally prevented the fusion (Buckland et al., J. Gen. Virol. 73: 1703 1992). None of the mutations affected the ability of MVF to form a tetramer.

[0028] It has been found that amino acid substitutions at residues a and d of a synthetic peptide representing the leucine zipper domain of GCN4 change the properties of oligomerization of the leucine zipper domain (Alber, Sixth Symposium of the Protein Society, San Diego CA). When all the remains in position a are changed to isoleucine, the leucine zipper continues to form a parallel dimer. When, in addition to this change, all leucine residues in position d are also exchanged for isoleucine, the resulting peptide spontaneously forms a trimeric parallel alpha helic packaging in solution. Substitution of all amino acids in position d with isoleucine and in position a with leucine results in a tetramerizing peptide. Peptides containing these substitutions are still called leucine zipper domains.

[0029] Fragments or derivatives of the intracellular domain of RANK are also included within the scope of the invention. Such fragments are prepared by any of the techniques mentioned herein and include peptides that are identical to the cytoplasmic domain of RANK as shown in SEQ ID NO: 6, and those that comprise a portion of the cytoplasmic region. All techniques used in the preparation of soluble forms can also be used in the preparation of fragments or analogs of the cytoplasmic domain (i.e., RTPCR techniques or use of selected restriction enzymes to prepare truncation). DNAs encoding all or a fragment of the intracytoplasmic domain will be useful for identifying other proteins that are associated with RANK signaling, for example, using the immunoprecipitation techniques described herein or another technique such as a double hybrid yeast system. (Rothe et al., Mentioned above).

[0030] The present invention also includes RANK with or without associated native pattern glycosylation. Proteins expressed in yeast or mammalian expression systems, for example, COS-7 cells, may be similar or slightly different in molecular weight and glycosylation pattern than native molecules, depending on the expression system. The expression of DNA encoding the proteins of the invention in bacteria such as E. coli provides non-glycosylated molecules. Analogues of functional mutants of the RANK protein can be produced which have N-glycosylation sites inactivated by oligonucleotide synthesis and ligation or by site specific mutagenesis techniques. These analogous proteins can be produced in a homogeneous form of reduced carbohydrates with good performance using yeast expression systems. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-A1-Z, where A1 is any amino acid except Pro and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for the covalent attachment of a carbohydrate. Said site can be eliminated by the substitution of Asn or the Z moiety with another amino acid, the deletion of Asn or Z, or the insertion of a non-Z amino acid between A1 and Z, or an amino acid other than Asn between Asn and A1.

[0031] RANK protein derivatives can also be obtained by mutations of the native RANK protein or subunits thereof. A mutated RANK protein, as mentioned herein, is a polypeptide homologous to a native RANK protein, respectively, but having an amino acid sequence different from that of the native protein due to one or a plurality of deletions, insertions or substitutions The effect of any mutation performed on a DNA encoding a mutated peptide can easily be determined by analyzing the ability of the mutated peptide to bind to its counter-structure in a specific way. In addition, the activity of analogs, muteins or derivatives of RANK can be determined by any of the assays described herein (for example, inhibition of the ability of RANK to activate transcription).

[0032] Analogs of the proteins of the invention can be constructed, for example, by making various substitutions of moieties or sequences or deleting terminal or internal moieties or sequences that are not needed for biological activity. For example, cysteine residues can be deleted or replaced with other amino acids to prevent the formation of incorrect intramolecular disulfide bonds after renaturation. Other approaches to mutagenesis involve the modification of adjacent dibasic amino acid residues to increase expression in yeast systems in which KEX2 protease activity is present.

[0033] When a deletion or insertion strategy is adopted, the potential effect of the deletion should be considered

or insertion on biological activity. Subunits of the proteins of the invention can be constructed by deletion of residues or terminal or internal sequences. Soluble forms of RANK can be readily prepared and tested for their ability to inhibit RANK-induced NF-activaciónB activation. Polypeptides corresponding to the cytoplasmic regions and fragments thereof (eg, a death domain) can be prepared by similar techniques. Additional guidelines are provided as to the types of mutations that can be made by a comparison of the RANK sequence with proteins having similar structures, as well as by performing a structural analysis of the RANK proteins of the invention.

[0034] In general, substitutions should be made conservatively; that is, the most preferred substitute amino acids are those that do not affect the biological activity of RANK (i.e., the ability of the proteins of the invention to bind antibodies against the corresponding native protein in a substantially equivalent manner, the ability to bind to the counter-structure in substantially the same way as the native protein, the ability to transduce a RANK signal, or the ability to induce NF-activaciónB activation after overexpression in transient transfection systems, for example). Examples of conservative substitutions include substitution of amino acids outside the domain or binding domains (ligand / receptor or antibody binding areas for the extracellular domain, or regions that interact with other intracellular proteins for the cytoplasmic domain), and amino acid substitution that do not alter the secondary and / or tertiary structure of the native protein. Other additional examples include the substitution of one aliphatic moiety with another, such as Ile, Val, Leu or Ala with each other, or substitutions of one polar moiety for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other of these conservative substitutions are well known, for example, substitutions of entire regions having similar hydrophobicity characteristics.

[0035] Of course, mutations in nucleotide sequences constructed for the expression of analogous proteins or fragments thereof must preserve the reading phase of the coding sequences and, preferably, will not create complementary regions that can hybridize to produce structures of Secondary mRNAs such as loops or hairpins that would adversely affect the translation of mRNA.

[0036] Not all mutations in the nucleotide sequence encoding a RANK protein or fragments thereof will be expressed in the final product, for example, nucleotide substitutions can be made to improve expression, mainly to avoid loops of secondary structure in the transcribed mRNA (see EPA 75.444A), or to provide codons that are more easily translated by the selected host, for example, the E. coli preferred codons well known for expression in E. coli.

[0037] Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, to select optimal mutant characteristics, random mutagenesis can be performed and the desired activity of the expressed mutated proteins can be selected. Mutations can be introduced into particular loci by synthesis of oligonucleotides containing a mutant sequence, flanked by restriction sites that allow binding to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired insertion, substitution or deletion of amino acids.

[0038] Alternatively, site-specific mutagenesis procedures directed to oligonucleotides can be employed to provide an altered gene having particular codons in accordance with the necessary replacement, deletion or insertion. Exemplary methods for performing the alterations described above by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and United States Patents No.

4,518,584 and 4,737,462 describe suitable techniques.

[0039] Other embodiments of the proteins of the invention include RANK polypeptides encoded by DNA capable of hybridizing with the DNA of SEQ ID NO: 5 under moderately stringent conditions (5 X SSC prewash solution, 0.5% SDS, 1.0 mM EDTA (pH 8.0)) and under hybridization conditions of 50 ° C, 5 X SSC, overnight, with the DNA sequences encoding RANK, or more preferably under stringent conditions (eg, hybridization in SSC 6 X at 63 ° C overnight; wash in SSC 3 X at 55 ° C) and with other sequences that are degenerated with respect to those encoding the RANK protein. In one embodiment, the RANK polypeptides have an identity of at least about 70% in the amino acid sequence with respect to the amino acid sequence of the native RANK protein set forth in SEQ ID NO: 6. In a preferred embodiment, the polypeptides RANK have an identity of at least about 80% in the amino acid sequence with respect to the native form of RANK; the most preferred polypeptides are those that have an identity of at least about 90% with native RANK.

[0040] The percent identity can 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 at the University of Wisconsin Genetics Computer Group (UWGCG). For fragments derived from the RANK protein, the identity is calculated based on the portion of the RANK protein that is present in the fragment.

[0041] The biological activity of RANK analogs or muteins can be determined by testing the ability of analogs or muteins to inhibit transcription activation, for example, as described in the examples herein. Alternatively, suitable assays can be used, for example an enzyme immunoassay or a point blot using an antibody that binds to native RANK, or to a soluble form of RANKL, to evaluate the activity of RANK analogs or muteins, as well. as assays that employ cells that express RANKL. Suitable assays also include, for example, signal transduction assays and methods that assess the ability of the RANK cytoplasmic region to associate with other intracellular proteins (i.e., TRAF 2 and 3) involved in signal transduction and will also be useful for evaluating the activity of analogs or muteins of RANK. Such methods are well known in the art.

[0042] Fragments of the nucleotide sequences of RANK are also useful. In one embodiment, said fragments comprise at least 17 consecutive nucleotides, preferably at least about 25 nucleotides, more preferably at least 30 consecutive nucleotides, of the RANK DNA described herein. DNA and RNA complements of said fragments are provided herein, together with both single-stranded and double-stranded forms of RANK DNA of SEQ ID NO: 5, and those encoding the aforementioned polypeptides. A RANK DNA fragment generally comprises at least about 17 nucleotides, preferably from about 17 to about 30 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).

[0043] The probes also find use in detecting the presence of RANK nucleic acids in in vitro assays and in procedures such as Northern and Southern blots. Cell types that express RANK can also be identified. Such procedures are well known and the person skilled in the art can choose a probe of suitable length, depending on the particular application desired. For PCR, 5 'and 3' primers corresponding to the end of a desired RANK DNA sequence are used to amplify that sequence, using conventional techniques.

[0044] Other useful fragments of RANK nucleic acids are antisense or sense oligonucleotides comprising a single stranded nucleic acid sequence (RNA or DNA) capable of binding to RANK (sense) mRNA target sequences or RANK DNA ( antisense). The ability to create an antisense or sense oligonucleotide based on a cDNA sequence for a given protein is described, for example, in Stein and Cohen, Cancer Res. 48: 2659, 1988 and van der Krol et al., BioTechniques 6: 958, 1988.

Uses of DNA, Proteins and Analogs

[0045] 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-activaciónB activation, as well as to inhibit the transduction of a signal through RANK. RANK compositions (both proteins and DNA) will also be useful in the development of both agonist and antagonist antibodies against RANK. The DNAs of the invention are useful for the expression of recombinant proteins and as probes for the analysis (quantitative or qualitative) of the presence or distribution of RANK transcripts.

[0046] The proteins of the invention will also be useful in the preparation of kits that are used to detect soluble RANK or RANKL, or monitor RANK related activity, for example, in patient samples. RANK proteins will also find utility in controlling RANK-related activity in other samples or compositions, as necessary when antagonists or mimetics of this activity are selected (eg, peptides or small molecules that inhibit or mimic, respectively, the interaction ). A variety of assay formats including (but not limited to) ELISA, point transfer, solid phase binding assays (such as those using a biosensor), rapid format assays and bioassays are useful in such kits.

[0047] The purified RANK according to the invention will facilitate the discovery of RANK inhibitors and, therefore, inhibitors of an inflammatory response (by inhibiting the activation of NF-B). The use of a purified RANK polypeptide in the selection of potential inhibitors is important and can practically eliminate the possibility of interference reactions with contaminants. Said screening assay may use the extracellular domain of RANK, the intracellular domain or a fragment of any of these polypeptides. Detection of the inhibitory activity of a molecule would typically involve the use of a soluble form of RANK from the extracellular domain in a screening assay to detect molecules capable of binding to RANK and inhibiting the binding, for example, of an agonist antibody or RANKL, or the use of a polypeptide derived from the intracellular domain in an assay to detect the inhibition of the interaction of RANK and other intracellular proteins involved in signal transduction.

[0048] In addition, in vitro systems can be used to determine the ability of molecules to antagonize or agonize RANK activity. Such methods include uses of RANK chimeras, for example, a chimera of the intracellular domain of RANK and an extracellular domain derived from a protein having a known ligand. The effects on signal transduction of various molecules can then be controlled using the known ligand to transduce a signal.

[0049] In addition, RANK polypeptides can also be used for the design based on the structure of RANK inhibitors. Such structure-based design is also known as "rational drug design." RANK polypeptides can be analyzed three-dimensionally, for example, by X-ray crystallography, nuclear magnetic resonance

or modeled by homology, all of them being well known methods. The use of RANK structural information in molecular modeling software systems to aid inhibitor design is also included in the invention. Said computer-assisted modeling and drug design can use information such as chemical conformational analysis, the electrostatic potential of the molecules, protein folding, etc. A particular method of the invention comprises analyzing the three-dimensional structure of RANK with respect to probable substrate binding sites, synthesizing a new molecule that incorporates a predictive reactive site, and testing the new molecule as described above.

Recombinant RANK expression

[0050] The proteins of the present invention are preferably produced by recombinant DNA methods by inserting a DNA sequence encoding a RANK protein or an analogue thereof into a recombinant expression vector and by expressing the DNA sequence in a recombinant expression system under conditions that promote expression. The 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 that is capable of being inserted into a recombinant expression vector and Expressed in a recombinant transcriptional unit.

[0051] Recombinant expression vectors include DNA fragments derived from cDNA or synthetics encoding RANK, or bioequivalent homologs, muteins or analogs thereof, operably linked to suitable transcription or translation regulatory elements derived from mammalian, microbial genes , viral or insects. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences that control transcription termination and translation, as described in detail below. Replication capacity can be additionally incorporated into a host, normally conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.

[0052] DNA regions are operatively linked when they are functionally related to each other. For example, the DNA of a signal peptide (secretory leader) is operably linked to a DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operatively 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 that it produces the translation. In general, operatively linked means contiguous and, in the case of secretory leaders, contiguous and in reading phase. The DNA sequences encoding RANK, or homologs or analogs thereof that are to be expressed in a microorganism, preferably will not contain introns that could prematurely terminate the transcription of the DNA into mRNA.

[0053] Expression vectors useful for bacterial use may comprise a selection marker and a bacterial replication origin 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 pGEM1 (Promega Biotec, Madison, WI, USA). These "skeleton" sections of pBR322 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 a species of E. coli (Bolivar et al., Gene 2: 95, 1977). pBR322 contains ampicillin and tetracycline resistance genes and, therefore, provides simple means to identify transformed cells.

[0054] Promoters commonly used in recombinant microbial expression vectors include the -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: 4051, 1980; and EPA 36,776) and the tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful bacterial expression system employs the phage PL promoter  and the thermolabile repressor cI857ts. Plasmid vectors available in the American Type Culture Collection that incorporate derivatives of the PL PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli strain RR1 ( ATCC 53082).

[0055] Suitable promoter sequences in yeast vectors include 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. 77: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 phosphoglycerate mutase, pyruvate kinase, triosaphosphate isomerase, phosphoglucose isomerase and glucokinase. Vectors and promoters suitable for use in yeast expression are further described in R. Hitzeman et al., EPA 73,657.

[0056] Preferred yeast vectors can be assembled using pBR322 DNA sequences for selection and replication in E. coli (Ampr gene and origin of replication) and yeast DNA sequences including a glucose repressible ADH2 promoter and a de factor secretion leader. The ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). The leader of the yeast factor dirige, which directs the secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. See, for example, 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.

[0057] Transcription control sequences and translation into expression vectors to be used in the transformation of vertebrate cells can be provided by viral sources. For example, commonly used promoters and enhancers come from Polyoma, Adenovirus 2, Simian Virus 40 (SV40) and human cytomegalovirus. DNA sequences from the SV40 viral genome can be used, for example, the origin of SV40, the early and late promoter, enhancer, splice site and polyadenylation site, to provide the other genetic elements necessary for the expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are easily obtained from the virus as a fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature 273: 113, 1978). Smaller or larger SV40 fragments can also be used, provided that the approximately 250 bp sequence that extends from the Hind III site to the BglI site located at the origin of viral replication is included. In addition, viral genomic promoters, control sequences and / or signal sequences may be used, provided that said control sequences are compatible with the host cell chosen. Illustrative vectors can be constructed as described by Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983).

[0058] A system useful for stable high level expression of mammalian receptor cDNA 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 RANK DNA expression is called pDC406 (McMahan et al., EMBO J. 10: 2821, 1991) and includes regulatory sequences derived from SV40, human immunodeficiency virus (HIV) and Epstein virus -Barr (EBV). Other preferred vectors include pDC409 and pDC410, which come from pDC406. pDC410 was obtained from pDC406 by replacing the origin of EBV replication with sequences encoding the SV40 large T antigen. pDC409 differs from pDC406 in that a Bgl II restriction site has been deleted outside the multiple cloning site, making the Bgl II site unique within the multiple cloning site.

[0059] A useful cell line that allows episomal replication of expression vectors, such as pDC406 and pDC409, which contains the origin of EBV replication, is CV-1 / EBNA (ATCC CRL 10478). The CV-1 / EBNA cell line was obtained by transfection of the CV-1 cell line with a gene encoding the Epstein-Barr virus nuclear antigen 1 (EBNA-1) and constitutively expressing EBNA-1 directed by the promoter / immediate early enhancer of human CMV.

Hosting Cells

[0060] Transformed host cells are cells that have been transformed or transfected with expression vectors constructed using recombinant DNA techniques and that contain sequences encoding the proteins of the present invention. Transformed host cells can express the desired protein (RANK or homologs or analogs thereof), but transformed host cells for the purpose of cloning or amplifying the DNA of the invention do not need to express the protein. The expressed proteins will preferably be secreted in the culture supernatant, depending on the selected DNA, but can be deposited on the cell membrane.

[0061] Suitable host cells for protein expression include prokaryotes, yeasts 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. Upper eukaryotic cells include established cell lines from mammals as described below. Cellless translation systems could also be used to produce proteins using RNA derived from the DNA constructs described herein. Cloning and expression vectors suitable for use with bacterial, fungal, yeast and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0062] Prokaryotic expression hosts may be used for the expression of RANK, or homologs or analogs thereof that do not require proteolytic processing and extensive disulfide. Prokaryotic expression vectors generally comprise one or more phenotypic selection markers, for example a gene that encodes proteins that confer antibiotic resistance or that provides an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host. . Prokaryotic hosts suitable for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genus Pseudomonas, Streptomyces and Staphylococcus, although others may also be used.

[0063] Recombinant RANK can also be expressed in yeast hosts, preferably from Saccharomyces species such as S. cerevisiae. Yeasts from other genera such as Pichia can also be used

or Kluyveromyces. Yeast vectors will generally contain an origin of replication of the 2 yeast plasmid, or an autonomous replication sequence (ARS), promoter, DNA encoding the protein, sequences for polyadenylation and termination of transcription and a selection gene. Preferably, the yeast vectors will include an origin of replication and a selection marker that allows the transformation of both yeast and

AND.

 coli, for example, the E. coli ampicillin resistance gene and the S. cerevisiae trp1 gene, which provides a selection marker for a mutant strain of yeast that lacks the ability to grow in tryptophan, and a derived promoter of a high-expression yeast gene to induce the transcription of a downstream structural sequence. Then, the presence of the trp1 lesion in the genome of the yeast host cell provides an effective environment to detect transformation by growth in the absence of tryptophan.

[0064] Suitable yeast transformation protocols are known to those skilled in the art; An exemplary technique is described by Hinnen et al., Proc. Natl Acad. Sci. USA 75: 1929, 1978, selection of Trp + transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 g / adenine ml and 20 /g / ml of uracil. Host strains transformed by vectors comprising the ADH2 promoter can be cultured for expression in a rich medium consisting of 1% yeast extract, 2% peptone and 1% glucose supplemented with 80 g / ml adenine and uracil at 80 /g / ml. ADH2 promoter derepression occurs after depletion of the glucose in the medium. Raw yeast supernatants are collected by filtration and maintained at 4 ° C before further purification.

[0065] Various mammalian or insect cell culture systems can be employed to express the recombinant protein. Baculovirus systems for the 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 monkey kidney cell lines, described by Gluzman (Cell 23: 175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, CV-1 / EBNA (ATCC CRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter and an enhancer linked to the gene to be expressed, and other 5 'or 3' non-transcribed flanking sequences, and 5 'or 3 untranslated sequences. ', such as necessary ribosome binding sites, a polyadenylation site, donor and splice acceptor sites, and transcription termination sequences.

RANK Recombinant Purification

[0066] Purified RANK and homologues and analogs thereof are prepared by culturing host / vector systems suitable for expressing the recombinant translation products of the DNAs of the present invention, which are then purified from the culture medium or cell extracts. For example, supernatants from systems that secrete the recombinant protein into the culture medium can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.

[0067] After the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix may comprise a protein or lecithin counter-structure or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be used, for example, a matrix or substrate having pendant diethylaminoethyl groups (DEAE). The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. Alternatively, a cation exchange stage can be used. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Molecular exclusion chromatography also provides a means to purify the proteins of the invention.

[0068] Affinity chromatography is a particularly preferred method for 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. In addition, a RANK protein comprising an oligomerization zipper domain can be purified on a resin that it comprises an antibody specific for the oligomerization zipper domain. Monoclonal antibodies against RANK protein may also be useful in purification by affinity chromatography, using methods that are well known in the art. A ligand can also be used to prepare an affinity matrix for affinity purification of RANK.

[0069] Finally, one or more stages of reverse phase high resolution liquid chromatography (RP-HPLC) can be employed using hydrophobic RP-HPLC media, for example, silica gel having pendant methyl groups or other aliphatic groups, for further purify a RANK composition. Some or all of the above purification steps may also be employed, in various combinations, to provide a homogeneous recombinant protein.

[0070] The recombinant protein produced in bacterial culture is normally isolated by initial extraction from cell sediments, followed by one or more concentration steps, saline displacement, aqueous ion exchange.

or molecular exclusion chromatography. Finally, high performance liquid chromatography (HPLC) can be used for the final purification steps. The microbial cells employed in the expression of the recombinant protein can be broken by any convenient method, including freeze-thaw cycles, sonication, mechanical breakdown or use of cell lysate agents.

[0071] The fermentation of yeasts that express the protein of the invention as a secreted protein greatly simplifies purification. The secreted recombinant protein resulting from large-scale fermentation can be purified by methods analogous to those described by Urdal et al. (J. Chromatog. 296: 171, 1984). This reference describes two stages of sequential reverse phase HPLC for the purification of recombinant human GM-CSF on a preparative HPLC column.

[0072] The protein synthesized in the recombinant culture is characterized by the presence of cellular components, including proteins, in amounts and of a character that depends on the purification steps taken to recover the protein of the invention from the culture. These components will normally come from yeast, prokaryotes or a higher non-human eukaryotic organism and are preferably present in harmless contaminant amounts, of the order of less than about 1 percent by weight. In addition, recombinant cell culture allows the production of the proteins of the invention without other proteins that may normally be associated with the proteins as found in nature in the species of origin.

Uses and Composition Management of RANK

[0073] The present invention provides methods for 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 together with soluble cytokine or cytokine receptors, or other immunoregulatory molecules is also contemplated.

[0074] For therapeutic use, the purified protein is administered to a patient, preferably a human being, for treatment in a manner appropriate for the indication. Thus, for example, RANK protein compositions administered to regulate immune function can be administered 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, together with physiologically acceptable carriers, excipients or diluents. Such vehicles will not be toxic to the recipients at the dosages and concentrations used.

[0075] Normally, the preparation of said protein compositions involves combining the protein of the invention with buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Exemplary suitable diluents are neutral buffered saline or saline mixed with non-specific serum albumin. Preferably, the product is formulated as a lyophilisate using appropriate excipient solutions (eg, sucrose) as diluents. Appropriate dosages can be determined in tests. The amount and frequency of administration will, of course, depend on factors such as the nature and severity of the indication to be treated, the desired response, the patient's condition and the like.

[0076] Soluble forms of RANK and other RANK antagonists such as monoclonal antagonist antibodies can be administered to inhibit RANK-induced induction of NF-B activity. NF-B is a transcription factor that is used considerably by the cells of the immune system, and plays a role in the inflammatory response. In this way, RANK signaling inhibitors will be useful in the treatment of conditions in which signaling through RANK has resulted in negative consequences, for example, toxic or septic shock, or graft versus host reactions. They can also be useful for interfering with the role of NF-enB in cell transformation. Tumor cells respond more to radiation when their NF-B is blocked; Thus, soluble RANK (or other RANK signaling antagonists) will be useful as an auxiliary therapy for diseases characterized by neoplastic cells expressing RANK.

[0077] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

[0078] The example describes the identification and isolation of a DNA encoding a new member of the TNF receptor superfamily. A partial cDNA insert with an expected open reading phase was identified that had some similarity with CD40 (a cell surface antigen present on the surface of both normal and neoplastic human B cells that, as demonstrated, plays an important role in the proliferation and differentiation of B cells; Stamenkovic et al., EMBO J. 8: 1403, 1989), in a database containing cDNA sequence information generated from dendritic cells (DC) derived from human bone marrow . The insert was cleaved from the vector by restriction endonuclease digestion, gel purified, labeled with 32P and used to hybridize with colony transfer spots generated from a DC cDNA library containing larger cDNA inserts. size using high stringency hybridization and washing techniques (hybridization in 5x SSC, 50% formamide at 42 ° C overnight, washing in 0.5x SSC at 63 ° C); Other suitable high stringency conditions are described 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 produced a clone called 9D-8A (SEQ ID NO: 1); subsequent analysis indicated that this clone contained everything except the 5 'end of a new cDNA, with an intron sequence provided at the 5' end (nucleotides 1-92 of SEQ ID NO: 1). Additional colony hybridizations were performed and a second clone was isolated. The second clone, called 9D-15C (SEQ ID NO: 3), contained the 5 ’end without intron interruption but not the full 3’ end. SEQ ID NO: 5 shows the nucleotide and amino acid sequence of an expected full length protein based on the alignment of the overlapping sequences of SEQ ID NO: 1 and 3.

[0079] The encoded protein was called RANK, for the acronym of NF-B receptor activator. The cDNA encodes an expected Type 1 transmembrane protein that has 616 amino acid residues, with an expected signal sequence of 24 amino acids (the computer predicted that the cleavage site is after Leu24), an extracellular domain of 188 amino acids, a transmembrane domain of 21 amino acids and a cytoplasmic tail of 383 amino acids. The extracellular region of RANK showed a significant amino acid homology (38.5% identity, 52.3% similarity) with CD40. A cloning vector (pBluescriptSK-) containing the human RANK sequence, called pBluescript: huRANK (in E. coli DH10B) was deposited in the American Type Culture Collection, Rockville, MD (ATCC) on December 20, 1996, under the terms of the Budapest Treaty, and received access number 98285.

EXAMPLE 2

[0080] This example describes the 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 IgG1 was constructed in the mammalian expression vector pDC409 (USSN 08 / 571,579). This expression vector encodes the leader sequence of the open reading phase of Cytomegalovirus (CMV) R27080 (SEQ ID NO: 9), followed by amino acids 33-213 of RANK, followed by a mutated form of the constant domain of human IgG1 that it has reduced affinity for Fc receptors (SEQ ID NO: 8; for the fusion protein, the Fc part of the construct consisted of Arg3 to Lys232). An alternative expression vector was also prepared that included amino acids 1-213 of RANK (using the native leader sequence) followed by the IgG1 mutein. It was found that the two expression vectors induced high levels of RANK / Fc fusion protein expression in transfected cells.

[0081] To obtain the RANK / Fc protein, a RANK / Fc expression plasmid is introduced by transfection into CV-1 / EBNA cells and the supernatants are collected for about a week. The RANK / Fc fusion protein is purified by means well known in the art for the purification of Fc fusion proteins, for example, by column chromatography of protein A sepharose according to the manufacturer's recommendations (i.e., Pharmacia, Uppsala, Sweden). SDS-polyacrylamide gel electrophoresis analysis indicated that the purified RANK / Fc protein migrated with a molecular weight of 55 kDa in the presence of a reducing agent, and at a molecular weight of 110 kDa in the absence of a reducing agent.

[0082] N-terminal amino acid sequencing of the purified protein obtained using the CMV leader R27080 showed a 60% cleavage after Ala20, a 20% cleavage after Pro22 and a 20% cleavage after Arg 28 ( which is the Furina cleavage site; the amino acid residues are relative to SEQ ID NO: 9); N-terminal amino acid analysis of the fusion protein expressed with the native leader showed excision predominantly after Gln25 (80% after Gln25 and 20% after Arg23; amino acid residues are relative to SEQ ID NO: 6 , Full length RANK). The two fusion proteins were able to bind a ligand for RANK in a specific way (i.e., they bound to the surface of various cell lines such as the murine thymoma cell line, EL4), indicating that the presence of additional amino acids in the N end of RANK does not interfere with your ability to join RANKL. In addition, the construction comprising the CMV leader encoded RANK starting at amino acid 33; Thus, it is expected that a RANK peptide having an N-terminus at an amino acid between Arg23 and Prot33, may even bind a ligand for RANK in a specific manner.

[0083] Other members of the TNF receptor superfamily have an amino acid region between the transmembrane domain and the ligand binding domain that is called the "spacer" region, which is not necessary for ligand binding. In RANK, it is predicted that amino acids between 196 and 213 form said spacer region. Therefore, it is expected that a soluble form of RANK that ends with an amino acid in this region retains 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 can be used as the C-terminus.

EXAMPLE 3

[0084] 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 described in US Patent 4,411,993. DNA encoding RANK as an immunogen can also be used, for example, as reviewed by Pardoll and Beckerleg in Immunity 3: 165, 1995. Such antibodies will likely be useful for interfering with RANK-induced signaling (antagonist or blocking antibodies) or in the induction of a signal by cross-linking of RANK (agonist antibodies), as components of diagnostic or research assays for RANK or the activity of RANK, or in affinity purification of RANK.

[0085] To immunize rodents, RANK immunogen is emulsified in an adjuvant (such as complete adjuvant

or incomplete Freund, alum or other adjuvant, such as R700 adjuvant from Ribi (Ribi, Hamilton, MT) and is injected in amounts ranging from 10 to 100 g subcutaneously in a selected rodent, for example, BALB mice / Lewis rats. DNA can be administered intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91: 9519, 1994)

or intramuscular (Wang et al., Proc. Natl. Acad. Sci. USA 90: 4156, 1993); It has been found that the saline solution is a suitable diluent for DNA-based antigens. Ten days to three weeks later, the immunized animals are reinforced with more immunogen and then periodically reinforced in a weekly, biweekly or every three weeks immunization schedule.

[0086] Serum samples are periodically extracted by retro-orbital blood sampling or tail tip excision to be tested by the dot-blot (antibody sandwich), ELISA (enzyme-linked immunosorbent assay). ), immunoprecipitation or other suitable assays, including FACS analysis. After 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, the splenocytes are collected and fused to a murine myeloma cell line (eg, NS1 or preferably Ag 8.653 [ATCC CRL 1580]). The hybridoma cell lines generated by this procedure are cultured 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 hybrids. myeloma and splenocyte splenocyte hybrids.

[0087] Hybridoma clones generated in this manner can be screened by ELISA for reactivity with RANK, for example, by adaptations of the techniques described by Engvall et al., Immunochem. 8: 871 (1971) and in U.S. Patent No. 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 fluid that contains high concentrations (> 1 mg / ml) of anti-RANK monoclonal antibody. The resulting monoclonal antibody can be purified by precipitation with ammonium sulfate followed by molecular exclusion chromatography. Alternatively, affinity chromatography based on the binding of an antibody to protein A or G protein can also be used, as well as affinity chromatography based on binding to the RANK protein.

[0088] Monoclonal antibodies were generated using the RANK / Fc fusion protein as an immunogen. These reagents were screened to confirm reactivity against the RANK protein. Using the methods described herein to control the activity of the mAbs, both blockers (i.e., antibodies that bind to RANK and inhibit the binding of a ligand to RANK) were isolated as non-blockers (i.e. antibodies that are bind to RANK and do not inhibit ligand binding).

EXAMPLE 4

[0089] This example illustrates the induction of NF-B activity by RANK in 293 / EBNA cells (the cell line was obtained by transfection of the 293 cell line with a gene encoding Epstein virus nuclear-1 antigen) Barr (EBNA-1) constitutively expressing EBNA-1 directed by the immediate early promoter / enhancer of human CMV). The activation of NF-B 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B activity by a gel delay assay using a 25 base pair oligonucleotide that included NFB binding sites. Two million cells were seeded in 10 cm plates two days before transfection with DNA and grown in DMEM-F12 medium containing 2.5% FBS (fetal bovine serum). DNA transfections were performed as described herein for IL-8 promoter / indicator assays.

[0090] Nuclear extracts were prepared by solubilization of isolated nuclei with 400 mM NaCl (Yao et al., Mentioned above). Oligonucleotides containing an NF-B binding site were hybridized and terminally labeled with 32P using T4 polynucleotide kinase DNA. The mobility change reactions contained 10 µg of nuclear extract, 4 µg of poly (dI-dC) and 15,000 cpm of labeled double stranded oligonucleotide and were incubated at room temperature for 20 minutes. The resulting protein-DNA complexes were resolved on a 6% native polyacrylamide gel in 0.25 X Tris-borate-EDTA buffer.

[0091] Overexpression of RANK resulted in the induction of NF-B activity as shown by an appropriate change in the mobility of the radioactive probe in the gel. Similar results were observed when RANK was induced by a ligand that binds to RANK and transduces a signal to the cells expressing the receptor (i.e., by co-transfection of the cells with human RANK and murine RANKL DNA; see Example 7 presented below), and this would be expected to occur when induction is done with agonist antibodies.

EXAMPLE 5

[0092] This example describes a gene promoter / indicator system based on the human Interleukin-8 (IL-8) promoter used to analyze the activation of gene transcription in vivo. It is known that induction of transcription of the human IL-8 gene by cytokines Interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-) is dependent on transcription factor binding sites NF-IL-6 and NF-B intact. The fusion of the IL-8 promoter that responds to cytokines with a cDNA encoding the murine IL-4 receptor (mIL-4R) allows to measure the activation of the promoter by detection of the heterologous indicator protein (mIL-4R) on the surface Transfected cell cell.

[0093] Human kidney epithelial cells (293 / EBNA) (through the DEAE / DEXTRAN method) are transfected with plasmids encoding: 1). the indicator / promoter construction (called pIL-8rep) and 2). the cDNA or interest. DNA concentrations are always kept constant by the addition of empty vector DNA. 293 / EBNA cells are grown at a density of 2.5 x 104 cells / ml (3 ml / well) in a 6-well plate and incubated for two days before transfection. Two days after transfection, the mIL-4 receptor is detected by a radioimmunoassay (RIA) as described below.

[0094] In one of these experiments, 293 / EBNA cells were co-transfected with DNA encoding RANK and with DNA encoding RANKL (see Example 7 presented below). Co-expression of this receptor and its counter-structure by cells results in the activation of the RANK signaling process. For these co-transfection studies, the DNA / well concentration for DEAE transfection was as follows: 40 ng of pIL-8rep [pBluescriptSK-vector (Stratagene)]; 0.4 ng of CD40 (DNA encoding CD40, a control receptor; pCDM8 vector); 0.4 ng of RANK (DNA encoding RANK; vector pDC409) and 1-50 ng of 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 performed using soluble RANKL or agonist antibodies to RANK to induce cells transfected with RANK.

[0095] For the specific RIA of mIL-4R, a monoclonal antibody reactive with mIL-4R is labeled with 125I and through a method of conjugation with Chloramine T; The resulting specific activity is typically 1.5 x 106 cpm / nmol. After 48 hours, the transfected cells are washed once with medium (DMEM / F12, 5% FBS). Non-specific binding sites are blocked by the addition of preheated binding medium containing 5% skimmed milk powder and incubation at 37 ° C / 5% CO2 in a tissue culture incubator for one hour. The blocking medium is decanted and binding buffer containing 125I anti-mIL-4R (clone M1; rat IgG1) is added to the cells and incubated with stirring at room temperature for 1 hour. After incubation of the cells with the radiolabeled antibody, the cells are washed considerably with binding buffer (2X) and twice with phosphate buffered saline (PBS). The cells are lysed in 1 ml of 0.5 M NaOH, and the total radioactivity is measured with a gamma counter.

[0096] Using this assay, 293 / EBNA cells co-transfected with DNA encoding RANK showed transcriptional activity, as shown by detection of muIL-4R on the cell surface. Overexpression of RANK resulted in the transcription of muIL-4R, as did the induction of RANK by RANKL. Similar results are observed when RANK is induced by agonist antibodies.

EXAMPLE 6

[0097] This example illustrates the association of RANK with TRAF proteins. The interaction of RANK with cytoplasmic TRAF proteins was demonstrated by co-immunoprecipitation assays essentially as described by Hsu et al. (Cell

84: 299; nineteen ninety six). In summary, 293 / EBNA cells were co-transfected with plasmids that direct the synthesis of RANK and TRAF2 or TRAF3 with the labeled epitope (FLAG; SEQ ID NO: 7). Two days after transfection, the surface proteins were labeled with biotin ester and the cells were lysed in a buffer containing 0.5% NP-40. RANK and the proteins associated with this receptor were immunoprecipitated with anti-RANK, washed thoroughly, 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 membrane probing with an antibody that specifically recognizes the FLAG epitope. TRAF 2 and 3 did not imunoprecipitate with anti-RANK in the absence of RANK expression.

EXAMPLE 7

[0098] This example describes the isolation of a ligand for RANK, called RANKL, by direct expression cloning. The ligand was essentially cloned as described in USSN 08 / 249-189, filed May 24, 1994.

[0099] For CD40L, in summary, a library was prepared from a clone of an EL-4 mouse thymoma cell line (ATCC TIB 39), designated EL-40.5, obtained by separation five times with the fusion protein Biotinylated CD40 / Fc in an FACS (fluorescence activated cell separator). The cDNA library was obtained using the conventional methodology; Plasmid DNA was isolated and transfected into subconfluent CV1-EBNA cells using a DEAE-dextran method. Transfectants were selected by slide autoradiography for RANKL expression using a two-stage method of binding with the RANK / Fc fusion protein as prepared in Example 2 followed by radioiodinated human goat anti-IgG antibody. A clone encoding a protein that specifically bound RANK was isolated and sequenced; the clone was named 11H. An expression vector containing the murine RANKL sequence, called pDC406: muRANK-L (in E. coli DH10B), was deposited in the American Type Culture Collection, Rockville, MD (ATCC) on December 20, 1996, according to the terms of the Budapest Treaty, and received accession number 98284. The nucleotide sequence and expected amino acid sequence of this clone are illustrated in SEQ ID NO: 10. This clone did not contain an initiating 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); it was found that the 5 ’region was identical to that of human RANKL as shown in SEQ ID NO: 12, amino acids 1 to 22, except for the substitution of a Thr with Gly in the remainder 9.

[0100] This ligand is useful for assessing the ability of RANK to bind RANKL by several different assays. For example, transfected cells expressing RANKL can be used in a FACS assay (or similar assay) to assess the ability of soluble RANK to bind to RANKL. In addition, soluble forms of RANKL can be prepared and used in assays that are known in the art (i.e., 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 RANK levels in a sample. Soluble RANKL is also useful for inducing NF-activaciónB activation and thus protecting RANK-expressing cells from apoptosis.

EXAMPLE 8

[0101] This example describes the isolation of a human RANK ligand (RANKL) using a PCR-based technique. Murine RANK ligand specific oligonucleotide primers were used in PCR reactions using first strand cDNA derived from the human cell line as templates. The primers corresponded to nucleotides 478-497 and the complement of nucleotides 858-878 of the murine RANK ligand (SEQ ID NO: 10). An amplified band of approximately 400 bp in length of a reaction using the KB human epidermoid cell line (ATCC CCL-17) was gel purified and its nucleotide sequence was determined; the sequence was 85% identical to the corresponding region of the murine RANK ligand, confirming that the fragment came from human RANKL.

[0102] To obtain full-length human RANKL cDNA, two human RANKL specific oligonucleotides derived from the nucleotide sequence of the KB PCR product were radiolabelled and used as hybridization probes to explore a human PBL cDNA library prepared in lambda gt10 (Stratagene, La Jolla, CA), substantially as described in US Patent 5,599,905, issued on February 4, 1997. Several positive hybridization plates were identified and purified, their inserts were subcloned into pBluescrip SK- (Stratagene, La Jolla, CA) and its nucleotide sequence was determined. It was found that an isolate, PBL3, encoded most of the expected human RANKL, but about 200 bp of the 5 ’coding region appeared to be missing. It was discovered that a second isolate, PBL5, encoded much of the expected human RANKL, including the entire 5 ’end and an additional 200 bp of the 5’ untranslated sequence.

[0103] The 5 ′ end of PBL5 and the 3 ′ end of PBL3 joined together to form a full-length cDNA encoding human RANKL. The predicted nucleotide and amino acid sequence of the full-length human RANK ligand is shown in SEQ ID NO: 12. The human RANK ligand shares an identity of 83% nucleotides and 84% amino acids with the RANK ligand. murine A plasmid vector containing the RANKL sequence, called pBluescript: huRANK-L (in E. coli DH10B) was deposited in the American Type Culture Collection, Rockville, MD (ATCC) on March 11, 1997 under the terms of Treaty of Budapest, and received access number 98354.

[0104] Murine and human RANKL are Type 2 transmembrane proteins. Murine RANKL contains a predicted intracellular domain of 48 amino acids, a transmembrane domain of 21 amino acids and an extracellular domain of 247 amino acids. Human RANKL contains an expected intracellular domain of 47 amino acids, a transmembrane domain of 21 amino acids and an extracellular domain of 249 amino acids.

EXAMPLE 9

[0105] This example describes the chromosomal mapping of human RANK using PCR-based 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 was located on human chromosome 18; the RANK ligand was located on human chromosome 13. A more detailed mapping was performed using a mapping panel with Genebridge 4 Radiation Hybrid Panel radiation hybrids (Research Genetics, Huntsville, AL; described in Walter, MA et al., Nature Genetics

7: 22-28, 1994). The data from this analysis were then submitted electronically to the MIT Radiation Hybrid Mapper (URL: http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) following the instructions contained therein. This analysis produced specific genetic marker names that, when submitted electronically to the NCBI Entrez browser (URL: http://www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=c&form=0) , produced the specific map locations. RANK was located on chromosome 18q22.1 and RANKL was located on chromosome 13q14.

EXAMPLE 10

[0106] 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 used to generate monoclonal antibodies against RANKL using conventional techniques, such as those described in US Patent 4,411,993. DNA encoding RANKL can also be used as an immunogen, for example, as reviewed by Pardoll and Berckerleg in Immunity 3: 165, 1995. Such antibodies will likely be useful for interfering with the signaling of RANKL (antagonist or blocking antibodies), as components. of diagnostic or research assays for RANKL or RANKL activity, or in affinity purification of RANKL.

[0107] To immunize rodents, a RANKL immunogen is emulsified in an adjuvant (such as Freund's complete or incomplete adjuvant, alum or other adjuvant, such as Ribi's R700 adjuvant (Ribi, Hamilton, MT)) and injected in amounts ranging from 10 to 100 subg subcutaneously in a selected rodent, for example, BALB mice / with Lewis rats. DNA can be administered intradermally (Raz et al., Proc. Natl. Acad Sci. USA 91: 9519, 1994) or intramuscularly (Wang et al., Proc. Natl. Acad Sci. USA 90: 4156, 1993 ); It has been observed that the saline solution is a suitable diluent for DNA-based antigens. Ten days to three weeks later, the immunized animals are reinforced with an additional immunogen and subsequently periodically reinforced with a weekly, biweekly or every three weeks immunization schedule.

[0108] Serum samples are taken periodically by retro-orbital extraction or excision of the tail tip to be assayed by a point transfer assay (antibody sandwich), ELISA (enzyme-linked immunoabsorbent assay), immunoprecipitation or other assays. suitable, including FACS analysis. After 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, the splenocytes are collected and fused to a murine myeloma cell line (eg, NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell lines generated by this procedure are cultured 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 hybrids of splenocyte-splenocyte.

[0109] Hybridoma clones generated in this manner can be screened by ELISA for reactivity with RANKL, for example, by adaptations of the techniques described by Engvall et al., Immunochem. 8: 871 (1971) and in US 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 fluid that contains high concentrations (> 1 mg / ml) of anti-RANK monoclonal antibody. The resulting monoclonal antibody can be purified by precipitation with ammonium sulfate followed by molecular exclusion chromatography. Alternatively, affinity chromatography based on antibody to protein A or G protein binding can be used, as well as affinity chromatography based on RANKL protein binding. Using the methods described herein to control the activity of mAbs, both blocking antibodies (i.e., antibodies that bind to RANKL and inhibit RANK binding) are isolated as non-blocking antibodies (i.e., antibodies that bind to RANKL and do not inhibit binding).

EXAMPLE 11

[0110] This example demonstrates that RANK expression can be positively regulated. Human peripheral blood T cells were purified by flow cytometry separation or by negative selection using antibody coated beads, and activated with anti-CD3 coated plates (OKT3, Dako) or phytohemagglutinin in the presence or absence of various cytokines, including Interleukin-4 (IL-4), Transformation Growth Factor- (TGF-) and other commercially available cytokines (IL1-, IL-2, IL-3, IL-6, IL-7, IL -8, IL-10, IL-12, IL-15, IFN-, TNF-). RANK expression was evaluated by FACS in an experiment over time from day 2 to day 8, using a mouse monoclonal antibody mAb144 (prepared as described in Example 3), as shown in the table presented below. . The results are expressed as ‘+’ to ‘++++’ referring to the relative increase in staining intensity with anti-RANK. Double labeling experiments were also performed using anti-RANK and anti-CD8 or anti-CD4 antibodies.

5

10

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twenty

25

30

Table 1: Positive regulation of RANK by Cytokines

Cytokine (concentration)

Results

IL-4 (50 ng / ml) TGF- (5 ng / ml) IL-4 (50 ng / ml) + TGF- (5 ng / ml) IL1- (10 ng / ml) IL-2 ( 20 ng / ml) IL-3 (25 ng / ml) IL-7 (20 ng / ml) IL-8 (10 ng / ml) IL-10 (50 ng / ml) IL-12 (10 ng / ml) IL-15 (10 ng / ml) IFN- (100 U / ml) TNF- (10 ng / ml)

+ + a ++ ++++ ----------

[0111] Of the cytokines tested, IL-4 and TGF- increased the level of RANK expression in both CD8 + cytotoxic T cells and CD4 + helper T cells from day 4 to day 8. The combination of IL-4 and TGF-  acted synergistically to positively regulate the expression of this receptor on activated T cells. This particular combination of cytokines is secreted by suppressor T cells, and is believed to be important in the generation of tolerance (reviewed in Mitchison and Sieper, Z. Rheumatol. 54: 141, 1995), involving the interaction of RANK in the regulation of an immune response towards tolerance or induction of an active immune response.

EXAMPLE 12

[0112] This example illustrates the influence of RANK.Fc and hRANKL on the growth of activated T cells. The addition of TGF to anti-CD3 activated human peripheral blood T cells induces proliferation arrest and ultimately death of most lymphocytes within the first days of culture. We tested the effect of RANK: RANKL interactions on T cells treated with TGF-iendo by adding RANK.Fc or soluble human RANKL to T cell cultures.

[0113] Human peripheral blood T cells (7 x 105 PBT) were cultured for six days in 24-well plates coated with anti-CD3 (OKT3, 5 µg / ml) and anti-Flag (M1, 5 µg / ml) in the presence of TGF (1 ng / ml) and IL-4 (10 ng / ml), with

or without RANK.Fc (10 g / nl) or soluble hRANKL (1 /g / ml) labeled with recombinant FLAG. The recovery of viable T cells was determined by triplicate blue counts in triplicate.

[0114] The addition of RANK.Fc significantly reduced the number of viable T cells recovered after six days, while soluble RANKL greatly increased the recovery of viable T cells (Figure 1). In this way, endogenous or exogenous RANKL increases the number of viable T cells generated in the presence of TGF. TGF, together with IL-4, has been implicated in the regulation of the immune response when it is secreted by the subset of TH3 / regulatory T cells. It is believed that these T cells mediate bystander suppression of effector T cells. Therefore, RANK and its ligand can act in an auto / paracrine manner to influence cell tolerance

T. In addition, it is known that TGF plays a role in the evasion of the immune system by certain pathogenic or opportunistic organisms. In addition to playing a role in the development of tolerance, RANK can also play a role in the evasion of the immune system by pathogens.

EXAMPLE 13

[0115] This example illustrates the influence of the interaction of RANK on CD1a + dendritic cells (DC). Functionally mature dendritic (DC) cells were generated in vitro from CD34 + bone marrow (BM) progenitors. In summary, human BM cells from normal healthy volunteers were fractionated by means of Ficoll medium and CD34 + cells were immuno-isolated using an anti-CD34 matrix column (Ceprate, CellPro). BM CD34 + cells were then cultured in human GM-CSF (20 ng / ml), human IL-4 (20 ng / ml), human TNF-((20 ng / ml), human Flt3L derived from CHO (FL; 100 ng / ml) in Super McCoy medium supplemented with 10% fetal bovine serum in a fully humidified incubator at 37 ° C (5% CO2) for 14 days. DC CD1a + and HLA-DR + were then separated using a FACStar Plus and used for the biological evaluation of RANK.

[0116] In human CD1a + DCs from CD34 + bone marrow cells, only a subset (20-30%) of CD1a + DCs expressed RANK on the cell surface, as assessed by flow cytometric analysis. However, the addition of CD40L to DC cultures resulted in a surface expression of RANK in most CD1a + DCs. It has been shown that CD40L activates DC by increasing the formation of in vitro clusters, inducing morphological changes of DC and positively regulating the expression of HLA-DR, CD54, CD58, CD80 and CD86.

[0117] The addition of RANKL to DC cultures significantly increased the degree of DC aggregation and cluster formation with respect to control cultures, similar to the effects observed with CD40L (Figure 2). Separate human CD1a + Dc were grown in a cytokine cocktail (GM-CSF, IL-4, TNF- and FL) (upper left panel), in cocktail plus CD40L (1 /g / ml) (upper right), in cocktail more RANKL (1 g / ml) (lower left) or in a cocktail plus RANKL heat inactivated (H) (1 g / ml) (lower right) in 24-well flat bottom culture plates in a medium of 1 ml culture for 48-72 hours and then photographed using an inversion microscope. An increase in DC aggregation and cluster formation with respect to control cultures was not apparent when heat inactivated RANKL was used, indicating that this effect was dependent on the biologically active protein. However, the initial phenotypic analysis of the expression of adhesion molecules indicated that the clustering induced by RANKL was not due to the higher levels of CD2, CD11a, CD54 or CD58.

[0118] The addition of RANKL to CD1a + DC increased its allo-stimulatory capacity in a mixed lymphocyte reaction (MLR) at least 3 to 10 times, comparable to DC cultured with CD40L (Figure 3). Allogeneic T cells (1x105) were incubated with varying numbers of irradiated DC (2000 rad) cultured as indicated above for Figure 2 in 96-well round bottom culture plates in 0.2 ml of culture medium for four days. The cultures were pulsed with 0.5 mCi of [3 H] -thymidine for eight hours and the cells were collected on fiberglass sheets for counting on a  counter in the gas phase. Background counts for T or DC cells cultured alone were <100 cpm. The values represent the mean  SD of triplicate cultures. Heat inactivated RANKL had no effect. The allo-stimulatory activity of DC did not increase further when RANKL and CD40L were used in combination, possibly due to the functional capacity of DC that had reached a maximum level with any cytokine alone. Neither RANKL nor CD40L increased the in vitro growth of DC with respect to the three-day culture period. Unlike CD40L, RANKL did not significantly increase HLA-DR expression levels or CD80 or CD86 expression.

[0119] RANKL can increase the formation of DC clusters and functional capacity without modulating known molecules involved in cell adhesion (CD18, CD54), antigen presentation (HLA-DR) or co-stimulation (CD86), being all these processes regulated by the signaling of CD40 / CD40L. The absence of an effect on the expression of these molecules suggests that RANKL can regulate DC function by one or more alternative routes other than CD40 / CD40L. Since CD40L regulates surface expression of RANK in DC generated in vitro and that CD40L is positively regulated in activated T cells during interactions of DC-T cells, RANK and its ligand can form an important part of the activation cascade that induces during DC-mediated T cell expansion. In addition, the culture of DC in RANKL results in lower levels of CD1b / c expression, and higher levels of CD83. These two molecules are similarly modulated during DC maturation by CD40L (Caux et al. J. Exp. Med. 180: 1263; 1994), indicating that RANKL induces DC maturation.

[0120] Dendritic cells are called "professional" antigen presenting cells, and have a high capacity to sensitize MHC-restricted T cells. There is increasing interest in the use of ex vivo dendritic cells as vaccine adjuvants for infectious diseases or tumors (see, for example, Romani, et al., J. Exp. Med., 180: 83, 1994). Therefore, an agent such as RANKL that induces DC maturation and increases the ability of dendritic cells to stimulate an immune response will probably be useful in immunotherapy of various diseases.

EXAMPLE 14

[0121] This example describes the isolation of the murine homologue of RANK, called muRANK. MuRANK was isolated by a combination of cross-species PCR and colony hybridization. The conservation of Cys residues in the Cys-rich pseudo-repeats of the extracellular domains of TNFR superfamily member proteins was exploited to design RANK-based PCR primers for use in murine first-chain cDNAs from various sources. Both the upstream chain sense sense primer and the downstream chain antisense primer were designed so that their 3 ′ ends end with Cys residues.

[0122] 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 409427 (amino acid coding region 124-130). Conventional PCR reactions were prepared and performed using these primers and first strand cDNA from various murine cell lines or tissue sources. Thirty reaction cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds and 72 ° C for 20 seconds were performed. PCR products were analyzed by electrophoresis and specific bands were seen in several samples. The band of a sample was gel purified and DNA sequencing revealed that the sequence between the primers was approximately 85% identical to the corresponding human RANK nucleotide sequence.

[0123] A plasmid-based cDNA library prepared from the FLE18 murine fetal liver epithelium line (one of the cell lines identified as positive in the PCR scan) with respect to full-length RANK cDNAs was screened using specific oligonucleotide probes for murine RANK derived from the murine RANK sequence determined by sequencing the PCR product. Two cDNAs were recombined, one encoding the 5 'end and the other encoding the 3' end of full-length murine RANK (based on sequence comparison with full-length human RANK) to generate a murine RANK cDNA of full length The nucleotide and amino acid sequences of muRANK are shown in SEQ ID NO: 14 and

fifteen.

[0124] The cDNA encodes an expected Type 1 transmembrane protein that has 625 amino acid residues, with an expected signal sequence of 30 amino acids, an extracellular domain of 184 amino acids, a transmembrane domain of 21 amino acids and a cytoplasmic tail of 390 amino acids. The extracellular region of muRANK had a significant amino acid homology (69.7% identity, 80.8% similarity) with huRANK. Those skilled in the art will recognize that the actual cleavage site may be different from that provided by computer; therefore, the N-terminus of RANK can be from amino acid 25 to amino acid 35.

[0125] Other members of the TNF receptor superfamily have an amino acid region between the transmembrane domain and the ligand binding domain which is called the "spacer" region, which is not necessary for ligand binding. In muRANK, it is predicted that amino acids between 197 and 214 form said spacer region. Therefore, it is expected that a soluble form of RANK that ends with an amino acid in this region retains the ability to bind a ligand for RANK in a specific manner. Preferred C-terminal amino acids are selected for soluble RANK peptides 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 used as the C-terminus.

EXAMPLE 15

[0126] This example illustrates the preparation of several different soluble forms of RANK and RANKL. Conventional cutting techniques with restriction enzymes and ligation were used, in combination with PCR-based isolation of fragments for which there were no convenient restriction sites. When the PCR was used, the PCR products were sequenced to find out if any mutation had been introduced; These mutations were not found.

[0127] In addition to the huRANK / Fc described in Example 2, another RANK / Fc fusion protein was prepared by DNA linkage 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 1-213 of full length murine RANK (SEQ ID NO: 15) to DNA encoding amino acids 3-232 of the Fc mutein (SEQ ID NO: 8).

[0128] A labeled, soluble, soluble poly-His version of huRANKL was prepared by DNA binding encoding the leader peptide of the immunoglobulin kappa chain (SEQ ID NO: 16) to DNA encoding a short version of the FLAG marker ( SEQ ID NO: 17), followed by codons encoding Gly Ser, and then a poly-His marker (SEQ ID NO: 18), followed by codons encoding Gly Thr Ser and DNA encoding amino acids 138-317 of the SEC ID NO: 13. A soluble poly-His labeled version of murine RANKL was prepared by DNA linkage encoding the CMV leader (SEQ ID NO: 9) to codons encoding Arg Thr Ser, followed by DNA encoding poly- His (SEQ ID NO: 18) and followed by DNA encoding amino acids 119-294 of SEQ ID NO: 11.

[0129] A soluble oligomeric form of huRANKL was prepared by DNA linkage encoding the CMV leader (SEQ ID NO: 9) to a codon encoding Asp followed by DNA ending in a "leucine" zipper of trimer formation (SEQ ID NO: 19), and then by codons encoding Thr Arg Ser followed by amino acids 138-317 of SEQ ID NO: 13.

[0130] These and other constructions are prepared by routine experimentation. The various DNAs are then inserted into a suitable expression vector and expressed. Particularly preferred expression vectors are those that 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; in USSN 08 / 785,150, now admitted, several useful vectors are described, for example, one of the expression vectors derived from  2A5-3 analyzed herein.

EXAMPLE 16

[0131] This example demonstrates that RANKL expression can be positively regulated in murine T cells. Mesenteric lymph node cells were obtained from C57BL / 6 mice and activated with plates coated with antiCD3, Concanavalin A (ConA) or forbol 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 for approximately 2 to 5 days. RANKL expression was evaluated in a three-color analysis by FACS, using antibodies against CD4, CD8 and CD45RB and RANK / Fc T-cell markers, prepared as described herein.

[0132] RANKL was not expressed in unstimulated murine T cells. T cells stimulated with anti-CD3, ConA or PMA / ionomycin, showed differential expression of RANKL: CD4 + / CD45RBLLo and CD4 + / CD45RBHi cells were positive for RANKL, but CD8 + cells were not. RANKL was not observed in B cells, similar to the results observed with human cells.

EXAMPLE 17

[0133] This example illustrates the effects of murine RANKL on cell proliferation and activation. Various cells or cell lines representing cells that play a role in an immune response (spleen, thymus and murine lymph nodes) were evaluated by culturing them in conditions that promoted their viability, in the presence or absence of RANKL. RANKL did not stimulate any of the cells tested to proliferate. A cell line, a macrophage cell line called RAW 264.7 (ATCC accession number TIB 71) showed some signs of activation.

[0134] RAW cells constitutively produce small amounts of TNF-. Incubation with human or murine RANKL increased the production of TNF- 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 prevented the production of TNF-, while a similar treatment of purified endotoxin (LPS) did not affect the ability of the LPS stimulate the production of TNF-. Despite the fact that RANKL activated the RAW T64.7 macrophage cell line for TNF- production, neither human RANKL nor murine RANKL stimulated nitric oxide production by these cells.

EXAMPLE 18

[0135] This example illustrates the effects of murine RANKL on thymus growth and development in fetal mice.

5

10

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twenty

25

30

35

40

Pregnant female 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, RANK / Fc was injected intraperitoneally (IP) in newborns on a daily basis, starting at a dose of 1 g and doubling the dose approximately every four days, to reach a final dosage of 4  g. Newborns were collected on days 1, 8 and 15 after birth, their scams and spleens were collected and examined for size, cellularity and phenotypic composition.

[0136] A slight reduction in thymus size was observed on day 1 in newborns of females injected with RANK / Fc; no similar size reduction was observed in control newborns. On day 8, the thymus size and cellularity had been reduced by approximately 50% in animals treated with RANK / Fc compared to mice treated with MSA. Phenotypic analysis showed that the relative proportions of different populations of T cells in the thymus of RANK / Fc mice were equal to those of control mice, indicating that the reduced cellularity was due to a global depression in the number of T cells thymic rather than due to a reduction in one or more specific populations. Newborns treated with RANK / Fc were not significantly different from control newborns on day 15 regarding their size, cellularity or thymic cell phenotype. No significant difference in spleen size, cellularity or composition was observed at any of the time points evaluated. The difference in cellularity on day 8 and not on day 15 may suggest that RANK / Fc can exert its effect early on thymic development.

EXAMPLE 19

[0137] This example demonstrates that the C-terminal region of the cytoplasmic domain of RANK is important for the binding of several different TRAF proteins. RANK contains at least two recognizable PXQX (X) T motifs that are likely TRAF docking sites. Therefore, 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.

[0138] Comparison of the murine and human RANK nucleotide sequence indicated that there were several conserved regions that could be important for TRAF binding. Therefore, a PCR-based technique was developed to facilitate the preparation of various C-terminal truncations that would retain the conserved regions. PCR primers were designed to introduce a termination codon and a restriction enzyme site at selected points, producing the truncations described in Table 1 shown below. Sequencing confirmed that no unwanted mutations had been introduced into the constructs.

[0139] Radiolabelled TRAF proteins (32S-Met, Cys) were prepared by in vitro translation using a commercially available reticulocyte lysate kit according to the manufacturer's instructions (Promega). Truncated GST fusion proteins were substantially purified as described in Smith and Johnson (mentioned above). In summary, E. coli was transfected with an expression vector encoding a fusion protein and induced to express the protein. The bacteria were lysed, insoluble material was removed and the fusion protein was isolated by precipitation with glutathione coated beads (Sepharose 4B, Pharmacia, Uppsala Sweden).

[0140] The beads were washed and incubated with various radiolabeled TRAF proteins. After the incubation and washing steps, the fusion protein / TRAF complexes were removed from the beads by boiling in 0.1% SDS

+ -mercaptoethanol, and were loaded in 12% SDS gels (Novex). The gels underwent autoradiography and the presence and absence of radiolabelled material was recorded. The results are shown in Table 2 provided below.

Table 2: Binding of Various TRAF Proteins to the RANK Cytoplasmic Domain [0141] These results indicate that TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6 bind to the most distal part of the cytoplasmic domain of RANK (between amino acid G544 and A616). TRAF6 also has a binding site between S339 and Y421. In this experiment, TRAF5 also bound to the cytoplasmic domain of RANK.

Truncation: C terminals

E206-S339 E206-Y421 E206-M476 E206-G544 Full length

TRAF 1

- - - - ++

TRAF2

 - - - - ++

TRAF3

 - - - - ++

TRAF4

 - - - - -

TRAF5

 - - - - +

TRAF6

 - + + + ++

SEQUENCE LIST

[0142]

(one)

 GENERAL INFORMATION:

(i)

 APPLICANT: Immunex Corporation

(ii)

 TITLE OF THE INVENTION: NF-KappaB receiver activator

(iii) NUMBER OF SEQUENCES: 19

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(B)

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(viii) MANDATORY / AGENT INFORMATION

(TO)

 NAME: Perkins, Patricia Anne

(B)

 REGISTRATION NUMBER: 34,693

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(ix) TELECOMMUNICATIONS INFORMATION

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(saw)

 ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS

(vii) IMMEDIATE SOURCE:

(TO)

 LIBRARY: DENDRITHIC CELLS DERIVED FROM BONE MARROW

(B)

 CLON: 9D-8A

(ix) CHARACTERISTICS:

(TO)

 NAME / KEY: CDS

(B)

 LOCATION: 93..1868

25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1

image 1

image 1

image 1

image 1

(2) INFORMATION FOR SEQ ID NO: 2

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 591 amino acids

(B)

 TYPE: amino acid

(D)

 TOPOLOGY: linear

10 (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2

image 1

image 1

image 1

(2) INFORMATION FOR SEQ ID NO: 3

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 1391 base pairs

(B)

TYPE: nucleic acid

(C)

 CHAIN TYPE: simple

(D)

 TOPOLOGY: linear

10

(ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETICAL: NO

15 (iv) ANTISENTIDO: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS

5 (vii) IMMEDIATE SOURCE:

(TO)

 LIBRARY: DENDRITHIC CELLS DERIVED FROM BONE MARROW

(B)

 CLON: 9D-15C

(ix) CHARACTERISTICS:

10 (A) NAME / KEY: CDS

(B) LOCATION: 39..1391

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3

image 1

image 1

(2) INFORMATION FOR SEQ ID NO: 4

(i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 451 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

(ii) TYPE OF MOLECULE: protein

10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4

image 1

image 1

(2) INFORMATION FOR SEQ ID NO: 5

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 3136 base pairs

(B)

TYPE: nucleic acid

(C)

 CHAIN TYPE: simple

(D)

 TOPOLOGY: linear

10

(ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(saw)

 ORIGINAL SOURCE:

5 (A) ORGANISM: HOMO SAPIENS

(vii) IMMEDIATE SOURCE:

(TO)

 LIBRARY: DENDRITHIC CELLS DERIVED FROM BONE MARROW

(B)

 CLON: FULL LENGTH RANK

10

(ix) CHARACTERISTICS:

(TO)

 NAME / KEY: CDS

(B)

 LOCATION: 39..1886

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

image 1

image 1

image 1

image 1

(2) INFORMATION FOR SEQ ID NO: 6

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 616 amino acids

(B)

TYPE: nucleic acid

(D)

 TOPOLOGY: linear

10 (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6

image 1

image 1

image 1

(2) INFORMATION FOR SEQ ID NO: 7

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 8 amino acids

(B)

 TYPE: amino acids

(C)

 TYPE OF CHAIN: not relevant

(D)

 TOPOLOGY: linear

10

(ii) TYPE OF MOLECULE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLON: FLAG peptide

fifteen

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7

image 1

(2) INFORMATION FOR SEQ ID NO: 8

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 232 amino acids

(B)

 TYPE: amino acid

(C)

 TYPE OF CHAIN: not relevant

(D)

 TOPOLOGY: linear

10

(ii)

 TYPE OF MOLECULE: Protein

(saw)

 ORIGINAL SOURCE:

(A) ORGANISM: Human

fifteen

(vii) IMMEDIATE SOURCE:

(B) CLON: IGg1 Fc mutein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8

image 1

(2) INFORMATION FOR SEQ ID NO: 9

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 31 amino acids

(B)

 TYPE: amino acid

(C)

 TYPE OF CHAIN: not relevant

(D)

 TOPOLOGY: linear

10

(ii) TYPE OF MOLECULE: peptide

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(saw)

 ORIGINAL SOURCE:

(A) ORGANISM: CMV (Leader R2780)

(ix)

 FEATURES:

(TO)

 OTHER INFORMATION: Met1-Arg28 is the real leader peptide; Arg29 reinforces the furina cleavage site; nucleotides encoding Thr30 and Ser31 add a Spe1 site

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9

(2)

 INFORMATION FOR SEQ ID NO: 10

(i)

 CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 1630 base pairs

(B)

TYPE: nucleic acid

(C)

 CHAIN TYPE: simple

(D)

 TOPOLOGY: linear

(ii)

 TYPE OF MOLECULE: cDNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(saw)

 ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus

(vii) IMMEDIATE SOURCE:

(TO)

 LIBRARY:

(B)

 CLON: RANKL

(ix)

 FEATURES:

(TO)

 NAME / KEY: CDS

(B)

 LOCATION: 3,884

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 10

(2)

 INFORMATION FOR SEQ ID NO: 11

image 1

image 1

image 1

image 1

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 294 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

10 (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11

image 1

(2) INFORMATION FOR SEQ ID NO: 12

(i) SEQUENCE CHARACTERISTICS:

53

(TO)

 LENGTH: 954 base pairs

(B)

TYPE: nucleic acid

(C)

 CHAIN TYPE: simple

(D)

 TOPOLOGY: linear

5

(ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETICAL: NO

10 (iv) ANTISENTIDO: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

15 (vii) IMMEDIATE SOURCE:

(TO)

 LIBRARY:

(B)

 CLON: huRANKL (full length)

(ix) CHARACTERISTICS:

20 (A) NAME / KEY: CDS

(B) LOCATION: 1,951

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12

image 1

image 1

(2) INFORMATION FOR SEQ ID NO: 13

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 317 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

10 (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13

image 1

(2)

 INFORMATION FOR SEQ ID NO: 14

(i)

 CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 1878 base pairs

(B)

TYPE: nucleic acid

(C)

 CHAIN TYPE: simple

(D)

 TOPOLOGY: linear

(ii)

 TYPE OF MOLECULE: cDNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(saw)

 ORIGINAL SOURCE:

(A) ORGANISM: Murino

(vii) IMMEDIATE SOURCE:

(TO)

 LIBRARY: Murine Fetal Liver Epithelium

(B)

 CLON: muRANK

(ix)

 FEATURES:

(TO)

 NAME / KEY: CDS

(B)

 LOCATION: 1..1875

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 14

(2)

 INFORMATION FOR SEQ ID NO: 15

(i)

 CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 625 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

(ii)

 TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 15

(2)

 INFORMATION FOR SEQ ID NO: 16

(i)

 CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 20 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

(ii)

 TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 16

(2)

 INFORMATION FOR SEQ ID NO: 17

image 1

image 1

image 1

image 1

image 1

image2

(i) SEQUENCE CHARACTERISTICS: 15 (A) LENGTH: 5 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

(ii)

 TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 17

twenty

image 1

(2)

 INFORMATION FOR SEQ ID NO: 18

(i)

 CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 6 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

(ii)

 TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 18

(2)

 INFORMATION FOR SEQ ID NO: 19

25

30

image 1

5 (i) SEQUENCE CHARACTERISTICS:

(TO)

 LENGTH: 33 amino acids

(B)

 TYPE: amino acids

(D)

 TOPOLOGY: linear

10 (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

image 1

REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is solely for the convenience of the reader. This list is not part of the European patent document. Although great care has been taken in the collection of references, errors or omissions cannot be excluded and the EPO disclaims all responsibility in this regard. Patent documents cited in the description

image 1

Non-patent bibliography cited in the description

image 1

image3

Claims (40)

1. Isolated DNA selected from the group consisting of:

(to)

 a DNA encoding a protein that has an amino acid sequence as set forth in SEQ ID NO: 6, where the protein has an amino terminus selected from the group consisting of an amino acid between amino acid 1 and amino acid 33, inclusive, of SEC ID No.: 6, and a carboxy terminus selected from the group consisting of an amino acid between amino acid 196 and amino acid 616, inclusive;

(b)

 DNA molecules capable of hybridizing with the DNA of (a) under stringent conditions, including 63 ° C, 6X SSC, overnight; washed in 3X SSC at 55 ° C, and encoding a biologically active activator receptor of NF-;B; Y

(C)

 DNA molecules encoding protein fragments encoded by the DNA of (a) or (b), where the fragments are biologically active and the biological activity is selected from the group consisting of: binding to TRAF 1, 2, 3, 5 or 6, specific binding to a soluble NF-B ligand activating receptor or an activating receptor of an NF-B ligand expressed in cells or immobilized on a surface, and binding to an NF-específicos specific antibody activating receptor B.

2.

Isolated DNA of claim 1, encoding an NF-polipB polypeptide activating receptor that is at least 80% identical in the amino acid sequence to a polypeptide of SEQ ID NO: 6 and having an activity shown by said polypeptide.

3.

Isolated DNA of claim 1 or 2, wherein the biological activity is selected from the group consisting of: binding to TRAF 1, 2, 3, 5 or 6, specific binding to a soluble receptor activating a NF-liga ligand B or an activating receptor of an NF-B ligand expressed in cells or immobilized on a surface, and binding to an NF-B-specific antibody activating receptor.

Four.

Isolated DNA of claim 1 or claim 2, which encodes a soluble receptor activator of an NF-B polypeptide.

5.

Isolated DNA of claim 4, further comprising a DNA encoding a polypeptide selected from the group consisting of an immunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG marker, a peptide comprising at least about 6 residues of His, a leucine zipper and combinations thereof.

6.

Recombinant expression vector comprising a DNA sequence according to any one of claims 1 to 5.

7.

Host cell transformed or transfected with an expression vector according to claim

6.

8.

Method for preparing an NF-B protein activator receptor, which comprises culturing a host cell according to claim 7 under conditions that promote the expression and recovery of the NF-B activating active receptor.

9.

Isolated DNA fragment selected from the group consisting of oligonucleotides of at least about 17 nucleotides in length, oligonucleotides of at least about 25 nucleotides in length and oligonucleotides of at least about 30 nucleotides in length, where the fragment is a fragment of nucleotide residues 39-1886 of SEQ ID NO: 5.

10.

Isolated NF-B polypeptide activator receptor selected from the group consisting of:

(to)

 a polypeptide comprising an amino acid sequence of amino acids 33 to 196 of SEQ ID NO: 6;

(b) a polypeptide comprising an amino acid sequence of amino acids 1 to 213 of SEQ ID NO: 6;

(C)

 a polypeptide comprising an amino acid sequence of amino acids 234 to 616 of SEQ ID NO: 6;

(d)

 an NF-B polypeptide activator receptor encoded by a DNA capable of hybridizing with a DNA encoding the protein from (a) to (c) under stringent conditions (63 ° C, 6X SSC, overnight; 3X SSC washing at 55 ° C) and which is biologically active;

(and)

 fragments of the polypeptides from (a) to (d) that are biologically active and the biological activity is selected from the group consisting of: induction of NF-B activity, binding to TRAF 1, 2, 3, 5 or 6 , specific binding to a soluble receptor activator of an NF-B ligand or to an activator receptor of an NF-B ligand expressed in

cells or immobilized as a surface and binding to an NF-específicosB specific antibody activating receptor.

eleven.

Isolated NF-B polypeptide activator receptor of claim 10, wherein the biological activity is selected from the group consisting of: induction of NF-B activity, binding to TRAF 1, 2, 3, 5 or 6 , specific binding to a soluble receptor activating an NF-B ligand or to an activating receptor for an NF-B ligand expressed in cells or immobilized as a surface and binding to an NF-B specific antibody activating receptor .

12.

Protein according to claim 10 or 11, which has an amino acid sequence with an identity of at least about 70%, an identity of at least about 80% or an identity of at least about 90% with SEQ ID NO. : 6.

13.

Protein according to any of claims 10 to 12, which is a soluble receptor activator of NF-B.

14.

Soluble NF-B protein activator receptor according to claim 13, further comprising a peptide selected from the group consisting of an immunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG marker, a peptide comprising at least about 6 His residues, a leucine zipper and combinations thereof.

fifteen.

Soluble receptor activator of the NF-B protein according to claim 14, comprising the Fc part of human IgG1 consisting of amino acids 3 to 232 of SEQ ID NO: 8.

16.

Immunoreactive antibody with an NF-B polypeptide activator receptor according to any one of claims 10 to 13.

17.

Antibody according to claim 16, which is a monoclonal antibody.

18.

Antibody according to claim 16 or claim 17, wherein said antibody binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6.

19.

Composition comprising a purified NF-activaB activator receptor encoded by a DNA of any one of claims 1 to 5 and a physiologically acceptable carrier, excipient or diluent.

twenty.

Composition comprising an antagonist antibody according to claim 16, claim 17

or claim 18 and a physiologically acceptable carrier, excipient or diluent.

twenty-one.

NF-B polypeptide activator receptor as defined in any of claims 10-15, or encoded by a DNA as defined in any of claims 1-5 or a composition as defined in claim 18 or claim 19 , for use in medicine.

22

Soluble NF-B polypeptide activator receptor as defined in any of claims 10-15,

or encoded by a DNA as defined in any of claims 1-5, for use in inhibiting the activation of NF-B. 23. NF-B polypeptide activator soluble receptor as defined in any of claims 10-15, or encoded by a DNA as defined in any of claims 1-5, for use in the treatment of a condition in which signaling through an NF-B activating receptor has resulted in negative consequences, such as a toxic or septic shock or a rejection of a graft against host. 24. NF-B polypeptide activator soluble receptor as defined in any of claims 10-15, or encoded by a DNA as defined in any of claims 1-5, for use in the treatment of a disease characterized by neoplastic cells expressing an NF-B activating receptor. 25. NF-B polypeptide activator soluble receptor as defined in any of claims 10-15, or encoded by a DNA as defined in any of claims 1-5, for use in the regulation of an immune or inflammatory response.

26.

Use of a soluble NF-B activator receptor according to any of claims 13-15, or encoded by a DNA as defined in any of claims 1-5, or of an NF antagonist monoclonal antibody activating receptor -B in the preparation of a medicament to inhibit the activation of NFB.

27.

Use of a soluble NF-B activator receptor according to any of claims 13-15, or encoded by a DNA as defined in any of claims 1-5, or of an NF antagonist monoclonal antibody activating receptor -B in the preparation of a medicament for the treatment of a condition in which signaling through the NF-B activating receptor has resulted in negative consequences, such as toxic or septic shock or graft rejection against host.

28.

Use of a soluble NF-B activator receptor according to any of claims 13-15, or encoded by a DNA as defined in any of claims 1-5, or of an NF antagonist monoclonal antibody activating receptor -B in the preparation of a medicament for the treatment of a disease characterized by neoplastic cells expressing the NF-B activating receptor.

29.

Use of a soluble NF-B activator receptor according to any of claims 13-15, or encoded by a DNA according to any of claims 1-5, or of an NF-antagonist monoclonal antibody activating receptor B in the preparation of a medicament to interfere in the role of NF-B in cell transformation.

30

Use of a soluble activator receptor of an NF-B polypeptide composition, wherein the NF-B activator receptor is encoded by a DNA as defined in any of claims 1-5, or of a monoclonal antibody activating receptor NF-B antagonists, in the preparation of a medication to regulate an immune or inflammatory response.

31.

Use of a soluble NF-B polypeptide activating receptor according to any one of claims 13 to 15, to screen an inhibitor of an NF-B activity-activating receptor induced by NF-B.

32

Use of a soluble form of an NF-B polypeptide activating receptor, encoded by a DNA according to any one of claims 1 to 5, derived from the extracellular domain, in a screening assay to detect molecules capable of binding to a receptor NF-B activator.

33.

Use of an NF-B polypeptide activator receptor, encoded by a DNA according to any one of claims 1 to 5, derived from the cytoplasmic domain, in an assay to detect inhibition of NF-B activator receptor interaction and other intracellular proteins involved in signal transduction.

3. 4.

Use according to claim 33, wherein the assay serves to detect inhibition of the association of the activating receptor of NF-yB and TRAF 1, 2, 3, 5 or 6.

35

Use of an activator receptor of the NF-B polypeptide sequence, encoded by a DNA according to any one of claims 1 to 5, in the design based on the structure of an activator receptor of an NF-B inhibitor.

36.

Use of an NF-B polypeptide activator receptor, encoded by a DNA according to any one of claims 1 to 5, to ascertain the ability of molecules to antagonize or agonize the NFB activating receptor.

37.

Use of an NF-B polypeptide activator receptor, encoded by a DNA according to any one of claims 1 to 5, in the development of agonist or antagonist antibodies to the NF-B activator receptor.

38.

Use of an NF-B polypeptide activating receptor, encoded by a DNA according to any one of claims 1 to 5, in a screening of NF-actividadB activity activating receptor antagonists.

39.

Kit for detecting a soluble receptor activating the NF-idoB polypeptide or for detecting a soluble receptor activating an NF-B ligand or for monitoring a receptor activating NF-B-mediated activity in patient samples, said kit comprising a soluble NF-B activator receptor according to any of claims 13-15, wherein said detection or monitoring employs an assay selected from the group consisting of ELISA, point transfer assay, solid phase binding assay (such as those using a biosensor), a rapid format test and a bioassay.