AU748167B2 - Novel nucleic acid and polypeptide - Google Patents

Description

WO 99/33967 PCT/US98/27688 NOVEL NUCLEIC ACID AND POLYPEPTIDE Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

INTRODUCTION

The field of this invention is polypeptide molecules which regulate cell 1 o function, nucleic acid sequences encoding the polypeptides, and methods of using the nucleic acid sequences and the polypeptides. The present invention provides for novel receptor molecules, their use and assay systems useful for identifying novel ligands that interact with these receptors.

BACKGROUND OF THE INVENTION The tumor necrosis factor receptor (TNFR) superfamily consists mostly of transmembrane proteins that elicit signal transduction in a variety of cells. Tumor necrosis factor-alpha (TNF-alpha) is a cytokine primarily produced by activated macrophages. TNF-alpha stimulates T-cell and B-cell proliferation and induces expression of adhesion molecules on endothelial cells. This cytokine also plays an important role in host defense to infection.

TNF-alpha activities are mediated through two distinct receptors, and TNFR-p75. These two receptors also mediate activities triggered by soluble lymphotoxin-alpha (LT-alpha) secreted mainly by 1 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 activated lymphocytes. Specific stimulation of TNFR-p55 induces TNF activities such as in vitro tumor cell cytotoxicity, expression of adhesion molecules on endothelial cells and keratinocytes, activation of sphingomyelinase with concomitant increases of ceramide, activation of NF-kappaB and induction of manganese superoxide dismutase mRNA. Specific stimulation of TNFR-p75 results in a proliferative response of mouse and human thymocytes and cytotoxic Tcells, fibroblasts and natural killer cells and in GM-CSF secretion in cells.

The identification of a new member of the TNFR superfamily that regulates bone resorption was recently reported. The newly identified protein was termed Osteoprotegerin (OPG) and was postulated to act as a humoral regulator of bone resorption by blocking the differentiation of osteoclasts, the cells responsible for bone resorption. (Simonet, et al., 1997, Cell 89:309-319; International Publication Number WO 97/23614 published 3 July 1997 in the name of Amgen, Inc.).

However, relatively little is known about the soluble factors that act physiologically to regulate osteoclast development.

Novel receptor molecules are often identified and isolated by searching for additional members of known families of receptors using, for example, PCR-based screens or computer searches of EST databases involving known regions of homology among the family members. (See, for example, Maisonpierre, et al., 1993, Oncogene 8:1631-1637).

Isolation of such so called "orphan" receptors, for which no ligand is known, and subsequent determination of the tissues in which such receptors are expressed, provides insight into the regulation of the 2 SUBSTITUTE SHEET (Rule 26) I \s.

growth, proliferation and regeneration of cells in target tissues. Further, such receptors may be used to isolate their cognate ligands, which may then be used to regulate the survival, growth and regeneration of cells expressing the receptor. Alternatively, in the case of soluble receptors (extracellular domain only), the receptor itself can behave as a ligand.

SUMMARY OF THE INVENTION The present invention is based on the identification of a novel mammalian receptor termed NTR-5. Specifically, a novel human receptor termed HUMAN NTR-5 and a novel mouse receptor termed MOUSE NTR-5 have been identified. Throughout this 10 description, reference to MAMMALIAN NTR-5 includes, but is not limited to, the *specific embodiments of HUMAN NTR-5 and MOUSE NTR-5 as described herein.

These receptors are related to osteoprotegerin (OPG) and to tumor necrosis factor receptor (TNPR). Based upon its homology to osteoprotegerin, it is expected that NTR-5 will be involved in the regulation of bone mass, and may be useful for regulating 15 development, proliferation and death of osteoblast or osteodclast cells or for regulating muscle metabolism and may be implicated in diseases or disorders of muscle.

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*The present invention provides an isolated nucleic acid molecule having a sequence selected from the group consisting of: the nudcleotide sequence comprising the coding region of the HUMAN NTR-5 as set forth in SEQ. NO. 3; a nudeotide sequence that hybridizes under stringent conditions to the nudcleotide sequence of and which encodes a molecule which regulates bone mass or muscle metabolism; or a nudeotide sequence which, but for the degeneracy of the genetic code would hybridize to a nudeotide sequence of or and which encodes a molecule which regulates bone mass or muscle metabolism.

The present invention also provides an isolated NTR-5 polypeptide encoded by a nucleic acid of the invention. In a specific embodiment the invention rdelates to an isolated polypeptide having the amino adcid sequence set forth in SEQ. ID No. 4.

The present invention also provides for a polypeptide that comprises the extracellular domain of an NTR-5 polypeptide of the invention fused to an immunoglobulin constant or Fc region as well as a nucleic acd which encodes such a polypeptide.

The invention further provides for vectors comprising an isolated nucleic acid molecule of the invention and host-vector systems for the production of a polypeptide of the invention comprising such a vector in a host cell. Suitable host cell include bacterial, yeast, insect or mammalian cells, preferably COS or CHO cells.

The invention also provides a method of producing NTR-5 polypeptide which comprises growing cells of a host-vector system of the invention, under conditions 10 permitting the production of the polypeptide and recovering the NTR-5 polypeptide so produced.

NTR-5 polypeptides comprising the extracellular and/or intracellular domain may be used in screening for drugs that interact with MAMMALIAN NTR-5. Novel agents that bind to the polypeptides described herein may mediate survival and differentiation 15 in cells naturally expressing polypeptides, but also may confer survival and proliferation when used to treat cells engineered to express the polypeptides. In particular embodiments, the extracellular domain of MAMMALIAN NTR-5 is utilized in screens for cognate ligands.

Preferred uses for the subject MAMMALIAN NTR-5 polypeptides include screening for agents that bind to the polypeptides. The agents may be biologically active agents (agonists), which activate the MAMMALIAN NTR-5 polypeptides or they may bind and block activation of the polypeptides (antagonists). Screening methods include incubating a MAMMALIAN NTR-5 polypeptide in the presence of a MAMMALIAN polypeptide-specific binding target and a candidate agent under conditions whereby, but for the presence of the agent, the polypeptide specifically binds the binding target at a reference affinity; detecting the binding affinity of the polypeptide to the binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the binding of the polypeptide to the binding target.

The invention further provides for antibodies which specifically bind the polypeptides of the invention.

The present invention also has diagnostic and therapeutic utilities. In particular, methods of detecting aberrancies in the function or expression of the polypeptides described herein may be used in the diagnosis of disorders. Manipulation of the polypeptides or agonists or antagonists which bind the polypeptides may be used in the treatment of diseases. The extracellular domain of the polypeptide may also be utilized as an agent to block the binding of a binding agent to its target.

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10 The present invention therefore provides pharmaceutical compositions comprising a polypeptide or an antibody of the invention and a carrier.

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DETAILED DESCRIPTION OF THE INVENTION The invention provides MAMIMALIAN NTR-5 polypeptides which include isolated MAMNALIAN NTR-5 polypeptides and recombinant polypeptides comprising a MAMMALIAN NTR-5 amino acid sequence, or a functional MAMMALIAN polypeptide domain thereof having an assay-discernable MAMMALIAN activity. Accordingly, the polypeptides may be deletion mutants of the disclosed MAMMALIAN NTR-5 polypeptide and may be provided as fusion products, with non-MAMMALIAN NTR-5 polypeptides. The subject MAMMALIAN polypeptides have MAMMALIAN NTR-5-specific activity or function.

i 10 A number of applications for MAMMALIAN NTR-5 polypeptides are suggested from their properties. MAMMALIAN NTR-5 polypeptides may be useful in the study and treatment of conditions similar to those which are treated using TNF. Furthermore, the MAMMALIAN NTR-5 cDNA may be useful as a diagnostic tool, such as through the use of oligonucleotides as primers in a PCR test to amplify those sequences having 15 similarities to the oligonucleotide primer, and to see how much MAMMALIAN mRNA is present in a particular tissue or sample. The isolation of MAMMALIAN NTR-5, of course, also provides the key to isolate its putative ligand, other MAMMALIAN NTR-5 binding polypeptides, and/or study its properties.

WO 99/33967 PCT/US98/27688 MAMMALIAN NTR-5-specific activity or function may be determined by convenient in vitro, cell based or in vivo assays. In vitro or cell based assays include but are not limited to binding assays and cell culture assays. In vivo assays include but are not limited to immune response, gene therapy and transgenic animals. Binding assays encompass any assay where the specific molecular interaction of a MAMMALIAN polypeptide with a binding target is evaluated. The binding target may be a natural binding target, or a nonnatural binding target such as a specific immune polypeptide such as an antibody, or a MAMMALIAN NTR-5-specific binding agent.

The claimed MAMMALIAN NTR-5 polypeptides may be isolated or pure an "isolated" polypeptide is one that is no longer accompanied by some of the material with which it is associated in its natural state, and 1 5 that preferably constitutes at least about and more preferably at least about 5% by weight of the total polypeptide in a given sample; a "pure" polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample.

The subject polypeptides may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see Molecular Cloning, A Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-lnterscience, NY).

7 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 The subject polypeptides find a wide variety of uses including but not limited to use as immunogens, targets in screening assays, bioactive reagents for modulating cell growth, differentiation and/or function.

For example, the invention provides methods for modifying the physiology of a cell comprising contacting the extracellular surface of the cell or medium surrounding the cell with an exogenous MAMMALIAN polypeptide under conditions whereby the added polypeptide specifically interacts with a component of the medium and/or the extracellular surface to effect a change in the physiology of the cell.

1 According to these methods, the extracellular surface includes plasma membrane-associated molecules. The term "exogenous MAMMALIAN polypeptide" refers to polypeptides not made by the cell or, if so, expressed at non-natural levels, times or physiologic locales.

Media, include, but not limited to, in vitro culture media and/or physiological fluids such as blood, synovial fluid and lymph. The polypeptides may be introduced, expressed, or repressed in specific populations of cells by any convenient way, including but not limited to, microinjection, promoter-specific expression of recombinant protein or targeted delivery of lipid vesicles.

The invention provides MAMMALIAN NTR-5-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. MAMMALIAN binding agents include MAMMALIAN antibodies (See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and also includes other binding agents identified with assays such as one-, twoand three-hybrid screens, and non-natural binding agents identified in 8 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCTIUS98/27688 screens of chemical libraries such as described below. Agents of particular interest modulate MAMMALIAN NTR-5 polypeptide function.

The invention provides MAMMALIAN NTR-5 nucleic acids, which find a wide variety of applications, including but not limited to, use as translatable transcripts, hybridization probes, PCR primers, or diagnostic nucleic acids, as well as use in detecting the presence of MAMMALIAN NTR-5 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional MAMMALIAN homologs and structural analogs.

The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, no longer accompanied by some of the material with which it is associated in its natural state, preferably constituting at least about more preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to a nucleotide(s) other than that to which it is joined on a natural chromosome. Nucleic acids comprising the nucleotide sequence disclosed herein and fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that to which it is joined on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is immediately flanked by a sequence other than that to which it is joined on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide, example, modified stability.

9 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 The sequence of the disclosed MAMMALIAN NTR-5 nucleic acid is used to obtain the deduced MAMMALIAN NTR-5 polypeptide sequence. Further, the sequence of the disclosed MAMMALIAN NTR-5 nucleic acid is optimized for selected expression systems (Holler, et al., (1993) Gene 136:323-328; Martin, et al., (1995) Gene 154:150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural MAMMALIAN NTR-5 encoding nucleic acid sequences ("GCG" software, Genetics Computer Group, Inc., Madison, WI).

MAMMALIAN NTR-5 encoding nucleic acids may be part of expression vectors and may be incorporated into recombinant host cells, for expression and screening, for transgenic animals, or for functional studies such as the efficacy of candidate drugs for diseases associated with MAMMALIAN NTR-5 polypeptide-mediated signal transduction.

Expression systems are selected and/or tailored to effect MAMMALIAN polypeptide structural and functional variants through alternative post-translational processing.

The invention also provides for nucleic acid hybridization probes and replication/amplification primers having a MAMMALIAN NTR-5 cDNA specific sequence and sufficient to effect specific hybridization with SEQ. NO. 1 or SEQ. NO. 3. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5x SSPE (0.18 M NaCI, 0.01 M NaPO 4 pH 7.7, 0.001 M EDTA) buffer at a temperature of 420C and remaining bound when subject to washing at 420C with 0.2x SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5x SSPE buffer at a temperature of 420C and remaining bound when subject to washing at SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 420C with 0.2x SSPE buffer at 420C. MAMMALIAN NTR-5 cDNA homologs can also be distinguished from one another using alignment algorithms, such as BLASTX (Altschul, et al., (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215:403-410).

MAMMALIAN NTR-5 hybridization probes find use in identifying wild-type and mutant alleles in clinical and laboratory samples.

Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. MAMMALIAN 1o NTR-5 nucleic acids are also used to modulate cellular expression or intracellular concentration or availability of active MAMMALIAN polypeptides. MAMMALIAN NTR-5 inhibitory nucleic acids are typically antisense- single stranded sequences comprising complements of the disclosed MAMMALIAN NTR-5 coding sequences. Antisense modulation of the expression of a given MAMMALIAN NTR-5 polypeptide may employ antisense nucleic acids operably linked to gene regulatory sequences.

Cells are transfected with a vector comprising a MAMMALIAN sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous MAMMALIAN NTR-5 encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration.

Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a given MAMMALIAN NTR-5 polypeptide may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted polypeptide. An enhancement in MAMMALIAN expression is effected by introducing into the targeted cell type 11 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCTIUS98/27688 MAMMALIAN NTR-5 nucleic acids which increase the functional expression of the corresponding gene products. Such nucleic acids may be MAMMALIAN NTR-5 expression vectors, vectors which upregulate the functional expression of an endogenous allele, or replacement vectors for targeted correction of mutant alleles. Techniques for introducing the nucleic acids into viable cells are known in the art and include, but are not limited to, retroviral-based transfection or viral coat protein-liposome mediated transfection.

1 0 The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of MAMMALIAN NTR-5 modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate the interaction of MAMMALIAN NTR-5 with a natural MAMMALIAN 1 5 binding target. A wide variety of assays for binding agents are provided including, but not limited to, protein-protein binding assays, immunoassays, or cell based assays. Preferred methods are amenable to automated, cost-effective, high throughput screening of chemical libraries for lead compounds.

In vitro binding assays employ a mixture of components including a MAMMALIAN NTR-5 polypeptide, which may be part of a fusion product with another peptide or polypeptide, a tag for detection or anchoring. The assay mixtures comprise a natural MAMMALIAN binding target. While native binding targets may be used, it is frequently preferred to use portions thereof as long as the portion provides binding affinity and avidity to the subject MAMMALIAN conveniently measurable in the assay. The assay mixture also 12 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds, preferably small organic compounds, and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents such as salts, buffers, neutral proteins, albumin, detergents, protease inhibitors, nuclease inhibitors, or antimicrobial agents may also be included. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. The mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the MAMMALIAN NTR-5 polypeptide specifically binds the binding target, portion or analog with a reference binding affinity. Incubation periods are chosen for optimal binding but are also minimized to facilitate rapid, high throughput screening.

After incubation, the agent-biased binding between the MAMMALIAN polypeptide and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may be effected by any number of methods that include, but are not limited to, precipitation or immobilization followed by washing by, e.g., membrane filtration or gel chromatography. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, or indirect detection such as an epitope tag or an enzyme. A variety of methods may be used to detect the label depending on the nature of the label and other assay 13 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 components, including but not limited to, through optical or electron density, radiative emissions, nonradiative energy transfers, or indirectly detected with, as a nonlimiting example, antibody conjugates. A difference in the binding affinity of the MAMMALIAN NTR-5 polypeptide to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the MAMMALIAN polypeptide to the corresponding binding target. A difference, as used herein, is statistically significant and preferably represents at 1o least a 50%, more preferably at least a 90% difference.

The invention provides for a method for modifying the physiology of a cell comprising an extracellular surface in contact with a medium, said method comprising the step of contacting said medium with an exogenous MAMMALIAN NTR-5 polypeptide under conditions whereby said polypeptide specifically interacts with at least one of the components of said medium to effect a change in the physiology of said cell.

The invention further provides for a method for screening for biologically active agents, said method comprising the steps of a) incubating a MAMMALIAN NTR-5 polypeptide in the presence of a MAMMALIAN NTR-5 polypeptide-specific binding target and a candidate agent, under conditions whereby, but for the presence of said agent, said polypeptide specifically binds said binding target at a reference affinity; b) detecting the binding affinity of said polypeptide to said binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity 14 SUBSTITUTE SHEET (Rule 26) indicates that said agent modulates the binding of said polypeptide to said binding target.

The present invention also provides for antibodies to the MAMMALIAN polypeptides described herein which are useful for detection of the polypeptides in, for example, diagnostic applications. For preparation of monoclonal antibodies directed toward MAMMALIAN NTR-5 polypeptides, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, *Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma 10 technique to produce human 0 *9 P e o .l Ooo*.

*p l WO 99/33967 PCT/US98/27688 monoclonal antibodies (Cole et al., 1985, in "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc. pp. 77-96) and the like are within the scope of the present invention.

The monoclonal antibodies for diagnostic or therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol.

92:3-16). Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).

Various procedures known in the art may be used for the production of polyclonal antibodies to the MAMMALIAN NTR-5 polypeptides described herein. For the production of antibody, various host animals can be immunized by injection with the MAMMALIAN NTR-5 polypeptides, or fragments or derivatives thereof, including but not limited to rabbits, mice and rats. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

16 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 A molecular clone of an antibody to a selected MAMMALIAN polypeptide epitope can be prepared by known techniques. Recombinant DNA methodology (see Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.

The present invention provides for antibody molecules as well as fragments of such antibody molecules. Antibody fragments which lo contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Antibody molecules may be purified by known techniques including, but not limited to, immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.

The following example is offered by way of illustration and not by way of limitation.

EXAMPLE I CLONING AND SEQUENCING OF THE HUMAN NTR-5 GENE Amino acid sequences of known human and mouse members of the TNF family were used as tblastn queries to search the NIH EST database of 17 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 random fragments of mRNA sequences (Altschul et al., (1990), Basic local alignment search tool, J. Mol. Biol. 215:403-10). Each query generated a list of hits, i.e. EST sequences with a substantial sequence similarity to the query sequence. Typically, the hits on top of the list corresponded to mRNA copies of the query protein, followed by ESTs derived from other members of the family and random-chance similarities.

A parser program was used to combine and sort all the hits from 1 o searches with all the members of the family. This allowed rapid subtraction of all the hits corresponding to known proteins. The remaining hits were analyzed for conservation of sequence motifs characteristic for the family. Additional database searches were performed to identify overlapping ESTs. Two mouse cDNA clones from the I.M.A.G.E. consortium were discerned to contain homologous sequence. Clone 472300 (the '300 clone) (GeneBank Accession No.

AA036247) and clone #427152 (the '152 clone) (GeneBank Accession No. AA003356) were obtained from Research Genetics, Inc. (Huntsville, AL) and sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City,

CA).

Both the '300 clone and the '152 clone contained a single coding frame encoding a 215 amino acid protein (SEQ. NO. 2) designated MOUSE as set forth below. MOUSE NTR-5 revealed sequence similarity to members of the TNF receptor family.

18 SUBSTITUTE SHEET (Rule 26) WO 99/33967 WO 9933967PCTIUS98/27688 20 30 40 so SEQ. NO. 1 -CT GGC ATG GAG TG TCC AAG GAA TOT GOC TMC GGC TAT GGG GAG OAT OCA CAG TOT OTO SEQ. NO. 2 Gly Met Giu Leu Ser Lys Giu Cys Oly Phe Gly Ty'r Gly Giu Asp Ala Gin Cys Val> 80 90 100 110 120 CCC TGC AGO CCG Pro Cys'Arg Pro CAC CGG TIC AAG GAA His Arg Phe Lys Giu GAC TOG Asp Trp GWT TM' CAG AAG WT MG CCA TOT GCG Gly Phe Gin Lys Cys Lys Pro Cys Ala> 160 GAC =O GCG =r OTOG AC C= =T CAG ADO WCC MAC TGC TCA CAC ACC AGT GAT GCT WTC Asp Cys Ala Leu Val Asn Arg Phe Gin Arg Ala Asn Cys Ser His Thr Ser Asp Ala Val> :0 230 240 =O GOG GAC TOC C= CCA OGA 7MT TAC CG MAG ACC AMA C= =1 G= TM' CAA GAC ATO Cys Gly Asp Cys Leu Pro Gly Phe Tyr Arg Lys Thr Lys Leu Val Gly Phe Gin Asp Met> 250 GAG TOT GTO CCC Giu Cys Val Pro 260 270 280 290 300 T1C OGA GAC CCA CCT Cys Gly Asp Pro Pro CC =CC TAC GMA CCA CAC TOT AMC ADC MUG Pro Pro Tyr Glu Pro His Cys Thr Ser Lys Val> 310 320 330 340 350 360 MAC Crr GMO AAO ATr TCC TCC ACC TCTCC AGC CCT CGG GAC ACG 0CG CT0 OCT GCC GM Asn Leu Val Lys Ile Ser Ser Thr Val Ser Ser Pro Arg Asp Thr Ala Leu Ala Ala Val> 370 380 390 400 410 420 ATC TOC A=T OCT CTG GCC AMO =P Cr Cr MC le Cys Ser Ala Leu Ala Thr Val Leu Leu Ala =r CTC ATC CrM TOT ATC TAC TGC toeu Leu Ile Leu Cys Val Ile Tyr Cys-> 430 440 450 460 MAG AGO CAG TTC ATO GAG AAD MAA CCC AGC TOT MAG CTC CCA TOCC CTC TO;T Cit ACT GTr0 Lys Arg Gin Phe Met Oiu Lys Lys Pro Ser Cys Lys Leu Pro Ser Leu Cys Leu Thr Val> 490 500 510 520 530 MAG MA OCT TOT TAG CAT TOT CAC CCA AGA OTT Cr1C MAG ACA CCT 0CC TOA GAC CrA AG L~YS The MOUSE NTR-5 cDNA as set forth in SEQ. NO. 1 above was used as a probe to 'screen Northern blots of human mRNA derived from heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.

Detectable expression levels of HUMAN NTR-5 were present in heart and 19 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 kidney. A human heart cDNA library (Stratagene Cat. 936208) was plated in six 243 mm x 243 mm plates at 2.5 x 105 plaques per plate, transferred to nitrocellulose using standard techniques known in the art, and probed with the MOUSE NTR-5 probe. The nitrocellulose filters were prehybridized for 30 minutes at 650C in 155 ml of hybridization buffer containing 0.5 M NaPO 4 (pH 1% bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA and 100 ng/ml sonicated, denatured salmon sperm DNA. The 32 P-ATP-radiolabeled MOUSE NTR-5 probe was added to the prehybridization mixture and hybridization was carried out 1 0 at 600C overnight. The filters were washed with 2x SSC several times at room temperature and for 30 minutes at 600C and exposed to x-ray film overnight at -800C. Two positive clones were obtained based on their ability to cross-hybridize with the MOUSE NTR-5 probe. One of these positive clones, human clone was amplified by PCR using 1 5 oligonucleotides specific to the T7/T3 vector components and the resultant PCR-amplified DNA fragment was sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The human clone #2 sequence contained an open reading frame encoding a 328 amino acid protein with sequence similarity to other members of the TNF receptor family, including features characteristic of membrane attached proteins predictive of a signal peptide sequence at amino acids 1-29, an extracellular domain at amino acids 30-169, a transmembrane motif at amino acids 170-193, and an intracellular domain at amino acids 194-328 (see SEQ. NO. 4).

SUBSTITUTE SHEET (Rule 26) WO 99/33967 WO 9933967PCTIUS98/27688 The nucleotide and deduced amino acid sequence of human clone #2 is set forth below.

SEQ. NO. 3 AMG GCr TrA MAA OTG CTA CTA GAA CAA GAG AAA ACG MI' T1'C ACT CTI' ?IA GTA TrA CrA SEQ. NO. 4 Met Ala Leu Lys Val Leu Leu Glu Gin Glu Lys Thr Phe Phe Thr Leu Leu Val Leu Leu> 80 90 100 110 120 GGC TAT 7MO TCA TGT AAA GTG ACT iWi OAA TCA GGA GAC TGT AGA CAG CAA OAA TTC AGG Gly Tyr Leu Ser Cys Lys Val Thr Cys Glu Ser Gly Asp Cys Arg Gin Gin Giu Phe Arg> 130 140 150 160 170 180 OAT CGG TCT 00k AAC TOT OTr CCC TGC MAC CAG TOT G CCA GGC ATG GAG ?1'G TCT AAG Asp Arg Ser Gly Asn Cys Val Pro Cys Asn Gin Cys Oly Pro Oly Met Glu Leu Ser Lys> 190 200 210 220 230 240 GAA TGT GGC TTC GGC TAT G GAG GAT GCA CAG TOT GTG ACG TGC COG CTG CAC AGG TTC Giu Cys Gly Phe Gly Tyr Gly Olu Asp Ala Gin Cys Val Thr Cys Arg Leu His Arg Phe> 250 MAG GAG GAC TOGG GGC Lys Giu Asp Trp, Oly 260 270 280 290 300 T1'C CAG MAA TOGC MhG CCC 7W CTG GAC TOC OCA GTro GTG MC CGC Phe Gin Lys Cys Lys Pro Cys Leu Asp Cys Ala Val Val Asn Arg> CAG MAG GCA MAT TOT TCA 0CC ACC ADT OAT GCC ATC TC =0G GAC =O TM' CCA 00k Phe Gin Lys Ala Asn Cys Ser Ala Thr Ser Asp Ala Ile Cys Giy Asp Cys Leu Pro Gly> 370 380 390 400 410 420 TTTAT ADO MAG ACO AMA CI'? GC GOC TI'? CMA GAC ATG GAG TOT G?0 CCT TO-T G0k GAC Phe Tyr Arg Lys Thr Lys Leu Val Gly Phe Gin Asp Met Oiu Cys Val Pro Cys Gly Asp> 430 440 450 460 470 480 CCT CCT CCT CCT TAC GMA CCG CAC TOT 0CC AGC AAO OTC MAC CTC O1'O MOG ATC OCO TCC Pro Pro Pro Pro Tyr Giu Pro His Cys Ala Ser Lys Vai Asn Leu Val Lys Ile Ala Ser> 490 500 510 520 530 540 ACG 0CC TCC AGC CCA CGG GAD ACO 000 CM0 OCT 0CC OTr ATC TOC ADC OCT CTG 0CC ACC Thr Ala Ser Ser Pro Arg Asp Thr Ala Leu Ala Ala Val Ile Cys Ser Ala Leu Ala Thr> 550 560 570 580 590 600 GTC CTG Cr0 0CC CT0 CrC ATC CTC TOT GTC ATC TAT TGT MAG AGA CAD TI'? ATO GAG MAG Val Leu Leu Ala Leu Leu Ile Leu Cys Val Ile Tyr Cys Lys Arg Gin Phe Met Giu Lys> 610 620 630 640 650 660 MAA CCC ADC TOG TCT cro COG 7TOO CAD CAC Al'? CAG TAC AAC 0CC TOT GAO Cr0 TCO TGI' Lys Pro Ser Trp, Ser Leu Arg Ser Gin Asp Ile Gln Tyr Asri Oly Ser 01u Leu Ser Cys> 21 SUBSTITUTE SHEET (Rule 26) WO 99/33967 PCT/US98/27688 1 5 670 TTT GAC AGA CCT CAG Phe Asp Arg Pro Gin 730 TCA GTG CAG ACC TGC Ser Val Gin Thr Cys 790 AGC CCC AAC CCG GCG Ser Pro Asn Pro Ala 850 AAG CTT AAA GAA CCT Lys Leu Lys Glu Pro 910 CCT TTG TTA GGC TTA Pro Leu Leu Gly Leu 970 680 CTC CAC GAA Leu His Glu 690 TAT GCC CAC Tyr Ala His 700 710 AGA GCC TGC TGC CAG TGC CGC Arg Ala Cys Cys Gin Cys Arg GGG CCG Gly Pro 800 ACT CTT Thr Leu 860 GCT TCT Ala Ser 920 TGG ACT Trp Thr 980 750 GTG CGC TIG CTC CCA Val Arg Leu Leu Pro 810 GGT TGT GGG GTG CAT Gly Cys Gly Val His 870 TTC TGC AGT AGA AGC Phe Cys Ser Arg Ser 930 760 TCC ATG Ser Met 820 TCT GCA Ser Ala 880 GTG TGC Val Cys 940 770 780 TGC TGT GAG GAG GCC TGC Cys Cys Glu Glu Ala Cys> 830 840 GCC AGT CTT CAG GCA AGG Ala Ser Leu Gin Ala Arg> 890 900 TGG AAC CCA AAG AGT ACT Trp Asn Pro Lys Ser Thr> 950 960 720 CGT GAC Arg Asp> GAG CAG TCT GGA CCT TGC ATG GCT TCT GGG GCA AAA ATA Glu Gin Ser Gly Pro Cys Met Ala Ser Gly Ala Lys Ile> AAT CTG AAC CAA ACT GAC GGC ATT TGA Asn Leu Asn Gln Thr Asp Gly Ile Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

22 SUBSTITUTE SHEET (Rule 26) Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

C,

22a

Claims (21)

1. An isolated nucleic acid molecule having a sequence selected from the group consisting of: the nucleotide sequence comprising the coding region of the HUMAN NTR-5 as set forth in SEQ. NO. 3; a nudeotide sequence that hybridizes under stringent conditions to the nucleotide sequence of and which encodes a molecule which regulates bone mass or muscle metabolism; or a nudeotide sequence which, but for the degeneracy of the genetic code would 10 hybridize to a nudeotide sequence of or and which encodes a molecule ,which regulates bone mass or muscle metabolism.

2. A vector which comprises a nucleic acid molecule of claim 1.

3. A vector according to claim 2, wherein the nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a 15 host cell. Sgo•

4. An isolated NTR-5 polypeptide encoded by a nucleic acid molecule of claim 1. Isolated HUMAN NTR-5 polypeptide having the amino add sequence set forth in SEQ. ID No. 4.

6. A host-vector system for the production of a polypeptide according to claim 4 or 5 which comprises a vector of claim 2 or 3, in a host cell.

7. A host-vector system according to claim 6, wherein the host cell is a bacterial, yeast, insect or mammalian cell.

8. A method of producing NTR-5 polypeptide which comprises growing cells of a host-vector system of claim 6 or 7, under conditions permitting the production of the polypeptide and recovering the NTR-5 polypeptide so produced.

9. A polypeptide produced by the method of claim 8. An antibody, which specifically binds a polypeptide of claim 4 or

11. An antibody according to claim 10, which is a monoclonal antibody.

12. A pharmaceutical composition comprising a polypeptide according to any one of claims 4, 5 or 9 and a carrier. o*

13. A pharmaceutical composition comprising an antibody according to claim 10 or 11, and a carrier. a.

14. A polypeptide according to any one of claims 4, 5 or 9 for use in a method of treatment of a human or animal body or in a method of diagnosis.

15. An antibody according to claim 10 or 11 for use in a method of treatment of a human or animal body or in a method of diagnosis.

16. A polypeptide comprising the extracellular portion of an NTR-5 polypeptide according to any one of claims 4, 5 or 9 fused to an immunoglobulin constant region.

17. A polypeptide according to claim 16, wherein the constant region is the human immunoglobulin gamma-1 constant region.

18. A polypeptide comprising the extracellular portion of an NTR-5 polypeptide according to claims 4, 5 or 9 fused to an immunoglobulin Fc region.

19. A polypeptide according to claim 18, wherein the Fc region is the human immunoglobulin gamma-1 Fc region. A nucleic acid encoding a polypeptide according to any one of claims 16 to 19.

21. A nucleic acid according to claim 1, which is substantially as hereinbefore described with reference to the Examples

22. A vector according to claim 2, which is substantially as hereinbefore described with reference to the Examples.

23. A polypeptide according to any one of claims 4, 5, 16 or 18 which is substantially as hereinbefore described with reference to the Examples.

24. A host-vector system according to claim 7, which is substantially as hereinbefore described with reference to the Examples. S SDATED: 28 March, 2002 PHILLIPS ORMONDE FITZPATRICK Attorneys for: REGENERON PHARMACEUTICALS, INC. a.