CA2299619A1 - Human orphan receptor ntr-1 - Google Patents

Abstract

The present invention provides for a nucleic acid sequence, designated as HUMAN NTR-1, that encodes a novel orphan receptor expressed in pancreas and fetal heart. The invention also provides assay systems that may be used to detect and/or measure ligands that bind the HUMAN NTR-1 gene product. The present invention also provides for diagnostic and therapeutic methods based on the interaction between NTR-1 and agents that initiate signal transduction through binding to NTR-1.

Description

WO 99/0773$ PCT/US98/16202 NOVEL OR~,'HAN RECEPTOR
This International Application claims priority of U.S. Provisional Application Serial No. 60/054,869 filed August 6, 1997. 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 function, nucleic acid sequences encoding the polypeptides, and methods of using the nucleic acid sequences and the polypeptides. The present invention provides for novel orphan receptor molecules, their use and assay systems useful for identifying novel ligands that interact with these receptors.
BACKGROUND OF THE INVENTION
2o 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.
TNT-alpha activities are mediated through two distinct receptors, TNFR-p55 and TNFR-p75. These two receptors also mediate activities triggered by soluble lymphotoxin-alpha (LT-alpha) secreted mainly by 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 proliferative response of mouse and human thymocytes and cytotoxic T cells, fibroblasts and natural killer cells and in GM-CSF
secretion in PC60 cells.
The identification of a new member of the TNFR superfamily that regulates to 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, W.S., 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 2o 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 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, the receptor itself can behave as a ligand.

SUMMARY OF THE INVENTION
The present invention provides for a novel orphan human receptor, termed HUMAN NTR-1, which is expressed in the pancreas, skeletal muscle, and fetal and adult hearts. The protein is related to osteoprotegerin (OPG) and to tumor necrosis factor receptor (TNFR). The present invention further provides for an isolated nucleic acid molecule encoding HUMAN
NTR-1. Based upon its homology to osteoprotegerin, it is expected that HUMAN NTR-1 will be involved in the regulation of bone mass, and may 1o be useful for regulating development, proliferation and death of osteoblast or osteoclast cells or for regulating muscle metabolism and may be implicated in diseases or disorders of muscle.
The present invention also provides for a protein or polypeptide that comprises the extracellular domain of HUMAN NTR-2 as well as the nucleic acid which encodes such extracellular domain. The invention further provides for vectors comprising an isolated nucleic acid molecule encoding HUMAN NTR-1 or its extracellular domain, which can be used to express HUMAN NTR-1 or its extracellular domain in bacteria, yeast, insect or mammalian cells, preferably COS or CHO cells.
The invention further provides for use of the HUMAN NTR-1 receptor or its extracellular or intracellular domain in screening for drugs that interact with HUMAN NTR-1. Novel agents that bind to the receptors) described herein may mediate survival and differentiation in cells naturally expressing the receptor, but also may confer survival and proliferation when used to treat cells engineered to express the receptor. In particular embodiments, the extracellular domain (soluble receptor) of HUMAN NTR-1 is utilized in screens for cognate ligands.

Preferred uses for the subject HUMAN NTR-1 polypeptides include screening for agents that bind to the receptor polypeptides. The agents may be biologically active agents (agonists), which activate the HUMAN NTR-1 receptor or they may bind and block activation of the receptor (antagonists).
Screening methods include incubating a HUMAN NTR-1 polypeptide in the presence of an extracellular HUMAN NTR-1 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 to 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 also provides for a nucleic acid probe capable of hybridizing i5 with a sequence included within the nucleic acid sequence encoding HUMAN NTR-1 useful for the detection of NTR-1 expressing tissue in humans and animals.
The invention further provides for antibodies directed to HUMAN NTR-1.
The present invention also has diagnostic and therapeutic utilities. In particular embodiments of the invention, methods of detecting aberrancies in the function or expression of the receptor described herein may be used in the diagnosis of disorders. In other embodiments, manipulation of the receptor or agonists or antagonists which bind this receptor may be used in the treatment of diseases. In further embodiments, the extracellular domain of the receptor is utilized as a blocking agent which blocks the binding of receptor to its target.

In a further embodiment of the invention, patients who suffer from an excess of NTR-1 may be treated by administering an effective amount of anti-sense RNA or anti-sense oligodeoxyribonucleotides corresponding to the HUMAN NTR-1 gene coding region, thereby decreasing expression of NTR-1.
DETAILED DESCRIPTIOI~OF THE INVENTION
The invention provides HUMAN NTR-1 polypeptide which includes isolated HUMAN NTR-1 polypeptide and recombinant polypeptides comprising a HUMAN NTR-1 amino acid sequence, or a functional HUMAN NTR-1 polypeptide domain thereof having an assay-discernable HUMAN NTR-1-specific activity. Accordingly, the polypeptides may be deletion mutants of the disclosed HUMAN NTR-1 polypeptides and may be provided as fusion products, e.g., with non - HUMAN NTR-1 polypeptides.
The subject HUMAN NTR-1 polypeptide domains have HUMAN NTR-1-specific activity or function.
A number of applications for HUMAN NTR-1 are suggested from its 2o properties. HUMAN NTR-1, may be useful in the study and treatment of conditions similar to those which are treated using TNF. Furthermore, the HUMAN NTR-1 cDNA may be useful as a diagnostic tool, such as through use of antibodies in assays for polypeptides in cell lines or use of oligonucleotides as primers in a PCR test to amplify those with sequence similarities to the oligonucleotide primer, and to see how much HUMAN
NTR-1 is present. The isolation of HUMAN NTR-1, of course, also provides the-key to isolate its putative ligand, other HUMAN NTR-1 binding polypeptides, and/or study its properties.

HUMAN NTR-1-specific activity or function may be determined by convenient in vi r , cell based, or inin vivo assays - e.g., 'n vi binding assays, cell culture assays, in animals (e.g., immune response, gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the specific molecular interaction of a HUMAN NTR-I polypeptide with a binding target is evaluated. The binding target may be a natural binding target, or a non-natural binding target such as a specific immune polypeptide such as an antibody, or a HUMAN NTR-1 specific agent such as those identified in assays described below.
to The claimed 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 that preferably constitutes at least about 0.5%, and more preferably at least about 5% by weight of the total 25 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 and polypeptide domains may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods 2o are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g., Molecular Cloning, A
Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ.
Assoc., Wiley-Interscience, NY).
The subject polypepddes find a wide variety of uses including use as immunogens, targets in screening assays, bioactive reagents for modulating cell growth, differentiation and/or function, etc. For example, the invention provides methods for modifying the physiology of a cell 3o comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous HUMAN NTR-1 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. According to these methods, the extracellular surface includes plasma membrane-associated receptors; the exogenous HUMAN NTR-1 refers to a polypeptide not made by the cell or, if so, expressed at non-natural levels, times or physiologic locales; and suitable media include in vitro culture media and physiological fluids such as blood, synovial fluid, etc. The polypeptides may be may be introduced, expressed, or repressed in specific populations of cells by any convenient way such as microinjection, promoter-specific expression of recombinant enzyme, targeted delivery of lipid vesicles, etc.
The invention provides HUMAN NTR-1-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. HUMAN NTR-1-specific binding agents include HUMAN NTR-1-specific receptors, such as somatically recombined protein receptors like specific antibodies or T-cell antigen receptors (See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring 2o Harbor Laboratory) and also includes other binding agents identified with assays such as one-, two- and three-hybrid screens, and non-natural binding agents identified in screens of chemical libraries such as described below.
Agents of particular interest modulate HUMAN NTR-1 function.
The invention provides HUMAN NTR-1 nucleic acids, which find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc., as well as use in detecting the presence of HUMAN NTR-1 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional HUMAN
3o NTR-1 homologs and structural analogs.

The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e., no longer accompanied by some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, 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 nucleotides) other than that which it is joined to 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 l0 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 i5 or nucleotide analogs to provide modified stability, etc.
The amino acid sequences of the disclosed HUMAN NTR-1 polypeptide is used to back translate HUMAN NTR-1 polypeptide-encoding nucleic acids optimized for selected expression systems (Holler, et al. (1993) Gene 136: 323-20 328; Martin, et al. (1995) Gene 154: 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural HUMAN NTR-1 encoding nucleic acid sequences ("GCG" software, Genetics Computer Group, Inc., Madison, WI). HUMAN NTR-1 encoding nucleic acids may be part of expression vectors and may be incorporated into 25 recombinant host cells, e.g., for expression and screening, for transgenic animals, for functional studies such as the efficacy of candidate drugs for disease associated with HUMAN NTR-1 mediated signal transduction, etc.
Expression systems are selected and/or tailored to effect HUMAN NTR-1 polypeptide structural and functional variants through alternative post-30 translational processing.

The invention also provides for nucleic acid hybridization probes and replication/amplification primers having a HUMAN NTR-1 cDNA specific sequence and sufficient to effect specific hybridization with SEQ. NO. 1.
Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0.18 M NaCI, 0.01 M NaP04, pH7.7, 0.001 M EDTA) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C
with 0.2 x SSPE; preferably hybridizing in a buffer comprising 50%
formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2x SSPE buffer at 42°C.
HUMAN
NTR-1 cDNA homologs can also be distinguished from other polypeptides using alignment algorithms, such as BLASTX (Altschul, et al. (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403-410).
~5 HUMAN NTR-1 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. HUMAN NTR-1 nucleic acids are also used to modulate cellular expression or intracellular concentration or availability of active 2o HUMAN NTR-1. HUMAN NTR-1 inhibitory nucleic acids are typically antisense - single stranded sequences comprising complements of the disclosed HUMAN NTR-1 coding sequences. Antisense modulation of the expression of a given HUMAN NTR-1 polypeptide may employ antisense nucleic acids operably linked to gene regulatory sequences. Cells are 25 transfected with a vector comprising a HUMAN NTR-1 sequence with a promoter sequence oriented such that transcription of the gene yields an anti~ense transcript capable of binding to endogenous HUMAN NTR-1 encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable 3o extrachromosomal maintenance or integration. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA
encoding a given HUMAN NTR-1 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 HUMAN NTR-1 expression is effected by introducing into the targeted cell type HUMAN NTR-1 nucleic acids which increase the functional expression of the corresponding gene products. Such nucleic acids may be HUMAN NTR-1 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 retroviral-based transfection, viral coat protein-liposome mediated transfection, etc.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of HUMAN NTR-1 modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate the interaction of HUMAN NTR-1 with a natural HUMAN NTR-1 binding target. A wide variety of assays for binding agents are provided including protein-protein binding assays, 2o immunoassays, cell based assays, etc. 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 HUMAN NTR-1 polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g., a tag for detection or anchoring, etc.
The~assay mixtures comprise a natural HUMAN NTR-1 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 3o to the subject HUMAN NTR-1 conveniently measurable in the assay. The assay mixture also 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, e.g., albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., 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 1o binding. The mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the HUMAN NTR-1 specifically binds the cellular 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 HUMAN NTR-1 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 2o unbound components. Separation may be effected by precipitation, immobilization, etc., 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, etc., or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label and -other assay components, e.g., through optical or electron density, radiative emissions, nonradiative energy transfers, or indirectly detected with antibody conjugates, etc. A difference in the binding affinity of the 3o HUMAN NTR-1 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 HUMAN NTR-1 polypeptide to the corresponding binding target. A difference, as used herein, is statistically significant and preferably represents at 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 HUMAN
1o NTR-1 polypeptide under conditions whereby said polypeptide specifically interacts with at least one of a component of said medium and said extracellular surface 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 HUMAN
NTR-1 polypeptide in the presence of an extracellular HUMAN NTR-1 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 2o 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 indicates that said agent modulates the binding of said polypeptide to said binding target.
One embodiment of the invention is an isolated HUMAN NTR-1 polypeptide comprising the amino acid sequence as set forth herein or a fragment thereof having HUMAN NTR-1-specific activity.

Another embodiment of the invention is a recombinant nucleic acid encoding HUMAN NTR-1 polypeptide comprising the amino acid sequence as set forth herein or a fragment thereof having HUMAN NTR-1-specific activity.
Still another embodiment is an isolated nucleic acid comprising a nucleotide sequence as set forth herein or a fragment thereof having at least 18 consecutive bases and sufficient to specifically hybridize with a nucleic acid having the sequence of set forth herein in the presence of natural 1o HUMAN NTR-1 cDNA.
The present invention also provides for antibodies to the HUMAN NTR-1 polypeptide described herein which are useful for detection of the polypeptide in, for example, diagnostic applications. For preparation of monoclonal antibodies directed toward this HUMAN NTR-1 polypeptide, 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 26:495-497), as well as the trioma technique, the human B-cell hybridoma 2o technique (Kozbor et al., 1983, Immunology Today 4_:72), and the EBV-hybridoma technique to produce human 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 epitopes of the HUMAN NTR-1 polypeptide described herein. For the production of antibody, various host animals can be immunized by injection with the HUMAN NTR-1 polypeptide, or a fragment or derivative thereof, including but not limited to rabbits, mice 1o and rats. Various adjuvants may be used to increase the immunological response, depending on the host species, and 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 Cor~mebacterium~arvum.
A molecular clone of an antibody to a selected HUMAN NTR-1 polypeptide epitope can be prepared by known techniques. Recombinant DNA
2o methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) 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 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 3o 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, g_g_, 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.
1o EXAMPLE 1' Cloning and Seauencin~ of HUMAN NTR-1 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 random fragments of mRNA sequences (Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David J. Lipman (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 2o family and random-chance similarities.
A parser program was used to combine and sort all the hits from 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 human cDNA clones, LD. Nos. 366305 (the '305 clone) and 592256 (the '256 clone) from the LM.A.G.E. consortium, were discerned to contain homologous sequence. These clones, GeneBank Accession Nos. AA025672 3o and AA155646, 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).
The '305 clone contained a stop codon and poly-A tail and thus was determined to encode the 3' end of the molecule. The '256 clone aligned with the 5' end of the '305 clone over an approximately 300 nucleotide stretch. Together, the overlapping clones encode a polypeptide of approximately 220 amino acids, but was missing about 80 amino acids from the 5' end as compared to Osteoprotegerin. Further, since there was no o coding sequence for a predicted signal peptide or for an initial methionine, the 5' end of the '256 clone was deemed incomplete. The 5' RACE procedure was then used to obtain a nucleotide sequence encoding 63 missing amino acids including the signal peptide. Using PCR primers flanking the coding sequence, a single fragment was constructed that contained the entire coding i5 sequence. The sequence of HUMAN NTR-1 was then further confirmed by sequencing. The nucleotide and deduced amino acid sequence of HUMAN
NTR-1 is set forth herein. Northern analysis revealed HUMAN NTR-1 transcript in adult lung, skeletal muscle, kidney, placenta and pancreas as well as in fetal heart and stomach.
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 3o invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

WO 99/07738 . PCTNS98/16202 Human NTR-1 Sequence i0_ a0 30 40 50 60 SCq ID~'1 ATG AG<i GCG CTG GAG GGG CCA GGC CTCi TCG CTQ CTG TGC CTQ GTti TT(i GC(t CTO CCT GCC
Seq 10#2 Met Arg Ala Leu Glu Gly Pro Gly Leu Ser Leu Leu Cys Leu Val Leu Ala Leu Pro Ala 70 80 90 100 iio iao CTG CTG CCG GTCi CC(i GCT OTA 'CGC GGA GTG GG GAA ACA CCC ACC TAC CCC TCiCi CGG GAC
Leu Leu Pro Val Pro Ala Val Arg Gly Val Ala Glu Thr Pro Thr Tyr Pro Trp Arg Asp GG GAG AG GGG' GAO CGO CTG GTO TGC GøC CAG TCiC CCC CG GGC ACC TTT GTG GG CGG
Ala Glu Thr Gly Glu Arg Leu Vai Cys Ala Gln Cys Pro Pro Gly Thr Phe Val Gln Arg i90 a00 aio aao a3o aao CCG TGC CGC CGA GAC AGC CCC ACCT ACG TGT GGC CCG T(iT CCA CCG CGC GC TAC ACG
GG
Pro Cys Arg Arg Asp Ser Pro Thr Thr Cys Gly Pro Cys Pro Pro Arg His Tyr Thr Gln a5o aso a7o aso ago 300 TTC TGG AAC TAC CTG C~1G CGC TGC CGC TAC TGC AAC GTC CTC TGC GGG GACi CGT GAG
GAG
Phe Trp Asn Tyr Leu Glu Arg Cys Arg Tyr Gys Asn Val Leu Cys Gly Glu Arg Glu Glu 310 3a0 330 340 350 ~ 360 GAG GG CGG GCT TGC GC GCC ACC CAC AAC CGT GCC TGC CGC TGC CGC ACC GGC TTC TTC
Glu Ala Arg.Ala Cys His Ala Thr His Asn Arg Ala Cys Arg Cys Arg Thr ply Phe Phe 370 380 390 400 4i0 4a0 GCG GC GCT GGT TTC TGC TTG CiAG GC GG TCG TGT CCA CCT GGT GCCvGGC GTfi ATT GCC
Ala His Ala Gly Phe Cys Leu Glu His Ala Ser Cys Pro Pro Gly Ala Gly Val Ile Ala CCG GGC ACC CCC AGC CAG AAC ACO GG TGC CAG CCC TGC CCC CG GGC ACC TTC TG C~C
Pro Gly Thr Pro-Ser Gin Asn Thr Gln Cys Gln Pro Cys Pro Pro Gly Thr Phe Ser Ala 490 500 510 5a0 530 540 AGC AGC TCC AGC TG GAG CAG TGC CAG CCC GC CGC AAC TGC ACG GCC CTti GGC CTG GCC
Ser Ser Ser Ser Ser Glu Oln Cys Gin Pro His Arg Asn Cys Thr Ala Leu Gly Leu Ala CTC A71~' GTG CG GGC TCT TCC TCC CAT GAC ACC CTG TGC ACC AGC'TGC ACT CGC TTC
CCC
Leu Asn Val Pro Gly Ser Ser Ser Hfs Asp Thr Leu Cys Thr Ser Cys Thr Oily Phe Pro 610 6a0 630 640 650 660 CTC AGC A~ AGG GTA CG GGA GCT GAG GAG TGT GAG CGT GCC GTC ATC GAC TTT GTG GCT
Leu Ser Thr Arg Val Pro Oly Ala Glu Glu Cys Giu Arg Ala Val Ile Asp Phe Val Ala 670 . 680 690 700 710 720 T'm GG GAC ATC TCC ATC AAG AGG CT<3 GO CGG CTG CTG CAG GCC CTC CAG GCC CCG GAG
Phe Gln Asp Ile Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu Ala Pro Glu GGC TGG GGT CCG ACA CCA AGG GCG GGC CGC GCG GCC TTG CAG CTC AAG CTG CGT CGG
CGG
Gly Trp Gly Pro Thr Pro Arg Ala Gly Arg Ala Ala Leu Gln Leu Lys Leu Arg Arg Arg CTC ACG GAG CTC CTG GGG GCG CAG GAC GGG GCG CTG CTG GTG CGG CTG CTG CAG GCG
CTG
Leu Thr Glu Leu Leu Gly Ala Gln Asp Gly Ala Leu Leu Val Arg Leu Leu Gln Ala Leu CGC CiTG GCC AGG ATG CCC GGG CTG GAG CGG AGC CTC CGT GAG CGC TTC CTC CCT GTG
CAC
Arg Val Ala Arg Met Pro Gly Leu Glu Arg Ser Val Arg Glu Arg Phe Leu Pro Val His TGA
~~~

Claims (18)

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule encoding HUMAN NTR-1.

2. An isolated nucleic acid molecule according to claim 1, having a sequence selected from the group consisting of:
(a) the nucleotide sequence comprising the coding region of the HUMAN NTR-1 as set forth in SEQ. NO. 1;
(b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) and which encodes a molecule having the biological activity of the HUMAN NTR-1; or (c) a nucleotide sequence which, but for the degeneracy of the genetic code would hybridize to a nucleotide sequence of (a) or (b), and which encodes a molecule having the biological activity of the HUMAN NTR-1.

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

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

5. A vector according to claim 3 or 4, which is a plasmid.

6. Isolated HUMAN NTR-1 polypeptide.

7. isolated HUMAN NTR-1 polypeptide, having the amino acid sequence as set forth in SEQ. NO. 2.

8. A host-vector system for the production of HUMAN NTR-1 which comprises a vector of claim 3 or 4, in a host cell.

9. A host-vector system according to claim 8, wherein the host cell is a bacterial, yeast, insect or mammalian cell.

10. A method of producing HUMAN NTR-1 which comprises growing cells of a host-vector system of claim 8 or 9, under condition NTR-1 so produced.

11. An antibody which specifically binds the HUMAN NTR-1 of claim 6 or 7.

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

13. A composition comprising HUMAN NTR-1 according to claim 6 or 7, and a carrier.

14. A composition comprising an antibody according to claim 11 or 12, and a carrier.

15. HUMAN NTR-1 according to claim 6 or 7, an antibody according to claim 11 or 12, or a composition according to claim 13 or 14, for use in a method of treatment of the human or animal body, or in a method of diagnosis.

16. A polypeptide produced by the method of claim 10.

17. A receptorbody comprising the extracellular portion of the HUMAN
NTR-1 receptor fused to an immunoglobulin constant region.

18. The receptorbody of claim 17, wherein the constant region is the human immunoglobulin gamma-1 constant region.