JP2001512667A - Human orphan receptor NTR-1 - Google Patents

Abstract

  (57) [Summary] The present invention provides a nucleic acid sequence designated human NTR-1, which encodes a novel orphan receptor expressed in pancreatic and fetal hearts. The invention also provides an assay system that can be used to detect and / or measure a ligand that binds to a human NTR-1 gene product. The invention also provides diagnostic and therapeutic methods based on the interaction between NTR-1 and an agent that initiates signal transduction via binding to NTR-1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This international application is related to US Provisional Application No. 60/054, filed on August 6, 1997.
, 869, claim priority. Throughout this application, various publications are referenced. The disclosures of these publications are incorporated herein by reference in their entirety.

INTRODUCTION The field of the invention is polypeptide molecules that regulate cell function, nucleic acid sequences encoding the polypeptide, and methods of using the nucleic acid sequence and the polypeptide. The present invention provides novel orphan receptor molecules, their uses, 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 primarily of transmembrane proteins that trigger signal transduction in various cells. Tumor necrosis factor α (TNF-α) is a cytokine produced primarily by activated macrophages. TNF-α stimulates T and B cell proliferation and induces the expression of adhesion molecules in endothelial cells. This cytokine also plays an important role in host defense against infection.

[0004] TNF-α activity is determined by two distinct receptors, TNFR-p55 and TNF.
It is mediated by R-p75. These two receptors also mediate the activity caused by soluble lymphotoxin α (LT-α), which is mainly secreted by activated lymphocytes. Specific stimulation of TNFR-p55 may include, for example, TNF activity such as in vitro tumor cytotoxicity, expression of adhesion molecules in endothelial cells and keratinocytes, activation of sphingomyelinase with a concomitant increase in ceramide,
Activation of NF-κB and manganese superoxide dismutase mRNA
Induction of induction. Specific stimulation of TNFR-p75 results in a proliferative response of mouse and human thymocytes and cytotoxic T cells, fibroblasts and natural killer cells, and GM-CSF secretion in PC60 cells.

[0005] The identification of a new member of the TNFR superfamily that regulates bone resorption has recently been reported. This newly identified protein is bone protegrin (Osteopr)
otegrin (OPG) and was hypothesized to act as a humoral regulator of bone resorption by blocking the differentiation of osteoclasts (the cells responsible for bone resorption). (Simonet, WS, et al., 1997, Cell 89:30.
9-319; Amgen, Inc. International Publication No. WO 97/23614 published on July 3, 1997). However, relatively little is known about soluble factors that act physiologically to regulate osteoclast development.

[0006] New receptor molecules are searched for additional members of the known family of receptors using, for example, PCR-based screening or computer searches of the EST database containing regions of known homology between members of the family. Are often identified and isolated by (For example, Maiso
npierre, et al., 1993, Oncogene 8: 1631-1637). Isolation of such a so-called "orphan" receptor (of which no ligand is known), and subsequent determination of the tissue in which such receptor is expressed, is dependent on the growth, proliferation and regeneration of cells in the target tissue. Provide insight into the regulation of Further, such receptors can be used to isolate these cognate ligands, which in turn can be used to regulate the survival, growth and regeneration of cells that express the receptor. Alternatively, in the case of a soluble receptor, this receptor itself can behave as a ligand.

SUMMARY OF THE INVENTION [0007] The present invention provides a novel orphan human receptor, designated human NTR-1, which is expressed in pancreas, skeletal muscle, muscle, and fetal and adult heart. You. This protein is related to bone protegrin (OPG) and to tumor necrosis factor receptor (TNFR). The present invention further provides an isolated nucleic acid molecule encoding human NTR-1. Based on its homology to bone protegrins, human NTR
-1 is involved in the regulation of bone mass and can be useful in regulating the development, proliferation and death of osteoblasts or osteoclasts or in regulating muscle metabolism, and muscle diseases or disorders May be related to

[0008] The invention also provides extracellular domains of human NTR-1 and proteins or polypeptides containing nucleic acids encoding such extracellular domains.
The invention further provides a vector comprising an isolated nucleic acid molecule encoding human NTR-1 or an extracellular domain thereof, which comprises a bacterial cell, a yeast cell, an insect cell, or a mammalian cell, preferably Human NT in COS or CHO cells
It can be used to express R-1 or its extracellular domain.

[0009] The present invention further provides the use of the human NTR-1 receptor or extracellular or intracellular domains thereof in screening for drugs that interact with human NTR-1. Novel agents that bind to the receptors described herein can mediate survival and differentiation in cells that naturally express the receptor, but also process cells that have been engineered to express the receptor When used to provide survival and growth. In certain embodiments, the extracellular domain of human NTR-1 (soluble receptor) is utilized in screening for cognate ligands.

[0010] Preferred uses of the human NTR-1 polypeptide of the present invention include screening for agents that bind to the receptor polypeptide. The drug is a biologically active drug (agonist), which activates the human NTR-1 receptor
Or it can bind to a receptor and block its activation (antagonist). The screening method involves the presence of an extracellular human NTR-1 polypeptide-specific binding target and a candidate drug under the condition that the polypeptide specifically binds to the binding target with a reference affinity in the absence of the drug. Below, human N
Incubating the TR-1 polypeptide; detecting the binding affinity of the polypeptide for the binding target and determining the drug-biased affinity, wherein the drug-biased affinity and the reference affinity The difference between comprises suggesting that the agent modulates the binding of the polypeptide to the binding target.

The present invention also provides a nucleic acid probe capable of hybridizing with a sequence contained in a nucleic acid sequence encoding human NTR-1 useful for detecting NTR-1 expressing tissues in humans and animals.

[0012] The present invention further provides antibodies directed to human NTR-1.

The present invention also has diagnostic and therapeutic efficacy. In certain embodiments of the present invention, the methods for detecting abnormalities in receptor function or expression described herein can be used in the diagnosis of this disorder. In other embodiments, manipulation of a receptor or agonist or antagonist that binds to the receptor can be used to treat a disease. In a further embodiment, the extracellular domain of the receptor is utilized as a blocking agent to block binding of the receptor to its target.

In a further embodiment of the invention, the patient suffering from an excess of NTR-1 is administered an effective amount of antisense RNA or antisense oligodeoxyribonucleotide consistent with the human NTR-1 gene coding region. Can reduce the expression of NTR-1.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to isolated human NTR-1 polypeptides, as well as human NTR-1.
Provided is a human NTR-1 polypeptide comprising an amino acid sequence or a recombinant polypeptide comprising the functional human NTR-1 polypeptide domain having human NTR-1 specific activity recognizable in an assay. Thus, the polypeptide can be a deletion mutant of the disclosed human NTR-1 polypeptide, and can be provided, for example, as a fusion product with a non-human NTR-1 polypeptide. Human NTR of the present invention
-1 polypeptide domain has human NTR-1 specific activity or function.

Many applications for human NTR-1 are suggested by its properties. Human NT
R-1 may be useful in studying and treating conditions similar to those treated using TNF. In addition, human NTR-1 cDNA can be used, for example, to use antibodies in assays for polypeptides in cell lines or to amplify those that have sequence similarity to oligonucleotide primers and how much human NTR-1 is present. The use of oligonucleotides as primers in PCR tests to find out may be useful as a diagnostic tool. Of course, isolation of human NTR-1 also depends on its putative ligand, other human NTR-
Provides clues for isolating the 1-binding polypeptide and / or studying its properties.

[0017] Human NTR-1 specific activity or function is a convenient in vitro, cell-based,
Or in vivo assays-for example, determined by in vitro binding assays, cell culture assays, animals (eg, immune responses, gene therapy, transgenics, etc.), and the like. Binding assays include any assay in which the specific molecular interaction between a binding target and a human NTR-1 polypeptide is evaluated. The binding target can 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 as identified in the assays described below. .

The polypeptides of the present invention may be isolated or pure—an “isolated” polypeptide is no longer associated with any of the materials with which it is associated in its natural state, and preferably "Pure" polypeptide comprises at least about 0.5%, and more preferably at least about 5%, of the total polypeptide weight in a given sample; the "pure" polypeptide is the weight of the total polypeptide in a given sample. At least about 90%, and preferably at least about 99%. The polypeptides and polypeptide domains of the present invention can be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for the biochemical synthesis, molecular expression, and purification of the compositions of the present invention, including, for example, Molecular
Cloning, A Laboratory Manual (Sambr
OK et al., Cold Spring Harbor Laboratory).
, Current Protocols in Molecular Biol
ogy (edited by Ausubel et al., Green Publ. Assoc.,
Wiley-Interscience, NY).

The polypeptides of the present invention find a wide variety of uses, including as immunogens, as targets in screening assays, as bioactive reagents to modulate cell proliferation, differentiation, and / or function, and the like. Find out. For example, the invention provides for exogenous human NTR-1 polypeptides under conditions in which the added polypeptide specifically interacts with components of the medium and / or the extracellular surface that affects changes in cell physiology. A method for regulating the physiology of a cell, including the extracellular surface, by contacting the cell with the medium or the medium surrounding the cell. According to these methods, the extracellular surface contains a plasma membrane associated receptor; exogenous human NTR-1 is not produced by the cell or is otherwise expressed at a non-natural level, stage, or physiological location And suitable media include in vitro culture media and physiological fluids such as blood, synovial fluid, and the like. Polypeptides can be introduced, expressed, or suppressed in a specific population of cells by any convenient method, such as microinjection, promoter-specific expression of a recombinant enzyme, targeted delivery of lipid vesicles, and the like.

The present invention provides human NTR-1 specific binding agents, methods for identifying and producing 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 protein receptor-like specific antibodies or T-cell antigen receptors that have been recombinant in somatic cells (eg, Harlow and Lane (1988) Antibody).
es, A Laboratory Manual, Cold Spring
See Harbor Laboratory) and also 1, 2,
And other binding agents identified in assays such as three-hybrid screening,
As well as non-natural binders identified in screening chemical libraries as described below. Certain drugs of interest modulate human NTR-1 function.

The present invention provides human NTR-1 nucleic acids, which are used as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, and the like, as well as human NTR-1 genes and gene transcripts. A wide variety of applications find use in detecting the presence and use in detecting or amplifying nucleic acids encoding additional human NTR-1 homologs and structural analogs.

The nucleic acids of the invention are synthetic / non-naturally occurring sequences and / or are isolated, that is to say no longer associated with some of their materials associated in their natural state, preferably in a predetermined fraction. It constitutes at least about 0.5%, more preferably at least about 5%, by weight of the total nucleic acid present, and usually recombination occurs with non-native sequences or naturally-linked nucleotides other than those linked on the native chromosome. Means containing an array.
Nucleic acids comprising the nucleotide sequences disclosed herein and fragments thereof,
A native flanking region of less than 10 kb, preferably less than 2 kb, immediately adjacent to sequences other than those linked on the natural chromosome, or immediately adjacent to sequences other than those linked on the natural chromosome. Such sequences or fragments are included at the terminus adjacent to. The nucleic acid is usually RNA or DNA, but is often advantageous for using nucleic acids containing other bases or nucleotide analogs to provide altered stability.

[0023] The amino acid sequence of the disclosed human NTR-1 polypeptide can be determined using selected expression systems (Holler et al. (1993) Gene 136: 323-328; Ma).
rtin et al. (1995) Gene 154: 150-166) used to reverse translate nucleic acids encoding human NTR-1 polypeptides, or a single copy of the nucleic acid sequence encoding native human NTR-1. Used to generate degenerate oligonucleotide primers and probes for use in isolation ("GCG" software, Genetics Computer)
Group, Inc. , Madison, WI). The nucleic acid encoding human NTR-1 can be part of an expression vector, and is a candidate drug for diseases associated with human NTR-1 mediated signal transduction, for example, for expression and screening, for transgenic animals, Can be incorporated into recombinant host cells, such as for functional studies such as the efficacy of E. coli. Expression systems are selected and / or adapted to affect human NTR-1 polypeptide structural and functional variants by alternative post-translational processing.

The present invention also has a human NTR-1 cDNA specific sequence and SEQ ID NO: 1.
Provide sufficient nucleic acid hybridization probes and replication / amplification primers to effect specific hybridization with Demonstrating specific hybridization generally requires stringent conditions, such as 5 × SSPE (0.18 M NaCl, 0.01 M NaP
Hybridization at a temperature of 42 ° C. in a buffer containing 30% formamide in buffer (O ₄ , pH 7.7, 0.001 M EDTA) and 0.2 × SS
Remains bound when washed at 42 ° C. with PE; preferably 5 × SS
Hybridizing at a temperature of 42 ° C. in a buffer containing 50% formamide in PE buffer and remaining bound when washing at 42 ° C. with 0.2 × SSPE buffer at 42 ° C. . The human NTR-1 cDNA homolog is also
LASTX (Altschul et al. (1990) Basic Local A
Lignent Search Tool, J.M. Mol. Biol.
215: 403-410) can be used to distinguish it from other polypeptides.

Human NTR-1 hybridization probes find use in identifying wild-type and mutant alleles in clinical and laboratory samples.
Variant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnostics. Human NTR-1 nucleic acids are also used to modulate cellular expression or intracellular concentration or availability of active human NTR-1. A human NTR-1 inhibitory nucleic acid is typically an antisense-single-stranded sequence that includes the complement of the disclosed human NTR-1 coding sequence.
Antisense regulation of the expression of a given human NTR-1 polypeptide can employ an antisense nucleic acid operably linked to a gene regulatory sequence. The cells are transfected with a vector containing the human NTR-1 sequence together with a promoter sequence oriented such that the transcription of the gene results in an antisense transcript capable of binding to mRNA encoding endogenous human NTR-1. Transcription of the antisense nucleic acid can be constitutive or inducible, and the vector can provide stable extrachromosomal maintenance or integration. Alternatively, the single-stranded antisense nucleic acid that binds to genomic DNA or mRNA encoding a given human NTR-1 polypeptide is present in or in the host at a concentration that results in a substantial decrease in expression of the targeted polypeptide. Can be administered to target cells that are temporarily isolated from. Enrichment in human NTR-1 expression is provided by introducing a human NTR-1 nucleic acid that increases the functional expression of the corresponding gene product into the targeted cell type. Such a nucleic acid can be a human NTR-1 expression vector, a vector that upregulates the functional expression of an endogenous allele, or a replacement vector for targeted modification of a mutant allele. Techniques for introducing nucleic acids into viable cells are known in the art,
And retrovirus-based transfection, virus coat protein-liposome mediated transfection, and the like.

The present invention provides efficient methods for identifying agents, compounds, and lead compounds for agents active at the level of human NTR-1 regulatable cell function. In general, these screening methods involve human NTR-1 binding to natural human NTR-1 binding targets.
Assaying for a compound that modulates the interaction of A wide variety of assays for binding agents are provided, including protein-protein binding assays, immunoassays, cell-based assays, and the like. The preferred method follows automated, cost-effective, high-throughput screening of chemical libraries for lead compounds.

The in vitro binding assay uses a mixture of components comprising a human NTR-1 polypeptide, which is a portion of the fusion product with another peptide or polypeptide, such as a tag for detection or immobilization. possible. The assay mixture contains the natural human NTR-1 binding target. Although native binding targets can be used, it is often preferred to use some of them, provided that they provide the binding affinity and avidity for human NTR-1 of the present invention that can be conveniently measured in an assay. .
The assay mixture also contains the candidate pharmacological agent. Candidate agents include many chemical classes, but 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 may also be included such as salts, buffers, neutral proteins (eg, albumin), detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like. The mixture components can be added in any order that provides the requisite binding, and the incubation can be performed at any temperature that facilitates optimal binding. The mixture is incubated under conditions in which human NTR-1 specifically binds a cell binding target, part, or analog with reference binding affinity, in the absence of the candidate pharmacological agent. The incubation period is selected for optimal binding, but is also minimized to facilitate rapid high throughput ratio screening.

After incubation, drug-biased binding between human NTR-1 and one or more binding targets is detected by any convenient method. For cell-free binding type assays, a separation step is often used to separate unbound and bound components. Separation is by precipitation, fixation, etc., and then, for example,
It can be effected by washing by membrane filtration or gel chromatography. For a cell-free binding assay, one of the components usually contains or is conjugated to a label. Labels can provide direct detection, such as radioactivity, luminescence, optical or electron density, or indirect detection, such as epitope tags, enzymes, and the like. Depending on the nature of the label and other assay components, such as, for example, detection by optical or electron density, radiative emission, non-radiative energy transfer, or indirectly with antibody conjugates, various Can be used. The difference in the binding affinity of a human NTR-1 polypeptide for a target in the absence of a drug as compared to the binding affinity in the presence of a drug indicates that the drug has a human NTR-1 binding activity to the corresponding binding target.
Fig. 7 illustrates modulating the binding of a polypeptide. As used herein, the difference is
It is statistically significant and preferably exhibits at least 50%, more preferably at least 90% difference.

The present invention provides a method of altering the physiology of a cell, including the extracellular surface, upon contact with a culture medium, the method comprising: Contacting an exogenous human NTR-1 polypeptide with the medium under conditions that specifically interact with the medium and at least one component of the extracellular surface.

The present invention further provides a method of screening for a biologically active agent, the method comprising the steps of: a) in the absence of the agent, wherein the polypeptide specifically binds a binding target with a reference affinity. Incubating the human NTR-1 polypeptide in the presence of an extracellular human NTR-1 polypeptide specific binding target and a candidate agent under binding conditions; b) determining the drug-biased affinity Detecting the binding affinity of the polypeptide for the binding target, wherein the difference between the drug-biased affinity and the reference affinity indicates that the agent is responsible for binding the polypeptide to the binding target. Adjusting, including adjusting.

One embodiment of the present invention is an isolated human NTR-1 polypeptide comprising an amino acid sequence described herein or a fragment thereof having human NTR-1 specific activity.

[0032] Another embodiment of the present invention provides a human NTR-comprising an amino acid sequence described herein.
A recombinant nucleic acid encoding one polypeptide or a fragment thereof having human NTR-1 specific activity.

[0033] Still other embodiments are isolated nucleic acids comprising a nucleotide sequence described herein, or native human NTR-1 having at least 18 contiguous bases.
A fragment thereof sufficient to specifically hybridize with a nucleic acid having a sequence described herein in the presence of the cDNA.

The present invention also provides antibodies to the human NTR-1 polypeptides described herein that are useful, for example, for detecting the polypeptide in diagnostic applications. For the preparation of monoclonal antibodies to the human NTR-1 polypeptide, any technique which provides for the production of antibody molecules by continuous cell lines in culture can be used.
See, for example, Kohler and Milstein (1975, Nature).
256: 495-497), as well as the trioma technology, the human B cell hybridoma technology (Kozbor et al., 19).
83, Immunology Today 4: 72) , and to produce human monoclonal antibodies EBV- hybridoma technique (Cole et al., 19
85, "Monoclonal Antibodies and Cancer"
Therapy, "Alan R. Liss, Inc. 77-96) are within the scope of the present invention.

[0035] Monoclonal antibodies for diagnostic or therapeutic use can be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be made by any of a number of techniques known in the art (eg, Teng et al., 1983, Proc. Natl.
Acad. Sci. U. S. A. 80: 7308-7312; Kozbo
R et al., 1983, Immunology Today 4: 72-79;
lsson et al., 1982, Meth. Enzymol. 92: 3-16
). Chimeric antibody molecules can be prepared that contain a mouse antigen binding domain together with a human constant region (Morrison et al., 1984, Proc. Natl. Ac.
ad. Sci. U. S. A. 81: 6851, Takeda et al., 19
85, Nature 314: 452).

Various procedures known in the art can be used for the production of polyclonal antibodies against an epitope of the human NTR-1 polypeptide described herein.
For the production of antibodies, various host animals can be immunized by injection of a human NTR-1 polypeptide, or a fragment or derivative thereof, including rabbits,
Includes but is not limited to mice and rats. Depending on the host species,
Various adjuvants can be used to increase the immunological response, including:
Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and BCG (Bacille Calmette- Guerin) and Cor
Includes, but is not limited to, potentially useful human adjuvants such as ynebacterium parvum.

[0037] Molecular clones of antibodies to the selected human NTR-1 polypeptide epitope can be prepared by known techniques. Recombinant DNA methodology (eg, Man
iatis et al., 1982, Molecular Cloning, AL.
laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New
York) can be used to construct a nucleic acid sequence encoding a monoclonal antibody molecule, or antigen-binding region thereof.

[0038] The present invention provides antibody molecules and fragments of such antibody molecules. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments are F (ab ') ₂ fragments that can be produced by pepsin cleavage of the antibody molecule; Fab' fragments that can be produced by reducing the disulfide bridges of the F (ab ') ₂ fragment; Includes, but is not limited to, Fab fragments that can be generated by treating an antibody molecule with papain and a reducing agent. Antibody molecules can be purified by known techniques, for example, immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.

The following examples are provided by way of illustration, and not by way of limitation.

Example 1 Cloning and Sequencing of the Human NTR-1 Gene The amino acid sequences of known human and mouse members of the TNF family were
Used as a tblastn question to search the NIH EST database for random fragments of NA sequences (Altschul, Stephen)
F. , Warren Gish, Webb Miller, Eugene
W. Myers, and David J. et al. Lipman (1990)
. Basic local alignment search tool.
J. Mol. Biol. 215: 403-10). Each question generated a list of hits, ie, EST sequences with substantial sequence similarity to the query sequence. Typically, the hit at the top of the list is the mR of the protein in question.
NA copies were followed by ESTs from the family and other members of the random-chance similarity.

Using the parser program, all of the hits from the search with all of the family members were combined and classified. This allowed for a rapid reduction of all hits corresponding to known proteins. The remaining hits were analyzed for conservation of sequence motifs characteristic of the family. An additional database search was performed to identify duplicate ESTs. Two human cDNA clones, I.P. M. A
. G. FIG. Identification numbers 366305 ('305 clone) and 592256 (' 256 clone) from the E Consortium were identified to contain homologous sequences. These clones, GeneBank accession numbers AA025672 and AA155
646 is Research Genetics, Inc. (Huntsville
e, AL) and obtained from ABI 373A DNA sequencer and Taq Video Terminator Cycle Sequence.
ing Kit (Applied Biosystems, Inc., Fost)
er City, CA).

The '305 clone contained a stop codon and a polyA tail and was therefore determined to encode the 3' end of the molecule. This '256 clone is this' 3
The 5 'end of the 05 clone was aligned over a length of about 300 nucleotides. Each other, the duplicated clone encodes a polypeptide of about 220 amino acids, but lacks about 80 amino acids from its 5 'end when compared to bone protegrin. In addition, the 5 'end of the' 256 clone was considered incomplete because there was no coding sequence for the expected signal peptide or for the first methionine. The 5 'RACE procedure was then performed on the 6' containing the signal peptide.
It was used to obtain a nucleotide sequence encoding three missing amino acids. Using the PCR primers flanking this coding sequence, a single fragment containing the entire coding sequence was constructed. The sequence of human NTR-1 was then further confirmed by sequencing. The nucleotide and deduced amino acid sequence of human NTR-1 is provided herein. Northern analysis revealed human NTR-1 transcripts in adult lung, skeletal muscle, kidney, placenta, and pancreas, and fetal heart and stomach.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be made without departing from the spirit or scope of the appended claims. What can be done will be readily apparent to those skilled in the art in light of the teachings of the present invention.

The present invention is not 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 and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/02 C 5/10 21/08 C12P 21/02 C12N 15/00 ZNAA // C12P 21/08 5/00 B (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Maciaskowski, Piotr Jay. United States New York 12569, Pleasant Valley, Box 404, Holter Lane (72) Inventor Morris New York 10956, New York, Bristol Court 3 (72) Inventor Valenzuela, David M. New York 10598, Yorktown Tsu, Giorudano drive 529 F-term (reference) 4B024 AA11 BA63 CA04 DA02 HA01 HA12 HA15 4B064 AG01 AG27 CA10 CA19 CC24 DA01 DA13 4B065 AA90X AA91X AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4H045 AA11 AA30 CA40 DA51 EA50 FA74

Claims (18)

[Claims] 1. An isolated nucleic acid molecule encoding human NTR-1. 2. The isolated nucleic acid molecule of claim 1, having a sequence selected from the group consisting of: (a) a nucleotide sequence comprising the coding region of human NTR-1 of SEQ ID NO: 1. (B) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) and encodes a molecule having the biological activity of human NTR-1; or (c) the degeneracy of the genetic code Otherwise, a nucleotide sequence that hybridizes to the nucleotide sequence of (a) or (b) and encodes a molecule having the biological activity of human NTR-1. 3. A vector comprising the nucleic acid molecule according to claim 1 or 2. 4. The vector of claim 3, wherein said nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. 5. The vector according to claim 3, which is a plasmid. 6. An isolated human NTR-1 polypeptide. 7. An isolated human NTR-1 polypeptide having the amino acid sequence of SEQ ID NO: 2. 8. A host-vector system for the production of human NTR-1, comprising a vector according to claim 3 or 4 in a host cell. 9. The host-vector system according to claim 8, wherein said host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. 10. A human NT comprising the step of growing cells of the host-vector system according to claim 8 or 9 under conditions in which NTR-1 is produced as described above.
A method for producing R-1. An antibody that specifically binds to human NTR-1 according to claim 6 or 7. 12. The antibody according to claim 11, which is a monoclonal antibody. 13. A composition comprising the human NTR-1 according to claim 6 and a carrier. 14. A composition comprising the antibody according to claim 11 or 12 and a carrier. 15. The human NTR-1 according to claim 6 or 7, the antibody according to claim 11 or 12, or the claim, for use in a method of treatment of the human or animal body or in a diagnostic method. Item 15. The composition according to Item 13 or 14. 16. A polypeptide produced by the method according to claim 10. 17. A receptor body comprising the extracellular portion of the human NTR-1 receptor fused to an immunoglobulin constant region. 18. The receptor according to claim 17, wherein the constant region is a human immunoglobulin γ-1 constant region.