JP2002508934A - Novel nucleic acids and polypeptides having homology to the TNF receptor - Google Patents

Abstract

  (57) [Summary] The present invention provides a nucleic acid sequence encoding a novel mammalian receptor polypeptide, designated NTR-5. The invention also provides an assay system that can be used to detect and / or measure a ligand that binds to a mammalian NTR-5 gene product. The invention also provides diagnostic and therapeutic methods based on the interaction between mammalian NTR-5 and an agent that initiates signal transduction via binding to mammalian NTR-5. In certain embodiments, the mammalian NTR-5 can be human NTR-5 or mouse NTR-5.

Description

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entirety as a result.

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 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 a variety of 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 TNF activity, eg, tumor cell injury in vitro, expression of adhesion molecules in endothelial cells and keratinocytes, activation of sphingomyelinase with concomitant increase in ceramide, activation of NF-κB, And manganese superoxide dismutase mRN
Induce the induction of A. 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)
otegerin) (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, issued 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, so-called "orphan" receptors (of which no ligand is known), and subsequent determination of the tissue in which such receptors are expressed, depends on the growth of cells in the target tissue, Provides insight into the regulation of growth and regeneration. 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 (extracellular domain only), the receptor itself may behave as a ligand.

SUMMARY OF THE INVENTION [0007] The present invention provides a novel mammalian receptor, termed NTR-5. In particular, the present invention provides a novel human receptor named human NTR-5. The present invention further provides a novel mouse receptor, termed mouse NTR-5. Throughout this description, references to mammalian NTR-5 include, but are not limited to, the specific embodiments of human and mouse NTR-5 described herein. These receptors are related to bone protegrin (OPG)
And it relates to tumor necrosis factor receptor (TNFR). The present invention further provides isolated nucleic acid molecules encoding mammalian NTR-5, and in particular, human NTR-5 or mouse NTR-5. Based on its homology to bone protegrins, NTR-5 is involved in regulating bone mass and regulating osteoblast or osteoclast development, proliferation and death, or regulating muscle metabolism And may be involved in muscle diseases or disorders.

[0008] The invention also provides polypeptides comprising the extracellular domain of mammalian NTR-5, as well as nucleic acids encoding such polypeptides. The invention further provides
Provided is a vector containing an isolated nucleic acid molecule encoding a mammalian NTR-5 or extracellular domain thereof, comprising a bacterial cell, a yeast cell, an insect cell, or a mammalian cell, preferably a COS or CHO cell. In which it can be used to express mammalian NTR-5 or its extracellular domain.

[0009] The present invention also provides polypeptides comprising the intracellular domain of mammalian NTR-5, as well as nucleic acids encoding such polypeptides. The invention further provides
Provided is a vector containing an isolated nucleic acid molecule encoding a mammalian NTR-5 or an intracellular domain thereof, comprising a bacterial cell, a yeast cell, an insect cell, or a mammalian cell, preferably a COS or CHO cell. Within, it can be used to express mammalian NTR-5 or an intracellular domain thereof.

[0010] The present invention further provides a method of screening for a drug that interacts with a mammalian NTR-5, wherein the mammalian NTR-5 comprises an extracellular and / or intracellular domain thereof.
5 provides the use of a polypeptide. Novel agents that bind to the polypeptides described herein can mediate survival and differentiation in cells that naturally express the polypeptide, but also use this agent to express the polypeptide When treated cells are treated as described above, they may confer viability and proliferation. In certain embodiments, the extracellular domain of mammalian NTR-5 is utilized in screening for cognate ligands.

[0011] Preferred uses of the mammalian NTR-5 polypeptide of the present invention include screening for agents that bind to the polypeptide. The agent is a biologically active agent (agonist), which activates a mammalian NTR-5 polypeptide.
Or can bind to a polypeptide and block its activation (antagonist). The screening method may be performed in the presence of a mammalian NTR-5 polypeptide-specific binding target and a candidate agent, provided that the polypeptide specifically binds to the binding target with a reference affinity in the absence of the agent. Incubating the mammalian NTR-5 polypeptide at a step; detecting the binding affinity of the polypeptide for the binding target and determining the drug-biased affinity, wherein the drug-biased affinity and Differences from the reference affinity include indicating that the agent modulates the binding of the polypeptide to the binding target.

[0012] The invention also provides nucleic acid probes capable of hybridizing to sequences contained within a nucleic acid sequence encoding mammalian NTR-5 useful for detecting NTR-5 expressing tissues in humans and animals.

[0013] The present invention further provides antibodies directed to mammalian NTR-5.

[0014] The invention also has diagnostic and therapeutic utilities. In certain embodiments of the invention, the methods for detecting abnormalities in the function or expression of a polypeptide described herein can be used in the diagnosis of this disorder. In other embodiments, manipulation of the polypeptide or agonist or antagonist that binds the polypeptide can be used to treat a disease. In a further embodiment, the extracellular domain of the polypeptide is utilized as an agent that blocks binding of the binding agent to its target.

[0015] In a further embodiment of the invention, the patient suffering from an excess of the mammalian NTR-5 polypeptide is an effective amount of an antisense RNA or antisense oligodeoxyribonucleotide corresponding to the mammalian NTR-5 gene coding region. It can be treated by administering nucleotides, thereby reducing the expression of mammalian NTR-5.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to isolated mammalian NTR-5 polypeptides, as well as mammalian NTR-5 polypeptides.
Mammalian NTR-5 polypeptides comprising a TR-5 amino acid sequence, or a recombinant polypeptide comprising a functional mammalian NTR-5 polypeptide domain thereof having assay recognizable mammalian NTR-5 specific activity. . Thus, the polypeptide can be a deletion mutant of the disclosed mammalian NTR-5 polypeptide, and can be provided, for example, as a fusion product with a non-mammalian NTR-5 polypeptide. The mammalian NTR-5 polypeptide of the present invention has mammalian NTR-5 specific activity or function.

[0017] Many applications for mammalian NTR-5 polypeptides are suggested by their properties. Mammalian NTR-5 polypeptides may be useful in the study and treatment of conditions similar to those treated using TNF. Furthermore, mammalian NTR-5
The cDNA can be used, for example, in PCR tests to amplify those sequences with similarity to the oligonucleotide primers and to know how much mammalian NTR-5 mRNA is present in a particular tissue or sample. It may be useful as a diagnostic tool by using oligonucleotides as primers. Of course, isolation of mammalian NTR-5 also provides clues to isolating its putative ligand, other mammalian NTR-5 binding polypeptides, and / or studying its properties.

[0018] Mammalian NTR-5 specific activity or function can be determined by convenient in vitro, cell-based, or in vivo assays. In vitro assays 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 include any assay in which a specific molecular interaction between a binding target and a mammalian NTR-5 polypeptide is evaluated. The binding target can be a natural binding target, or a non-natural binding target, such as a specific immune polypeptide (eg, an antibody), or a mammalian NTR-5 specific agent.

The claimed mammalian NTR-5 polypeptide can be isolated or pure—an “isolated” polypeptide is no longer associated with some of the materials with which it is associated in its natural state. And preferably comprises at least about 0.5%, and more preferably at least about 5% of the total polypeptide weight in a given sample; "pure" polypeptides in a given sample Constitute at least about 90%, and preferably at least about 99%, of the total polypeptide by weight. The polypeptides of the invention can 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 compositions of the present invention, including, for example, Molecula
r Cloning, A Laboratory Manual (Sambro
Ok et al., Cold Spring Harbor Laboratory, Co.
ld Spring Harbor, NY), Current Protocol
ls in Molecular Biology (edited by Ausubel et al., Gr.
eene Publ. Assoc. , Wiley-Interscience,
NY).

The polypeptides of the present invention may be used in a wide variety of forms, including but not limited to, as immunogens, as targets in screening assays, and as bioactive agents to modulate cell proliferation, differentiation, and / or function. Find various uses. For example, the present invention relates to a method for treating the extracellular surface of a cell or the cell under conditions in which the added polypeptide specifically interacts with components of the medium and / or the extracellular surface to affect a change in the physiology of the cell. Provide a method for modulating the physiology of a cell, comprising contacting the medium surrounding the cell with an exogenous mammalian NTR-5 polypeptide. According to these methods, the extracellular surface contains plasma membrane associated molecules. The term "exogenous mammalian NTR-5 polypeptide" refers to a polypeptide that is not produced by a cell or otherwise expressed at a non-natural level, time, or physiological location. Media include, but are not limited to, in vitro culture media and / or physiological fluids such as blood, synovial fluid, and lymph. The polypeptide can be introduced, expressed, expressed in a specific population of cells by any convenient method, including but not limited to microinjection, promoter-specific expression of recombinant proteins, or targeted delivery of lipid vesicles. Or it can be suppressed.

[0021] The present invention provides mammalian NTR-5 specific binding agents, methods for identifying and producing such agents, and their use in diagnosis, therapy, and pharmaceutical development. The mammalian NTR-5 specific binding agent is a mammalian NTR-5 specific antibody (
See, for example, Harlow and Lane (1988) Antibodies, A.
Laboratory Manual, Cold Spring Harbo
r Laboratory, Cold Spring Harbor, NY), and also other binding agents identified in assays such as 1, 2, and 3 hybrid screening, and chemical libraries as described below. Non-natural binding agents identified in the screening of Certain agents of interest modulate mammalian NTR-5 polypeptide function.

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

The nucleic acid of the invention is a synthetic / non-natural sequence and / or an isolated sequence,
That is, it is no longer associated with some of its naturally associated materials and preferably comprises at least about 0.5%, more preferably at least about 5%, by weight of total nucleic acid present in a given fraction. And usually recombination is meant to include a non-native sequence or sequence that is linked to nucleotides, other than that the non-native sequence or sequence is linked on the natural chromosome. Nucleic acids comprising the nucleotide sequences disclosed herein and fragments thereof may be located at the terminus immediately adjacent to a sequence other than that to which the sequence or fragment is ligated on the native chromosome, or at which the sequence or fragment is located on the native chromosome Such a sequence or fragment is included at the terminus adjacent to a native flanking region of less than 10 kb, preferably less than 2 kb, immediately flanking sequences other than those linked above. Nucleic acids are usually RNA
Alternatively, it is advantageous to use nucleic acids that are DNA, but often include other bases or nucleotide analogs, for example, to provide altered stability.

[0024] The sequence of the disclosed mammalian NTR-5 nucleic acid is used to obtain a deduced mammalian NTR-5 polypeptide sequence. Further, the disclosed mammalian NTR-
The sequence of the 5 nucleic acids was determined using a selected expression system (Holler et al. (1993) Gene 1).
36: 323-328; Martin et al. (1995) Gene 154: 150.
-166) or used to generate degenerate oligonucleotide primers and probes for use in isolating nucleic acid sequences encoding native mammalian NTR-5 ("GCG"). Software, Gene
tics Computer Group, Inc. , Madison, WI)
. The nucleic acid encoding mammalian NTR-5 can be part of an expression vector, and for example, for expression and screening, for transgenic animals,
Alternatively, they can be incorporated into recombinant host cells for functional studies such as the efficacy of candidate drugs for diseases associated with mammalian NTR-5 mediated signal transduction.
Expression systems are selected and / or adapted to affect mammalian NTR-5 polypeptide structural and functional variants through alternative post-translational processing.

The present invention also provides nucleic acid hybridization probes and replication / amplification primers having a mammalian NTR-5 cDNA specific sequence and sufficient to effect specific hybridization with SEQ ID NO: 1 or SEQ ID NO: 3. I will provide a. Demonstrating specific hybridization generally requires stringent conditions, such as 5 × SSPE (0.18 M NaC
1, hybridizing at a temperature of 42 ° C. in a buffer containing 30% formamide in 0.01 M NaPO ₄ , pH 7.7, 0.001 M EDTA) buffer, and 42 ° C. with 0.2 × SSPE Remains bound when washed with; preferably in buffer containing 50% formamide in 5 × SSPE buffer.
Hybridizing at a temperature of 0 ° C. and remaining bound when washing at 42 ° C. in 0.2 × SSPE buffer at 42 ° C. Mammal NTR
The -5 cDNA homolog is also known as BLASTX (Altschul et al. (1990).
) Basic Local Alignment Search Tool, J
. Mol. Biol. 215: 403-410) can be distinguished from one another using an alignment algorithm.

[0026] Mammalian NTR-5 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 diagnosis. Mammal NTR
-5 nucleic acids are also used to regulate the cellular expression or intracellular concentration or availability of an active mammalian NTR-5 polypeptide. A mammalian NTR-5 inhibitory nucleic acid is typically an antisense single-stranded sequence that includes the complement of the disclosed mammalian NTR-5 coding sequence. Antisense regulation of expression of a given mammalian NTR-5 polypeptide may utilize an antisense nucleic acid operably linked to a gene regulatory sequence. The cells are transfected with a vector containing a mammalian NTR-5 sequence having a promoter sequence oriented such that transcription of the gene results in an antisense transcript capable of binding to mRNA encoding endogenous mammalian NTR-5. You. Transcription of the antisense nucleic acid can be constitutive or inducible, and the vector can provide stable extrachromosomal maintenance or integration. Alternatively, the given mammal N
Single-stranded antisense nucleic acid that binds to genomic DNA or mRNA encoding a TR-5 polypeptide can be expressed in the host or transiently at a concentration that results in a substantial decrease in expression of the targeted polypeptide. Can be administered to the released target cells. Enhancement of mammalian NTR-5 expression is effected by introducing a mammalian NTR-5 nucleic acid that increases the functional expression of the corresponding gene product into the targeted cell type. Such a nucleic acid can be a mammalian NTR-5 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 include, but are not limited to, retrovirus-based transfection, or virus coat protein-liposome-mediated transfection.

The present invention provides efficient methods for identifying agents, compounds, and lead compounds for agents active at the level of mammalian NTR-5 regulatable cell function. Typically,
These screening methods involve assaying for a compound that modulates mammalian NTR-5 interaction with a native mammalian NTR-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. The preferred method follows automated, cost-effective, high-throughput screening of chemical libraries for lead compounds.

The in vitro binding assay utilizes a mixture of components comprising a mammalian NTR-5 polypeptide, which is a portion of the fusion product with another peptide or polypeptide, eg, a tag for detection or immobilization. Can be The assay mixture contains the native mammalian NTR-5 binding target. Although native binding targets can be used, it is often not possible to use some of them, as long as they provide the binding affinity and avidity for the mammalian NTR-5 of the invention that can be conveniently measured in the assay. preferable. 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. . Various other reagents may also be included such as salts, buffers, neutral proteins (eg, albumin), detergents, protease inhibitors, nuclease inhibitors, or 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 comprises, in the absence of the candidate pharmacological agent, a binding target wherein the mammalian NTR-5 polypeptide has a reference binding affinity;
Incubate under conditions that specifically bind the part or analog. The incubation period is selected for optimal binding, but is also minimized to facilitate rapid high-throughput screening.

After incubation, drug-biased binding between the mammalian NTR-5 polypeptide 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 bound from unbound components. Separation can be effected by a number of methods, including but not limited to precipitation or immobilization, followed by washing, for example, by membrane filtration or gel chromatography. For a cell-free binding assay, one of the components usually contains or is conjugated to a label. The label can provide direct detection, such as radioactivity, luminescence, optical or electron density, or indirect detection, such as an epitope tag or enzyme. Various methods depend on the nature of the label, and other assay components (optical or electron density, emission,
(Including, but not limited to, non-radiative energy transfer, or by way of non-limiting example, detection indirectly using an antibody conjugate). The difference in binding affinity of a mammalian NTR-5 polypeptide for a target in the absence of a drug as compared to the binding affinity in the presence of the drug indicates that the drug has a mammalian NTR-5 to the corresponding binding target. Fig. 7 illustrates modulating the binding of a polypeptide. As used herein, a difference is statistically significant and preferably exhibits a difference of at least 50%, more preferably at least 90%.

[0030] The present invention provides a method of altering the physiology of a cell, including the extracellular surface, which is in contact with a culture medium, wherein the method comprises modifying the polypeptide to affect a change in the physiology of the cell. Contacting the exogenous mammalian NTR-5 polypeptide with the medium under conditions that specifically interact with at least one component of the medium.

The present invention further provides a method of screening for a biologically active agent, the method comprising: a) in the absence of the agent, the polypeptide identifies the binding target with reference affinity. Incubating a mammalian NTR-5 polypeptide in the presence of a mammalian NTR-5 polypeptide-specific binding target and a candidate agent under conditions that specifically bind; b) determining a 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 drug has a binding affinity for the polypeptide to the binding target. The step of modulating the binding of

[0032] One embodiment of the present invention is an isolated mammalian NTR-5 polypeptide or a fragment thereof having mammalian NTR-5 specific activity comprising an amino acid sequence described herein.

Another embodiment of the present invention is directed to a mammalian NT comprising an amino acid sequence described herein.
A recombinant nucleic acid encoding an R-5 polypeptide or a fragment thereof having mammalian NTR-5 specific activity.

[0034] Yet another embodiment is an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3 as described herein, or the sequence of a native mammalian NTR-5 having at least 18 contiguous bases and Is a fragment that can specifically hybridize with a nucleic acid having

The invention also provides antibodies to a mammalian NTR-5 polypeptide described herein that are useful, for example, for detecting the polypeptide in diagnostic applications. For preparation of monoclonal antibodies to mammalian NTR-5 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 2).
56: 495-497), as well as the trioma technology, the human B cell hybridoma technology (Kozbor et al., 1983).
, Immunology Today 4:72), and the EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al., 1985, "
Monoclonal Antibodies and Cancer The The
rapy ", Alan R.A. Lis, Inc. 77-96) are within the scope of the present invention.

[0036] 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; Kozbor et al., 1983.
, Immunology Today 4: 72-79; Olsson et al., 19
82, Meth. Enzymol. 92: 3-16). Chimeric antibody molecules can be prepared that include a mouse antigen binding domain together with a human constant region (Morrison
Et al., 1984, Proc. Natl. Acad. Sci. U. S. A. 81: 6
851, Takeda et al., 1985, Nature 314: 452).

Various procedures known in the art can be used for the production of polyclonal antibodies against the mammalian NTR-5 polypeptide described herein. For the production of antibodies, various host animals can be immunized by injection of a mammalian NTR-5 polypeptide, or a fragment or derivative thereof, including, but not limited to, rabbits, mice, and rats. Various adjuvants can be used to increase the immunological response, depending on the host species, including Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surfaces such as lysolecithin Actives, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and BCG
(Bacille Calmette-Guerin) and Coryneba
Includes, but is not limited to, potentially useful human adjuvants such as C. terium parvum.

[0038] Molecular clones of antibodies to the selected mammalian NTR-5 polypeptide epitope can be prepared by known techniques. Recombinant DNA methodology (eg, M
aniatis et al., 1982, Molecular Cloning, A La.
boratemanual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY) can be used to construct a nucleic acid sequence encoding a monoclonal antibody molecule, or antigen-binding region thereof.

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 antibody molecules; Fab' fragments that can be produced by reducing the disulfide bridges of F (ab ') ₂ fragments, and papain and Includes, but is not limited to, Fab fragments that can be generated by treating an antibody molecule with a reducing agent. Antibody molecules can be purified by known techniques including, but not limited to, 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-5 Gene The amino acid sequences of known human and mouse members of the TNF family were
Used as a tblastn query to search the NIH EST database for random fragments of NA sequences (Altschul et al., (1990).
Basic local alignment search tool, J. Am.
Mol. Biol. 215: 403-10). Each query generated a list of hits, ie, EST sequences with substantial sequence similarity to the query sequence. Typically, hits at the top of the list are mRNA copies of the query protein,
Then E from other members of the family and random-chance similarities
ST supported.

Using the parser program, all members of the family and all of the hits from the search were combined and sorted. 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. I. M. A. G. FIG. 2 from E Consortium
Two mouse cDNA clones were identified as containing homologous sequences. Clone # 4
72300 ('300 clone) (GeneBank accession number AA036247)
) And clone # 427152 ('152 clone) (GeneBank accession number AA003356) were purchased from Research Genetics, Inc. (
Huntsville, AL) and ABI 373A DNA
sequencer and Taq Video Terminator C
Cycle Sequencing Kit (Applied Biosystem)
ms, Inc. , Foster City, CA).

Both the '300 and' 152 clones contained the mouse N
Mouse NTR-5, which contained one coding frame encoding a 215 amino acid protein designated as TR-5 (SEQ ID NO: 2), showed sequence similarity to members of the TNF receptor family.

[0044]

Embedded image The mouse NTR-5 cDNA shown in SEQ ID NO: 1 was used for the heart, brain, placenta, lung,
It was used as a probe to screen Northern blots of human mRNA from liver, skeletal muscle, kidney, and pancreas. Detectable expression levels of human NTR-5 were present in heart and kidney. Human heart cDNA library (Str
atagene Cat. # 936208) to six 243 mm x 243 mm
Plates are plated with 2.5 × 10 ⁵ plaques per plate, transferred to nitrocellulose using standard techniques known in the art, and
Probed with 5 probes. The nitrocellulose filter was washed with 0.5M NaP
155 ml of hybridization buffer containing salmon sperm DNA denatured by O ₄ (pH 7), 1% bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA and 100 ng / ml sonication Prehybridization was performed in the solution for 30 minutes at 65 ° C. ^{A 32} P-ATP-radiolabeled mouse NTR-5 probe was added to the prehybridization mixture, and hybridization was performed at 60 ° C. overnight. This filter
Washed several times with 2 × SSC at room temperature and 30 minutes at 60 ° C., and exposed to x-ray film at −80 ° C. overnight. 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 # 2, was amplified by PCR using an oligonucleotide specific for the T7 / T3 vector component, and the resulting PCR
The amplified DNA fragment was subjected to ABI 373A DNA sequencer and Taq Diodexy Terminator Cycle Sequence.
sing Kit (Applied Biosystems, Inc., Fos)
ter City, CA). The human clone # 2 sequence
An open reading frame encoding a 328 amino acid protein,
This protein has a signal peptide sequence of amino acids 1-29, amino acids 30-
Sequences for other members of the TNF receptor family, including the extracellular domain of 169, a transmembrane motif of amino acids 170-193, and the characteristic properties of the putative membrane-bound protein of the intracellular domain of amino acids 194-328. It had similarity (see SEQ ID NO: 4).

The nucleotide and deduced amino acid sequence of human clone # 2 is shown below.

[0046]

Embedded image Although 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 can be made without departing from the spirit or scope of the appended claims. Obtaining is readily apparent to one 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 description. Such modifications are intended to fall within the scope of the appended claims.

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Claims (20)

[Claims] 1. An isolated nucleic acid molecule encoding human NTR-5. 2. The isolated nucleic acid molecule of claim 1, comprising: (a) a nucleotide sequence comprising the coding region of human NTR-5 as set forth in SEQ ID NO: 3; (b) (a) A nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 and encodes a molecule having the biological activity of human NTR-5; or (c) without the degeneracy of the genetic code, (a) or A nucleic acid molecule having a sequence selected from the group consisting of: a nucleotide sequence that hybridizes to the nucleotide sequence of (b) and encodes a molecule having the biological activity of human NTR-5. 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. An isolated nucleic acid molecule encoding a mammalian NTR-5. 6. An isolated mammalian NTR-5 polypeptide. 7. An isolated human NTR-5 polypeptide encoded by the nucleic acid molecule of claim 2. 8. A host-vector system for the production of human NTR-5, 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 host-vector system according to claim 8 or 9 under conditions that allow for the production of human NTR-5 polypeptide and recover the human NTR-5 polypeptide produced. A method for producing a human NTR-5 polypeptide, comprising the step of: An antibody that specifically binds to the NTR-5 polypeptide 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-5 polypeptide according to claim 7, and a carrier. 14. A composition comprising the antibody according to claim 11 or 12, and a carrier. 15. A human NTR-5 polypeptide according to claim 7, an antibody according to claim 11 or 12, or a 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 mammalian NTR-5 fused to an immunoglobulin constant region.
A polypeptide comprising the extracellular portion of the polypeptide. 18. The polypeptide of claim 17, wherein said constant region is a human immunoglobulin γ-1 constant region. 19. A mammalian NTR-5 fused to an immunoglobulin Fc region.
A polypeptide comprising the extracellular portion of the polypeptide. 20. The polypeptide according to claim 19, wherein said Fc region is a human immunoglobulin γ-1 Fc region.