JP2002517977A - NTN-2 member of the TNF ligand family - Google Patents

Abstract

  (57) [Summary] Provided are human NTN-2 polypeptides and related nucleic acids. Human NTN-2 polypeptides comprising a human NTN-2 domain having specific human NTN-2 activity are included. Polypeptides can be produced recombinantly from host cells transformed with the nucleic acids of the present invention. Also provided are specific binding agents and methods of making and using the compositions of the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION All publications, patents, and patent applications cited herein are specifically and individually stating that each individual publication, patent, or patent application is to be incorporated by reference. , Is incorporated herein by reference. INTRODUCTION Field of the Invention The field of the invention is polypeptide molecules that modulate cellular functions, nucleic acid sequences encoding the polypeptide, and methods of using the nucleic acid sequence and the polypeptide. Background Tumor necrosis factor α (TNF-α) is a cytokine produced primarily by activated macrophages. TNF-α stimulates T and B cell proliferation and induces expression of adhesion molecules in endothelial cells. This cytokine also plays an important role in host defense against infection. TNF-α activity is mediated by two different receptors, TNFR-p55 and TNFR-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 includes, for example, TNF activity such as in vitro tumor cytotoxicity, expression of adhesion molecules in endothelial cells and keratinocytes, activation of sphingomyelinase with concomitant increase in ceramide, activation of NF-κB , As well as manganese superoxide dismutase mRNA. 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. TNF has the ability to selectively kill or inhibit malignant cell lines that are not matched by some other combination of cytokines, especially in combination with gamma interferon (IFN-γ). However, clinical trials of cancer patients with TNF-α antitumor therapy were disappointing, as the toxic side effects of TNF prevented obtaining effective dose levels in humans. These toxic side effects were due to TNF binding to the TNFR-p75 receptor, whereas the cytotoxic activity in malignant cells was due to TNF binding to the TNFR-p55 receptor. SUMMARY OF THE INVENTION The present invention is a molecule that is homologous to tumor necrosis factor (TNF). The invention provides methods and compositions relating to this molecule, human NTN-2 polypeptide, and related nucleic acids. Polypeptides comprising a human NTN-2 specific domain and having human NTN-2 specific activity are included. This polypeptide can be produced recombinantly from host cells transformed with the nucleic acids of the invention. The present invention is directed to binding agents such as specific antibodies, as well as diagnostics (eg, gene hybridization screening for human NTN-2 transcripts), treatments (eg, gene therapy to regulate human NTN-2 gene expression). And methods for making and using the compositions of the present invention in the biopharmaceutical industry (eg, reagents for screening chemical libraries for lead pharmacological agents). Preferred uses for the human NTN-2 polypeptides of the present invention include those in which the added polypeptide specifically interacts with extracellular surfaces that affect changes in media components and / or cell physiology. Modifying the physiology of the cell, including the extracellular surface, by contacting the exogenous human NTN-2 polypeptide with the cell or the medium surrounding the cell. Also preferred is a method of screening for a biologically active agent, which comprises the step of extracellular human NTN-2 polyprotein under conditions in which the polypeptide specifically binds the binding target with reference affinity in the absence of the agent. Incubating the human NTN-2 polypeptide in the presence of the peptide-specific binding target and the candidate drug; detecting the binding affinity of the polypeptide for the binding target to determine the drug-biased affinity And a step wherein the difference between the drug-biased affinity and the reference affinity indicates that the agent modulates the binding of the polypeptide to the binding target. Based on homology to TNF, human NTN-2 is expected to be a mediator of immune-regulatory and inflammatory responses, closely related to disease development. It may be useful for regulating the development, proliferation, and death of lymphoid, hematopoietic, and other lineage cells. Also, human NTN-2 can help control septic shock, autoimmune diseases, and graft-host disease. In addition, human NTN-2 polypeptide can be used to identify its receptor. BRIEF DESCRIPTION OF THE FIGURES FIG. 1-Northern analysis of various human tissue-specific RNAs using a 608 nucleotide fragment of the human NTN-2 sequence as a probe. Lanes 1-8 are as follows: heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to native human NTN-2 polypeptides and human NTN-2 amino acid sequences or functional human NTN-2 polypeptide domains having assayable human NTN-2 specific activity. Human NTN-2 polypeptides, including recombinant polypeptides. Thus, the polypeptide can be a deletion mutant of the disclosed native human NTN-2 polypeptide, and can be provided, for example, as a fusion product with a non-human NTN-2 polypeptide. The human NTN-2 polypeptide domain of the present invention has human NTN-2 specific activity or function. Many applications for human NTN-2 are suggested by its properties. Human NTN-2 may be useful for studying and treating conditions similar to those treated using TNF. In addition, human NTN-2 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 NTN-2 is present The use of oligonucleotides as primers in PCR tests to make them may be useful as diagnostic tools. Of course, isolation of human NTN-2 also provides clues to isolating its putative receptor, other human NTN-2 binding polypeptides, and / or studying its antagonist properties. Human NTN-2 specific activity or function is determined by convenient in vitro, cell-based, or in vivo assays—eg, in vitro binding assays, cell culture assays, animals (eg, immune response, gene therapy, transgenics, etc.). Can be done. Binding assays include any assay in which the specific molecular interaction between a binding target and a human NTN-2 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 NTN-2 specific agent as identified in the assays described below. A polypeptide of the present invention can be isolated or pure-an "isolated" polypeptide no longer accompanies some of the materials with which it is associated in its natural state, and preferably in a given sample. "Pure" polypeptide comprises at least about 0.5%, and more preferably at least about 5%, of the total polypeptide by weight; at least about 90%, by weight, of the total polypeptide in a given sample , And preferably at least about 99%. The polypeptides and polypeptide domains of the present invention can be synthesized, produced by recombinant techniques, or purified from cells. A variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression, and purification of the compositions of the invention, including, for example, Molecular Cloning, A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory). See Current Protocols in Molecular Biology (Ed. Ausubel et al., Greene Publ. Assoc., Wiley-Interscience, NY). The polypeptides of the present invention find various uses, including as immunogens, as targets in screening assays, as bioactive reagents to modulate cell proliferation, differentiation, and / or function, and the like. For example, the present invention provides for exogenous human NTN-2 polypeptides under conditions in which the added polypeptide specifically interacts with components of the culture medium and / or the extracellular surface affecting changes in cell physiology. A method for regulating the physiology of a cell, including the extracellular surface, by contacting the cell with a medium or a medium surrounding the cell. According to these methods, the extracellular surface contains a plasma membrane associated receptor; exogenous human NTN-2 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 natural and non-natural human NTN-2 specific binding agents, methods for identifying and producing such agents, and their use in diagnosis, therapy, and drug development. Human NTN-2 specific binding agents include human NTN-2 specific receptors such as somatically recombinant protein receptor-like antibodies or T cell antigen receptors (eg, Harlow and Lane (1988) Antibodies , A Laboratory Manual, Cold Spring Harbor Laboratory), and also other natural binders 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 drugs of interest modulate human NTN-2 function. The present invention provides human NTN-2 nucleic acids, which are used as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc., and detect the presence of human NTN-2 gene and gene transcripts Various applications find use in detecting and amplifying nucleic acids encoding additional human NTN-2 homologs and structural analogs. The nucleic acids of the invention are synthetic / non-naturally occurring sequences and / or are isolated, ie, no longer associated with some of their materials associated in their natural state, and preferably have the total amount present in a given fraction. It comprises at least about 0.5%, more preferably at least about 5%, by weight of the nucleic acid, and usually the recombination comprises a non-native sequence or a native sequence linked to nucleotides other than those linked on the native chromosome. means. Nucleic acids comprising the nucleotide sequences disclosed herein and fragments thereof may be directly adjacent to sequences other than those linked on the native chromosome, or directly linked to sequences other than those linked on the native chromosome. Such a sequence or fragment is included at the end adjacent to an intact flanking region of less than 10 kb, preferably less than 2 kb. The nucleic acid is usually RNA or DNA, but is often advantageous for use with nucleic acids containing other bases or nucleotide analogs to provide altered stability. The amino acid sequence of the disclosed human NTN-2 polypeptide was optimized for a selected expression system (Holler et al. (1993) Gene 136: 323-328; Martin et al. (1995) Gene 154: 150-166). Used to reverse translate a nucleic acid encoding a human NTN-2 polypeptide, or to generate degenerate oligonucleotide primers and probes for use in isolating a nucleic acid sequence encoding a native human NTN-2 ("GCG" software, Genetics Computer Group, Inc., Madison, WI). The nucleic acid encoding human NTN-2 can be part of an expression vector and, for example, for expression and screening, for transgenic animals, for candidate drugs for diseases associated with human NTN-2 mediated signaling 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 NTN-2 polypeptide structural and functional variants by selective post-translational processing. The invention also provides nucleic acid hybridization probes and replication / amplification primers having a human NTN-2 cDNA specific sequence and sufficient to effect specific hybridization with SEQ ID NO: 1. Prove specific hybridization generally requires stringent conditions, for example, 5 × SSPE a _{(0.18M NaCl, 0.01M NaPO 4,} pH 7.7,0.001M EDTA) 30% formamide in the buffer solution Hybridizing at a temperature of 42 ° C. in a buffer containing, and remaining bound when washing at 42 ° C. with 0.2 × SSP E; preferably 50% formamide in 5 × SSPE buffer Hybridization at a temperature of 42 ° C. in a buffer containing solution and remaining bound when washing at 42 ° C. with 0.2 × SSPE buffer at 42 ° C. The human NTN-2 cDNA homolog is also distinguished from other polypeptides using an alignment algorithm such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403-410). Can be done. Human NTN-2 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 ratio clinical diagnosis. Human NTN-2 nucleic acids are also used to modulate cellular expression or intracellular concentration or availability of active human NTN-2. A human NTN-2 inhibitory nucleic acid is typically an antisense-single-stranded sequence that includes the complement of the disclosed native human NTN-2 coding sequence. Antisense regulation of the expression of a given human NTN-2 polypeptide can employ an antisense nucleic acid operably linked to a gene regulatory sequence. Cells are transfected with a vector containing human NTN-2 sequences with a promoter sequence oriented so that transcription of the gene results in an antisense transcript capable of binding to mRNA encoding endogenous human NTN-2. Transcription of the antisense nucleic acid can be constitutive or inducible, and the vector can provide stable extrachromosomal maintenance or integration. Alternatively, a single-stranded antisense nucleic acid that binds to genomic DNA or mRNA encoding a given human NTN-2 polypeptide is present in the host or at a concentration that results in a substantial decrease in expression of the targeted polypeptide. Enrichment in human NTN-2 expression, which can be administered to target cells that are temporarily isolated from, introduces human NTN-2 nucleic acid into the targeted cell type that increases the functional expression of the corresponding gene product Brought by. Such a nucleic acid can be a human NTN-2 expression vector, a vector that up-regulates 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 retrovirus-based transfection, virus coat protein-liposome-mediated transfection, and the like. The present invention provides an effective method of identifying an agent, compound, or lead compound for an agent active at the level of human NTN-2 regulatable cell function. Generally, these screening methods involve assaying for compounds that modulate human NTN-2 interaction with a natural human NTN-2 binding target. Various assays for binding agents are provided, including protein-protein binding assays, immunoassays, cell-based assays, and the like. A preferred method follows automated, cost-effective, high-throughput ratio screening of chemical libraries for lead compounds. In vitro binding assays use a mixture of components that include a human NTN-2 polypeptide, which can be part of a fusion product with another peptide or polypeptide, such as a tag for detection or immobilization. The assay mixture contains the natural human NTN-2 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 NTN-2 of the present invention that can be conveniently measured in an assay. . The assay mixture also contains the candidate pharmacological agent. Candidate agents comprise a number of chemical classes, but typically they are organic compounds, preferably small organic compounds, and are obtained from a 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 in the absence of the candidate pharmacological agent, human NTN-2 specifically binds a cell binding target, part, or analog having a reference binding affinity. 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 NTN-2 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 can be performed by precipitation, fixation, and the like, and then by washing, eg, 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. Various methods for detecting the label depending on the nature of the label and the assay components, such as, for example, by optical or electron density, radiative emission, non-radiative energy transfer, or indirectly detected with the antibody conjugate, Can be used. The difference in binding affinity of a human NTN-2 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 NTN-2 polypeptide that has a corresponding binding target. And modulates binding. As used herein, differences are statistically significant, and preferably represent at least 50%, more preferably at least 90%, differences. The present invention provides a method of altering the physiology of a cell, including the extracellular surface, in contact with a medium, the method comprising: And contacting the exogenous human NTN-2 polypeptide with the medium under conditions that specifically interact with at least one component of the extracellular surface. The 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, a condition under which the polypeptide specifically binds the binding target with reference affinity. Incubating the human NTN-2 polypeptide in the presence of an extracellular human NTN-2 polypeptide-specific binding target and a candidate agent; b) binding to this binding target to determine drug-biased affinity Detecting the binding affinity of the polypeptide, wherein the difference between the drug-biased affinity and the reference affinity is that the agent modulates the binding of the polypeptide to the binding target. And the step of One embodiment of the present invention is an isolated human NTN-2 polypeptide comprising an amino acid sequence described herein, or a fragment thereof having human NTN-2 specific activity. Another embodiment of the present invention is a recombinant nucleic acid encoding a human NTN-2 polypeptide comprising an amino acid sequence described herein or a fragment thereof having human NTN-2 specific activity. Yet another embodiment is an isolated nucleic acid comprising a nucleotide sequence described herein, or described herein in the presence of a native human NTN-2 cDNA having at least 18 contiguous bases and Is a fragment thereof sufficient to specifically hybridize with a nucleic acid having the sequence The present invention also provides antibodies to the human NTN-2 polypeptides described herein that are useful, for example, for detecting polypeptides in diagnostic applications. For the preparation of monoclonal antibodies to the human NTN-2 polypeptide, any technique which provides for the production of antibody molecules by continuous cell lines in culture can be used. For example, Ko Hler and Milstein: originally developed the hybridoma technique by (1975, Nature 256 495-497), as well as the trioma technique, the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72 ), and human monoclonal EBV-hybridoma techniques for producing antibodies (Cole et al., 1985, "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc. pages 77-96) and the like are within the scope of the invention. Monoclonal antibodies for diagnostic or therapeutic uses 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. USA. 80: 730 8-7312; Kozbor et al., 1983). Olmsson 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. Acad. Sci. USA. 81: 6851, Takeda et al., Nature 314: 452). Various procedures known in the art can be used for the production of polyclonal antibodies against the epitopes of the human NTN-2 polypeptide described herein. For the production of antibodies, various host animals, including but not limited to rabbits, mice, and rats, can be immunized by injection of a human NTN-2 polypeptide, or a fragment or derivative thereof. 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 Including, but not limited to, active substances, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum Not done. Molecular clones of antibodies to the selected human NTN-2 polypeptide epitope can be prepared by known techniques. Recombinant DNA methodology (see, eg, Maniatis et al., 1998, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) encodes a monoclonal antibody molecule, or antigen-binding region thereof. Used to construct a nucleic acid sequence. 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; 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, for example, immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof. The following examples are provided for illustrative purposes and do not provide a limitation. Example 1-Cloning and Sequencing of Partial Human NTN-2 Coding Sequence The amino acid sequences of all known human and mouse members of the TNF family were used as tbl astn queries to search the NIH EST database for 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 question generated a list of hits, ie, EST sequences with substantial sequence similarity to the query sequence. Typically, hits at the top of the list corresponded to mRNA copies of the protein in question, followed by ESTs from other members of the family and random-chance similarities. The parser program was used to combine and sort all of the hits from searches with all of the family members. 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. Additional database searches were performed to identify duplicate ESTs. The partial nucleotide and deduced amino acid sequence of human NTN-2 was determined as follows: Using the nucleotide sequence of SEQ ID NO: 1 as a question, an additional database search was performed to identify overlapping ESTs. Two additional clones from the IMAGE consortium were identified as containing homologous sequences. These clones, Genebank accession numbers AA166695 and T87299, were purchased from Research Genetics, Inc. (Huntsville, AL) and sequenced using an ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). Alignment of two additional clones with SEQ ID NO: 1 showed a total length of 680 nucleotides. Oligonucleotides were designed based on the partial human sequence and used for reverse transcription reactions and as primers for PCR. A 608 nucleotide long sequence was obtained and used as a probe to isolate the full length sequence as described below. Example 2 -Isolation and sequencing of a full length cDNA clone encoding human NTN-2 A human placental cDNA library in λgt-10 was obtained from Clontech Laboratories, Inc. (Pa lo Alto, CA). Plaques were plated at a density of 1.25 × 10 ^6/20 × 20cm plate, and replica filters taken following standard procedures (Sam brook et al., Molecular Cloning: A Laboratory Manual, Second Edition, pp. 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The filters were screened with a probe corresponding to nucleotides 216-824 of the hNTN-2 sequence shown in SEQ ID NO: 3 at normal stringency (2xSSC, 65 ° C). The probe was hybridized at 65 ° C. in a hybridization solution containing 0.5 mg / ml salmon sperm DNA to reduce non-specific binding of the probe to the filter. Filters were washed at 65 ° C. with 2 × SSC and exposed to X-ray film overnight. Five positive clones showing strong hybridization signals were selected and also produced fragments when PCR amplified using oligos from the cDNA vector. hNTN-2 sequencing The coding regions from each of the five clones were sequenced using an ABI 373A DNA sequencer and a Taq Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The nucleotide and deduced amino acid sequences of the full length hNTN-2 coding sequence from one of the clones are described below: Example 3 Tissue-Specific Expression of hNTN-2 A fragment corresponding to nucleotides 216-824 of the hNTN-2 sequence shown in SEQ ID NO: 3 was radiolabeled and used for Northern analysis of various human tissue-specific RNAs. . Northern blots containing poly A + RNA from several human tissues were obtained from Clontech Laboratories, Inc. (Palo Alto, Calif.) And 0.5% NaPO4 (pH 7), 1% bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA, and 100 ng / ml sonicated denatured salmon sperm DNA. Hybridized at 65 ° C to a radiolabeled hNTN-2 probe in the presence. Filters were washed with 2 × SSC, 0.1% SDS at 65 ° C. and autoradiographed at −70 ° C. for 16 hours with screen and X-ray film intensifier. The hNTN-2 probe strongly hybridized to the 2.7 kb transcript in human heart, placenta, pancreas, and lung tissue (FIG. 1) and weakly hybridized to RNA from brain and liver. Weaker levels of expression could also be found in skeletal muscle and kidney. High expression of hNTN-2 in heart tissue may suggest that the present invention can be used to treat heart disease. The expression of hNTN-2 in lung and pancreatic tissues may suggest that the present invention can be used to treat lung and / or pancreatic related diseases. Although the present 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. This will be readily apparent to one skilled in the art in light of the teachings of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 C07K 16/24 C07K 14/52 19/00 16/24 C12N 1/19 19/00 1 / 21 C12N 1/19 C12P 21/08 1/21 C12Q 1/68 A 5/10 C12N 15/00 ZNAA C12P 21/08 5/00 A C12Q 1/68 A61K 37/02 (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, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, M , 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, GW, 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 Valenzuela, David New York 11010, Franklin Square, Grunge Street 216

Claims (1)

[Claims]   1. An isolated nucleic acid molecule encoding human NTN-2.   2. 2. The isolated of claim 1, having a sequence selected from the group consisting of: Nucleic acid molecule:   (a) a nucleotide sequence comprising the coding region of human NTN-2 as set forth in SEQ ID NO: 3 ;   (b) hybridizing to the nucleotide sequence of (a) under stringent conditions; A nucleotide sequence encoding a molecule having the biological activity of human NTN-2; Or   (c) If there is no degeneracy in the genetic code, the nucleotide sequence of (a) or (b) A nucleic acid that encodes a molecule that redisys and has the biological activity of human NTN-2 Reotide sequence.   3. A vector comprising the nucleic acid molecule according to claim 1.   4. The nucleic acid molecule has an expression control sequence capable of directing its expression in a host cell. 4. The vector of claim 3, which is operably linked.   5. The vector according to claim 3 or 4, which is a plasmid.   6. An isolated human encoded by the nucleic acid molecule according to claim 1 or 2. A NTN-2 polypeptide.   7. Single chick human NTN-2 having an amino acid sequence as set forth in SEQ ID NO: 4 Polypeptide.   8. A human NTN-2 comprising the vector according to claim 3 or 4 in the host cell. Host-vector system for the production of   9. The claim, wherein the host cell is a bacterial, yeast, insect, or mammalian cell. A host-vector system according to claim 8.   10. 10. A host according to claim 8 or 9 under conditions allowing the production of cerberus. Growing the cells of the vector system, and the human NTN-2 thus produced; A method for producing human NTN-2, comprising a step of collecting.   11. An antibody that specifically binds human NTN-2 according to claim 6 or 7.   12. The antibody according to claim 11, which is a monoclonal antibody.   13. The human NTN-2 according to claim 6 or 7, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a rear.   14． An antibody according to claim 11 or 12, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:   15. In methods of treatment of the human or animal body or in diagnostic methods A human NTN-2 according to claim 6 or 7, for use, according to claim 11 or 12. 15. The antibody of claim 13 or the composition of claim 13 or 14.   16. A polypeptide produced by the method of claim 10.   17． A ligand form comprising human NTN-2 fused to an immunoglobulin constant region.   18. 18. The immunoglobulin constant region is the Fc portion of human IgG1. The ligand body according to any one of the above.   19. In methods of treatment of the human or animal body or in diagnostic methods 19. A ligand body according to claim 17 or 18 for use.   20. A polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4.