JP2002504818A - NTN-2 members of the 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                    NTN-2 member of the ligand family   All publications, patents, and patent applications cited herein are in each individual publication. Specifically and individually that the work, patent, or patent application should be incorporated by reference. , Is incorporated herein by reference.                                   Introduction Field of the invention   The field of the invention is polypeptide molecules that regulate cellular functions, Nucleic acid sequence to be encoded, and the use of the nucleic acid sequence and the polypeptide Is the law.background   Tumor necrosis factor α (TNF-α) is mainly produced by activated macrophages. Are cytokines. TNF-α stimulates T and B cell proliferation, and Induces expression of adhesion molecules in endothelial cells. This cytokine is also Plays an important role in host defense.   TNF-α activity is mediated by two different receptors, TNFR-p55 and TNFR-p75. Through. These two receptors are also mainly separated by activated lymphocytes. Mediates activity caused by soluble lymphotoxin alpha (LT-α) I do. Specific stimulation of TNFR-p55 may be associated with TNF, e.g., in vitro tumor cytotoxicity. Activity, expression of adhesion molecules in endothelial cells and keratinocytes, ceramide associated Activation of sphingomyelinase, activation of NF-κB, and manganese in increased Induces superoxide dismutase mRNA induction. The specific stimulation of TNFR-p75 , Mouse and human thymocytes and cytotoxic T cells, fibroblasts and Nazi Proliferative response of neural killer cells, as well as GM-CSF secretion in PC60 cells .   TNF, especially in combination with gamma interferon (IFN-γ), Selectively kill or kill malignant cell lines that are not matched by some other combination Has the ability to inhibit. However, the toxic side effects of TNF may be The clinical trial of cancer patients with TNF-α antitumor therapy Was disappointing. These toxic side effects are associated with TNF binding to the TNFR-p75 receptor. The cytotoxic activity in malignant cells was due to the TNFR-p55 receptor Due to the binding of TNF to TNF.                                Summary of the Invention   The present invention is a molecule that is homologous to tumor necrosis factor (TNF). The present invention Provided are methods and compositions relating to offspring, human NTN-2 polypeptides, and related nucleic acids I do. Poly-containing human NTN-2-specific domain and having human NTN-2-specific activity Peptides. This polypeptide is a host cell transformed with the nucleic acid of the present invention. Vesicles can be produced recombinantly. The invention relates to binding agents, such as specific antibodies, as well as diagnostics. Fragmentation (eg, a gene hybridization screen for human NTN-2 transcripts) Gene therapy to regulate human NTN-2 gene expression ), And the biopharmaceutical industry (eg, chemical libraries for derived pharmacological agents) Production and Use of the Compositions of the Invention in Reagents for Screening Provide a way to   Preferred uses for the human NTN-2 polypeptides of the present invention include added polypeptides. Extracellular surface that affects changes in media components and / or cell physiology Under conditions that specifically interact with the exogenous human NTN-2 polypeptide and cells or Physiology of cells, including extracellular surfaces, by contacting the medium around the cells Modifying the function. Screening for biologically active drugs A method is also preferred, where the polypeptide has a reference affinity in the absence of the agent. Under conditions that specifically bind the binding target, extracellular human NTN-2 polypeptide-specific binding Incubate human NTN-2 polypeptide in the presence of target and candidate agents Step: Binding of polypeptide to binding target to determine biased affinity with drug Detecting affinity affinity, wherein the difference between the drug-biased affinity and the reference affinity Demonstrating that the agent modulates binding of the polypeptide to the binding target. I do.   Based on homology to TNF, human NTN-2 was closely associated with the development of the disease, It is expected to be a mediator of immune regulation and inflammatory response. Lymphoid, Useful for regulating the expression, proliferation, and death of cells of hematopoiesis, and other lineages obtain. In addition, human NTN-2 has been shown to treat septic shock, autoimmune disease, and graft- It can help control the main disease. In addition, human NTN-2 polypeptide has its receptor Can be used to identify                             BRIEF DESCRIPTION OF THE FIGURES   Figure 1-Using a 608 nucleotide fragment of the human NTN-2 sequence as a probe Analysis of various human tissue-specific RNAs. Lanes 1 to 8 are as follows Are: 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, as well as human NTN-2 amino acid sequences, Or its functional human NTN-2 polyprotein having recognizable human NTN-2 specific activity. Provide a human NTN-2 polypeptide containing a recombinant polypeptide containing a peptide domain Offer. Thus, the polypeptide lacks the disclosed native human NTN-2 polypeptide. As a fusion product with a non-human NTN-2 polypeptide. Can be provided. The human NTN-2 polypeptide domain of the present invention is human NTN-2 specific. Have activity or function.   Many applications for human NTN-2 are suggested by its properties. Human NTN-2 Can be useful for studying and treating symptoms similar to those treated using TNF . In addition, human NTN-2 cDNA can be used, for example, for polypeptides in cell lines. Use of antibodies in assays or against oligonucleotide primers To amplify those with sequence similarity and how much human NTN-2 is present Oligonucleotides as primers in PCR tests to find out Can be useful as a diagnostic tool through the use of a code. Of course, human NTN-2 Separation also involves isolating its putative receptor, another human NTN-2 binding polypeptide, You And / or provide clues to study its antagonist properties.   Human NTN-2 specific activity or function can be conveniently performed in vitro, cell-based, or Are in vivo assays-e.g., in vitro binding assays, cell culture assays, (Eg, immune response, gene therapy, transgenic, etc.) Can be determined. Binding assays are based on specific binding between the binding label and the human NTN-2 polypeptide. Include any assays in which offspring interactions are evaluated. The binding target is a natural binding A target, or a non-native binding target, such as a specific immune polypeptide such as an antibody, or 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. Ripeptides no longer accompany some of their naturally associated substances, and And preferably at least about 0.5% by weight of the total polypeptide in a given sample, And more preferably at least about 5%; a "pure" polypeptide Peptides comprise at least about 90% of the weight of total polypeptide in a given sample, and Preferably at least about 99%. Polypeptides and polypeptides of the invention Domains can be synthesized, produced by recombinant techniques, or purified from cells. Can be A variety of molecular and biochemical methods are available for the biochemical synthesis, Can be used for expression and purification, for example, Molecular Cloning, A Laborat ory Manual (Sambrook et al., Cold Spring Harbor Laboratory), Current Protocols  in Molecular Biology (edited by Ausubel et al., Greene Publ. Assoc., Wiley-Interscien ce, NY).   The polypeptides of the present invention can be used as immunogens in screening assays. Biological activity to regulate cell growth, differentiation, and / or function, etc. Various uses are found, including use as reagents. For example, the present invention Polypeptide may affect changes in media components and / or cell physiology Exogenous human NTN-2 polypeptide under conditions that specifically interact with the extracellular surface Contacting the cells or the medium surrounding the cells Methods for regulating the physiology of cells. According to these methods, The extracellular surface contains plasma membrane associated receptors; exogenous human NTN-2 is Not produced or otherwise unnatural levels, timing or physiological location And a suitable medium is an in vitro culture medium or the like. And physiological fluids such as blood, synovial fluid and the like. The polypeptide is Injection, promoter-specific expression of recombinant enzymes, targeting of lipid vesicles Introduced in a specific population of cells by any convenient method such as controlled delivery , Expressed, or suppressed.   The present invention identifies natural and unnatural human NTN-2 specific binding agents, such agents And methods of making and its use in diagnosis, therapy and pharmaceutical expression I will provide a. Human NTN-2 specific binding agent is a recombinant protein receptor in the body NTN-2 specific receptor such as a T cell-like antibody or T cell antigen receptor (Eg, Harlow and Lane (1988) Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory), and also 1, 2, and Non-natural binders identified in assays such as and hybrid screening , And identified by screening chemical libraries as described below Contains non-natural binders. Certain drugs of interest modulate human NTN-2 function.   The present invention provides a human NTN-2 nucleic acid, which comprises a translatable transcript, a hybridizer. Use as an immobilization probe, PCR primer, diagnostic nucleic acid, etc., and Detecting the presence of the human NTN-2 gene and gene transcript and additional human N To detect or amplify nucleic acids encoding TN-2 homologs and structural analogs Various applications are found, including uses in   The nucleic acids of the invention are synthetic / non-naturally occurring sequences and / or are isolated, That is, some of the materials that are associated in their natural state are no longer associated, preferably At least about 0.5%, more preferably less than about 0.5% by weight of total nucleic acid present in a given fraction At least about 5%, and usually recombination occurs on non-native sequences or on native chromosomes. It is meant to include the native sequence linked to nucleotides other than the link. Book Nucleic acids comprising the nucleotide sequences disclosed herein and fragments thereof, A chromosome that is immediately adjacent or naturally adjacent to a sequence other than the one linked on the natural chromosome Less than 10 kb, preferably 2 kb immediately adjacent to sequences other than those ligated above At the end adjacent to the less native flanking region, such a sequence or Or fragments. Nucleic acids are usually RNA or DNA, but often Nucleotides to provide altered stability This is advantageous for using logs and the like.   The amino acid sequence of the disclosed human NTN-2 polypeptide can be selected from selected expression systems (Hol (ler et al. (1993) Gene 136: 323-328; Martin et al. (1995) Gene 154: 150-166). Used to reverse translate nucleic acids encoding human NTN-2 polypeptides optimized for Used in the isolation of nucleic acid sequences encoding natural human NTN-2 Used to generate degenerate oligonucleotide primers and probes for ("GCG" software, Genetics Computer Group, Inc., Madison, WI) . The nucleic acid encoding human NTN-2 can be part of an expression vector, and Human NTN-, for transgenic animals, for expression and screening Functional, such as the efficacy of candidate drugs for diseases associated with 2-mediated signaling It can be incorporated into recombinant host cells, such as for research. Expression systems are based on alternative post-translational Processing affects human NTN-2 polypeptide structural and functional variants Selected and / or adapted to do so.   The present invention also has a human NTN-2 cDNA specific sequence and Sufficient nucleic acid hybridization to effect hybridization Provide lobe and replication / amplification primers. Specific hybridization , Generally requires stringent conditions, eg, 5 × SSPE (0.18M NaCl, 0.01M NaPO_Four, PH 7.7, 0.001M EDTA) 30% formaldehyde in buffer Hybridization in a buffer containing amide at a temperature of 42 ° C., and 0.2 × SSP Remains bound when washing at 42 ° C. with E; preferably 5 × SSPE buffer Hybridization at a temperature of 42 ° C in a buffer containing 50% formamide in solution And remains bound when washed at 42 ° C with 0.2 × SSPE buffer at 42 ° C It is. The human NTN-2 cDNA homolog is also available from BLASTX (Altschul et al. (1990) Basic Lo cal Alignment Search Tool, J. Mol. Biol. 215: 403-410) Can be distinguished from other polypeptides using a consensus algorithm.   Human NTN-2 hybridization probe is useful in clinical and laboratory samples Find use in identifying wild-type and mutant alleles. Mutation Alleles are allele-specific oligonucleotides for high-throughput ratio clinical diagnosis. Used to generate otide (ASO) probes. Human NTN-2 nucleic acids are also active To regulate cellular expression or intracellular concentration or availability of neutral human NTN-2. Used. The human NTN-2 inhibitory nucleic acids typically comprise the disclosed native human NTN-2 encoding Antisense-single-stranded sequence containing the complement of the sequence. Prescribed human NTN-2 polypeptide Antisense regulation of peptide expression can be achieved by an operably linked gene regulatory sequence. Chisense nucleic acids can be used. In cells, gene transcription encodes endogenous human NTN-2 Promoter arrangement to obtain an antisense transcript that can bind to the mRNA Transfected with a vector containing the human NTN-2 sequence along with the sequence. Antise Transcription of the sense nucleic acid can be constitutive or inducible, and the vector Extra-color body maintenance or incorporation may be provided. Alternatively, a given human NTN-2 polypeptide Single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding Concentration in the host at a concentration that results in a substantial decrease in the expression of the targeted polypeptide. Alternatively, it can be administered to target cells that are temporarily isolated from the host. Human NTN-2 expression Enrichment targets human NTN-2 nucleic acids that increase the functional expression of the corresponding gene product. As a result of introduction into the targeted cell type. Such a nucleic acid is Human NTN-2 expression vector, up-regulates functional expression of endogenous alleles Vector, or replacement vector for targeted modification of a mutant allele -Can be. Techniques for introducing nucleic acids into viable cells are known in the art. Known and retroviral-based transfection, viral Protein-liposome-mediated transfection and the like.   The present invention relates to a drug active at the level of human NTN-2 regulatable cell function, An effective method for identifying agents, compounds, and lead compounds is provided. Generally, these Screening methods modulate human NTN-2 interaction with natural human NTN-2 binding targets Assaying for a compound of interest. Protein-protein binding For binding agents, including assays, immunoassays, cell-based assays, etc. Are provided. A preferred method is to use a chemical compound for the lead compound. Follow Ibrary's automated, cost-effective, high-throughput screening.   In vitro binding assays use a mixture of components containing the human NTN-2 polypeptide. This is for other peptides or polypeptides, for example, for detection or immobilization. May be part of a fusion product with a tag or the like. The assay mixture contains native human NTN- Includes 2 binding targets. Intact binding targets can be used, but some are not Conveniently measurable binding affinity and avidity for human NTN-2 It is often preferred to use some of them as long as they provide Assay mix The article also includes a candidate pharmacological agent. Candidate drugs include many chemical classes, Typically, these are organic compounds, preferably small organic compounds, and Obtained from a variety of sources, including libraries of synthetic or natural compounds. Salt, buffer Agent, neutral protein (eg, albumin), detergent, protease Various other reagents such as inhibitors, nuclease inhibitors, antimicrobial agents, etc. May be included. The mixture components can be added for any purpose that provides the requisite binding, The incubation can then be performed at any temperature that facilitates optimal binding. Mixed The compound shows that in the absence of the candidate pharmacological agent, human NTN-2 has a reference binding affinity. Under conditions that specifically bind the cell-binding target, part, or analog It is private. The incubation period is chosen for optimal binding, It is also minimized to facilitate rapid high throughput ratio screening.   After incubation, drug bias between human NTN-2 and one or more binding targets The binding is detected by any convenient method. Cell-free binding type assay For the separation step, to separate unbound and bound components Often used. Separation is effected by sedimentation, fixation, etc. This can be done by bran filtration or washing by gel chromatography. Nothing For cell binding assays, one of the components usually contains or is labeled with a label. Be combined. Labels can be directly associated with radioactivity, fluorescence, optical or electron density, etc. Detection, or indirect detection, such as an epitope or enzyme, may be provided. An example For example, by optical or electron density, radiative emission, non-radiative energy transfer, or Depends on the nature of the label and assay components, such as indirect detection with antibody conjugate Various methods can be used to detect the label. Binding in the presence of the drug Binding of human NTN-2 polypeptide to its target in the absence of drug compared to affinity The difference in affinity indicates that the agent binds human NTN-2 polypeptide to the corresponding binding target. Indicates adjustment. As used herein, differences are statistically significant. , And preferably, they show a difference of at least 50%, more preferably at least 90%.   The present invention provides a method for modifying a physiological function of a cell including an extracellular surface by contacting with a medium. Providing the method, wherein the polypeptide affects the change in physiology of the cell. Specifically interact with the medium and at least one component of the extracellular surface to exert Under operating conditions, exogenous human NTN-2 polypeptide is contacted with this medium. Process.   The present invention further provides methods for screening for biologically active agents. The method comprises the steps of: a) in the absence of the agent, the polypeptide has a reference affinity Under conditions that specifically bind the binding target, extracellular human NTN-2 polypeptide-specific binding Incubate human NTN-2 polypeptide in the presence of target and candidate agents Step b) the polypeptide to the binding target to determine drug-biased affinity The step of detecting the binding affinity of the peptide, where the drug-biased affinity and reference The difference between the affinities indicates that the agent modulates the binding of the polypeptide to this binding target. To indicate that the   One embodiment of the present invention provides an isolated method comprising the amino acid sequence described herein. A human NTN-2 polypeptide or a fragment thereof having human NTN-2 specific activity is there.   Another embodiment of the present invention provides a human NTN-2 polypeptide comprising an amino acid sequence described herein. A recombinant nucleic acid encoding a polypeptide or a fragment thereof having human NTN-2 specific activity. It is a fragment.   In yet another embodiment, the isolated comprises a nucleotide sequence described herein. Nucleic acids, or of natural human NTN-2 cDNA having at least 18 contiguous bases and To specifically hybridize with a nucleic acid having the sequence described herein in the presence Enough of that fragment.   The present invention also relates to the present invention, which is useful for detecting polypeptides, for example, in diagnostic applications. Antibodies to the human NTN-2 polypeptides described herein are provided. This human NTN-2 For preparation of monoclonal antibodies to the polypeptide, see Continuous Cell Culture in Cultures. Any technique that provides for the production of antibody molecules by the vesicle strain can be used. For example, Ko hler and Milstein (1975, Nature256: 495-497). The hybridoma technique, the trioma technique, the human B cell hybridoma technique ( Kozbor et al., 1983, Immunology TodayFour: 72), and human monoclonal antibodies EBV-hybridoma technique for production (Cole et al., 1985, Monoclonal Antibodi es and Cancer Therapy, "Alan R. Liss, Inc. Pages 77-96), etc. It is in the box.   Monoclonal antibodies for diagnostic or therapeutic use are human monoclonal antibodies Alternatively, it can be a chimeric human-mouse (or other species) monoclonal antibody. Hi Monoclonal antibodies can be raised by any of a number of techniques known in the art. (Eg, Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 730). 8-7312: Kozbor et al., 1983, Immunology Today 4: 72-79; Olsson et al., 1982, Meth. E nzymol. 92: 3-16). Texture containing mouse antigen-binding domain with human constant region Can be prepared (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81 : 6851, Takeda et al., 1985, Nature 314: 452).   Various procedures known in the art include the human NTN-2 polypeptides described herein. Can be used for the production of polyclonal antibodies against the epitopes of the antibodies. antibody For the production of the various host animals, human NTN-2 polypeptide, or its Can be immunized by injection of a fragment or derivative, including rabbits, mice, And rats, but are not limited to these. Depending on the host species, Various adjuvants may be used to increase compliance, including Freund's (Complete and incomplete), inorganic gels such as aluminum hydroxide, lysoresit Surfactants, pluronic polyols, polyanions, peptides, Oil emulsion, keyhole limpet hemocyanin, dinitrophenol, and BCG (Bacille Calmette-Guerin) andCorynebacterium parvumPotentially like Including but not limited to useful human adjuvants.   The molecular clone of the antibody against the selected human NTN-2 polypeptide epitope is Can be prepared by known techniques. Recombinant DNA methodology (eg, Maniatis et al., 1) 982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laborato ry, Cold Spring Harbor, New York) is a monoclonal antibody molecule Or a nucleic acid sequence encoding the antigen binding region thereof. .   The invention provides for antibody molecules and fragments of such natal molecules. Minute Antibody fragments which contain the idiotype of the offspring can be produced by known techniques. You. For example, such fragments are produced by pepsin cleavage of antibody molecules. F (ab ')_TwoFragment; F (ab ')_TwoReduces disulfide bridges in fragments Fragments that can be produced by performing the method; and papain and a reducing agent. Fab fragments that can be generated by treating antibody molecules with It is not limited to these. Antibody molecules can be obtained by known techniques, such as immunoadsorption or immunoassay. Like affinity chromatography, HPLC (high performance liquid chromatography) Can be purified by various chromatographic methods, 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 Partial Human NTN-2 Coding Sequence   Amino acid sequences of all known human and mouse members of the TNF family , Tbl to search NIH EST database for random fragments of mRNA sequences Used as astn question (Altsschul, Stephen F., Warren Gish, Webb Miller Eugene W .; Myers, and David J. Lipman (1990). Basic local alignment se arch tool. J. Mol. Biol. 215: 403-10). Each question is a list of hits, That is, an EST sequence having substantial sequence similarity to the query sequence was generated. Typical The hit at the top of the list is the mRNA copy of the protein in question, then Compatible with ESTs from other members of the family and random-chance similarities did.   Use the parser program to search from all of the family members All of the kits were combined and classified. This corresponds to a known protein. All the quick reductions of hits that were possible. The rest of the hits will be The conservation of the symbolic sequence motif was analyzed. Perform an additional database search , Duplicate ESTs were identified. Partial nucleotide and predicted amino acid sequence of human NTN-2 The columns were determined as follows:   Using the nucleotide sequence of SEQ ID NO: 1 as a question, additional database A search was performed to identify overlapping ESTs. Two from the I.M.A.G.E.Consortium Additional clones were identified as containing homologous sequences. These clones, Geneba nk accession numbers AA166695 and T87299 were purchased from Research Genetics, Inc. (Huntsville, A L), and ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle  Sequence using Sequencing Kit (Applied Biosystems, Inc., Foster City, CA) Were determined. An alignment of the two additional clones with SEQ ID NO: 1 The total length of leotide is indicated. Oligonucleotides are designed based on partial human sequences. And use it as a primer for the reverse transcriptase reaction and for PCR. Used. A 608 nucleotide long sequence was obtained and the full length sequence was Used as a probe for release. Example 2Isolation and sequencing of a full-length cDNA clone encoding human NTN-2   The human placenta cDNA library in λgt-10 was purchased from Clontech Laboratories, Inc. (Pa lo Alto, CA). Plaque, 1.25 x 10⁶/ 20 × 20cm plate density And replica filters were collected according to standard procedures (Sam brook et al., Molecular Cloning: A Laboratory Manual, Second Edition, p. 8.46, Cold Sprin. g Harbor Laboratory, Cold Spring Harbor, New York). Filter the array number A probe corresponding to nucleotides 216 to 824 of the hNTN-2 sequence shown in Screening was performed at a lingency (2 × SSC, 65 ° C.). Connect the probe 0.5 mg / ml salmon sperm DNA to reduce non-specific binding of the probe to Hybridization was carried out at 65 ° C. in a hybridization solution containing the mixture. fill The filters were washed with 2 × SSC at 65 ° C. and exposed to X-ray film overnight. Strong c Five positive clones showing an hybridization signal were selected and also cD Fragment produced when PCR amplified using oligo from NA vector .hNTN-2 Sequencing   The coding region from each of the five clones was analyzed using an ABI 373A DNA sequencer and And Taq Dydeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., (Foster City, CA). Full length hNTN-2 from one of the clones The nucleotide and deduced amino acid sequences of the coding sequence are described below: Example 3-Tissue-specific expression of hNTN-2   A fragment corresponding to nucleotides 216 to 824 of the hNTN-2 sequence shown in SEQ ID NO: 3 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 Lab. oratories, Inc. (Palo Alto, CA) and 0.5 M NaPO4 (pH 7), 1% bovine blood Clear albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA, and 100 ng / ml ultrasound A radiolabeled hNTN-2 probe was added at 65 ° C in the presence of the treated denatured salmon sperm DNA. I hybridized. Wash the filter with 2x SSC, 0.1% SDS at 65 ° C, For 16 hours at -70 ° C with screening and x-ray film enhancement Geography.   hNTN-2 probe transcribes 2.7 kb in human heart, placenta, pancreas, and lung tissue Strongly hybridize to the organism (Figure 1) and weakly to RNA from brain and liver. I hybridized. Weaker levels of expression may also be found in skeletal muscle and kidney Was. The high expression of hNTN-2 in heart tissue makes the present invention useful for treating heart disease. It could suggest that it could be used. HNTN-2 expression in lung and pancreatic tissues Shows that the invention can be used to treat lung and / or pancreas related diseases I can insist.   The invention has been described in some detail by way of illustration and example for purposes of clarity of understanding. Although described in detail, certain changes and modifications are intended to be within the scope of the appended claims or What can be done without departing from the scope will be apparent to those skilled in the art in light of the teachings of the present invention. It is readily apparent.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 11/00 C07K 16/24 C07K 14/525 19/00 16/24 C12N 1/19 19/00 1 / 21 C12N 1/19 C12P 21/02 C 1/21 21/08 5/10 C12N 15/00 ZNAA C12P 21/02 5/00 B 21/08 A61K 37/02 (81) Designated countries 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, MD) , RU, TJ, TM), L, 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 United States 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. An isolated human NTN-2 having an amino acid sequence as set forth in SEQ ID NO: 4. Polypeptide.   8. Production of human NTN-2, comprising the vector of claim 3 or 4 in a host cell. A host-vector system for production.   9. The host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. The host-vector system of claim 8, wherein   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. 4. The ligand body according to 1.   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.