EP1242580A1 - Procedes et substances relatifs a des polypeptides de type semaphorine et a des polynucleotides - Google Patents

Procedes et substances relatifs a des polypeptides de type semaphorine et a des polynucleotides

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Publication number
EP1242580A1
EP1242580A1 EP00989452A EP00989452A EP1242580A1 EP 1242580 A1 EP1242580 A1 EP 1242580A1 EP 00989452 A EP00989452 A EP 00989452A EP 00989452 A EP00989452 A EP 00989452A EP 1242580 A1 EP1242580 A1 EP 1242580A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
protein
cells
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00989452A
Other languages
German (de)
English (en)
Other versions
EP1242580A4 (fr
Inventor
Bryan J. Boyle
George Y. Yeung
Matthew C. Arterburn
Nancy K. Mize
Y. Tom Tang
Chenghua Liu
Radoje T. Drmanac
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nuvelo Inc
Original Assignee
Hyseq Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/653,274 external-priority patent/US6635742B1/en
Application filed by Hyseq Inc filed Critical Hyseq Inc
Priority claimed from PCT/US2000/035158 external-priority patent/WO2001053466A1/fr
Publication of EP1242580A1 publication Critical patent/EP1242580A1/fr
Publication of EP1242580A4 publication Critical patent/EP1242580A4/fr
Withdrawn legal-status Critical Current

Links

Definitions

  • plexins which contain a distantly related sema domain
  • neuropilins have been characterized as semaphorin receptors.
  • This invention further provides cloning or expression vectors comprising at least a fragment of the polynucleotides set forth above and host cells or organisms transformed with these expression vectors.
  • Useful vectors include plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.
  • the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
  • the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
  • Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • a host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
  • polypeptides ofthe invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.
  • the invention also provides compositions comprising a polypeptide of the invention.
  • kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
  • the invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g. , bind to) the polypeptides of the invention.
  • the semaphorin-like polypeptide of SEQ ID NO: 4 is an approximately 1086-amino acid secreted, transmembrane protein with a predicted molecular mass of approximately 121 kDa unglycosylated.
  • Protein database searches with the BLASTX algorithm Altschul S.F. et al. , J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol. 21:403-10 (1990), herein incorporated by reference
  • SEQ LD NO: 4 is homologous to human KIAA1479 and human Semaphorin Y proteins.
  • GSCs germ line stem cells
  • primordial stem cells primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.
  • primordial germ cells PGCs
  • PGCs primordial germ cells
  • PGCs primordial germ cells
  • PGCs primordial germ cells
  • the PGCs, the GSCs and the ES cells are capable of self-renewal. Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.
  • the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.
  • fifteen-mer segments can be used.
  • the probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.
  • operably linked refers to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.
  • operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.
  • pluripotent refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.
  • translated protein coding portion means a sequence which encodes for the full length protein which may include any leader sequence or a processing sequence.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitations of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
  • an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell.
  • a sequence may be natarally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.
  • additional exemplary stringent hybridization conditions include washing in 6X SSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55 °C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).
  • polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 4, 6-8, 11 or 13 or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. Also included are the cDNA inserts of any of the clones identified herein.
  • a polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
  • operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO:4, 6-8, 11 or 13 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-3, 5 or 12 are additionally provided.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g. , an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. , phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acerylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-iso ⁇ entenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5 ' -me
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.
  • ribozymes e.g. , hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
  • oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g. , Krol et al , 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g. , Zon, 1988, Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g. , a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • the present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.
  • host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.
  • the present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomycespom.be, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, ox any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.
  • the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
  • the invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 4, 6-8, 11 or 13 or the corresponding full length or mature protein; and "substantial equivalents" thereof (e.g., with at least about 65% , at least about
  • the present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.
  • the protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.
  • the mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.
  • the sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.
  • proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which it is expressed.
  • Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
  • the present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.
  • degenerate variant is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g. , an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.
  • Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
  • the polypeptide or protein is purified from bacterial cells which natarally produce the polypeptide or protein.
  • One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g. , Scopes, Protein
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A- agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or irnmunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification.
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g. , silica gel having pendant methyl or other aliphatic groups
  • RP- HPLC reverse-phase high performance liquid chromatography
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.
  • the polypeptides of the invention include analogs (variants).
  • the polypeptides of the invention include semaphorin-like analogs.
  • Transgenic ammals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.
  • Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.
  • Transgenic animals, preferably non-human mammals are produced using methods as described in U.S. Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference.
  • polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.
  • Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments. These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.
  • compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.
  • a polypeptide of the present invention may also exhibit angiogenic activity.
  • anaphylaxis serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.
  • the efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al. , Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • a polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.
  • tamor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g. , a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and ⁇ 2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • polypeptide of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pitaitary. See, for example, U.S. Pat. No. 4,798,885.
  • a polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.
  • the activity of a polypeptide of the invention may, among other means, be measured by the following methods.
  • Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91 :562-572, 1972; Ling et al., Natare 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Natare 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T- cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
  • Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic compositions e.g.
  • compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
  • Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer. For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.
  • compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the
  • An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.
  • Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (
  • therapeutic compositions of the invention may be used for prophylactic treatment of cancer.
  • hereditary conditions and/or environmental situations e.g. exposure to carcinogens
  • In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.
  • These in vitro models include proliferation assays of cultared tamor cells, growth of cultared tamor cells in soft agar (see Freshney, (1987) Cultare of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY Ch 18 and Ch 21), tamor systems in nude mice as described in Giovanella et al. , J. Natl. Can. Inst., 52: 921-30 (1974), mobility and invasive potential of tamor cells in Boy den Chamber assays as described in Pilkington et al.
  • a polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods.
  • radioisotopes include, but are not limited to, tritium and carbon-14 .
  • colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.
  • toxins include, but are not limited, to ricin.
  • Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.
  • Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structaral analogs of known compounds or compounds that are identified as "hits" or "leads” via natural product screening.
  • the sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtares for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.
  • Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998).
  • Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries.
  • the binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.
  • toxins e.g., ricin or cholera
  • the toxin- binding molecule complex is then targeted to a tamor or other cell by the specificity of the binding molecule for a polypeptide of the invention.
  • the binding molecules may be complexed with imaging agents for targeting and imaging purposes.
  • BIAcore assays can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
  • infectious lesions in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
  • Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.
  • therapeutics which elicit any of the following effects may be useful according to the invention:
  • neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g. , weakness, motor neuron conduction velocity, or functional disability.
  • motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio- Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
  • disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary
  • polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.
  • Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.
  • the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.
  • the amount of polypeptide administered per dose will be in the range of about O.Ol ⁇ g/kg to 100 mg/kg of body weight, with the preferred dose being about 0. l ⁇ g/kg to 10 mg/kg of patient body weight.
  • semaphorin-like polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.
  • the vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.
  • cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors are administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • the polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.
  • a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.
  • Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models. Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.
  • protein or other active ingredient of the present invention When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95 % protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • protein or other active ingredient of the present invention When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable protein or other active ingredient solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • the compounds may be formulated for parenteral administration by injection, e.g. , by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g. , in ampules or in multi- dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use.
  • Such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.
  • Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.
  • the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.
  • the amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the natare of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response. Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about 0.1 ⁇ g to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g. , bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • IGF I insulin like growth factor I
  • the addition of other known growth factors, such as IGF I may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell cultare (i.e. , the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.
  • Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90% .
  • the effective local concentration of the drug may not be related to plasma concentration.
  • An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 ⁇ g/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 ⁇ g/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g. , lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Natare.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al. , 1988; and Presta, Curr. Op. Struct. BioL. 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al. , 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al. , 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. BioL. 227:381 (1991); Marks et al., J. Mol. BioL.
  • nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in cultare, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co- transfected into a suitable host organism.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al.. J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule.
  • Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tamor targets.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991). Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.
  • bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • Fc ⁇ R Fc receptors for IgG
  • Fc ⁇ R such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • V function so as to enhance, e.g., the effectiveness of the antibody in treating cancer.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemofherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemofherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • toxin e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof
  • a radioactive isotope i.e., a radioconjugate
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 2,2 Bi, 131 I, 13 Tn, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis- azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • a nucleotide sequence of the present invention can be recorded on computer readable media.
  • computer readable media refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium.
  • a skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.
  • the choice of the data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.
  • the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • a skilled artisan can readily adapt any number of data processor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
  • nucleotide sequences SEQ ID NO: 1-3, 5 or 12 or a representative fragment thereof; or a nucleotide sequence at least 95 % identical to any of the nucleotide sequences of the SEQ ID NO: 1-3, 5 or 12 in computer readable form a skilled artisan can routinely access the sequence information for a variety of purposes.
  • Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.
  • the examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. BioL 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.
  • ORFs open reading frames
  • Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.
  • the minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.
  • data storage means refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.
  • search means refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.
  • a variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA).
  • EMBL Smith-Waterman
  • BLASTN BLASTN
  • BLASTA NPOLYPEPTIDEIA
  • a "target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.
  • a skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.
  • the most preferred sequence length of a target sequence is from about 10 to 100 amino acids, or from about 30 to 300 nucleotide residues.
  • searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing may be of shorter length.
  • a target structaral motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
  • target motifs include, but are not limited to, enzyme active sites and signal sequences.
  • Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
  • fragments of the present invention can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.
  • Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 3:173 (1979); Cooney et al. , Science 15241:456 (1988); and Dervan et al. , Science 251:1360 (1991)) or to the mRNA itself (antisense - Olmno, J. Neurochem.
  • the present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.
  • methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a .complex is detected, a polynucleotide of the invention is detected in the sample.
  • Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.
  • methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.
  • such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.
  • Conditions for incubating a nucleic acid probe or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and natare of the nucleic acid probe or antibody used in the assay.
  • One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol.
  • test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.
  • the test sample used in the above-described method will vary based on the assay format, natare of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.
  • kits which contain the necessary reagents to carry out the assays of the present invention.
  • the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.
  • a compartment kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross- contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe.
  • Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • labeled nucleic acid probes labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
  • novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g. , where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection). See, e.g., Kunkel et al., U.S. Pat. NO. 5,413,778.
  • Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.
  • the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in the SEQ ID NO: 1-3, 5 or 12, or bind to a specific domain of the polypeptide encoded by the nucleic acid.
  • said method comprises the steps of:
  • such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.
  • Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.
  • Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).
  • compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).
  • Compounds, such as compounds identified via the methods of the invention can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.
  • the agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.
  • the agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.
  • agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.
  • agents may be rationally selected or designed.
  • an agent is said to be "rationally selected or designed" when the agent is chosen based on the configuration of the particular protein.
  • one skilled in the art can readily adapt currentiy available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides," In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.
  • one class of agents of the present invention can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.
  • One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.
  • Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 3:173 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene
  • Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.
  • Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with natarally occurring nucleotide sequences.
  • the hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-3, 5 or 12. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-3, 5 or 12 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.
  • any suitable hybridization technique can be employed, such as, for example, in situ hybridization.
  • PCR as described in US Patents Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.
  • probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.
  • the probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.
  • Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.
  • the nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.
  • the nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY.
  • Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 198 If). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.
  • the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals. 5.20 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
  • CovaLink NH is a polystyrene surface grafted with secondary amino groups ( > NH) that serve as bridge-heads for further covalent coupling.
  • CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink exclusively at the 5 '-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussenet al, (1991) Anal Biochem 198(1) 138-42.
  • the oligonucleotide terminus must have a 5 '-end phosphate group. It is, perhaps, even possible for biotin to be covalendy bound to CovaLink and then streptavidin used to bind the probes. More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95°C and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (l-Melmr), is then added to a final concentration of 10 mM l-Melm?.
  • a ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.
  • An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.
  • addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directiy on a glass surface, as described by Fodor et al. (1991) Science 251(4995) 767-73 , incorporated herein by reference.
  • Probes may also be immobilized on nylon supports as described by Van Nesset al. (1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal Biochem 169(1) 104-8; all references being specifically incorporated herein.
  • DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directiy from genomic DNA or cDNA by PCR or other amplification methods. Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.
  • advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2- 5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed).
  • Each of the subarrays may represent replica spotting of the same samples.
  • a selected gene segment may be amplified from 64 patients.
  • the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8 x 12 cm membrane.
  • Subarrays may contain 64 samples, one from each patient. Where the 96 subarrays are identical, the dot span may be 1 mm 2 and there may be a 1 mm space between subarrays.
  • a plurality of novel nucleic acids were obtained from a cDNA library prepared from fetal liver-spleen (Hyseq clone identification numbers 5688868 (SEQ ID NO: 1)) using standard PCR, sequencing by hybridization sequence signature analysis, and Sanger sequencing techniques.
  • the inserts of the library were amplified with PCR using primers specific for vector sequences flanking the inserts. These samples were spotted onto nylon membranes and interrogated with oligonucleotide probes to give sequence signatures.
  • the clones were clustered into groups of similar or identical sequences, and single representative clones were selected from each group for gel sequencing. The 5' sequence of the amplified inserts was then deduced using the reverse Ml 3 sequencing primer in a typical Sanger sequencing protocol.
  • SEQ ID NO: 2 The nucleic acid of the present invention, designated as SEQ ID NO: 2 was assembled using SEQ ID NO: 1 as a seed. Then a recursive algorithm was used to extend the seed into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 114, gb pri 114, and UniGene version 101) that belong to this assemblage. The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage. Inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95% .
  • the nearest neighbor result for the assembled contigs were obtained by a FASTA version 3 search against Genpept release 114, using FASTXY algorithm.
  • FASTXY is an improved version of FASTA alignment which allows in-codon frame shifts.
  • the nearest neighbor result showed the closest homologue for each assemblage from Genpept (and contains the translated amino acid sequences for which the assemblage encodes).
  • the nearest neighbor results is set forth below:
  • a polypeptide (SEQ ID NO:4) was predicted to be encoded by SEQ ID NO:3 as set forth below.
  • the polypeptide was predicted using a software program called BLASTX which selects a polypeptide based on a comparison of translated novel polynucleotides to known polypeptides.
  • the initial methionine starts at position 434 of SEQ ID NO: 3 and the putative stop codon, TAG, begins at position 3692 of the nucleotide sequence.
  • Figure 1 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4) and the human KIAA1479 protein (SEQ ID NO: 9) indicating that the two sequences share 100% similarity over 429 amino acid residues of SEQ ID NO: 4 and 100% identity over the same 429 amino acid residues of SEQ ID NO: 4.
  • Figure 2 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e. SEQ ID NO: 4) and the human semaphorin Y protein (SEQ ID NO: 10) indicating that the two sequences share 71 % similarity over 540 amino acid residues of SEQ ID NO: 4 and 52% identity over the same 540 amino acid residues of SEQ ID NO: 4.
  • a predicted approximately sixteen-residue signal peptide is encoded from approximately residue 1 to residue 16 inclusive of SEQ ID NO: 4 (SEQ ID NO: 6).
  • the signal peptide region was predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • a predicted approximately twenty-nine-residue transmembrane peptide is encoded from approximately residue 671 to residue 699 inclusive of SEQ ID NO: 4 (SEQ ID NO: 7).
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • EXAMPLE 4 A. Expression of SEQ ID NO: 4 in cells Chinese Hamster Ovary (CHO) cells or other suitable cell types are grown in DMEM
  • SEQ ID NO: 1-3, 5 or 12 The expression of SEQ ID NO: 1-3, 5 or 12 in various tissues is analyzed using a semi- quantitative polymerase chain reaction-based technique.
  • Human cDNA libraries are used as sources of expressed genes from tissues of interest (adult bladder, adult brain, adult heart, adult kidney, adult lymph node, adult liver, adult Jung, adult ovary, adult placenta, adult rectum, adult spleen, adult testis, bone marrow, thymus, thyroid gland, fetal kidney, fetal liver, fetal liver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage).
  • Gene-specific primers are used to amplify portions of the SEQ ID NO: 1-3, 5 or 12 sequence from the samples.
  • Amplified products are separated on an agarose gel, transferred and chemically linked to a nylon filter.
  • the filter is then hybridized with a radioactively labeled ( 33 P-dCTP) double-stranded probe generated from SEQ ID NO: 1-3, 5 or 12 using a Klenow polymerase, random-prime method.
  • the filters are washed (high stringency) and used to expose a phosphorimaging screen for several hours. Bands indicate the presence of cDNA including SEQ ID NO: 1-3, 5 or 12 sequences in a specific library, and thus mRNA expression in the corresponding cell type or tissue.

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  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention se rapporte à de nouveaux polynucléotides et à des polypeptides codés par ces polynucléotides et à des mutants ou variants de ceux-ci, qui correspondent à un nouveau polypeptide de type sémaphorine sécrété humain. Ces polynucléotides comprennent des séquences d'acide nucléique isolées à partir d'une bibliothèque d'ADNc provenant du foie/de la rate d'un foetus (numéros d'identification de clones Hyseq 5688868 (SEQ ID:1)). D'autres aspects de cette invention concernent des vecteurs contentant des processus pour produire de nouveaux polypeptides de type sémaphorine sécrétés humains, et des anticorps spécifiques de ces polypeptides.
EP00989452A 1999-12-23 2000-12-22 Procedes et substances relatifs a des polypeptides de type semaphorine et a des polynucleotides Withdrawn EP1242580A4 (fr)

Applications Claiming Priority (9)

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US47127599A 1999-12-23 1999-12-23
US471275 1999-12-23
US48872500A 2000-01-21 2000-01-21
US488725 2000-01-21
US55231700A 2000-04-25 2000-04-25
US552317 2000-04-25
US09/653,274 US6635742B1 (en) 2000-01-25 2000-08-31 Antibodies specific for semaphorin-like polypeptides
US653274 2000-08-31
PCT/US2000/035158 WO2001053466A1 (fr) 1999-12-23 2000-12-22 Procedes et substances relatifs a des polypeptides de type semaphorine et a des polynucleotides

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WO1999032622A2 (fr) * 1997-12-19 1999-07-01 Zymogenetics, Inc. Semaphorine zsmf-3
EP1385953A2 (fr) * 2000-12-08 2004-02-04 Curagen Corporation Proteines et acides nucleiques codant pour elles

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ARTIGIANI S. ET AL.: "Plexins, semaphorins, and scatter factor receptors: a common root for cell guidance signals ?" IUBMB LIFE, vol. 48, no. 5, 1999 - 1999, pages 477-482, XP009007733 *
DATABASE EMBL [Online] 194970 bp, 9 November 1999 (1999-11-09) "Homo sapiens chromosome 15 clone RP11-485p3 map 15" retrieved from EBI Database accession no. AC013334 XP002234326 *
DATABASE EMBL [Online] 2770 bp, 17 November 1997 (1997-11-17) "Mus musculus semaphorin VIa mRNA, complete cds" retrieved from EBI Database accession no. AF030430 XP002234329 -& ZHOU L. ET AL.: "Cloning and expression of a novel murine semaphorin with structural similarity to insect semaphorin I." MOL CELL NEUROSCI., vol. 9, no. 1, 1997, pages 26-41, XP002109255 *
DATABASE EMBL [Online] 3364 bp, 15 November 2002 (2002-11-15) "Human semaphorin-like gene #5" retrieved from EBI Database accession no. ABS64384 XP002234332 -& DATABASE EMBL [Online] 1088 aa, 15 November 2002 (2002-11-15) "Human semaphorin-like protein #5" retrieved from EBI Database accession no. ABG79177 XP002234433 -& WO 02 64791 A (CURAGEN CORP) 22 August 2002 (2002-08-22) *
DATABASE EMBL [Online] 3432 bp, 2 September 1998 (1998-09-02) "Human semaphorin Y encoding cDNA" retrieved from EBI Database accession no. AAV28915 XP002234327 -& DATABASE EMBL [Online] 930 aa, 2 September 1998 (1998-09-02) "Human semaphorin Y" retrieved from EBI Database accession no. AAW57260 XP002234328 *
DATABASE EMBL [Online] RNA, 5924 pb, 23 May 2000 (2000-05-23) "Homo sapiens mRNA for KIAA1479" retrieved from EBI Database accession no. AB040912 XP002234330 -& DATABASE EMBL [Online] 1022 aa, 1 October 2000 (2000-10-01) "Hypothetical protein KIAA1479" retrieved from EBI Database accession no. Q9P249 XP002234430 *
DATABASE EMBL [Online] RNA, HUM, 1588 bp, 29 September 2000 (2000-09-29) "Homo sapiens cDNA FLJ11598 fis, clone HEMBA1003866, moderately similar to Mus musculus semaphorin VIa mRNA" retrieved from EBI Database accession no. AK021660 XP002234331 *
See also references of WO0153466A1 *

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CA2395763A1 (fr) 2001-07-26
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AU2595501A (en) 2001-07-31
AU2596501A (en) 2001-07-31
CA2395736A1 (fr) 2001-07-26
EP1254256A2 (fr) 2002-11-06
AU783303B2 (en) 2005-10-13
EP1254256A4 (fr) 2005-01-19

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