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WO1999002664A1 - Convertase n-arginine dibasique humaine - Google Patents

Convertase n-arginine dibasique humaine Download PDF

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Publication number
WO1999002664A1
WO1999002664A1 PCT/US1998/014022 US9814022W WO9902664A1 WO 1999002664 A1 WO1999002664 A1 WO 1999002664A1 US 9814022 W US9814022 W US 9814022W WO 9902664 A1 WO9902664 A1 WO 9902664A1
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Prior art keywords
polypφtide
nrd convertase
convertase
nrd
seq
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PCT/US1998/014022
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English (en)
Inventor
Sanjay Kumar
Stephanie Van Horn
Michael William Lark
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Smithkline Beecham Corporation
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Publication of WO1999002664A1 publication Critical patent/WO1999002664A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to NRD convertase family, hereinafter referred to as h-NRD convertase. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides. BACKGROUND OF THE INVENTION
  • NRDs N-arginine dibasic convertase class
  • metalloendopeptidases which have been suggested to activate numerous proteins including somatostatin-28 and dynorphins A and B.
  • the metallo-NRDs are related to protease m from E. coli and to insulinase.
  • Furin has been proposed to activate this family of membrane type matrix metalloproteinases as well as other proteins including TGF-b [Dubois, et al. J. Biol. Chem. 270(18): 10618-10624, 1995] and endopeptidase-24.18 [Milhiet, et al., Biochem. J. 309: 683- 688, 1995].
  • Furin cleaves C-terminal to the Arg-X-Arg/Lys-Arg basic sequence and has been shown to activate an mtMMP-related enzyme, stromelysin-3, at this site [Pel and Weiss, Nature 375: 244-247, 1995].
  • furin is the enzyme responsible for activation of membrane type matix metalloproteinases in vivo. It is possible that furin, or another endopeptidase such as an NRD could be responsible for this activation in vivo.
  • the present invention describes a novel member of the NRD family of endopeptidases. This indicates that the NRD convertase family has an established, proven history as therapeutic targets.
  • NRD convertase family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease.
  • diseases including, but not limited to, chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoprolife
  • the invention relates to h-NRD convertase polypeptides and recombinant materials and methods for their production.
  • Another aspect of the invention relates to methods for using such h-NRD convertase polypeptides and polynucleotides.
  • Such uses include the treatment of chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg flammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • autoimmune diseases eg flammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AIDS, metabolic
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with h-NRD convertase imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate h-NRD convertase activity or levels.
  • h-NRD convertase refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or an allelic variant thereof.
  • h-NRD convertase activity or h-NRD convertase polypeptide activity or "biological activity of the h-NRD convertase or h-NRD convertase polypeptide” refers to the metabolic or physiologic function of said h-NRD convertase including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said h-NRD convertase.
  • h-NRD convertase gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or allelic variants thereof and/or their complements.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polyp ⁇ tide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polyp ⁇ tide refers to any p ⁇ tide or protein comprising two or more amino acids joined to each other by peptide bonds or modified p ⁇ tide bonds, i.e., p ⁇ tide isosteres.
  • Polyp ⁇ tide refers to both short chains, commonly referred to as peptides, oligop ⁇ tides or oligomers, and to longer chains, generally referred to as proteins. Polyp ⁇ tides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in a polyp ⁇ tide, including the p ⁇ tide backbone, the amino acid side- chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polyp ⁇ tide. Also, a given polypeptide may contain many types of modifications.
  • Polyp ⁇ tides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • Variant is a polynucleotide or polyp ⁇ tide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
  • Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polyp ⁇ tide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polyp ⁇ tide. Generally, differences are limited so that the sequences of the reference polyp ⁇ tide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polyp ⁇ tide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polyp ⁇ tide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non- naturally occurring variants of polynucleotides and polyp ⁇ tides may be made by mutagenesis techniques or by direct synthesis.
  • "Identity” is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING:
  • identity is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J. , et al , Nucleic Acids Research (1984) 12(1): 387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al , J Molec Biol (1990) 215:403).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence exc ⁇ t that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
  • a polynucleotide having a nucleotide sequence at least 95 % identical to a reference nucleotide sequence up to 5 % of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5 % of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polyp ⁇ tide having an amino acid sequence having at least, for example, 95 % "identity" to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polyp ⁇ tide is identical to the reference sequence exc ⁇ t that the polyp ⁇ tide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2.
  • up to 5 % of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5 % of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the present invention relates to h-NRD convertase polypeptides (or h-NRD convertase proteins) .
  • the h-NRD convertase polypeptides include the polyp ⁇ tide of SEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polyp ⁇ tides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO: 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99 % are highly preferred.
  • h- NRD convertase polyp ⁇ tides having the amino acid sequence which have at least 80% identity to the polyp ⁇ tide having the amino acid sequence of SEQ ID NO: 2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95 % identity to SEQ LD NO:2. Furthermore, those with at least 97- 99 % are highly preferred.
  • h-NRD convertase polyp ⁇ tide exhibit at least one biological activity of h-NRD convertase.
  • the h-NRD convertase polyp ⁇ tides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • Fragments of the h-NRD convertase polyp ⁇ tides are also included in the invention.
  • a fragment is a polyp ⁇ tide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned h-NRD convertase polyp ⁇ tides.
  • fragments may be "free-standing," or comprised within a larger polyp ⁇ tide of which they form a part or region, most preferably as a single continuous region.
  • R ⁇ resentative examples of polyp ⁇ tide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of h-NRD convertase polyp ⁇ tide.
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
  • Preferred fragments include, for example, truncation polyp ⁇ tides having the amino acid sequence of h-NRD convertase polyp ⁇ tides, exc ⁇ t for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha- helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Other preferred fragments are biologically active fragments.
  • Biologically active fragments are those that mediate h-NRD convertase activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
  • polyp ⁇ tide fragments retain the biological activity of the h- NRD convertase, including antigenic activity.
  • fragment is that having the amino acid sequence of SEQ ID NO: 4.
  • Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions — i.e. , those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • the h-NRD convertase polyp ⁇ tides of the invention can be prepared in any suitable manner.
  • Such polyp ⁇ tides include isolated naturally occurring polyp ⁇ tides, recombinantly produced polyp ⁇ tides, synthetically produced polyp ⁇ tides, or polyp ⁇ tides produced by a combination of these methods. Means for preparing such polyp ⁇ tides are well understood in the art.
  • h-NRD convertase polynucleotides include isolated polynucleotides which encode the h-NRD convertase polyp ⁇ tides and fragments, and polynucleotides closely related thereto. More specifically, h-NRD convertase polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a h-NRD convertase polyp ⁇ tide of SEQ ED NO: 2, and polynucleotides having the particular sequences of SEQ ID NOS: 1 and 3.
  • h-NRD convertase polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the h-NRD convertase polyp ⁇ tide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to that of SEQ ED NO:l over its entire length.
  • polynucleotides at least 90% identical are particularly preferred, and those with at least 95 % are especially preferred.
  • h-NRD convertase polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 to hybridize under conditions useable for amplification or for use as a probe or marker.
  • the invention also provides polynucleotides which are complementary to such h-NRD convertase polynucleotides.
  • h-NRD convertase of the invention is structurally related to other proteins of the NRD convertase family, as shown by the results of sequencing the cDNA encoding human h-NRD convertase.
  • the cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 162 to 3614) encoding a polyp ⁇ tide of 1151 amino acids of SEQ ID NO:2.
  • the amino acid sequence of Table 2 (SEQ ID NO:2) has about 93.6% identity (using BestFit) in 1151 amino acid residues with rat NRD convertase (GENBANK Accession # L27124) (A.R. Herotti et al. , Proc. Natl. Acad. Sci., USA.
  • amino acid sequence of Table 1 (SEQ ID NO: 2) has about 33.4% identity to rat insulin-degrading enzyme (GENBANK Accession ft X67269) over 981 amino acids residues (H. Baumeister et al., FEES Lett. 317:250-254, 1993).
  • the nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 89.4% identity (using BestFit) in 3684 nucleotide residues with rat NRD convertase (GENBANK Accession # L27124) (A.R. Pierotti et al. , Proc. Natl. Acad. Sci., USA. 91:6078-6082, 1994).
  • nucleotide sequence of Table 1 (SEQ ED NO:l) has about 53.3 % identity to drosophila insulin-degrading enzyme (GENBANK Accession ft M58465) over 843 nucleotide base residues (W. Kuo et al., Mol. Endocrinol. 4:1580-1591, 1990).
  • h-NRD convertase polyp ⁇ tides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polyp ⁇ tides and polynucleotides, and their utility is obvious to anyone skilled in the art.
  • a nucleotide sequence of a human h-NRD convertase (SEQ ID NO: 1).
  • One polynucleotide of the present invention encoding h-NRD convertase may be obtained using standard cloning and screening, from a cDNA hbrary derived from mRNA in cells of human primary dendritic cells, osteoblasts, osteoarthritic cartilage, spleen, helper T cells and retinoic acid-treated NTRA2 cells using the expressed sequence tag (EST) analysis (Adams, M.D., et al Science (1991) 252:1651-1656; Adams, M.D. et al , Nature, (1992) 555:632-634; Adams, M.D. , et al , Nature (1995) 377 Supp:3-174).
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
  • the nucleotide sequence encoding h-NRD convertase polyp ⁇ tide of SEQ ID NO:2 may be identical to the polyp ⁇ tide encoding sequence contained in Table 1 (nucleotide number 162 to 3614 of SEQ ID NO: 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polyp ⁇ tide of SEQ ID NO:2.
  • the polynucleotide may include the coding sequence for the mature polyp ⁇ tide or a fragment thereof, by itself; the coding sequence for the mature polyp ⁇ tide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion p ⁇ tide portions.
  • a marker sequence which facilitates purification of the fused polyp ⁇ tide can be encoded.
  • the marker sequence is a hexa-histidine p ⁇ tide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polynucleotides encoding h-NRD convertase variants comprising the amino acid sequence of h-NRD convertase polyp ⁇ tide of Table 2 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.
  • Table 3 SEQ ID NO: 3 encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).
  • a partial nucleotide sequence of a human h-NRD convertase (SEQ ID NO: 3).
  • a partial amino acid sequence of a human h-NRD convertase (SEQ ID NO: 4).
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 80 % , and preferably at least 90 % , and more preferably at least 95 % , yet even more preferably 97-99 % identity between the sequences.
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ED NO: 1 or a fragment thereof (including that of SEQ ID NO: 3), may be used as hybridization probes for cDNA and genomic DNA, to isolate full- length cDNAs and genomic clones encoding h-NRD convertase polyp ⁇ tide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the h-NRD convertase gene.
  • Such hybridization techniques are known to those of skill in the art.
  • these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent.
  • the probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
  • to obtain a polynucleotide encoding h-NRD convertase polyp ⁇ tide, including homologs and orthologs from species other than human comprises the st ⁇ s of screening an appropriate hbrary under stingent hybridization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment thereof (including that of SEQ ED NO: 3), and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • Such hybridization techniques are well known to those of skill in the art.
  • h- NRD convertase polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO: 3). Also included with h-NRD convertase polyp ⁇ tides are polyp ⁇ tide comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition.
  • Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C.
  • 5xSSC 150mM NaCl, 15mM trisodium citrate
  • 50 mM sodium phosphate pH7.6
  • 5x Denhardt's solution 10 % dextran sulfate
  • 20 microgram/ml denatured, sheared salmon sperm DNA followed by washing the filters in 0. lx SSC at about 65°C.
  • polynucleotides and polyp ⁇ tides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
  • the present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polyp ⁇ tides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al. , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • R ⁇ resentative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
  • bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, sta
  • Such systems include, among others, chromosomal, ⁇ isomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast ⁇ isomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviiuses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • viruses such as baculoviiuses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides to produce a polyp ⁇ tide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al. , MOLECULAR CLONING, A LABORATORY MANUAL (supra).
  • secretion signals may be incorporated into the desired polyp ⁇ tide. These signals may be endogenous to the polyp ⁇ tide or they may be heterologous signals.
  • the polyp ⁇ tide be produced at the surface of the cell.
  • the cells may be harvested prior to use in the screening assay. If h-NRD convertase polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polyp ⁇ tide is recovered.
  • h-NRD convertase polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polyp ⁇ tide is denatured during isolation and or purification.
  • This invention also relates to the use of h-NRD convertase polynucleotides for use as diagnostic reagents. Detection of a mutated form of h-NRD convertase gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susc ⁇ tibility to a disease which results from under-expression, over-expression or altered expression of h-NRD convertase. Individuals carrying mutations in the h-NRD convertase gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled h-NRD convertase nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobihty of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing.
  • an array of oligonucleotides probes comprising h-NRD convertase nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g. , genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.
  • the diagnostic assays offer a process for diagnosing or determining a susc ⁇ tibility to chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease through detection of mutation in the h- NRD convertase gene by the methods described.
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease through detection
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease
  • methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of h-NRD convertase polyp ⁇ tide or h-NRD convertase mRNA.
  • Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as an h- NRD convertase polyp ⁇ tide, in a sample derived from a host are well-known to those of skill in the art.
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and EEISA assays.
  • the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, A DS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, which comprises:
  • a h-NRD convertase polynucleotide preferably the nucleotide sequence of SEQ ED NO: 1, or a fragment thereof ;
  • b a nucleotide sequence complementary to that of (a);
  • a h-NRD convertase polypeptide preferably the polyp ⁇ tide of SEQ ID NO: 2, or a fragment thereof ;
  • an antibody to a h-NRD convertase polypeptide, preferably to the polyp ⁇ tide of SEQ ID NO: 2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first st ⁇ in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).
  • genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • linkage analysis coinheritance of physically adjacent genes.
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • polyp ⁇ tides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the h-NRD convertase polyp ⁇ tides.
  • immunospecific means that the antibodies have substantial! greater affinity for the polyp ⁇ tides of the invention than their affinity for other related polyp ⁇ tides in the prior art.
  • Antibodies generated against the h-NRD convertase polyp ⁇ tides can be obtained by administering the polyp ⁇ tides or ⁇ itope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al. ,
  • Antibodies against h-NRD convertase polyp ⁇ tides may also be employed to treat chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with h-NRD convertase polyp ⁇ tide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • h-NRD convertase polyp ⁇ tide, or a fragment thereof adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering h-NRD convertase polyp ⁇ tide via a vector directing expression of h-NRD convertase polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases. Further aspect of the invention relates to an immunological/vaccine formulation
  • composition which, when introduced into a mammalian host, induces an immunological response in that mammal to a h-NRD convertase polyp ⁇ tide wherein the composition comprises a h-NRD convertase polyp ⁇ tide or h-NRD convertase gene.
  • the vaccine formulation may further comprise a suitable carrier. Since h-NRD convertase polyp ⁇ tide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will d ⁇ end on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the h-NRD convertase polyp ⁇ tide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the h-NRD convertase polyp ⁇ tide of the present invention.
  • polyp ⁇ tides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
  • These agonists or antagonists may be natural or modified substrates, ligands, enzymes, rec ⁇ tors, etc. , as the case may be, of the polyp ⁇ tide of the present invention; or may be structural or functional mimetics of the polyp ⁇ tide of the present invention.
  • h-NRD convertase polyp ⁇ tides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate h-NRD convertase polyp ⁇ tide on the one hand and which can inhibit the function of h-NRD convertase polyp ⁇ tide on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs.
  • Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs.
  • screening procedures may involve using appropriate cells which express the h-NRD convertase polyp ⁇ tide or respond to h-NRD convertase polyp ⁇ tide of the present invention.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • Cells which express the h-NRD convertase polyp ⁇ tide (or cell membrane containing the expressed polyp ⁇ tide) or respond to h-NRD convertase polyp ⁇ tide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for h-NRD convertase activity.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the h-NRD convertase polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the h-NRD convertase polyp ⁇ tide, using detection systems appropriate to the cells bearing the h-NRD convertase polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • the assays may simply comprise the st ⁇ s of mixing a candidate compound with a solution containing a h-NRD convertase polyp ⁇ tide to form a mixture, measuring h-NRD convertase activity in the mixture, and comparing the h-NRD convertase activity of the mixture to a standard.
  • the h-NRD convertase cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of h-NRD convertase mRNA and protein in cells.
  • an ELISA may be constructed for measuring secreted or cell associated levels of h-NRD convertase protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of h-NRD convertase (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the h-NRD convertase protein may be used to identify membrane bound or soluble rec ⁇ tors, if any, through standard rec ⁇ tor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the h-NRD convertase is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a p ⁇ tide sequence suitable for detection or purification, and incubated with a source of the putative rec ⁇ tor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
  • a radioactive isotope eg 1251
  • chemically modified eg biotinylated
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
  • binding assays can be used to identify agonists and antagonists of h-NRD convertase which compete with the binding of h-NRD convertase to its receptors, if any. Standard methods for conducting screening assays are well understood in the art.
  • Examples of potential h-NRD convertase polyp ⁇ tide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, enzymes, rec ⁇ tors, etc., as the case may be, of the h-NRD convertase polyp ⁇ tide, e.g., a fragment of the ligands, substrates, enzymes, rec ⁇ tors, etc.; or small molecules which bind to the polyp ⁇ tide of the present invention but do not ehcit a response, so that the activity of the polyp ⁇ tide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, rec ⁇ tors, substrates, enzymes, etc. for h-NRD convertase polyp ⁇ tides; or compounds which decrease or enhance the production of h- NRD convertase polypeptides, which comprises: (a) a h-NRD convertase polyp ⁇ tide, preferably that of SEQ ED NO:2;
  • kits may comprise a substantial component.
  • This invention provides methods of treating abnormal conditions such as, chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AEDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymph ⁇ roliferative disorders), atherosclerosis, and Alzheimers disease, related to both an excess of and insufficient amounts of h-NRD convertase polyp ⁇ tide activity.
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AEDS, metabohc and other bone diseases (eg osteoporosis)
  • cancer eg lymph ⁇ roliferative disorders
  • atherosclerosis
  • h-NRD convertase polyp ⁇ tide comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acc ⁇ table carrier in an amount effective to inhibit the fimction of the h-NRD convertase polyp ⁇ tide, such as, for example, by blocking the binding of ligands, substrates, enzymes, rec ⁇ tors, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of h-NRD convertase polyp ⁇ tides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous h-NRD convertase polyp ⁇ tide may be administered. Typical embodiments of such competitors comprise fragments of the h-NRD convertase polyp ⁇ tide.
  • soluble forms of h-NRD convertase polyp ⁇ tides still capable of binding the ligand in competition with endogenous h-NRD convertase polyp ⁇ tide may be administered.
  • Typical embodiments of such competitors comprise fragments of the h- NRD convertase polyp ⁇ tide.
  • expression of the gene encoding endogenous h-NRD convertase polyp ⁇ tide can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or s ⁇ arately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988).
  • oligonucleotides which form triple helices with the gene can be supplied.
  • oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • h-NRD convertase For treating abnormal conditions related to an under-expression of h-NRD convertase and its activity, several approaches are also available.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound which activates h-NRD convertase polyp ⁇ tide, i.e., an agonist as described above, in combination with a pharmaceutically acc ⁇ table carrier, to thereby alleviate the abnormal condition.
  • gene therapy may be employed to effect the endogenous production of h-NRD convertase by the relevant cells in the subject.
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polyp ⁇ tide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polyp ⁇ tide in vivo.
  • gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based
  • P ⁇ tides such as the soluble form of h-NRD convertase polyp ⁇ tides, and agonists and antagonist p ⁇ tides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polyp ⁇ tides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
  • Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible.
  • Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like. The dosage range required depends on the choice of p ⁇ tide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner.
  • Suitable dosages are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
  • Polyp ⁇ tides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polyp ⁇ tide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • Example 1 An EST encoding the human homologue of rat NRD convertase was identified through a search of a commercial EST database. The full length sequence of this gene shares 89.4 % identity over 3684 nucleotides and 93.3 % identity over 1151 amino acid residues with rat NRD convertase (A.R. Pierotti et al., Proc. Natl. Acad. Sci., USA. 91:6078-6082, 1994). Therefore, this gene was named h-NRD convertase. The clone encoding h-NRD convertase was found in a human primary dendritic cells cDNA hbrary. It was also found in human osteoblasts, osteoarthritic cartilage, spleen, helper T cells and retinoic acid-treated NTRA2 cells cDNA libraries.
  • Tyr Arg Tyr lie Lys Leu Gin Asn Gly Leu Gin Ala Leu Leu lie Ser 115 120 125
  • Glu Val lie Gly Glu Ala Leu Asn Gin Leu Val Pro Gin Lys Ala Asn 595 600 605 Leu Val Leu Leu Ser Gly Ala Asn Glu Gly Lys Cys Asp Leu Lys Glu 610 615 620
  • Lys Lys lie Glu Glu Phe Leu Ser Ser Phe Glu Glu Lys lie Glu Asn
  • 1045 1050 1055 lie Glu Ala Leu Lys Ser Phe Ser Lys Ser Asp Leu Val Asn Trp Phe
  • ACGAATTCCC CCCCCCGGCG AGAGGGAGAC TGGGTTGGGG GAGGGGTTCA GGCCTGTTCC 60 CCGCGGCTGC GGCAGCACCA GGGCCGGCCG CCACCGCCTC TAGAACGCGG AGGAGGTGGG 120
  • Tyr Arg Tyr lie Lys Leu Gin Asn Gly Leu Gin Ala Leu Leu lie Ser 115 120 125

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Abstract

L'invention concerne des polypeptides et polynucléotides h-NRD convertase et des procédés de production de tels polypeptides par des techniques de recombinaison. L'invention concerne également des procédés d'utilisation des polypeptides et polynucléotides h-NRD convertase dans la conception de protocoles destinés au traitement de l'inflammation chronique et aiguë, de l'arthrite, de l'arthrose, de la septicémie, de maladies auto-immunes (par exemple, la maladie intestinale inflammatoire, le psoriasis), d'un rejet de greffe, d'une réaction du greffon contre l'hôte, d'une infection, d'un accident cérébrovasculaire, de l'ischémie, d'une atteinte respiratoire aiguë, de troubles rénaux, de la resténose, d'une lésion cérébrale, du SIDA, de maladies métaboliques et d'autres maladies osseuses (par exemple, l'ostéoporose), d'un cancer (par exemple, les syndromes lymphoprolifératifs), de l'athérosclérose et de la maladie d'Alzheimer, entre autres, ainsi que des méthodes diagnostiques pour de tels états pathologiques.
PCT/US1998/014022 1997-07-07 1998-07-07 Convertase n-arginine dibasique humaine WO1999002664A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DRAOUI M., ET AL.: "EXPRESSION AND RETINOID MODULATION OF N-ARGININE DIBASIC CONVERTASE AND AN AMINOPEPTIDASE-B IN HUMAN NEUROBLASTOMA CELL LINES.", JOURNAL OF NEURO-ONCOLOGY., KLUWER, BOSTON., US, vol. 31., no. 01/02., 1 January 1997 (1997-01-01), US, pages 99 - 106., XP002912137, ISSN: 0167-594X, DOI: 10.1023/A:1005745717231 *
GEORGIOU G.: "OPTIMIZING THE PRODUCTION OF RECOMBINANT PROTEINS IN MICROORGANISMS", HEWLETT-PACKARD JOURNAL., HEWLETT-PACKARD CO. PALO ALTO., US, vol. 34., no. 08., 1 August 1988 (1988-08-01), US, pages 1233 - 1248., XP002912141 *
JOULIE C., ET AL.: "HUMAN AND RAT TESTIS EXPRESS TWO MRNA SPECIES ENCODING VARIANTS OF NRD CONVERTASE, A METALLOENDOPEPTIDASE OF THE INSULINASE FAMILY.", BIOCHEMICAL JOURNAL, PORTLAND PRESS LTD., GB, vol. 327., 1 November 1997 (1997-11-01), GB, pages 773 - 779., XP002912139, ISSN: 0264-6021 *
SAMBROOK J., FRITSCH E. F., MANIATIS T.: "MOLECULAR CLONING. LABORATORY MANUAL.", 1 January 1987, NEW YORK, COLD SPRING HARBOUR PRESS., US, article "CONSTRUCTION AND ANALYSIS OF CDNA LIBRARIES.", pages: 8.01 - 8.86 + 17.01, XP002912138, 016613 *

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