WO1999046391A2 - Nouvelles molecules d'acides nucleiques et de proteines pdsp et utilisations correspondantes - Google Patents

Nouvelles molecules d'acides nucleiques et de proteines pdsp et utilisations correspondantes Download PDF

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Publication number
WO1999046391A2
WO1999046391A2 PCT/US1999/005416 US9905416W WO9946391A2 WO 1999046391 A2 WO1999046391 A2 WO 1999046391A2 US 9905416 W US9905416 W US 9905416W WO 9946391 A2 WO9946391 A2 WO 9946391A2
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pdsp
nucleic acid
seq
polypeptide
protein
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PCT/US1999/005416
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WO1999046391A3 (fr
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Douglas A. Holtzman
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Millennium Biotherapeutics, Inc.
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Priority to AU33541/99A priority Critical patent/AU3354199A/en
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Publication of WO1999046391A3 publication Critical patent/WO1999046391A3/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)

Definitions

  • the serine proteases are an extensive family of proteases so named because they have an unusually reactive serine residue at their active sites.
  • the serine protease family includes the chymotrypsins, trypsins, elastases, subtilisins, and thrombins, all of which have similar amino acid sequences at their active sites and share a common catalytic mechanism, but which differ primarily in their substrate specificity. For example, the
  • the catalytic activity is provided by a charge relay system involving an aspartic acid residue hydrogen bonded to a histidine, which itself is hydrogen bonded to a serine.
  • the amino acids involved in this charge relay system are commonly referred to as the catalytic
  • the conserved amino acid sequences in the vicinity of the conserved histidine and serine residues are commonly referred to as the "histidine active site” and the “serine active site”, respectively. See e.g., Brenner, S. (1988) Nature 334:528-530 and Rawlings, N.D. and Barrett, A.J. (1994) Meth. Enzymol. 244:19-61.
  • glandular proteins One highly homologous subfamily of serine proteases, termed the glandular subfamily of serine proteases, termed the glandular subfamily of serine proteases
  • the present invention is based, at least in part, on the discovery of novel trypsin family serine protease molecules, referred to herein as Prostate-derived Serine Protease ("PDSP") nucleic acid and protein molecules.
  • PDSP Prostate-derived Serine Protease
  • the PDSP molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding PDSP proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of PDSP- encoding nucleic acids.
  • a PSP nucleic acid molecule is 75% homologous to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a complement thereof.
  • the isolated nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:3, or a complement thereof.
  • the nucleic acid molecule further comprises nucleotides 1-243 of SEQ ID NO:l.
  • the nucleic acid molecule further comprises nucleotides 1123-1539 of SEQ ID NO:l.
  • an isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1. In yet another preferred embodiment, an isolated nucleic acid molecule has the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a complement thereof.
  • a PDSP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2.
  • a PDSP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 60% homologous to the amino acid sequence of SEQ ID NO:2.
  • an isolated nucleic acid molecule encodes the amino acid sequence of human PDSP.
  • the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2.
  • an isolated nucleic acid molecule of the present invention encodes a protein, preferably a PDSP protein, which includes a histidine active site. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a PDSP protein, which includes a serine active site. In another embodiment, an isolated nucleic acid molecule of the present invention encodes protein, preferably a PDSP protein, which includes a histidine active site and a serine active site. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a PDSP protein, which includes a histidine active site, a serine active site and a signal sequence, and, preferably, is secreted. In yet another embodiment, a PDSP nucleic acid molecule encodes a PDSP protein and is a naturally occurring nucleotide sequence.
  • nucleic acid molecules preferably PDSP nucleic acid molecules, which specifically detect PDSP nucleic acid molecules relative to nucleic acid molecules encoding non-PDSP proteins.
  • a nucleic acid molecule is at least 550, 551-600, 601-650, 651-700, 701 -750, or 751 -800 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:l, the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a complement thereof.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a PDSP nucleic acid.
  • Another aspect of the invention provides a vector comprising a PDSP nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention also provides a method for producing a protein, preferably a PDSP protein, by culturing in a suitable medium, a host cell of the invention containing a recombinant expression vector such that the protein is produced.
  • Another aspect of this invention features isolated or recombinant PDSP proteins and polypeptides.
  • an isolated protein, preferably a PDSP protein includes a histidine active site.
  • an isolated protein preferably a PDSP protein
  • an isolated protein, preferably a PDSP protein includes a histidine active site, a serine active site, and is, preferably, secreted.
  • an isolated protein, preferably a PDSP protein has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2.
  • a protein, preferably a PDSP protein has an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:2.
  • the invention features fragments of the proteins having the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15 contiguous amino acids of the amino acid sequence of SEQ ID NO:2, or an amino acid or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession No. 98544.
  • a protein preferably a PDSP protein, has the amino acid sequence of SEQ ID NO:2.
  • Another embodiment of the invention features an isolated protein, preferably a PDSP protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 75% homologous to a nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:3, or a complement thereof.
  • This invention further features an isolated protein, preferably a PDSP protein, which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, or a complement thereof.
  • the proteins of the present invention can be operatively linked to a non-PDSP polypeptide to form fusion proteins.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably PDSP proteins.
  • the PDSP proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting PDSP expression in a biological sample by contacting the biological sample with an agent capable of detecting a PDSP nucleic acid molecule, protein or polypeptide such that the presence of a PDSP nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of PDSP activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of PDSP activity such that the presence of PDSP activity is detected in the biological sample.
  • the invention provides a method for modulating PDSP activity comprising contacting a cell capable of expressing PDSP with an agent that modulates PDSP activity such that PDSP activity in the cell is modulated.
  • the agent inhibits PDSP activity.
  • the agent stimulates PDSP activity.
  • the agent is an antibody that specifically binds to a PDSP protein.
  • the agent modulates expression of PDSP by modulating transcription of a PDSP gene or translation of a PDSP mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a PDSP mRNA or a PDSP gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by aberrant PDSP protein or nucleic acid expression or activity by administering an agent which is a PDSP modulator to the subject.
  • the PDSP modulator is a PDSP protein.
  • the PDSP modulator is a PDSP nucleic acid molecule.
  • the PDSP modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant PDSP protein or nucleic acid expression is a proliferative or differentiative disorder.
  • the present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) abe ⁇ ant modification or mutation of a gene encoding a PDSP protein; (ii) mis-regulation of said gene; and (iii) aberrant post-translational modification of a PDSP protein, wherein a wild-type form of said gene encodes an protein with a PDSP activity.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a PDSP protein, by providing a indicator composition comprising a PDSP protein having PDSP activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on PDSP activity in the indicator composition to identify a compound that modulates the activity of a PDSP protein.
  • Figure I depicts the cDNA sequence and predicted amino acid sequence of human PDSP-1.
  • the nucleotide sequence corresponds to nucleic acids 1 to 1539 of SEQ ID NO:l.
  • the amino acid sequence corresponds to amino acids 1 to 293 of SEQ ID NO:2.
  • the histidine active site is indicated in italics and underlined.
  • the serine active site is indicated in bold and underlined.
  • the conserved cysteine residues are underlined.
  • Figure 2 depicts an alignment of the amino acid sequence of human PDSP-1 with the amino acid sequences of the following proteins: Enamel matrix serine proteinase 1 precursor, GenBank Accession No. U76256; Stratum Corneum Chymotryptic Enzyme Precursor ("SCCE"), Accession No.P49862; rat trypsinogen I, Accession No. P00762; bovine cationic trypsinogen I, Accession No. P00760, and murine neuropsin, Accession No. 1020091.
  • SCE Stratum Corneum Chymotryptic Enzyme Precursor
  • the present invention is based on the discovery of novel molecules, referred to herein as PDSP protein and nucleic acid molecules, which comprise a family of molecules having certain conserved structural and functional features.
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin.
  • Members of a family may also have common functional characteristics.
  • the isolated proteins of the present invention preferably
  • PDSP proteins are proteins having an amino acid sequence of about 250-340, preferably about 260-330, more preferably about 270-320, more preferably about 280-310, and even more preferably about 290-300 amino acid residues in length, of which at least about 6-14, preferably 8-12, and more preferably 10 amino acids are cysteine residues which are preferably conserved between family members and are capable of forming disulfide bonds.
  • cys73 and cysl78, cys93 and cysl09, cysl85 and cys251, cys217 and cys231, and cys241 and cys266 of SEQ ID NO:2 are capable of forming disulfide bonds.
  • residues are highly conserved among serine protease family members.
  • a PDSP family member is identified based on the presence of at least one "histidine active site " in the protein or corresponding nucleic acid molecule.
  • the term "histidine active site” refers to a protein site consisting of at least about 3-9, preferably about 4-8, more preferably about 5-7, and even more preferably at least about 6 amino acid residues in length, of which at least one amino acid residue is a histidine residue which is conserved among trypsin family serine proteases and is part of the catalytic triad.
  • a catalytic triad consists of the amino acid residues histidine, serine and aspartate, which are conserved among members of the serine protease protein family and provide the charge relay system involved in the catalytic activity of these proteases.
  • the aspartate residue is hydrogen bonded to the histidine which is, itself, hydrogen bonded to the serine.
  • his 108, and ser245 comprise the catalytic traid of the human PDSP protein set forth in SEQ ID NO:2.
  • a histidine active site has the amino acid sequence [LIVM]-[ST]-A-[STAG]-H-C.
  • a PDSP protein is human PDSP-1 having a histidine active site containing about amino acid residues 104-109 of SEQ ID NO:2.
  • the histidine active site is further described in PROSITE Document, Accession No. PDOC00124 (http://expasy.hcuge.ch/cgi-bin/get-prodoc-entry?PDOC00124) and as PROSITE Accession No. PSOO 134.
  • a PDSP family member is identified based on the presence of at least one "serine active site " in the protein or corresponding nucleic acid molecule.
  • the term "serine active site” refers to a protein site consisting of at least about 9-15, preferably about 10-14, more preferably about 11-13, and even more preferably at least about 12 amino acid residues in length, of which at least one amino acid residue is a serine residue which is conserved among trypsin family serine proteases and is part of the catalytic triad.
  • a PDSP protein is a human PDSP-1 having a serine active site containing about amino acid residues 239-250 of SEQ ID NO:2.
  • the serine active site is further described as PROSITE Accession No. PS00135.
  • a PDSP protein has at least one histidine active site, and/or at least one serine active site, and, preferably, a signal sequence.
  • a PDSP has both a histidine active site and a serine active site, and, preferably a signal sequence.
  • a signal sequence refers to a peptide of about 20-30 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains a majority of hydrophobic amino acid residues.
  • a signal sequence contains at least about 15-45 amino acid residues, preferably about 20-40 amino acid residues, more preferably about 25-35 amino acid residues, and more preferably about 28-32 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55- 60% hydrophobic amino acid residues (e.g., Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tyrosine, Tryptophan, or Proline).
  • Such a "signal sequence” also referred to in the art as a “signal peptide" serves to direct a protein containing such a sequence to a lipid bilayer.
  • a PDSP-1 protein contains a signal sequence of about amino acids 1-29 of SEQ ID NO:2.
  • the "signal sequence” is cleaved during processing of the mature protein.
  • the mature PDSP-1 protein corresponds to amino acids 30 to 293 of SEQ ID NO:2 or to amino acids 1 to 264 of SEQ ID NO:4.
  • a PDSP protein has at least one histidine active site, and/or at least one serine active site, and a propeptide.
  • a propeptide refers to a peptide of about 5-70 amino acid residues in length which occurs at the N-terminus of secretory proteases. The propetide is cleaved from the precursor protein sequence to thereby result in an active form of the protease.
  • the propeptide is about 60-70 amino acid residues, preferably about 50-59 amino acid residues, more preferably about 40-49 amino acid residues, and more preferably about 30-39, 20-29, 10-19, or 5-9 amino acid residues in length.
  • the exsistence of a propeptide can be determined by, for example, comparing the electrophoretic mobility of a PDSP protein synthesized by recombinant technology to the electrophoretic mobility of a PDSP protein experimentally purified from a biological sample, e.g., a human tissue sample.
  • a PDSP-1 protein contains a putative propeptide of about amino acids 30-66 of SEQ ID NO:2. Accordingly, following cleavage of this propeptide, an active PDSP-1 protein corresponds to amino acids 67 to 293 of SEQ ID NO:2 or to amino acids 1-227 of SEQ ID NO:5.
  • one embodiment of the invention features an PDSP protein having at least a histidine active site and a propeptide.
  • Another embodiment features an PDSP protein having at least a serine active and a propeptide.
  • Another embodiment features a PDSP protein having at least a histidine active site but lacking a propeptide.
  • Yet another embodiment features a PDSP protein having at least a histidine active site but lacking a propeptide.
  • Such a PDSP protein is a catalytically active PDSP protein.
  • Isolated proteins of the present invention preferably PDSP proteins, have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or are encoded by a nucleotide sequence sufficiently homologous to SEQ ID NO:l or SEQ ID NO:3.
  • the term "sufficiently homologous” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least about 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently homologous.
  • amino acid or nucleotide sequences which share at least 50%, preferably 60%, more preferably 70-80, or 90-95% homology and share a common functional activity are defined herein as sufficiently homologous.
  • a PDSP activity refers to an activity exerted by a PDSP protein, polypeptide or nucleic acid molecule as determined in vivo, or in vitro, according to standard techniques.
  • a PDSP activity is a direct activity, such as an association with a PDSP- target molecule.
  • a target molecule is a molecule with which a PDSP protein binds or interacts in nature, such that PDSP-mediated function is achieved.
  • a PDSP target molecule can be a PDSP protein or polypeptide of the present invention or a non-PDSP molecule.
  • a PDSP target molecule can be a non-PDSP protein molecule.
  • a PDSP target molecule is a PDSP binding partner.
  • a PDSP "binding partner" is a molecule with which a PDSP protein interacts in nature such that PDSP activity is modulated.
  • a PDSP binding partner can be a serine protease inhibitor.
  • a PDSP target is PDSP substrate.
  • a "PDSP" substrate is a molecule with which a PDSP protein interacts in nature such that the PDSP substrate is cleaved by the enzymatic activity of the PDSP protein.
  • a PDSP activity is an indirect activity, such as an activity mediated by interaction of the PDSP protein with a PDSP target molecule such that the target molecule modulates a downstream cellular activity (e.g., cleavage of a PDSP substrate can modulate the activity of that substrate on a receptor-mediated cellular signaling pathway).
  • a PDSP activity is at least one or more of the following activities: (i) interaction of a PDSP protein with a PDSP target molecule; (ii) interaction of a PDSP protein with a PDSP target molecule, wherein the PDSP target is a serine protease inhibitor; (iii) interaction of a PDSP protein with a PDSP target molecule, wherein the PDSP target is a PDSP substrate; (iv) cleavage of a PDSP substrate; and (v) cleavage of a PDSP substrate, wherein the PDSP substrate is a growth factor.
  • a PDSP activity is at least one or more of the following activities: (1) modulation of growth factor biosynthesis, either in vitro or in vivo; (2) generation of biologically active peptides, either in vitro or in vivo; (3) regulation of cellular proliferation (e.g., proliferation of prostate cells); (4) degradation of high molecular weight seminal proteins; (5) degradation of growth factor binding proteins; (6) degradation of intercellular cohesive structures; (7) regulation of cellular differentiation (e.g., differentiation of prostate cells); (8) regulation of metastasis (e.g., metastasis of cancerous prostatic cells); and (9) regulation of prostate development.
  • another embodiment of the invention features isolated PDSP proteins and polypeptides having a PDSP activity.
  • Preferred proteins are PDSP proteins having at least a serine active site and, preferably, a PDSP activity. Additional preferred proteins are PDSP proteins having at least a histidine active site and, preferably, a PDSP activity.
  • the isolated protein further comprises a signal sequence.
  • the isolated protein is a PDSP protein having a serine active site, a histidine active site, a PDSP activity, preferably an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO:2, and optionally a signal sequence and/or propeptide.
  • the human PDSP-1 cDNA which is approximately 1540 nucleotides in length, encodes a protein which is approximately 293 amino acid residues in length.
  • the human PDSP-1 protein has at least a histidine active site.
  • a histidine active site includes, for example, about amino acids 104-109 of SEQ ID NO:2.
  • the human PDSP-1 protein further has at least a serine active site.
  • a serine active site includes, for example, about amino acids 239-250 of SEQ ID NO:2.
  • the human PDSP-1 protein is predicted to be a secreted protein which further contains a signal sequence at about amino acids 1-29 of SEQ ID NO:2.
  • a signal peptide can be made, for example, utilizing the computer algorithm SIGN ALP (Nielsen, et al. (1997) Protein Engineering 10:1-6).
  • SIGN ALP Computer algorithm
  • the human PDSP-1 protein is predicted to be secreted in an inactive form containing a propeptide.
  • a propeptide can be found at least for example, at about amino acids 30-66 of SEQ ID NO:2. Accordingly, a proenzyme form of PDSP- 1 includes about 264 amino acid residues. Likewise, a processed enzyme form of PDSP-1 includes about 227 amino acid residues.
  • nucleic acid molecules that encode PDSP proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify PDSP-encoding nucleic acids (e.g. , PDSP mRNA) and fragments for use as PCR primers for the amplification or mutation of PDSP nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated PDSP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, the nucleotide sequence of SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • PDSP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:
  • nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to PDSP nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1.
  • the sequence of SEQ ID NO:l corresponds to the human PDSP-1 cDNA.
  • This cDNA comprises sequences encoding the human PDSP-1 protein (i.e., "the coding region”, from nucleotides 244- 1122), as well as 5' untranslated sequences (nucleotides 1-243) and 3' untranslated sequences (nucleotides 1123-1539).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO:l (e.g., nucleotides 244-1122, corresponding to SEQ ID NO:3).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a portion of any of these nucleotide sequences.
  • Accession Number 98544 is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 60-65%, preferably at least about 70-75%, more preferable at least about 80-85%, and even more preferably at least about 90-95% or more homologous to the nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a portion of any of these nucleotide sequences.
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1 , SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a PDSP protein.
  • the nucleotide sequence determined from the cloning of the PDSP-1 genes allows for the generation of probes and primers designed for use in identifying and/or cloning other PDSP family members, as well as PDSP homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense sequence of SEQ ID N :l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, of an anti- sense sequence of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or of a naturally occurring mutant of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544.
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater that 550, 551-600, 015-650, 651-700, 701-750, or 751-800 nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1 , SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544.
  • Probes based on the PDSP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a PDSP protein, such as by measuring a level of a PDSP- encoding nucleic acid in a sample of cells from a subject e.g., detecting PDSP mRNA levels or determining whether a genomic PDSP gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a PDSP protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, which encodes a polypeptide having a PDSP biological activity (the biological activities of the PDSP proteins have previously been described), expressing the encoded portion of the PDSP protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the PDSP protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, due to degeneracy of the genetic code and thus encode the same PDSP proteins as those encoded by the nucleotide sequence shown in SEQ ID NO: 1 , SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2.
  • SEQ ID NO: 1 SEQ ID NO: 1
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the PDSP proteins may exist within a population (e.g. , the human population). Such genetic polymorphism in the PDSP genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a PDSP protein, preferably a mammalian PDSP protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a PDSP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in PDSP genes that are the result of natural allelic variation and that do not alter the functional activity of a PDSP protein are intended to be within the scope of the invention.
  • nucleic acid molecules encoding other PDSP family members e.g., PDSP-2
  • PDSP-2 PDSP family members
  • nucleic acid molecules encoding other PDSP family members e.g., PDSP-2
  • a nucleotide sequence which differs from the PDSP-1 sequences of SEQ ID NO: 1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544 are intended to be within the scope of the invention.
  • a PDSP-2 cDNA can be identified based on the nucleotide sequence of human PDSP-1.
  • nucleic acid molecules encoding PDSP proteins from different species and thus which have a nucleotide sequence which differs from the PDSP sequences of SEQ ID NO: 1 , SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544 are intended to be within the scope of the invention.
  • an mouse PDSP cDNA can be identified based on the nucleotide sequence of a human PDSP.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the PDSP cDNAs of the invention can be isolated based on their homology to the PDSP nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544.
  • the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1- 6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of the PDSP sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, thereby leading to changes in the amino acid sequence of the encoded PDSP proteins, without altering the functional ability of the PDSP proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non- essential” amino acid residues can be made in the sequence of SEQ ID NO: 1 , SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of PDSP (e.g., the sequence of SEQ ID NO:2) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the PDSP proteins of the present invention are predicted to be particularly unamenable to alteration (e.g.
  • amino acid residues that are defined by the PDSP histidine active site and PDSP serine active site signature motifs are particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the PDSP proteins of the present invention and other members of the serine protease or kallikrein protein families are not likely to be amenable to alteration.
  • nucleic acid molecules encoding PDSP proteins that contain changes in amino acid residues that are not essential for activity. Such PDSP proteins differ in amino acid sequence from SEQ ID NO:2 yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:2.
  • the protein encoded by the nucleic acid molecule is at least about 65-70% homologous to SEQ ID NO:2, more preferably at least about 75-80% homologous to SEQ ID NO:2, even more preferably at least about 85-90% homologous to SEQ ID NO:2, and most preferably at least about 95% homologous to SEQ ID NO:2.
  • An isolated nucleic acid molecule encoding a PDSP protein homologous to the protein of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a PDSP protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a PDSP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for PDSP biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a mutant PDSP protein can be assayed for the ability to (1) modulate growth factor biosynthesis, either in vitro or in vivo; (2) generate biologically active peptides, either in vitro or in vivo; (3) regulate cellular proliferation (e.g., proliferation of prostate cells); (4) degrade high molecular weight seminal proteins; (5) degrade growth factor binding proteins; (6) degrade intercellular cohesive structures; (7) regulate cellular differentiation (e.g., differentiation of prostate cells); (8) regulate metastasis (e.g., metastasis of cancerous prostatic cells); and (9) regulate prostate development.
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g. , complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire PDSP coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding PDSP.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human PDSP-1 corresponds to SEQ ID NO:3).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding PDSP.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of PDSP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of PDSP mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PDSP mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 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'-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a PDSP protein to thereby inhibit expression of the protein, e.g. , by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • 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.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an 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 PDSP mRNA transcripts to thereby inhibit translation of PDSP mRNA.
  • a ribozyme having specificity for a PDSP-encoding nucleic acid can be designed based upon the nucleotide sequence of a PDSP-1 cDNA disclosed herein (i.e., SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98544).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PDSP- encoding mRNA. See, e.g., Cech et al. U.S. Patent No.
  • PDSP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • PDSP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the PDSP (e.g., the PDSP promoter and/or enhancers) to form triple helical structures that prevent transcription of the PDSP gene in target cells. See generally, Helene, C.
  • the PDSP nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • 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 B. et al. (1996) supra; Perry-O'Keefe et al PNAS 93: 14670-675.
  • PNAs of PDSP nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of PDSP nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA- directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry- O'Keefe supra).
  • PNAs of PDSP can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of PDSP nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a step wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl Acad. Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134, published April 25, 1988).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl Acad. Sci. US. 86:6553-6556; Lemaitre et al.
  • 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, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • Isolated PDSP Proteins and Anti-PDSP Antibodies One aspect of the invention pertains to isolated PDSP proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-PDSP antibodies.
  • native PDSP proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • PDSP proteins are produced by recombinant DNA techniques.
  • a PDSP protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the PDSP protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of PDSP protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of PDSP protein having less than about 30% (by dry weight) of non-PDSP protein (also referred to herein as a "contaminating protein”), more preferably less than about 20% of non-PDSP protein, still more preferably less than about 10% of non-PDSP protein, and most preferably less than about 5% non- PDSP protein.
  • non-PDSP protein also referred to herein as a "contaminating protein”
  • contaminating protein also preferably substantially free of culture medium, i.e. , culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of PDSP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of PDSP protein having less than about 30% (by dry weight) of chemical precursors or non-PDSP chemicals, more preferably less than about 20% chemical precursors or non-PDSP chemicals, still more preferably less than about 10% chemical precursors or non-PDSP chemicals, and most preferably less than about 5% chemical precursors or non-PDSP chemicals.
  • Biologically active portions of a PDSP protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the PDSP protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include less amino acids than the full length PDSP proteins, and exhibit at least one activity of a PDSP protein.
  • biologically active portions comprise a domain or motif with at least one activity of the PDSP protein.
  • a biologically active portion of a PDSP protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a PDSP protein comprises at least a serine active site. In another embodiment, a biologically active portion of a PDSP protein comprises at least a histidine active site. In another embodiment a biologically active portion of a PDSP protein comprises at least a serine active site and a histidine active site.
  • a preferred biologically active portion of a PDSP protein of the present invention may contain at least one of the above-identified structural domains.
  • a more preferred biologically active portion of a PDSP protein may contain at least two of the above-identified structural domains.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native PDSP protein.
  • the PDSP protein has an amino acid sequence shown in SEQ ID NO:2.
  • the PDSP protein is substantially homologous to SEQ ID NO:2, and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the PDSP protein is a protein which comprises an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO: 5 and retains the functional activity of the PDSP proteins of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5, respectively.
  • the protein is at least about 70% homologous to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5, more preferably at least about 80% homologous to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5, even more preferably at least about 90% homologous to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5, and most preferably at least about 95% or more homologous to SEQ ID NO:2.
  • the protein is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% homologous to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the PDSP amino acid sequence of SEQ ID NO:2 having 293 amino acid residues, at least 88, preferably at least 117, more preferably at least 147, even more preferably at least 176, and even more preferably at least 205, 234 or 264 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity”
  • the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithim.
  • a preferred, non- limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. ( 1990) J. Mol. Biol. 215 :403- 10.
  • Gapped Gapped
  • BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Research 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST e.g., XBLAST and NBLAST
  • Another preferred, non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the invention also provides PDSP chimeric or fusion proteins. As used herein, a
  • PDSP "chimeric protein” or “fusion protein” comprises a PDSP polypeptide operatively linked to a non-PDSP polypeptide.
  • a "PDSP polypeptide” refers to a polypeptide having an amino acid sequence corresponding to PDSP
  • a non-PDSP polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the PDSP protein, e.g. , a protein which is different from the PDSP protein and which is derived from the same or a different organism.
  • the PDSP polypeptide can correspond to all or a portion of a PDSP protein.
  • a PDSP fusion protein comprises at least one biologically active portion of a PDSP protein. In another preferred embodiment, a PDSP fusion protein comprises at least two biologically active portions of a PDSP protein.
  • the term "operatively linked" is intended to indicate that the PDSP polypeptide and the non-PDSP polypeptide are fused in-frame to each other. The non-PDSP polypeptide can be fused to the N-terminus or C- terminus of the PDSP polypeptide.
  • the fusion protein is a GST-PDSP fusion protein in which the PDSP sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant PDSP.
  • the fusion protein is a PDSP protein containing a heterologous signal sequence at its N-terminus.
  • the native PDSP signal sequence i.e, about amino acids 1 to 29 of SEQ ID NO:2
  • the native PDSP signal sequence can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of PDSP can be increased through use of a heterologous signal sequence.
  • the PDSP fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the PDSP fusion proteins can be used to affect the bioavailability of a PDSP substrate.
  • Use of PDSP fusion proteins may be useful therapeutically for the treatment of proliferative disorders (e.g., prostate cancer).
  • the PDSP-fusion proteins of the invention can be used as immunogens to produce anti-PDSP antibodies in a subject, to purify PDSP ligands and in screening assays to identify molecules which inhibit the interaction of PDSP with a PDSP substrate.
  • a PDSP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a PDSP- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the PDSP protein.
  • the present invention also pertains to variants of the PDSP proteins which function as either PDSP agonists (mimetics) or as PDSP antagonists.
  • Variants of the PDSP proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a PDSP protein.
  • An agonist of the PDSP proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a PDSP protein.
  • An antagonist of a PDSP protein can inhibit one or more of the activities of the naturally occurring form of the PDSP protein by, for example, competitively inhibiting the protease activity of a PDSP protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the PDSP protein.
  • variants of a PDSP protein which function as either PDSP agonists (mimetics) or as PDSP antagonists can be identified by screening combinatorial libraries of mutants, e.g. , truncation mutants, of a PDSP protein for PDSP protein agonist or antagonist activity.
  • a variegated library of PDSP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of PDSP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PDSP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of PDSP sequences therein.
  • a degenerate set of potential PDSP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of PDSP sequences therein.
  • methods which can be used to produce libraries of potential PDSP variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential PDSP sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al ( ⁇ 9%4) Annu. Rev. Biochem. 53:323; Itakura et al (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
  • libraries of fragments of a PDSP protein coding sequence can be used to generate a variegated population of PDSP fragments for screening and subsequent selection of variants of a PDSP protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a PDSP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the PDSP protein.
  • PDSP proteins The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify PDSP variants (Arkin and Yourvan (1992) PNAS 59:7811-7815; Delgrave et al (1993) Protein Engineering 6(3):327-331). In one embodiment, cell based assays can be exploited to analyze a variegated
  • a library of expression vectors can be transfected into a cell line which ordinarily synthesizes and secretes PDSP.
  • the transfected cells are then cultured such that PDSP and a particular mutant PDSP are secreted and the effect of expression of the mutant on PDSP activity in cell supernatants can be detected, e.g., by any of a number of enzymatic assays.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of PDSP activity, and the individual clones further characterized.
  • An isolated PDSP protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind PDSP using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length PDSP protein can be used or, alternatively, the invention provides antigenic peptide fragments of PDSP for use as immunogens.
  • the antigenic peptide of PDSP comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of PDSP such that an antibody raised against the peptide forms a specific immune complex with PDSP.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of PDSP that are located on the surface of the protein, e.g., hydrophilic regions.
  • a PDSP immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed PDSP protein or a chemically synthesized PDSP polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic PDSP preparation induces a polyclonal anti-PDSP antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as PDSP.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind PDSP.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of PDSP.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular PDSP protein with which it immunoreacts.
  • Polyclonal anti-PDSP antibodies can be prepared as described above by immunizing a suitable subject with a PDSP immunogen.
  • the anti-PDSP antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized PDSP.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against PDSP can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al (1981) J. Immunol. 127:539-46; Brown et al (1980) J. Biol Chem .255:4980-83; Yeh et al (1976) PNAS 76:2927-31 ; and Yeh et al. ( 1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds PDSP.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind PDSP, e.g., using a standard ELISA assay.
  • a monoclonal anti-PDSP antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with PDSP to thereby isolate immunoglobulin library members that bind PDSP.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al.
  • recombinant anti-PDSP antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al U.S. Patent No. 4,816,567; Cabilly et al.
  • An anti-PDSP antibody e.g., monoclonal antibody
  • An anti-PDSP antibody can be used to isolate PDSP by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-PDSP antibody can facilitate the purification of natural PDSP from cells and of recombinantly produced PDSP expressed in host cells.
  • an anti-PDSP antibody can be used to detect PDSP protein (e.g.
  • Anti- PDSP antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • vectors preferably expression vectors, containing a nucleic acid encoding a PDSP protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adeno viruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., PDSP proteins, mutant forms of PDSP proteins, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of PDSP proteins in prokaryotic or eukaryotic cells.
  • PDSP proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in En ⁇ ymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • fusion proteins can be utilized in PDSP activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for PDSP proteins, for example.
  • a PDSP fusion protein expressed in a retro viral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • nucleic acid sequence of the nucleic acid is altered into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the PDSP expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933- 943), pJRY88 (Schultz et al, (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • PDSP proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • viral regulatory elements For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to PDSP mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a PDSP protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a PDSP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (/ ' . e. , express) a PDSP protein.
  • the invention further provides methods for producing a PDSP protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a PDSP protein has been introduced) in a suitable medium such that a PDSP protein is produced.
  • the method further comprises isolating a PDSP protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which PDSP-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous PDSP sequences have been introduced into their genome or homologous recombinant animals in which endogenous PDSP sequences have been altered.
  • Such animals are useful for studying the function and/or activity of a PDSP and for identifying and/or evaluating modulators of PDSP activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous PDSP gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal .
  • a transgenic animal of the invention can be created by introducing a PDSP- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retro viral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the PDSP-1 cDNA sequence of SEQ ID NO:l can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of a human PDSP-1 gene such as a mouse or rat PDSP-1 gene, can be used as a transgene.
  • a PDSP-1 gene homologue such as a PDSP-2 gene can be isolated based on hybridization to the PDSP-1 cDNA sequences of SEQ ID NO:l, SEQ ID NO:3, or the DNA insert of the plasmid deposited with ATCC as Accession Number 98544 (described further in subsection I above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a PDSP transgene to direct expression of a PDSP protein to particular cells.
  • transgenic animals via embryo manipulation and microinjection, particularly animals such as mice
  • methods for generating transgenic animals via embryo manipulation and microinjection have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al, U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals.
  • a transgenic founder animal can be identified based upon the presence of a PDSP transgene in its genome and/or expression of PDSP mRNA in tissues or cells of the animals.
  • transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a transgene encoding a PDSP protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of a PDSP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the PDSP gene.
  • the PDSP gene can be a human gene (e.g., the cDNA of SEQ ID NO:3), but more preferably, is a non- human homologue of a human PDSP gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO: 1).
  • a mouse PDSP gene can be used to construct a homologous recombination vector suitable for altering an endogenous PDSP gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous PDSP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous PDSP gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous PDSP protein).
  • the altered portion of the PDSP gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the PDSP gene to allow for homologous recombination to occur between the exogenous PDSP gene carried by the vector and an endogenous PDSP gene in an embryonic stem cell.
  • flanking PDSP nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced PDSP gene has homologously recombined with the endogenous PDSP gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system of bacteriophage PI.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. ( 1991 ) Science 251 :1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g. , by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810- 813.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the recontructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • Pharmaceutical Compositions e.g., a somatic cell
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a PDSP protein or anti-PDSP antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a PDSP protein or anti-PDSP antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • 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 LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g. , therapeutic and prophylactic).
  • a PDSP protein of the invention has one or more of the following activities: (i) interaction of a PDSP protein with a PDSP target molecule; (ii) interaction of a PDSP protein with a PDSP target molecule, wherein the PDSP target is a serine protease inhibitor; (iii) interaction of a PDSP protein with a PDSP target molecule, wherein the PDSP target is a PDSP substrate; (iv) cleavage of a PDSP substrate; and (v) cleavage of a PDSP substrate, wherein the PDSP substrate is a growth factor, and can thus be used in, for example, (1) modulation of growth factor biosynthesis, either in vitro or in vivo; (2) generation of biologically active peptides, either in vitro or in vivo; (3) regulation of cellular proliferation (e.g., proliferation of prostate cells); (4) degradation of high molecular weight seminal proteins; (5) degradation of growth factor binding
  • the isolated nucleic acid molecules of the invention can be used, for example, to express PDSP protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect PDSP mRNA (e.g. , in a biological sample) or a genetic alteration in a PDSP gene, and to modulate PDSP activity, as described further below.
  • PDSP proteins can be used to treat disorders characterized by insufficient or excessive production of a PDSP substrate or production of PDSP inhibitors.
  • the PDSP proteins can be used to screen for naturally occurring PDSP substrates, to screen for drugs or compounds which modulate PDSP activity, as well as to treat disorders characterized by insufficient or excessive production of PDSP protein or production of PDSP protein forms which have decreased or aberrant activity compared to PDSP wild type protein.
  • the anti-PDSP antibodies of the invention can be used to detect and isolate PDSP proteins, regulate the bioavailability of PDSP proteins, and modulate PDSP activity.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to PDSP proteins, have a stimulatory or inhibitory effect on, for example, PDSP expression or PDSP activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of PDSP substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to PDSP proteins, have a stimulatory or inhibitory effect on, for example, PDSP expression or PDSP activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of PDSP substrate.
  • the invention provides assays for screening candidate or test compounds which are substrates of a PDSP protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a PDSP protein or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses a PDSP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate PDSP activity determined. Determining the ability of the test compound to modulate PDSP activity can be accomplished by monitoring the bioactivity of a PDSP substrate (e.g., a growth factor or other bioactive peptide).
  • a PDSP substrate e.g., a growth factor or other bioactive peptide.
  • the cell for example, can be of mammalian origin or a yeast cell.
  • Determining the ability of the test compound to modulate PDSP activity can be accomplished, for example, by coupling the PDSP substrate with a radioisotope or enzymatic label such that binding of the PDSP substrate to its cognate receptor can be determined by detecting the labeled PDSP substrate in a complex.
  • compounds e.g., PDSP substrates
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a compound with its cognate receptor without the labeling of either the compound or the receptor. McConnell, H. M. et al. (1992) Science 257:1906- 1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light- addressable potentiometric sensor
  • the assay comprises contacting a cell which expresses a PDSP protein or biologically active portion thereof, with a PDSP substrate to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to modulate the activity of the PDSP protein or biologically active portion thereof, wherein determining the ability of the test compound to modulate the activity of the PDSP protein or biologically active portion thereof, comprises determining the ability of the test compound to modulate a biological activity of the PDSP expressing cell (e.g. , determining the ability of the test compound to modulate proliferation or metastasis of the cell).
  • the assay comprises contacting a cell which is responsive to a PDSP protein or biologically active portion thereof, with a PDSP protein or biologically-active portion thereof, to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to modulate the activity of the PDSP protein or biologically active portion thereof, wherein determining the ability of the test compound to modulate the activity of the PDSP protein or biologically active portion thereof comprises determining the ability of the test compound to modulate a biological activity of the PDSP-responsive cell (e.g., determining the ability of the test compound to modulate proliferation or metastasis of the cell).
  • an assay is a cell-based assay comprising contacting a cell expressing a PDSP target molecule (e.g., a PDSP substrate) with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the PDSP target molecule. Determining the ability of the test compound to modulate the activity of a PDSP target molecule can be accomplished, for example, by determining the ability of the PDSP protein to bind to or interact with the PDSP target molecule.
  • a PDSP target molecule e.g., a PDSP substrate
  • Determining the ability of the test compound to modulate the activity of a PDSP target molecule can be accomplished, for example, by determining the ability of the PDSP protein to bind to or interact with the PDSP target molecule.
  • Determining the ability of the PDSP protein to bind to or interact with a PDSP target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the PDSP protein to bind to or interact with a PDSP target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e.
  • a reporter gene comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • a target-regulated cellular response for example, cellular proliferation or metastisis.
  • an assay of the present invention is a cell-free assay in which a PDSP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the PDSP protein or biologically active portion thereof is determined. Binding of the test compound to the PDSP protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the PDSP protein or biologically active portion thereof with a known compound which binds PDSP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a PDSP protein, wherein determining the ability of the test compound to interact with a PDSP protein comprises determining the ability of the test compound to preferentially bind to PDSP or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a PDSP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the PDSP protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of a PDSP protein can be accomplished, for example, by determining the ability of the PDSP protein to bind to a PDSP target molecule by one of the methods described above for determining direct binding. Determining the ability of the PDSP protein to bind to a PDSP target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S.
  • BIOS Biomolecular Interaction Analysis
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g. , BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • determining the ability of the test compound to modulate the activity of a PDSP protein can be accomplished by determining the ability of the PDSP protein to further modulate the activity of a downstream effector (e.g., a growth factor mediated signal transduction pathway component) of a PDSP target molecule (e.g., a growth factor).
  • a downstream effector e.g., a growth factor mediated signal transduction pathway component
  • a PDSP target molecule e.g., a growth factor
  • the cell-free assay involves contacting a PDSP protein or biologically active portion thereof with a known compound which binds the PDSP protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the PDSP protein, wherein determining the ability of the test compound to interact with the PDSP protein comprises determining the ability of the PDSP protein to preferentially bind to or modulate the activity of a PDSP target molecule.
  • the cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g. PDSP proteins or biologically active portions thereof or receptors to which PDSP targets bind).
  • isolated proteins e.g. PDSP proteins or biologically active portions thereof or receptors to which PDSP targets bind.
  • a membrane-bound form an isolated protein e.g., a cell surface receptor
  • non-ionic detergents such as n-oc
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/ PDSP fusion proteins or glutathione-S- transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads
  • the test compound or the test compound and either the non-adsorbed target protein or PDSP protein are then combined with the test compound or the test compound and either the non-adsorbed target protein or PDSP protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of PDSP binding or activity determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • either a PDSP protein or a PDSP target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated PDSP protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g. , biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with PDSP protein or target molecules but which do not interfere with binding of the PDSP protein to its target molecule can be derivatized to the wells of the plate, and unbound target or PDSP protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the PDSP protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the PDSP protein or target molecule.
  • modulators of PDSP expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of PDSP mRNA or protein in the cell is determined.
  • the level of expression of PDSP mRNA or protein in the presence of the candidate compound is compared to the level of expression of PDSP mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of PDSP expression based on this comparison. For example, when expression of PDSP mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of PDSP mRNA or protein expression.
  • the candidate compound when expression of PDSP mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of PDSP mRNA or protein expression.
  • the level of PDSP mRNA or protein expression in the cells can be determined by methods described herein for detecting PDSP mRNA or protein.
  • the PDSP proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No.
  • PDSP-binding proteins proteins which bind to or interact with PDSP
  • PDSP-binding proteins proteins which bind to or interact with PDSP
  • Such PDSP-binding proteins are also likely to be involved in the propagation of signals by the PDSP proteins or PDSP targets as, for example, downstream elements of a PDSP-mediated signaling pathway.
  • PDSP-binding proteins are likely to be PDSP inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a PDSP protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the PDSP protein.
  • a reporter gene e.g., LacZ
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g. , a PDSP modulating agent, an antisense PDSP nucleic acid molecule, a PDSP-specific antibody, or a PDSP-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the PDSP nucleotide sequences, described herein, can be used to map the location of the PDSP genes on a chromosome. The mapping of the PDSP sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • PDSP genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the PDSP nucleotide sequences. Computer analysis of the PDSP sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the PDSP sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P.
  • Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the PDSP nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.
  • Other mapping strategies which can similarly be used to map a 9o, lp, or lv sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) PNAS, 87:6223-27), pre-screening with labeled flow- sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the PDSP sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the PDSP nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the PDSP nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • the noncoding sequences of SEQ ID NO:l can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene.
  • the amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • the sequences of the present invention can be used to provide polynucleotide reagents, e.g.
  • PCR primers targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • identity marker i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NO: 1 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the PDSP nucleotide sequences or portions thereof, e.g. , fragments derived from the noncoding regions of SEQ ID NO: 1 , having a length of at least 20 bases, preferably at least 30 bases.
  • the PDSP nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such PDSP probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such PDSP probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g. , PDSP primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • PDSP primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • diagnostic assays for determining PDSP protein and/or nucleic acid expression as well as PDSP activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant PDSP expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with PDSP protein, nucleic acid expression or activity. For example, mutations in a PDSP gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with PDSP protein, nucleic acid expression or activity.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of PDSP in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of PDSP protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting PDSP protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes PDSP protein such that the presence of PDSP protein or nucleic acid is detected in the biological sample.
  • a compound or an agent capable of detecting PDSP protein or nucleic acid e.g., mRNA, genomic DNA
  • a preferred agent for detecting PDSP mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to PDSP mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length PDSP nucleic acid, such as the nucleic acid of SEQ ID NO: 1 (or that of SEQ ID NO:3, or the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a portion thereof), such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to PDSP mRNA or genomic DNA.
  • a full-length PDSP nucleic acid such as the nucleic acid of SEQ ID NO: 1 (or that of SEQ ID NO:3, or the DNA insert of the plasmid deposited with ATCC as Accession Number 98544, or a portion thereof), such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to PDSP mRNA or genomic DNA.
  • a preferred agent for detecting PDSP protein is an antibody capable of binding to PDSP protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect PDSP mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of PDSP mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of PDSP protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of PDSP genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of PDSP protein include introducing into a subject a labeled anti-PDSP antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting PDSP protein, mRNA, or genomic DNA, such that the presence of PDSP protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of PDSP protein, mRNA or genomic DNA in the control sample with the presence of PDSP protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of PDSP in a biological sample can comprise a labeled compound or agent capable of detecting PDSP protein or mRNA in a biological sample; means for determining the amount of PDSP in the sample; and means for comparing the amount of PDSP in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect PDSP protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant PDSP expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with PDSP protein, nucleic acid expression or activity such as prostate cancer.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing prostate cancer.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant PDSP expression or activity in which a test sample is obtained from a subject and PDSP protein or nucleic acid (e.g, mRNA, genomic DNA) is detected, wherein the presence of PDSP protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant PDSP expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant PDSP expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for prostate cancer.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant PDSP expression or activity in which a test sample is obtained and PDSP protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of PDSP protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant PDSP expression or activity.)
  • the methods of the invention can also be used to detect genetic alterations in a PDSP gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by an aberrant proliferative response.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a PDSP-protein, or the mis-expression of the PDSP gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a PDSP gene; 2) an addition of one or more nucleotides to a PDSP gene; 3) a substitution of one or more nucleotides of a PDSP gene, 4) a chromosomal rearrangement of a PDSP gene; 5) an alteration in the level of a messenger RNA transcript of a PDSP gene, 6) aberrant modification of a PDSP gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a PDSP gene, 8) a non- wild type level of a PDSP-protein, 9) allelic loss of a PDSP gene, and 10) inappropriate post-translational modification of a PDSP-protein.
  • a preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al (1988) Science 241:1077-1080; and Nakazawa et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a PDSP gene under conditions such that hybridization and amplification of the PDSP-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a PDSP gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in PDSP can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al (1996) Nature Medicine 2: 753- 759).
  • genetic mutations in PDSP can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential ovelapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the PDSP gene and detect mutations by comparing the sequence of the sample PDSP with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463).
  • any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl Biochem. Biotechnol 38:147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the PDSP gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230: 1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type PDSP sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in PDSP cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a PDSP sequence e.g., a wild-type PDSP sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in PDSP genes.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control PDSP nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a PDSP gene.
  • any cell type or tissue in which PDSP is expressed may be utilized in the prognostic assays described herein.
  • Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a PDSP protein e.g., modulation of angiogenesis or of an inflammatory response
  • agents e.g., drugs, compounds
  • a PDSP protein e.g., modulation of angiogenesis or of an inflammatory response
  • the effectiveness of an agent determined by a screening assay as described herein to increase PDSP gene expression, protein levels, or upregulate PDSP activity can be monitored in clinical trials of subjects exhibiting decreased PDSP gene expression, protein levels, or downregulated PDSP activity.
  • the effectiveness of an agent determined by a screening assay to decrease PDSP gene expression, protein levels, or downregulate PDSP activity can be monitored in clinical trials of subjects exhibiting increased PDSP gene expression, protein levels, or upregulated PDSP activity.
  • the expression or activity of a PDSP gene, and preferably, other genes that have been implicated in, for example, a proliferative disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes including PDSP, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates PDSP activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • PDSP activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of PDSP and other genes implicated in the proliferative disorder, respectively.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of PDSP or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a PDSP protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the PDSP protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the PDSP protein, mRNA, or genomic DNA in the preadministration sample with the PDSP protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g
  • increased administration of the agent may be desirable to increase the expression or activity of PDSP to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of PDSP to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • PDSP expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant PDSP expression or activity.
  • treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market.
  • the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype”.)
  • a drug e.g., a patient's "drug response phenotype", or "drug response genotype”.
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the PDSP molecules of the present invention or PDSP modulators according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant PDSP expression or activity, by administering to the subject a PDSP or an agent which modulates PDSP expression or at least one PDSP activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant PDSP expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the PDSP aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a PDSP, PDSP agonist or PDSP antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the present invention are further discussed in the following subsections.
  • the modulatory method of the invention involves contacting a cell with a PDSP or agent that modulates one or more of the activities of PDSP protein activity associated with the cell.
  • An agent that modulates PDSP protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a PDSP protein (e.g., a PDSP substrate), a PDSP antibody, a PDSP agonist or antagonist, a peptidomimetic of a PDSP agonist or antagonist, or other small molecule.
  • the agent stimulates one or more PDSP activities.
  • stimulatory agents include active PDSP protein and a nucleic acid molecule encoding PDSP that has been introduced into the cell.
  • the agent inhibits one or more PDSP activites.
  • inhibitory agents include antisense PDSP nucleic acid molecules, anti-PDSP antibodies, and PDSP inhibitors.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g. , upregulates or downregulates) PDSP expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a PDSP protein or nucleic acid molecule as therapy to compensate for reduced or aberrant PDSP expression or activity.
  • Stimulation of PDSP activity is desirable in situations in which PDSP is abnormally downregulated and or in which increased PDSP activity is likely to have a beneficial effect.
  • stimulation of PDSP activity is desirable in situations in which a PDSP is downregulated and/or in which increased PDSP activity is likely to have a beneficial effect.
  • inhibition of PDSP activity is desirable in situations in which PDSP is abnormally upregulated and/or in which decreased PDSP activity is likely to have a beneficial effect.
  • the PDSP molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on PDSP activity (e.g., PDSP gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g, prostate cancer) associated with aberrant PDSP activity.
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a PDSP molecule or PDSP modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a PDSP molecule or PDSP modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23(10-11) :983- 985 and Linder, M.W., Clin Chem, 1997, 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymo ⁇ hisms (SNPs) in the human genome.
  • SNP single nucleotide polymo ⁇ hisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response.
  • a gene that encodes a drugs target e.g., a PDSP protein or PDSP receptor of the present invention
  • all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite mo ⁇ hine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., a PDSP molecule or PDSP modulator of the present invention
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a PDSP molecule or PDSP modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • Example 1 Identification And Characterization of human PDSP-1 cDNA
  • the identification and characterization of the gene encoding human PDSP-1 (also referred to as "TANGO 114") is described. Isolation of the human PDSP-1 cDNA
  • the invention is based, at least in part, on the discovery of the murine and human genes encoding a novel protein, referred to herein as PDSP-1.
  • human prostate epithelial cells obtained from Clonetics Co ⁇ oration; San Diego, CA
  • Prostate Epithelial Growth Media PrEGM; Clonetics
  • PrEGM Prostate Epithelial Growth Media
  • CHI cycloheximide
  • Complementary DNA was directionally cloned into the expression plasmid pMET7 using the Sail and Notl sites in the polylinker to construct a plasmid library. Transformants were picked and grown up for single-pass sequencing.
  • prostate cDNA was ligated into the Sall/Notl sites of the ZipLox vector (Gibco BRL) for construction of a lambda phage cDNA library.
  • PDSP-1 has a pro-domain that is removed prior to activation. Furthermore, PDSP-1 contains the conserved "catalytic triad" (His-108, Asp-153, and Ser-245) necessary for protease activity.
  • the nucleotide sequence encoding the human PDSP-1 protein is shown in Figure 1 and is set forth as SEQ ID NO: 1.
  • the full length protein encoded by this nucleic acid is comprised of about 293 amino acids and has the amino acid sequence shown in Figure 1 and set forth as SEQ ID NO:2.
  • SEQ ID NO:l The coding portion (open reading frame) of SEQ ID NO:l is set forth as SEQ ID NO:3.
  • Clone jthqb020f07, comprising the entire coding region of human PDSP-1 has been deposited with the ATCC and has Accession No.98544.
  • Each of these proteins is a serine protease and contains both a serine active site and a histidine active site although PDSP-1 shares no greater than 60% identity with any of these serine proteases.
  • An alignment of human PDSP-1 and the above-described proteins is presented in Figure 2.
  • PDSP-1 shares significant homology with the human kallekreins including hKl , hK2, and hK3 or Prostate Specific Antigen. (Accession Nos. P06870, P20151 , and P07288, respectively.)
  • This Example describes the tissue distribution of PDSP mRNA, as determined by Northern blot hybridization.
  • Northern blot hybridizations with the various RNA samples were performed under standard conditions and washed under stringent conditions, i.e., 0.2xSSC at 65°C. In each sample, the probe hybridized to a single RNA of about 1.4 kb. The results of hybridization of the probe to various mRNA samples are described below. The expression of PDSP-1 was analyzed using Northern blot hybridization.
  • a 428 base pair (bp) DNA probe was generated using PCR with oligonucleotide primers pDH30RPOl (5' GGT GGT TAT AAC TCA GGC C3' (SEQ ID NO:6)); corresponding to nucleotides 42-60 in PDSP-1 and pDH30FPOl (5 * GCA TAT CGC AGT CGG ATC 3' (SEQ ID NO:7)); reverse and complement to nucleotides 452-469 in PDSP-1.
  • the DNA was radioactively labeled with 32P-dCTP using the Prime-It kit (Stratagene, La Jolla, CA) according to the instructions of the supplier.
  • PDSP-1 is expressed as an ⁇ 1.4 kb transcript in testis. Lower levels of the 1.4 kb transcript are also seen in prostate, lung and kidney. In addition, a -2.0 kb transcript was also seen in spleen.

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Abstract

L'invention se rapporte à de nouvelles molécules d'acides nucléiques, de protéines et de polypeptides PDSP. L'invention se rapporte non seulement à des protéines PDSP complètes, isolées, mais également à des protéines de fusion PDSP isolées, à des peptides antigéniques et à des anticorps anti-PDSP. L'invention se rapporte également à des molécules d'acides nucléiques PDSP, à des vecteurs d'expression recombinés contenant une telle molécule d'acide nucléique, à des cellules hôtes dans lesquelles les vecteurs d'expression ont été introduits et à des animaux transgéniques non humains dans lesquels un gène PDSP a été introduit ou disloqué. L'invention se rapporte également à des méthodes de diagnostic, de criblage et de thérapie mettant en oeuvre les compositions décrites ci-dessus.
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EP1297117A1 (fr) * 2000-06-20 2003-04-02 Human Genome Sciences, Inc. Polynucleotides codant les serine proteases humaines
EP1297117A4 (fr) * 2000-06-20 2004-12-15 Human Genome Sciences Inc Polynucleotides codant les serine proteases humaines
WO2001098467A2 (fr) * 2000-06-23 2001-12-27 Bayer Aktiengesellschaft Regulation de la serine protease humaine de type prostasine
WO2001098467A3 (fr) * 2000-06-23 2002-04-04 Bayer Ag Regulation de la serine protease humaine de type prostasine

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