WO1998007735A1 - Inhibiteur de l'activateur du plasminogene derive du pancreas - Google Patents

Inhibiteur de l'activateur du plasminogene derive du pancreas Download PDF

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Publication number
WO1998007735A1
WO1998007735A1 PCT/US1996/013283 US9613283W WO9807735A1 WO 1998007735 A1 WO1998007735 A1 WO 1998007735A1 US 9613283 W US9613283 W US 9613283W WO 9807735 A1 WO9807735 A1 WO 9807735A1
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Prior art keywords
amino acid
polypeptide
seq
papai
plasminogen activator
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PCT/US1996/013283
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English (en)
Inventor
Jian Ni
Reiner L. Gentz
Steven M. Ruben
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Human Genome Sciences, Inc.
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Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to PCT/US1996/013283 priority Critical patent/WO1998007735A1/fr
Priority to AU73579/96A priority patent/AU7357996A/en
Publication of WO1998007735A1 publication Critical patent/WO1998007735A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a novel member of the serine protease inhibitor (serpin) superfamily of proteins, in particular the plasminogen activator inhibitor (PAI) protein family. More specifically, isolated nucleic acid molecules are provided encoding the pancreas-derived plasminogen activator inhibitor (PAPAI) protein. Plasminogen activator inhibitor polypeptides are also provided.
  • the present invention further relates to methods for treating physiologic and pathologic disease conditions and diagnostic methods for detecting pathologic disorders.
  • the mammalian serine protease inhibitors are a superfamily of single chain proteins that contain a conserved structure of approximately 370 amino acids and generally range between 40 and 60 kDa in molecular mass.
  • serpins also known as ⁇ ,-proteinase inhibitor
  • ⁇ ,-proteinase inhibitor is a characteristic member of the serpin family in that it is a single chain glycoprotein of nearly 400 amino acid residues that functions by forming a tight 1 : 1 complex with its cognate protease, neutrophil (leucocyte) elastase, which subsequently slowly dissociates so yield active enzyme and inactive cleaved inhibitor
  • the reactive center of the se ⁇ ins is typically formed by an X-Ser that acts as a substrate for the target serine protease: ⁇ ,-antitrypsin has a Met-Ser reactive center with the methionine residue providing a putative cleavage site for neutrophil elastase.
  • the majority of se ⁇ ins function as protease inhibitors and so are involved in regulation of several proteinase-activated physiological processes, such as blood coagulation, fibrinolysis, complement activation, extracellular matrix turnover, cell migration and prohormone activation (Potempa, J. et al., J. Biol. Chem.
  • se ⁇ ins inhibit proteolytic events by forming a 1:1 stoichiometric complex with the active site of their cognate proteinases, which is resistant to denaturants (Cohen, A.B. et al, Biochemistry 77:392-400 (1987).
  • the se ⁇ ins include, but are not limited to, ⁇ r antitrypsin
  • PAI-1 plasminogen activator inhibitor 2
  • PAI-2 plasminogen activator inhibitor 2
  • ⁇ plasminogen activator inhibitor 2
  • ⁇ plasminogen activator inhibitor 2
  • ⁇ plasminogen activator inhibitor 2
  • ⁇ plasminogen activator inhibitor 2
  • ⁇ plasminogen activator inhibitor 2
  • ⁇ plasminogen activator inhibitor 2
  • ⁇ plasminogen activator inhibitor 2
  • ⁇ ⁇ 2 -antiplasmin
  • Plasmin a trypsin-like protease
  • tissue-type plasminogen activator also known as tissue plasminogen activator
  • urokinase tissue-type plasminogen activator
  • Plasminogen activation is a highly regulated process. Precise, coordinated, spatial and temporal regulation is afforded by the interaction of a variety of mechanisms. These mechanisms include (1) inhibition by specific plasmin and plasminogen activator inhibitors; (2) binding of plasminogen, plasminogen activators, and inhibitors to fibrin, extracellular matrix proteins, and specific cell surface receptors; (3) release of tissue plasminogen activator and inhibitors from intracellular storage granules; (4) regulation of gene expression of plasminogen activators and inhibitors; (5) an autocrine feedback loop whereby plasmin-mediated activation of latent forms of growth factors regulates the expression of activators and inhibitors; and (6) clearance of free and inhibitor- bound activators via receptors (Bachmann, F.
  • PAI-1 and PAI-2 plasminogen activator inhibitors 1 and 2
  • PAI-1 and PAI-2 regulate mitogenesis, adhesion of myeloid cells, fusion of myoblasts, and migration of endothelial cells (Fazioli, F. et al, Trends Pharm. Sci. 75:25-29 (1995)).
  • PAI-1 and PAI-2 are involved in many physiological and pathological processes, including normal pregnancy, preeclampsia, intrauterine growth retardation, wound healing, tumor cell invasion and metastasis, inflammation and arthritis, inflammatory bowel disease, appendicitis, complications from systemic lupus erythematosus, ovulation and prostatic involution and osteonecrosis (Kruithof, E. .O. et al, Blood 86:4007 (1995)).
  • plasminogen activator inhibitors play in physiologic and pathologic processes, there is a continuing need for the isolation and characterization of novel plasminogen activator inhibitors.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the pancreas-derived plasminogen activator inhibitor
  • PAPAI polypeptide having the amino acid sequence is shown in Figure 1 [SEQ ID NO:2] or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 97657 on July 12, 1996.
  • the nucleotide sequence determined by sequencing the deposited PAPAI clone, which is shown in Figure 1 [SEQ ID NO: 1 ] contains an open reading frame encoding a polypeptide of 392 amino acid residues, including an initiation codon at positions 67-69, with a leader sequence of about 14 amino acid residues, and a deduced molecular weight of about 44.5 kDa.
  • the amino acid sequence of the /07735 mature PAPAI protein is shown in Figure 1, amino acid residues 15-392 [SEQ ID NO:2].
  • one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of : (a) a nucleotide sequence encoding the PAPAI polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2]; (b) a nucleotide sequence encoding the mature PAPAI polypeptide having the amino acid sequence at positions 15-392 in Figure 1 [SEQ ID NO:2]; (c) a nucleotide sequence encoding the PAPAI polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
  • nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d) or (e), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d) or (e), above.
  • This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
  • An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a PAPAI polypeptide having an amino acid sequence in (a), (b), (c) or (d), above.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of PAPAI polypeptides or peptides by recombinant techniques.
  • the invention further provides an isolated PAPAI polypeptide having amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the PAPAI polypeptide having the complete 392 amino acid sequence, including the leader sequence shown in Figure 1 [SEQ ID NO: 2]; (b) the amino acid sequence of the mature PAPAI polypeptide (without the leader) having the amino acid sequence at positions 15-392 in Figure 1 [SEQ ID NO:2]; (c) the amino acid sequence of the PAPAI polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No.
  • polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in (a), (b), (c) or (d) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 97%, 98% or 99% identical to those above.
  • An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a PAPAI polypeptide having an amino acid sequence described in (a), (b), (c) or (d), above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a PAPAI polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
  • the invention provides an isolated antibody that binds specifically to a PAPAI polypeptide having an amino acid sequence described in (a), (b), (c) or (d) above.
  • PAPAI gene expression For a number of pathologic disorders, such as tumor invasion and metastasis, significant alterations (increases or decreases) in level of PAPAI gene expression can be detected in a sample of tissue or bodily fluid. Increased or decreased levels of PAPAI gene expression can be measured, in such a sample, relative to a "standard" PAPAI gene expression level, i.e., the PAPAI expression level in a tissue or bodily fluid from an individual not having the disorder.
  • the present invention provides a diagnostic method useful during diagnosis of such disorders, which involves assaying the expression level of the gene encoding the PAPAI protein in tissue or bodily fluid from an individual and comparing the gene expression level with a standard PAPAI gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of a pathologic disorder, such as tumor invasion and metastasis, hemorrhage in liver disease, and preeclampsia.
  • a pathologic disorder such as tumor invasion and metastasis, hemorrhage in liver disease, and preeclampsia.
  • the PAPAI protein inhibits the plasminogen activator system when administered to an individual.
  • the plasminogen activator system is responsible for the degradation of intravascular blood clots, while also contributing to extracellular proteolysis in a wide variety of physiological processes (e.g. wound healing, cell migration, tissue remodeling, angiogenesis, trophoblast implantation, ovulation and fetal development) and pathological processes (e.g. tumor invasion and metastasis, intrauterine growth retardation, preeclampsia, and acute and chronic inflammation).
  • physiological processes e.g. wound healing, cell migration, tissue remodeling, angiogenesis, trophoblast implantation, ovulation and fetal development
  • pathological processes e.g. tumor invasion and metastasis, intrauterine growth retardation, preeclampsia, and acute and chronic inflammation.
  • methods are provided for inhibiting the plasminogen activator system, which involve administering an inhibitory amount of PAPAI either alone or together with one or more plasminogen activ
  • Figure 1 shows the nucleotide [SEQ ID NO:l] and deduced amino acid [SEQ ID NO:2] sequences of pancreas-derived plasminogen activator inhibitor (PAPAI) protein.
  • the protein has a leader sequence of about 14 amino acid residues (underlined) and a deduced molecular weight of about 44.5 kDa.
  • the predicted amino acid sequence of the mature PAPAI protein is also shown in Figure 1 [SEQ ID NO:2].
  • Figure 2 shows the regions of similarity between the amino acid sequences of the PAPAI protein (HPASD5OP protein) and human plasminogen activator inhibitor 1 (PAI-l) [SEQ ID NO:3] and human plasminogen activator inhibitor 2 (PAI-2) [SEQ ID NO:4].
  • Figure 3 shows an analysis of the predicted alpha, beta, turn, and coil regions, and the predicted hydrophilicity, amphipathic nature, flexible regions, antigenic index, and surface probability plot of the of the polypeptide of Figure 1 [SEQ ID NO: 2].
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding PAPAI polypeptide, having the amino acid sequence shown in Figure 1 [SEQ ID NO:2], which was determined by sequencing a cloned cDNA.
  • the PAPAI protein of the present invention shares sequence homology with human plasminogen activator inhibitor 1 (PAI- 1 ) ( Figure 2) [SEQ ID NO:3] and human plasminogen activator 2 (PAI-2) ( Figure 2) [SEQ ID NO:4].
  • nucleotide sequence shown in Figure 1 was obtained by sequencing the HPASD5OP clone, which was deposited on July 12, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession number 97657. Accordingly, in one embodiment of the present invention, isolated nucleic acid molecules are provided which encode the PAPAI protein.
  • PAPAI is a novel member of the plasminogen activator inhibitor subfamily.
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleotide sequence set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T).
  • nucleic acid molecule or polynucleotide a sequence of deoxyribonucleotides
  • RNA molecule or polynucleotide the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).
  • RNA molecule having the sequence of SEQ ID NO:l set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U.
  • nucleic acid molecule of the present invention encoding a PAPAI polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
  • standard cloning and screening procedures such as those for cloning cDNAs using mRNA as starting material.
  • nucleic acid molecule described in Figure 1 [SEQ ID NO:l] was discovered in a cDNA library derived from human pancreatic tissue.
  • the determined nucleotide sequence of the PAPAI cDN A of Figure 1 [SEQ ID NO : 1 ] contains an open reading frame encoding a protein of 392 amino acid residues, with an initiation codon at positions 67-69 of the nucleotide sequence in Figure 1 [SEQ ID NO:l], a predicted leader sequence of about 14 amino acid residues, and a deduced molecular weight of about 44.5 kDa.
  • the amino acid sequence of the predicted mature PAPAI is shown in Figure 1 [SEQ ID NO:2] from amino acid residue 15 to residue 392.
  • the PAPAI protein shown in Figure 1 [SEQ ID NO:2] is about 67 % and 68 % identical to PAI-l and PAI-2, respectively ( Figure 2).
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment
  • recombinant DNA molecules contained in a vector are considered isolated for the pu ⁇ oses of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) with an initiation codon at positions 67-69 of the nucleotide sequence shown in Figure 1 [SEQ ID NO:l] DNA molecules comprising the coding sequence for the mature PAPAI protein shown in Figure 1 (last 378 amino acids) [SEQ ID NO:2]; and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the PAPAI protein.
  • ORF open reading frame
  • SEQ ID NO:l DNA molecules comprising the coding sequence for the mature PAPAI protein shown in Figure 1 (last 378 amino acids) [SEQ ID NO:2]
  • the invention provides isolated nucleic acid molecules encoding the PAPAI polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97657 on
  • this nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA clone.
  • the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 1 [SEQ ID NO:l] or the nucleotide sequence of the PAPAI cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
  • Such isolated molecules, particularly DNA molecules are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the PAPAI gene in human tissue, for instance, by Northern blot analysis.
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC Deposit 97657.
  • stringent hybridization conditions is intended overnight incubation at 42 °C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • polynucleotides hybridizing to a larger portion of the reference polynucleotide e.g., the deposited cDNA clone
  • a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 [SEQ ID NO:l].
  • such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, the entire disclosure of which is hereby inco ⁇ orated herein by reference.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of the PAPAI cDNA shown in Figure 1 [SEQ ID NO: 1 ]), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • isolated nucleic acid molecules comprising a polynucleotide encoding an epitope-bearing portion of the PAPAI protein.
  • isolated nucleic acid molecules of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 20 to about 30 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 45 to about 50 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 60 to about 90 in Figure
  • nucleic acid molecules of the present invention which encode a PAPAI polypeptide may include, but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the about 14 amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non- coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 56:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37: 767 (1984).
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the PAPAI protein. Variants may occur naturally, such as a natural allelic variant. By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the PAPAI protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecules encoding the mature PAPAI protein having the amino acid sequence shown in Figure 1 [SEQ ID NO:2] or the mature
  • nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the full-length PAPAI polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2], including the predicted leader sequence; (b) a nucleotide sequence encoding the mature pancreas-derived plasminogen activator inhibitor polypeptide (full-length polypeptide with the leader removed) having the amino acid sequence at positions 15-392 in Figure 1 [SEQ ID NO:2]; (c) a nucleotide sequence encoding the full- length PAPAI polypeptide having the complete amino acid sequence including the leader encoded by the cDNA clone contained in ATCC Deposit No.
  • nucleotide sequence having a nucleotide sequence at least, for example, 95%) "identical" to a reference nucleotide sequence encoding a PAPAI polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the PAPAI polypeptide.
  • nucleotide having a nucleotide sequence at least 95%) identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5 1 or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 90%o, 95%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in Figure 1 or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
  • Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the present application is directed to nucleic acid molecules at least 90%, 95%o, 97%), 98%) or 99% identical to the nucleic acid sequence shown in Figure 1 [SEQ ID NO:l] or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having PAPAI activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having PAPAI activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having PAPAI activity include, inter alia, (1) isolating the PAPAI gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the PAPAI gene, as described in Verma et al, Human Chromosomes r A Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting PAPAI mRNA expression in specific tissues.
  • a polypeptide having PAPAI activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the PAPAI protein of the invention (either the full-length protein or, preferably, the mature protein), as measured in a particular biological assay.
  • Assays of plasminogen activator activity are well-known to those in the art. These assays can be used to measure plasminogen activator activity of partially purified or purified native or recombinant protein. For example, an l25 I fibrin lysis assay can be used (Lyon, P.B. et al., The Prostate 27:179-186 (1995);
  • 125 I fibrinogen is placed into 96-weIl, flat bottom culture plates at a concentration of 10 ⁇ g/cm 2 in a volume of 10-30 ⁇ l. Dried plates are exposed to 100 ⁇ l volume of RPMI medium containing 10% fetal bovine serum.
  • tissue plasminogen activator Sigma, St. Louis, MO.
  • cells were used in place of tissue plasminogen activator, 1 x 10 5 cells, washed twice with phosphate-buffered saline, are placed into each test well.
  • each test well is placed 1 ⁇ g of human plasminogen and 1 to 10 ⁇ g of the partially purified or purified native or recombinant test protein, in 5-50 ⁇ l of phosphate buffered saline.
  • Control wells receive an equal volume (5-50 ⁇ l) of phosphate buffered saline.
  • Each sample is assayed in duplicate or triplicate, with background values of radioactive release determined from the duplicate sample wells without plasminogen.
  • Additional control wells include media alone, media plus plasminogen, media plus plasminogen and plasminogen activator, and wells containing up to 200 ⁇ l of 0.25% bovine trypsin to determine the maximal releasable radioactivity.
  • Plasminogen-dependent fibrinolysis is defined as the difference in supernatant radioactivity between plasminogen-containing and plasminogen-free wells. Plasminogen activator-dependent fibrinolysis is expressed as a percentage of the maximal releasable radioactivity per well.
  • telomere length is determined by averaging the total trypsinizable radioactivity in three fibrin-coated wells. It will be apparent to one of ordinary skill in the art that the amounts of reactants and reactant conditions may have to be modified in order to practice the assay.
  • PAPAI inhibits plasminogen activators such as urokinase and tissue plasminogen activator.
  • a polypeptide having PAPAI protein activity includes polypeptides that exhibit the plasminogen activator inhibiting activity, in the above-described assay and in a dose-dependent manner.
  • a polypeptide having PAPAI protein activity will exhibit substantially similar dose-dependence in a given activity as compared to the PAPAI protein (i.e., the candidate polypeptide will exhibit greater activity or not more than about tenfold less and, preferably, not more than about twofold less activity relative to the reference PAPAI protein).
  • nucleic acid molecules having a sequence at least 90%, 95%, 97%), 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figure 1 [SEQ ID NO:l] will encode a polypeptide "having PAPAI protein activity.”
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • the present invention also relates to vectors which include the isolated
  • DNA molecules of the present invention host cells which are genetically engineered with the recombinant vectors, and the production of PAPAI polypeptides or fragments thereof by recombinant techniques.
  • Recombinant constructs may be introduced into host cells using well known techniques such infection, transduction, transfection, transvection, electroporation and transformation.
  • the vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • vectors comprising cis-acting control regions to the polynucleotide of interest.
  • Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA,
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
  • CHO, COS and Bowes melanoma cells CHO, COS and Bowes melanoma cells; and plant cells.
  • Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ⁇ trc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • bacterial promoters suitable for use in the present invention include the E. coli lacl and lacL promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the
  • HSV thymidine kinase promoter the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • retroviral LTRs such as those of the Rous sarcoma virus (RSV)
  • metallothionein promoters such as the mouse metallothionein-I promoter.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be inco ⁇ orated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage.
  • peptide moieties may be added to the polypeptide to facilitate purification.
  • the PAPAI protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the invention further provides an isolated PAPAI polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in Figure 1 [SEQ ID NO:2], or a peptide or polypeptide comprising a portion of the above polypeptides.
  • the terms "peptide” and “oligopeptide” are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least to amino acids coupled by peptidyl linkages.
  • polypeptide is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.
  • the invention further includes variations of the PAPAI polypeptide which show substantial PAPAI polypeptide activity or which include regions of PAPAI protein such as the protein portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of the PAPAI polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene ⁇ 57. * 31-40 (1988).
  • polypeptides of the present invention include the polypeptide encoded by the deposited cDNA including the leader, the mature polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein), the polypeptide of Figure 1 [SEQ ID NO:2] including the leader, the polypeptide of Figure 1 [SEQ ID NO:2] minus the leader, as well as polypeptides which have at least 90%) similarity, more preferably at least 95%) similarity, and still more preferably at least 97%o, 98% or 99%> similarity to those described above.
  • polypeptides of the present invention include polypeptides at least 80% identical, more preferably at least 90%) or 95%o identical, still more preferably at least 97%, 98%o or 99%o identical to the polypeptide encoded by the deposited cDNA, to the polypeptide of Figure 1 [SEQ ID NO:2], and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
  • %> similarity for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489, 1981) to find the best segment of similarity between two sequences.
  • polypeptide having an amino acid sequence at least, for example, 95%o "identical" to a reference amino acid sequence of a PAPAI polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the PAPAI polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 90%, 95%), 97%), 98%> or 99%» identical to, for instance, the amino acid sequence shown in Figure 1 [SEQ ID NO:2] or to the amino acid sequence encoded by deposited cDNA clone can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • polypeptides of the present invention can be used to raise polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting PAPAI protein expression as described below or as agonists and antagonists capable of enhancing or inhibiting PAPAI protein function.
  • polypeptides can be used in the yeast two-hybrid system to "capture" PAPAI protein binding proteins which are also candidate agonist and antagonist according to the present invention.
  • the yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989).
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661.
  • 18 of 20 peptides designed according to these guidelines containing 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
  • a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein.
  • the antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes post-translational processing.
  • the peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about
  • Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein.
  • the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate PAPAI-specific antibodies include: a polypeptide comprising amino acid residues from about 20 to about 30 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 45 to about 50 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 60 to about 90 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 125 to about 135 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 160 to about 175 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 220 to about 225 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 250 to about 260 in Figure 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 320 to about 330 in Figure 1
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention.
  • a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies.
  • Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis. For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Nat Acad. Sci. USA 52:5131 -5135. This "Simultaneous Multiple Peptide Synthesis
  • Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al. Proc. Natl Acad. Sci. USA 52:910-914; and Bittle, F. J. et al, J. Gen. Virol. 66:2347-2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine may be coupled to carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde.
  • Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adso ⁇ tion to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • Immunogenic epitope-bearing peptides of the invention i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. For instance, Geysen et al, supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art.
  • the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined.
  • peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method.
  • PAPAI is involved in inhibition of the plasminogen activator system.
  • pathologic disorders including tumor invasion and metastasis, inflammation, and complications of pregnancy, it is believed that significantly altered levels of PAPAI gene expression can be detected in body tissue or fluids taken from an individual having such a disorder.
  • the level of PAPAI gene expression is measured relative to a "standard" PAPAI gene expression level, i.e., the PAPAI expression level in tissue or fluids from an individual not having the pathologic disorder.
  • the invention provides a diagnostic method useful during diagnosis of a pathologic disorder, which involves assaying the expression level of the gene encoding the PAPAI protein in tissue or body fluid from an individual and comparing the gene expression level with a standard PAPAI gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of a pathologic disorder.
  • PAPAI vascular endothelial growth factor
  • Angiogenesis the growth of new vascular tissue, is regulated by a balance between coagulation and fibrinolysis. Angiogenesis is essential for the expansion of a primary tumor and is also required for the growth of established matastases at distant sites (Holmgren, L. et al, Nature Medicine 7:149-153 (1995)).
  • the level of PAI-l in tumors indicates the level of vascularization, and highly vascularized tumors have higher chances of invasion and metastasis (Fazioli, F. et al, Trends Pharm. Sci. 75:25- 29 (1995)).
  • Substantial alterations in PAPAI expression or activity can be used to predict whether hemorrhage is likely to occur in patients who suffer from hepatic illnesses.
  • Alcoholic cirrhosis, primary biliary cirrhosis, and liver cancer are all diseases which are accompanied by hemorrhage due to fibrinolytic bleeding. That is, the bleeding which occurs is not due to injury, but rather is due to a dysregulated fibrinolytic system.
  • the overall level of plasminogen activator activity represents a balance between the relative levels of activator and inhibitor.
  • Changes in PAPAI activity, or a difference in the ratio of plasminogen activator to PAPAI can serve as indicators of imminent hemorrhage in patients who suffer from alcoholic cirrhosis, primary biliary cirrhosis, and liver cancer.
  • the invention further provides a method for predicting whether hemorrhage will occur in such patients.
  • the terms "hemorrhage,” “alcoholic cirrhosis,” “primary biliary cirrhosis,” “liver cancer, and “fibrinolytic bleeding” are well understood by those of ordinary skill in the art. For example, see Leiper, K. et al, J. Clin. Pathol 47:214-217 (1994).
  • Preeclampsia is a clinical syndrome that affects women in the third trimester of pregnancy. The syndrome is characterized by hypertension and proteinuria. The etiology of this obstetric complication is unknown. However, it is associated with fibrin deposition in the subendothelium of the renal glomerulus and in the decidua segments of spiral arteries. In fatal cases of eclampsia, widespread fibrin deposition has been a prominent histologic finding.
  • Changes in PAPAI activity, or a difference in the ratio of plasminogen activator to PAPAI can serve as indicators of an imminent advance from the pre-eclamptic to the eclamptic state in patients who are at risk for eclampsia.
  • the invention further provides a method for predicting whether a pre-eclamptic patient is at risk for developing eclampsia.
  • preeclampsia is well understood by those of ordinary skill in the art. For example, see Koh, C.L. et al, Gynecol Obstet. Invest. 55:214-221 (1993). Alterations in PAPAI expression can be assayed at the level of messenger
  • RNA transcription or protein expression RNA transcription or protein expression.
  • Suitable assay techniques are disclosed below.
  • PAPAI inhibitory activity can be assayed using a spectrophotometric plasminogen activator inhibitor assay.
  • Such an assay is well- known to those of ordinary skill in the art. For example, see Erikkson, E. et al, Thrombosis Research 50:91-101 (1988).
  • measuring the expression level of the gene encoding the PAPAI protein is intended qualitatively or quantitatively measuring or estimating the level of the PAPAI or the level of the mRNA encoding the PAPAI protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the PAPAI protein level or mRNA level in a second biological sample).
  • the PAPAI protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard PAPAI protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
  • a standard PAPAI protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, a cell line, a tissue culture, or other source which contains PAPAI protein or mRNA, secretes mature PAPAI protein, or expresses the PAPAI receptor.
  • Biological samples include normal tissue or cells and tumor cells (whether malignant or benign).
  • Biological samples include body fluids, including whole blood, serum, plasma, urine, saliva, tears, pulmonary secretions, gastrointestinal secretions, fecal material, lymph fluid, synovial fluid, and cerebrospinal fluid. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
  • Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol- chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 762.156-159 (1987). Levels of mRNA encoding the PAPAI protein are then assayed using any appropriate method. These include Northern blot analysis, SI nuclease mapping, the polymerase chain reaction (PCR), reverse transcription (RT) in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the Iigase chain reaction (RT-LCR).
  • PCR polymerase chain reaction
  • RT reverse transcription
  • RT-LCR reverse transcription in combination with the Iigase chain reaction
  • RNA is prepared from a biological sample as described above.
  • an appropriate buffer such as glyoxal/dimethyl sulfoxide/sodium phosphate buffer
  • the filter is prehybridized in a solution containing formamide, SSC, Denhardt ' s solution, denatured salmon sperm, SDS, and sodium phosphate buffer.
  • PAPAI protein cDNA labeled according to any appropriate method such as the 32 P-multiprimed DNA labeling system (Amersham) is used as probe. After hybridization overnight, the filter is washed and exposed to x-ray film.
  • cDNA for use as probe according to the present invention is described in the sections above and will preferably at least 15 bp in length.
  • SI mapping can be performed as described in Fujita et al, Cell 49:357- 367 (1987).
  • probe DNA for use in SI mapping, the sense strand of above-described cDNA is used as a template to synthesize labeled antisense DNA.
  • the antisense DNA can then be digested using an appropriate restriction endonuclease to generate further DNA probes of a desired length.
  • Such antisense probes are useful for visualizing protected bands corresponding to the target mRNA (i.e., mRNA encoding the PAPAI protein).
  • Northern blot analysis can be performed as described above.
  • levels of mRNA encoding the PAPAI protein are assayed using the RT-PCR method described in Makino et al, Technique 2.295-301
  • this method involves adding total RNA isolated from a biological sample in a reaction mixture containing a RT primer and appropriate buffer. After incubating for primer annealing, the mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of the RNA, the RT products are then subject to PCR using labeled primers. Alternatively, rather than labeling the primers, a labeled dNTP can be included in the PCR reaction mixture.
  • PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of the appropriate bands (corresponding to the mRNA encoding the PAPAI protein) is quantified using an imaging analyzer. RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art. Variations on the RT-PCR method will be apparent to the skilled artisan. Any set of oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as described in the sections above.
  • Assaying PAPAI protein levels in a biological sample can occur using any art-known method.
  • Preferred for assaying PAPAI protein levels in a biological sample are antibody-based techniques.
  • PAPAI protein expression in tissues can be studied with classical immunohistological methods. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological staining of tissue section for pathological examination is obtained. Tissues can also be extracted, e.g., with urea and neutral detergent, for the liberation of
  • PAPAI protein for Western-blot or dot/slot assay Jalkanen, M., et al, J. Cell Biol. 101.-976-985 (1985)); Jalkanen, M., et al, J. Cell . Biol. 705:3087-3096 (1987)).
  • quantitation of PAPAI protein can be accomplished using isolated PAPAI protein as a standard.
  • This technique can also be applied to body fluids. With these samples, a molar concentration of PAPAI protein will aid to set standard values of PAPAI protein content for different body fluids, like serum, plasma, urine, synovial fluid, spinal fluid, etc.
  • the normal appearance of PAPAI protein amounts can then be set using values from healthy individuals, which can be compared to those obtained from a test subject.
  • PAPAI protein-specific monoclonal antibodies can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify the PAPAI protein. The amount of
  • PAPAI protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • a linear regression computer algorithm Such an ELISA for detecting a tumor antigen is described in Iacobelli et al, Breast Cancer Research and Treatment 77: 19-30 (1988).
  • two distinct specific monoclonal antibodies can be used to detect PAPAI protein in a body fluid. In this assay, one of the antibodies is used as the immunoadsorbent and the other as the enzyme-labeled probe.
  • the above techniques may be conducted essentially as a "one-step” or “two-step” assay.
  • the "one-step” assay involves contacting PAPAI protein with immobilized antibody and, without washing, contacting the mixture with the labeled antibody.
  • the "two-step” assay involves washing before contacting the mixture with the labeled antibody.
  • Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
  • Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate.
  • Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available.
  • Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction.
  • radioisotopes such as iodine ( 125 I, I 1 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( l ,2 In), and technetium ( 9 Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • PAPAI protein can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of PAPAI protein include those detectable by X-radiography, NMR or ESR.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be inco ⁇ orated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a PAPAI protein-specific antibody or antibody portion which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, *3* I, U2 In, " m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined.
  • a radioisotope for example, *3* I, U2 In, " m Tc
  • m Tc radio-opaque substance, or a material detectable by nuclear magnetic resonance
  • the labeled antibody or antibody portion will then preferentially accumulate at the location of cells which contain PAPAI protein.
  • In vivo tumor imaging is described in S. W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Portions" (Chapter 13 in Tumor Imaging: The
  • PAPAI protein-specific antibodies for use in the present invention can be raised against the intact PAPAI protein or an antigenic polypeptide portion thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
  • a carrier protein such as an albumin
  • antibody As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody portions (such as, for example, Fab and F(ab') 2 portions) which are capable of specifically binding to
  • Fab and F(ab') 2 portions lack the Fc portion of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:3X6-325 (1983)).
  • the antibodies of the present invention may be prepared by any of a variety of methods.
  • cells expressing the PAPAI protein or an antigenic portion thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • a preparation of PAPAI protein is prepared and purified as described above to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • the antibodies of the present invention are monoclonal antibodies (or PAPAI protein binding portions thereof).
  • monoclonal antibodies can be prepared using hybridoma technology (Kohler et al, Nature 256:495 (1975); Kohler et al, Eur. J. Immunol. 6:51 1 (1976); Kohler et al, Eur. J. Immunol. 6:292 (1976); Hammerling et al, In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).
  • such procedures involve immunizing an animal (preferably a mouse) with a PAPAI protein antigen or, more preferably, with a PAPAI protein- expressing cell.
  • Suitable cells can be recognized by their capacity to bind PAPAI protein antibody.
  • Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10%> fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 ⁇ g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland.
  • SP 2 O parent myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al (Gastroenterology 50:225-232 (1981)).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the PAPAI antigen.
  • additional antibodies capable of binding to the PAPAI protein antigen may be produced in a two-step procedure through the use of anti- idiotypic antibodies.
  • PAPAI protein specific antibodies are used to immunize an animal, preferably a mouse.
  • the splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the PAPAI protein-specific antibody can be blocked by the PAPAI protein antigen.
  • Such antibodies comprise anti-idiotypic antibodies to the PAPAI protein-specific antibody and can be used to immunize an animal to induce formation of further PAPAI protein-specific antibodies.
  • Fab and F(ab') 2 and other portions of the antibodies of the present invention may be used according to the methods disclosed herein. Such portions are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab portions) or pepsin (to produce F(ab') 2 portions). Alternatively, PAPAI protein-binding portions can be produced through the application of recombinant DNA technology or through synthetic chemistry.
  • chimeric monoclonal antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Cabilly et al, U.S. Patent No. 4,816,567; Taniguchi et al, EP 171496; Morrison et al, EP 173494; Neuberger et al, WO 8601533; Robinson et al, WO
  • suitable labels for the PAPAI protein-specific antibodies of the present invention are provided below.
  • suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.
  • radioisotopic labels examples include 3 H, '"In, 125 I, l31 1, 32 P, 35 S, , C, 51 Cr, "To, 58 Co, 59 Fe, 75 Se, ,52 Eu, 90 Y, 67 Cu, 2,7 Ci, 2, l At, 2,2 Pb, 47 Sc, l ⁇ 9 Pd, etc.
  • ' ' 'In is a preferred isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the 125 I or 131 I-labeled monoclonal antibody by the liver.
  • this radionuclcotide has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med.
  • non-radioactive isotopic labels examples include I57 Gd, 55 Mn,
  • fluorescent labels examples include an 152 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.
  • Suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.
  • chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.
  • nuclear magnetic resonance contrasting agents examples include heavy metal nuclei such as Gd, Mn, and Fe.
  • Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al. (Clin. Chim. Ada 70:1-31 (1976)), and Schurs et al. (Clin. Chim. Acta 57:1-40 (1977)). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are inco ⁇ orated by reference herein.
  • the nucleic acid molecules of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymo ⁇ hisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • the cDNA herein disclosed is used to clone genomic DNA of a PAPAI protein gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially.
  • the genomic DNA then is used for in situ chromosome mapping using well known techniques for this pu ⁇ ose. Typically, in accordance with routine procedures for chromosome mapping, some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified portion.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of portions from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow- sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with probes from the cDNA as short as 50 or 60 bp.
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. This assumes 1 megabase mapping resolution and one gene per 20 kb. Therapeutic Uses of PAPAI
  • the PAPAI-protein is believed to be involved in inhibition of the plasminogen activator system and plays a role in a wide variety of physiologic and pathologic processes. Accordingly, the PAPAI protein has application to any physiologic or pathologic disease condition in which abnormal activity of the plasminogen activator system is implicated and has pathological or physiological consequences.
  • a large number of disease conditions are associated with modifications of the plasminogen activator system (Kruithof et al, Thromb. Haemost. 59:7 (1988)). Examples of such disease conditions include, but are not limited to: complications of pregnancy, such as preeclampsia and intrauterine growth retardation (Halligan et al, Br. Obstet.
  • the invention also provides a method of treatment of an individual in need of an increased level of activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated PAPAI polypeptide of the invention, particularly a mature form of the PAPAI protein of the invention, effective to increase the PAPAI activity level in such an individual.
  • the invention provides a method for treating or preventing tumor invasion and metastasis in cancers including, but not limited to, leukemia, lung cancer, breast cancer, endometrial and ovarian cancer, melanoma, and gastrointestinal cancers, including pancreatic cancer and colorectal cancer.
  • the invention provides a method for treating or preventing coagulation disorders including, but not limited to, arterial thrombi, venous thrombi, disseminated intravascular coagulation, and excessive bleeding caused by the administration of a pharmaceutical plasminogen activator (such as urokinase or tissue plasminogen activator).
  • a pharmaceutical plasminogen activator such as urokinase or tissue plasminogen activator
  • HIV-1 Human Immunodeficiency Virus 1
  • HIV-2 hepatitis A, hepatitis B, hepatitis C, hepatitis E, hepatitis F, and hepatitis G.
  • PAPAI polypeptides for treating an individual in need of an increased level of PAPAI activity can be determined empirically for each condition where administration of PAPAI is indicated.
  • the polypeptide having PAPAI activity my be administered in pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the pharmaceutical compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutical ly effective dose level for any particular patient will depend upon a variety of factors including the type and degree of the response to be achieved; the specific composition an other agent, if any, employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the composition; the duration of the treatment; drugs (such as a chemotherapeutic agent) used in combination or coincidental with the specific composition; and like factors well known in the medical arts.
  • the PAPAI composition to be used in the therapy will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with PAPAI alone), the site of delivery of the PAPAI composition, the method of administration, the scheduling of administration, and other factors known to practitioners.
  • the "effective amount" of PAPAI for pu ⁇ oses herein is thus determined by such considerations.
  • the total pharmaceutically effective amount of the PAPAI administered parenterally per dose will be in the range of about 1 ⁇ g kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day. If given continuously, the PAPAI is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump.
  • An intravenous bag solution or bottle solution may also be employed.
  • a course of PAPAI treatment to affect the fibrinolytic system appears to be optimal if continued longer than a certain minimum number of days, 7 days in the case of the mice.
  • the length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
  • the PAPAI is also suitably administered by sustained-release systems.
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules.
  • Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U.
  • Sustained-release PAPAI compositions also include liposomally entrapped PAPAI. Liposomes containing PAPAI are prepared by methods known per se: DE 3,218,121; Epstein, et al, Proc. Natl.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal PAPAI therapy.
  • the PAPAI is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a pharmaceutically acceptable carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
  • the formulations are prepared by contacting the PAPAI uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation.
  • the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
  • carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
  • the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbi
  • PAPAI to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
  • Therapeutic PAPAI compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • PAPAI ordinarily will be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10- ml vials are filled with 5 ml of sterile-filtered 1%> (w/v) aqueous PAPAI solution, and the resulting mixture is lyophilized.
  • the infusion solution is prepared by reconstituting the lyophilized PAPAI using bacteriostatic Water-for-Injection.
  • Dosaging may also be arranged in a patient specific manner to provide a predetermined concentration of an PAPAI activity in the blood, as determined by an RIA technique, for instance. Thus patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
  • compositions of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically
  • pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the DNA sequence encoding the mature PAPAI protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the PAPAI protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences, respectively.
  • the 5' oligonucleotide primer has the sequence 5' CGC CCA TGG GAA GTC AAG CCT CAA G 3' [SEQ ID NO:5] containing the underlined Nco I restriction site (which encodes a start ATG within the Nco I site), followed by 16 nucleotides complementary to bp 110-125 of the antisense strand of the PAPAI protein coding sequence set out in Figure 1 [SEQ ID NO:l].
  • the 3' primer has the sequence 5 * CGC AAG CTT TCA CTT CCT TTT
  • restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE-60, which is used for bacterial expression in these examples. (Qiagen, Chatsworth, CA, 91311).
  • the amplified PAPAI protein DNA and the vector pQE-60 are both digested with Nco I and Hind III and the digested DNAs are subsequently ligated together. Insertion of the PAPAI protein DNA into the pQE-60 restricted vector places the PAPAI protein coding region downstream of and operably linked to the vector's promoter and in-frame with an initiating AUG appropriately positioned for translation of PAPAI protein.
  • the ligation mixture is transformed into competent E. coli cells using standard procedures. Such procedures are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described here.
  • This strain which is only one of many that are suitable for expressing PAPAI protein, is available commercially from Qiagen.
  • Transformants are identified by their ability to grow on LB plates in the presence of ampicillin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA is confirmed by restriction analysis.
  • Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250.
  • the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
  • Isopropyl-B-D-thiogalactopyranoside (“IPTG”) are then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lad repressor.
  • Cells subsequently are incubated further for 3 to 4 hours.
  • Cells are then harvested by centrifugation and disrupted, by standard methods.
  • Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein are solubilized from the inclusion bodies into 8M urea.
  • the 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein.
  • PBS 2X phosphate buffered saline
  • the protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered.
  • the sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 micrograms per mL.
  • vectors used for the transient expression of the PAPAI protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g., COS cells) which express the T-antigen required for the initiation of viral DNA synthesis. Any other suitable mammalian cell line can also be utilized for this pu ⁇ ose.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). Cellular signals may, however, also be used (e.g., human actin, promoter).
  • LTRs long terminal repeats
  • CMV cytomegalovirus
  • Suitable expression vectors for use in practicing the present invention include , for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • Mammalian host cells that may be used include human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and C 127 cells, Cos 1, Cos 7 and CV1 African green monkey cells, quail QCl-3 cells, mouse L cells and Chinese hamster ovary cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • the co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded protein.
  • the DHFR dihydrofolate reductase
  • GS glutamine synthase
  • the expression vectors pC 1 and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology 438:4470 (1985)) plus a fragment of the CMV-enhancer (Boshart et al, Cell 47:521-530 (1985)).
  • Plasmid pCl is used for the expression of PAPAI protein.
  • Plasmid pCl is a derivative of the plasmid pSV2-dr.fr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the S V40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt,
  • Plasmid pC 1 contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al. , Cell 41:521 -530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Pvull, and Nrul.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • the plasmid contains translational stop codons in all three reading frames followed by the 3' intron and the polyadenylation site of the rat preproinsulin gene.
  • Other high efficient promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
  • the plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding PAPAI protein in the deposited polynucleotide is amplified using PCR oligonucleotide primers specific to the carboxyl terminal sequence of the PAPAI protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
  • the 5' primer has the sequence 5' CGC GGA TCC .
  • the 3' primer has the full length sequence CGC GGT ACC TCA CTT CCT TTT ATC TCC CTG [SEQ ID NO:8], containing the underlined Asp718 restriction site, followed by 8 nucleotides complementary to bp 1250-1267 of the sense strand of the PAPAI protein coding sequence set out in Figure 1 [SEQ ID NO: 1 ], and a stop codon.
  • the restrictions sites are convenient to restriction enzyme sites in the CHO expression vector CHO-1.
  • the amplified PAPAI protein DNA and the vector CHO- 1 both are digested with BamH I and Asp718 and the digested DN As subsequently ligated together. Insertion of the PAPAI protein DNA into the BamH I/Asp718 digested vector places the PAPAI protein coding region downstream of and operably linked to the vector's promoter.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 1 1099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the PAPAI-encoding fragment.
  • Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection.
  • 5 ⁇ g of the expression plasmid CI are cotransfected with 0.5 ⁇ g of the plasmid pSVneo using the lipofectin method (Feigner et al, supra).
  • the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M). The same procedure is repeated until clones grow at a concentration of 100 ⁇ M.
  • the expression plasmid, pPAPAI HA is made by cloning a cDNA encoding PAPAI into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
  • the expression vector pcDNAI/amp contains: (1) an E.coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • a DNA fragment encoding the entire PAPAI precursor and an HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37: 767 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is as follows.
  • the PAPAI cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of PAPAI in E. coli.
  • primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of PAPAI in E. coli.
  • one of the primers contains a hemagglutinin tag ("HA tag”) as described above.
  • Suitable primers include that following, which are used in this example.
  • the 5' primer has the sequence 5' CGC GGA TCC GCC ATC ATG GAC ACA
  • the 3' primer has the full length sequence CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GT ⁇ GGG ATT TGT CAC TCT TCC [SEQ ID NO:9], containing the underlined Xba I restriction site, an HA tag, and 18 nucleotides complementary to bp 1225-1242 of the sense strand of the PAPAI protein coding sequence set out in Figure 1 [SEQ ID NO: 1].
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with BamH I and Xba I and then Iigated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning
  • the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the PAPAI -encoding fragment.
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of PAPAI by the vector.
  • PAPAI HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al, Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35 S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, l%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
  • Example 3 Cloning and expression of the PAPAI protein in a baculovirus expression system
  • the cDN A sequence encoding the soluble extracellular domain of PAPAI protein receptor protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
  • the 5' primer has the sequence 5" CGC GGA TCC GCC ATC ATG GAC ACA ATC TTC TTG 3' [SEQ ID NO:7] containing the underlined BamH I restriction enzyme site followed by 18 bases (bp 67-84) complementary to the antisense strand of the PAPAI protein coding sequence of Figure 1 [SEQ ID NO:l].
  • Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding PAPAI protein receptor provides an efficient signal peptide.
  • An efficient signal for initiation of translation in eukaryotic cells as described by Kozak, M., J Mol Biol. 196:947-950 (1987), may be located, as appropriate, in the vector portion of the construct.
  • the 3' primer has the full length sequence CGC GGT ACC TCA CTT CCT TTT ATC TCC CTG [SEQ ID NO:8], containing the underlined Asp718 restriction followed by nucleotides complementary to bp 1250-1267 of the sense strand of the PAPAI protein set out in Figure 1 [SEQ ID NO:8]
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with BamHI and Asp718 and again is purified on a 1% agarose gel. This fragment is designated herein F2.
  • the vector pA2 is used to express the PAPAI protein in the baculovirus expression system, using standard methods, such as those described in Summers et al, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
  • This expression vector contains the strong polyhedrin promoter of the Autographa califomica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
  • AcMNPV Autographa califomica nuclear polyhedrosis virus
  • the beta- galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin sequences are flanked at both sides by viral sequences for cellmediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
  • baculovirus vectors could be used in place of pA2, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
  • Such vectors are described, for example, in Luckow et al. , Virology 770:31-39 (1989). Suitable vectors will be readily apparent to the skilled artisan.
  • the plasmid is digested with the restriction enzymes BamH I and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
  • Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
  • E. coli HB 101 cells are transformed with ligation mix and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human PAPAI protein gene by digesting DNA from individual colonies using Bam HI and Asp718 and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein as pBacPAPAI.
  • plasmid pBacPAPAI 5 ⁇ g of plasmid pBacPAPAI is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method described by
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra.
  • An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques.
  • a detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, MD, at pages 9-10.
  • the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an
  • V-PAPAI A clone containing properly inserted PAPAI protein receptor is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-PAPAI.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS.
  • the cells are infected with the recombinant baculovirus V-
  • PAPAI multiplicity of infection
  • MOI multiplicity of infection
  • RNA is size resolved by electrophoresis through a 1 % agarose gel under strongly denaturing conditions. RNA is blotted from the gel onto a nylon filter, and the filter then is prepared for hybridization to a detectably labeled polynucleotide probe.
  • the antisense strand of the coding region of the cDNA insert in the deposited clone is labeled to a high specific activity.
  • the cDNA is labeled by primer extension, using the Prime-It kit, available from Stratagene. The reaction is carried out using 50 ng of the cDNA, following the standard reaction protocol as recommended by the supplier.
  • the labeled polynucleotide is purified away from other labeled reaction components by column chromatography using a Select-G-50 column, obtained from 5-Prime - 3-Prime, Inc. of 5603 Arapahoe Road, Boulder, CO 80303.
  • the labeled probe is hybridized to the filter, at a concentration of 1 ,000,000 cpm/ml, as described in Kreider et al ., Molecular and Cellular Biology,
  • ADDRESSEE STERNE, KESSLER, GOLDSTEIN & FOX, P.L.L.C.
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO : 3 :
  • MOLECULE TYPE cDNA

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Abstract

L'invention concerne un membre nouveau de la famille des protéines inhibitrices de l'activateur du plasminogène. En particulier, des molécules d'acide nucléique isolées codent la protéine inhibitrice de l'activateur du plasminogène dérivée du pancréas. L'invention concerne également des polypeptides inhibiteurs de l'activateur du plasminogène dérivés du pancréas, des vecteurs, des cellules hôtes et des procédés de recombinaison utilisés pour les produire. L'invention se rapporte en outre à des méthodes de traitement de maladies ou de troubles physiologiques et à des méthodes diagnostiques de détection de troubles pathologiques.
PCT/US1996/013283 1996-08-16 1996-08-16 Inhibiteur de l'activateur du plasminogene derive du pancreas WO1998007735A1 (fr)

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PCT/US1996/013283 WO1998007735A1 (fr) 1996-08-16 1996-08-16 Inhibiteur de l'activateur du plasminogene derive du pancreas
AU73579/96A AU7357996A (en) 1996-08-16 1996-08-16 Pancreas-derived plasminogen activator inhibitor

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PCT/US1996/013283 WO1998007735A1 (fr) 1996-08-16 1996-08-16 Inhibiteur de l'activateur du plasminogene derive du pancreas

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999011786A1 (fr) * 1997-09-01 1999-03-11 Otsuka Pharmaceutical Co., Ltd. GENE pancpin ET COMPOSITION DE THERAPIE GENIQUE
EP2230252A1 (fr) 2006-03-13 2010-09-22 The Johns Hopkins University Augmentation de la thromborésistance endothéliale
WO2012090997A1 (fr) 2010-12-27 2012-07-05 京都府公立大学法人 CELLULES SPi ET LEUR PROCÉDÉ DE PRODUCTION
WO2013100208A1 (fr) 2011-12-28 2013-07-04 京都府公立大学法人 Normalisation d'une culture de cellules endothéliales de la cornée
EP2862867A2 (fr) 2005-10-25 2015-04-22 The Johns Hopkins University Procédés et compositions pour le traitement du syndrome de Marfan et troubles associés
WO2015064768A1 (fr) 2013-10-31 2015-05-07 京都府公立大学法人 Médicament thérapeutique pour des maladies associées à la mort cellulaire du réticulum endoplasmique dans l'endothélium de la cornée
US10882903B2 (en) 2015-05-18 2021-01-05 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for treating an alphavirus infection

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4923807A (en) * 1984-05-18 1990-05-08 New England Medical Center Hospitals Inc. Arg-Serpin human plasminogen activator inhibitor designated PAI-2
US5470970A (en) * 1991-02-28 1995-11-28 Dana-Farber Cancer Institute, Inc. Maspin, a serpin with tumor suppresing activity

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US4923807A (en) * 1984-05-18 1990-05-08 New England Medical Center Hospitals Inc. Arg-Serpin human plasminogen activator inhibitor designated PAI-2
US5470970A (en) * 1991-02-28 1995-11-28 Dana-Farber Cancer Institute, Inc. Maspin, a serpin with tumor suppresing activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AM. J. TROP. MED. HYG., Vol. 401, No. 1, KRISHNAMURTI et al., "Stimulation of Plasminogen Activator Inhibitor Activity in Human Monocytes Infected with Dengue Virus", pages 102-107. *
EUR. J. BIOCHEM., 1992, Vol. 209, URANO et al., "A Substrate-Like form of Plasminogen-Activator-Inhibitor Type 1", pages 985-992. *
J. CLINICAL PATHOLOGY, 1994, Vol. 47, LEIPER et al., "Tissue Plasminogen Activator, Plasminogen Activator Inhibitors and Activator-Inhibitor Complex in Liver Disease", pages 214-217. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999011786A1 (fr) * 1997-09-01 1999-03-11 Otsuka Pharmaceutical Co., Ltd. GENE pancpin ET COMPOSITION DE THERAPIE GENIQUE
EP2862867A2 (fr) 2005-10-25 2015-04-22 The Johns Hopkins University Procédés et compositions pour le traitement du syndrome de Marfan et troubles associés
EP2230252A1 (fr) 2006-03-13 2010-09-22 The Johns Hopkins University Augmentation de la thromborésistance endothéliale
WO2012090997A1 (fr) 2010-12-27 2012-07-05 京都府公立大学法人 CELLULES SPi ET LEUR PROCÉDÉ DE PRODUCTION
WO2013100208A1 (fr) 2011-12-28 2013-07-04 京都府公立大学法人 Normalisation d'une culture de cellules endothéliales de la cornée
EP3553169A1 (fr) 2011-12-28 2019-10-16 Kyoto Prefectural Public University Corporation Normalisation de culture de cellules endothéliales cornéennes
WO2015064768A1 (fr) 2013-10-31 2015-05-07 京都府公立大学法人 Médicament thérapeutique pour des maladies associées à la mort cellulaire du réticulum endoplasmique dans l'endothélium de la cornée
EP3804760A1 (fr) 2013-10-31 2021-04-14 Kyoto Prefectural Public University Corporation Médicament thérapeutique pour des maladies liées à la mort cellulaire du réticulum endoplasmique dans l'endothélium cornéen
US10882903B2 (en) 2015-05-18 2021-01-05 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for treating an alphavirus infection

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