WO1997012984A1 - COMPOSE PRESENTANT UNE HOMOLOGIE DE SEQUENCE AVEC UNE PHOSPHOLIPASE A2 ASSOCIEE A UNE LIPOPROTEINE (Lp-PLA2) OU ACETYLE HYDROLASE DU FACTEUR D'ACTIVATION DES PLAQUETTES - Google Patents

COMPOSE PRESENTANT UNE HOMOLOGIE DE SEQUENCE AVEC UNE PHOSPHOLIPASE A2 ASSOCIEE A UNE LIPOPROTEINE (Lp-PLA2) OU ACETYLE HYDROLASE DU FACTEUR D'ACTIVATION DES PLAQUETTES Download PDF

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Publication number
WO1997012984A1
WO1997012984A1 PCT/GB1995/002320 GB9502320W WO9712984A1 WO 1997012984 A1 WO1997012984 A1 WO 1997012984A1 GB 9502320 W GB9502320 W GB 9502320W WO 9712984 A1 WO9712984 A1 WO 9712984A1
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WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
dna
sequence
seq
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PCT/GB1995/002320
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English (en)
Inventor
Christopher Donald Southan
Simon Quentyn John Rice
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Smithkline Beecham Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Plc filed Critical Smithkline Beecham Plc
Priority to AU35728/95A priority Critical patent/AU3572895A/en
Priority to PCT/GB1995/002320 priority patent/WO1997012984A1/fr
Priority to EP95932839A priority patent/EP0853673A1/fr
Priority to JP9514042A priority patent/JPH11514228A/ja
Priority to AU72169/96A priority patent/AU7216996A/en
Priority to EP96933431A priority patent/EP0859834A2/fr
Priority to PCT/EP1996/004268 priority patent/WO1997012963A2/fr
Priority to JP51396697A priority patent/JP2002515728A/ja
Priority to CA002233300A priority patent/CA2233300A1/fr
Publication of WO1997012984A1 publication Critical patent/WO1997012984A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the use of inhibitors of a polypeptide in the therapy.
  • the present invention also relates to the polypeptide, to polynucleotides encoding the polypeptide, to recombinant host cells transformed with DNA encoding the polypeptide and to the use of the polypeptide in identifying compounds which are potentially useful in therapy.
  • Lipoprotein Associated Phospholipase A2 (Lp-PLA2) is also known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase).
  • PAF acetyl hydrolase Platelet Activating Factor Acetyl Hydrolase
  • Lp-PLA2 is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso- phosphatidylcholine and an oxidatively modified fatty acid. Both of these products of Lp-PLA2 action are potent chemoattractants for circulating monocytes.
  • this enzyme is thought to be responsible for the accumulation of cells loaded with cholesterol ester in the arteries, causing the characteristic 'fatty streak' associated with the early stages of atherosclerosis. Inhibition of the Lp-PLA2 enzyme would therefore be expected to stop the build up of this fatty streak (by inhibition of the formation of lysophosphatidylcholine), and so be useful in the treatment of atherosclerosis.
  • Lp-PLA2 inhibitors may also have a general application in any disorder that includes lipid peroxidation in conjunction with the enzyme activity, for example in addition to conditions such as atherosclerosis and diabetes other conditions such as rheumatoid arthritis, stroke, myocardial infarction, reperfusion injury, acute and chronic inflammation and various neuropsychiatric illnesses (e.g., schizophrenia, ref. Psychopharmacology Bulletin, 31: 159-165, 1995).
  • conditions such as atherosclerosis and diabetes other conditions such as rheumatoid arthritis, stroke, myocardial infarction, reperfusion injury, acute and chronic inflammation and various neuropsychiatric illnesses (e.g., schizophrenia, ref. Psychopharmacology Bulletin, 31: 159-165, 1995).
  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a novel lipase. The invention also relates to inhibiting the action of such polypeptides.
  • polypeptide which is a lipase having the amino acid sequence given in SEQ ID NO 1 , or a fragments, analogs or derivative thereof.
  • the polypeptide of the present invention is of human origin.
  • polypeptide(s) will be used to refer to the lipase and its fragments, analogs and derivatives
  • polynucleotides (DNA or RNA) which encode such polypeptides.
  • polynucleotide which encodes for the polypeptide having the amino acid sequence of SEQ ID NO 1.
  • the invention provides a polynucleotide having the DNA sequence given in SEQ ID NO 2.
  • cDNA molecules showing extended identity sections with the cDNA of SEQ ID NO 2 have been identified in cDNA libraries from the following tissues: foetal heart, pineal gland, activated T cells, microvascular endothelial cells and secondary breast tumour.
  • the polynucleotide of SEQ ID NO 2 was discovered in a cDNA library derived from prostate (benign possible hype ⁇ lasia). It is structurally related to the lipase family. It contains an open reading frame encoding a protein of about 393 amino acid residues.
  • the protein exhibits the highest degree of homology to Lp- PLA 2 (WO95/00649, WO 95/09912) with 40% identity and 60% similarity over a 390 amino acid stretch. Although the overall identity is only 40% the residues identified for the catalytic triad in Lp-PLA2 (WO 95/09912) are conserved between the two polypeptides implying that they are likely to have a similar biochemical function.
  • the positions of the Ser, Asp.and His, are underlined below. SEQ ID NO 1, is the lower seqence and Lp-PLA2 the upper sequence. Vertical lines indicate identical residues. 238 DIDHGKPVKNAIRLKFDMEQLKDSIDREKIAVIGH ⁇ FGGATVIQTLSEDQ 287 : : .
  • I .. I I • : I : I I : . I I .. : : I I : I I I I I I I I . I . I .. : 202 EVTAGQTVFNIFPGGLDLMTLKGNIDMSRVAVMGHiSFGGATAILALAKET 251
  • Tissue sources of this enzyme suggest a role in the biology of the vasculature as well as certain cancers.
  • an inhibitor of this polypeptide could find utility in disease states such as atherosclerosis, hypertension, endothelial dysfunction, myocardial infarction, reperfusion injury, and certain cancers.
  • the homology to Lp-PLA2 suggests that this novel enzyme could play similar roles and as such inhibitors may find utility in atherosclerosis, myocardial infarction, reperfusion injury, acute and chronic inflammation, rheumatoid arthritis, stroke, diabetes and neuropsychiatric illnesses.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence shown in SEQ ID NO 1 or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the DNA of SEQ ID NO I.
  • the present invention includes variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of the polypeptide having the amino acid sequence of SEQ ID NO 1.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same polypeptide as shown in SEQ ID NO 1 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence of SEQ ID NO 2.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotide which encodes for the polypeptide of SEQ ID NO 1 may include: only the coding sequence for the polypeptide; the coding sequence for the polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention therefore includes polynucleotides, wherein the coding sequence for the polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues.
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
  • the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove- described polynucleotides .
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • the polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the polypeptide of SEQ ID NO 1.
  • fragment when referring to the polypeptide of SEQ ID NO 1, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of SEQ ID NO 1 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded by the genetic code
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the polypeptide is preferably in purified form. By purified form is meant at least 80%, more preferably 90%, still more preferably 95% and most preferably 99% pure with respect to other protein contaminants.
  • the DNA of the present invention also makes possible the development by homologous recombination or "knockout" stategies (Kapecchi, Science, 244,: 1288- 1292 (1989) of animals that fail to express, or express a variant form of this enzyme
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • a process for producing the polypeptide of the invention by recombinant techniques by expresssing a polynucleotide encoding said polypeptide in a host and recovering the expressed product.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a cosmid, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Suitable expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter for example, LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • the protein sequences of the present invention can be expressed using, for example, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lad, IacZ, T3, T7, gpt, lambda PR, PL and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • Modification of the coding sequences may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. Modification of the coding sequences may also be performed to alter codon usage to suit the chosen host cell, for enhanced expression.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired character ⁇ istics, e.g., stabilization or simplified purification of expressed recombinant product.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage ⁇ (E. coli), pBR322 (£. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E.
  • Yeast expression vectors are also known in the art. See, e.g., U.S. Patent
  • pSV2neo (as described in J. Mol. Appl. Genet. 1:327-341) which uses the SV40 late promoter to drive expression in mammalian cells or pCDNAlneo, a vector derived from pCDNAl(Mol. Cell Biol. 7:4125-29) which uses the CMV promoter to drive expression. Both these latter two vectors can be employed for transient or stable(using G418 resistance) expression in mammalian cells.
  • Insect cell expression systems e.g., Drosophila
  • Polypeptides can be expressed in host cells under the control of appropriate promoters.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the present invention relates to host cells containing the above-described vectors.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • prokaryotes for example bacterial cells, such as E.
  • coli coli, Streptomyces, Salmonella typhimurium and eukaryotes for example fungal cells, such as yeast, insect cells such as Drosophila and Spodoptera frugiperda, mammalian cells such as CHO, COS or Bowes melanoma, plant cells, etc.
  • fungal cells such as yeast
  • insect cells such as Drosophila and Spodoptera frugiperda
  • mammalian cells such as CHO, COS or Bowes melanoma, plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the polypeptide of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed. The polypeptide is then isolated from the host cells and purified. If the expression system secretes the polypeptide into growth media, the polypeptide can be purified directly from the media. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. Where the polypeptide is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product. Polypeptide expressed in bacterial hosts such as E. coli may require isolation from inclusion bodies and refolding. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the polypeptide can be recovered and purified from recombinant cell cultures by 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. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be glycosylated or may be non- glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
  • the polypeptide of the present invention is also useful for identifying other molecules which have similar biological activity.
  • An example of a screen for this is isolating the coding region of the lipase gene by using the known DNA sequence to synthesize an oligonucleotide probe or as a probe itself. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA polynucleotide
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Recombinant polypeptides refer to polypeptides produced by recombinant
  • Synthetic polypeptides are those prepared by chemical synthesis.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • double-stranded DNA molecule refers to the polymeric form of deoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in a double- stranded helix, both relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear
  • DNA molecules e.g., restriction fragments
  • viruses e.g., viruses, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the sense strand of DNA.
  • a DNA “coding sequence of or a “nucleotide sequence encoding” a particular protein is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Within the promoter sequence will be found a transcription initiation site
  • control sequences refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (i.e., the transcription and translation) of a coding sequence in a host cell.
  • a control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a “host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence. A cell has been "transformed” by exogenous DNA when such exogenous
  • Exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomai element, such as a plasmid.
  • a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA or polypeptide sequences are "substantially homologous” or “substantially the same” when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule and includes allelic variations.
  • substantially homologous also refers to sequences showing identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that pa ⁇ icular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., "Current Protocols in Mol. Biol.” Vol. I & II, Wiley Interscience.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
  • compositions comprise a therapeutically effective amount of the active agent, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
  • compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
  • the polypeptides or polynucleotides of the present invention is administered in an amount which is effective for treatment and/or prophylaxis of the specific indication.
  • the amounts and dosage regimens of active agent administered to a subject will depend on a number of factors such as the mode of administration, the nature of the condition being treated and the judgment of the prescribing physician.
  • sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Chromosome marking reagents based on actual sequence data 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.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA 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 fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • 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 (FISH) of a cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as sho ⁇ as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • 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). Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymo ⁇ hisms.
  • the polypeptides of the invention or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • This invention further provides a method of screening compounds to identify those compounds which inhibit the polypeptide comprising contacting isolated polypeptide with a test compound and measuring the rate of turnover of an enzyme substrate as compared with the rate of turnover in the absence of test compound.
  • the invention also relates to compounds identified thereby.
  • This invention also provides transgenic non-human animals comprising a polynucleotide encoding a polypeptide of the invention. Also provided are methods for use of said transgenic animals as models for mutation and SAR (structure/activity relationship) evaluation as well as in drug screens.
  • the present invention is also directed to inhibitor molecules of the polypeptides of the present invention, and their use in reducing or eliminating the function of the polypeptide.
  • an inhibitor is an antibody or in some cases, an oligonucleotide which binds to the polypeptide.
  • An example of an inhibitor is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of polypeptide.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide (Okano, J.
  • oligonucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide.
  • an inhibitor is a small molecule which binds to and occupies the catalytic site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include but are not limited to small peptides or peptide-like molecules.
  • the inhibitors of the invention are formulated in accordance with standard pharmaceutical practice.
  • the inhibitors which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and, lozenges.
  • a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures.
  • pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
  • a sterile aqueous carrier or parenterally acceptable oil for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
  • the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
  • a typical suppository formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
  • a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
  • composition is in unit dose form such as a tablet or capsule.
  • dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of an inhibitor of the invention.
  • the daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day.
  • an oral dose of between 1 mg and 500 mg preferably between 1 mg and 250 mg
  • an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day.
  • the compounds will be administered for a period of continuous therapy.
  • Plasmids are designated by a lower case p preceded and or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • Olionucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized.
  • Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase.
  • a synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • Poly A+ (mRNA) was isolated from human prostate (benign possible hype ⁇ lasia) using standard methods (ref Maniatis et al). First strand cDNA was primed using an oligo dT primer.
  • the cDNA library was constructed with the Stratagene ZAP-cDNA synthesis kit, packaged with Gigpack 11 gold packaging extract and amplified in XL1- blue MRF bacterial cells. The cDNA inserts were cloned unidirectionally into the vector.
  • the phage clone containing the EST was excised from the ⁇ Unizap XR bacteriophage vector into the Bluescript phagemid (according to the Stratagene manual) for characterisation.
  • the insert of 1823bp was manually sequenced on both strands (using the Amersham -USB Sequenase 2.0 DNA sequencing kit) by primer walking (SEQ ID 2).
  • the cDNA has an open reading frame with the potential to code for a polypeptide of 393 amino acids (SEQ ID 1).
  • the predicted MW for the full reading frame is 44143Da.
  • CAGTCTAGGA TCATAACCGT TCTTGGTTCT GTTCATCGGA GTCAAACTGA CTTTGCTTTT GTGACTGGCA ACTTGATTGG TAAATTCTTC TCCACTGAAA
  • M is defined as either A or C, where the actual base is unclear from either DNA strand.

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Abstract

On décrit des polynucléotides codant le polypeptide de SEQ ID N0 1, lequel présente plus ou moins 40 % d'homologie avec la phospholipase A2 associée à une lipoprotéine (Lp-PLA2) ou acétyle hydrolase du facteur d'activation des plaquettes (PAF).
PCT/GB1995/002320 1995-09-29 1995-09-29 COMPOSE PRESENTANT UNE HOMOLOGIE DE SEQUENCE AVEC UNE PHOSPHOLIPASE A2 ASSOCIEE A UNE LIPOPROTEINE (Lp-PLA2) OU ACETYLE HYDROLASE DU FACTEUR D'ACTIVATION DES PLAQUETTES WO1997012984A1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
AU35728/95A AU3572895A (en) 1995-09-29 1995-09-29 Compound having sequence homology with lipoprotein associated phospholipase a2 (lp-pla2)/paf acetyl hydrolase
PCT/GB1995/002320 WO1997012984A1 (fr) 1995-09-29 1995-09-29 COMPOSE PRESENTANT UNE HOMOLOGIE DE SEQUENCE AVEC UNE PHOSPHOLIPASE A2 ASSOCIEE A UNE LIPOPROTEINE (Lp-PLA2) OU ACETYLE HYDROLASE DU FACTEUR D'ACTIVATION DES PLAQUETTES
EP95932839A EP0853673A1 (fr) 1995-09-29 1995-09-29 COMPOSE PRESENTANT UNE HOMOLOGIE DE SEQUENCE AVEC UNE PHOSPHOLIPASE A2 ASSOCIEE A UNE LIPOPROTEINE (Lp-PLA2) OU ACETYLE HYDROLASE DU FACTEUR D'ACTIVATION DES PLAQUETTES
JP9514042A JPH11514228A (ja) 1995-09-29 1995-09-29 リポ蛋白結合ホスホリパーゼＡ２（Ｌｐ−ＰＬＡ２）ＰＡＦアセチルヒドロラーゼに対して配列相同性を有する化合物
AU72169/96A AU7216996A (en) 1995-09-29 1996-09-26 A paf-acetylhydrolase and use in therapy
EP96933431A EP0859834A2 (fr) 1995-09-29 1996-09-26 Paf-acetylhydrolase paf et son utilisation en therapie
PCT/EP1996/004268 WO1997012963A2 (fr) 1995-09-29 1996-09-26 Paf-acetylhydrolase paf et son utilisation en therapie
JP51396697A JP2002515728A (ja) 1995-09-29 1996-09-26 新規用途
CA002233300A CA2233300A1 (fr) 1995-09-29 1996-09-26 Paf-acetylhydrolase paf et son utilisation en therapie

Applications Claiming Priority (1)

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PCT/GB1995/002320 WO1997012984A1 (fr) 1995-09-29 1995-09-29 COMPOSE PRESENTANT UNE HOMOLOGIE DE SEQUENCE AVEC UNE PHOSPHOLIPASE A2 ASSOCIEE A UNE LIPOPROTEINE (Lp-PLA2) OU ACETYLE HYDROLASE DU FACTEUR D'ACTIVATION DES PLAQUETTES

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0816504A2 (fr) * 1996-06-28 1998-01-07 Suntory Limited Facteur d'activation des plaquettes acétylhydrolase, et gène codant celui-ci
WO1999009147A1 (fr) * 1997-08-13 1999-02-25 Icos Corporation Version tronquee de l'acetyl-hydrolase du facteur d'activation plaquettaire
US7052862B2 (en) 1993-06-25 2006-05-30 Smithkline Beecham Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
CN106957824A (zh) * 2016-12-27 2017-07-18 南京诺唯赞医疗科技有限公司 耐脂蛋白干扰的抗人Lp‑PLA2蛋白的单克隆抗体及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995000649A1 (fr) * 1993-06-25 1995-01-05 Smithkline Beecham Plc Phospholipase a2 associee a une lipoproteine, inhibiteurs de cette phospholipase et son utilisation a des fins diagnostiques et therapeutiques
WO1995009921A1 (fr) * 1993-10-06 1995-04-13 Icos Corporation Facteur d'activation des plaquettes-acetylhydrolase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995000649A1 (fr) * 1993-06-25 1995-01-05 Smithkline Beecham Plc Phospholipase a2 associee a une lipoproteine, inhibiteurs de cette phospholipase et son utilisation a des fins diagnostiques et therapeutiques
WO1995009921A1 (fr) * 1993-10-06 1995-04-13 Icos Corporation Facteur d'activation des plaquettes-acetylhydrolase

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7052862B2 (en) 1993-06-25 2006-05-30 Smithkline Beecham Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US7217535B2 (en) 1993-06-25 2007-05-15 Smithkline Beecham P.L.C. Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US7416853B2 (en) 1993-06-25 2008-08-26 Smithkline Beecham P.L.C. Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
EP0816504A2 (fr) * 1996-06-28 1998-01-07 Suntory Limited Facteur d'activation des plaquettes acétylhydrolase, et gène codant celui-ci
EP0816504A3 (fr) * 1996-06-28 1999-01-07 Suntory Limited Facteur d'activation des plaquettes acétylhydrolase, et gène codant celui-ci
US6323017B1 (en) 1996-06-28 2001-11-27 Suntory Limited Platelet activating factor acetylhydrolase, and gene thereof
WO1999009147A1 (fr) * 1997-08-13 1999-02-25 Icos Corporation Version tronquee de l'acetyl-hydrolase du facteur d'activation plaquettaire
CZ297603B6 (cs) * 1997-08-13 2007-02-07 Icos Corporation Purifikovaný a izolovaný polypeptidový fragment PAF-AH, zpusob jeho produkce a pouzití, farmaceutická kompozice, izolovaný polynukleotid, DNA vektor a hostitelská bunka
CN106957824A (zh) * 2016-12-27 2017-07-18 南京诺唯赞医疗科技有限公司 耐脂蛋白干扰的抗人Lp‑PLA2蛋白的单克隆抗体及其应用
CN106957824B (zh) * 2016-12-27 2018-02-23 南京诺唯赞医疗科技有限公司 耐脂蛋白干扰的抗人Lp‑PLA2蛋白的单克隆抗体及其应用

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