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EP0859834A2 - Paf-acetylhydrolase paf et son utilisation en therapie - Google Patents

Paf-acetylhydrolase paf et son utilisation en therapie

Info

Publication number
EP0859834A2
EP0859834A2 EP96933431A EP96933431A EP0859834A2 EP 0859834 A2 EP0859834 A2 EP 0859834A2 EP 96933431 A EP96933431 A EP 96933431A EP 96933431 A EP96933431 A EP 96933431A EP 0859834 A2 EP0859834 A2 EP 0859834A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
dna
sequence
cell
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96933431A
Other languages
German (de)
English (en)
Inventor
Christopher Donald Southan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Ltd
Original Assignee
SmithKline Beecham Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/GB1995/002320 external-priority patent/WO1997012984A1/fr
Priority claimed from GBGB9617781.1A external-priority patent/GB9617781D0/en
Application filed by SmithKline Beecham Ltd filed Critical SmithKline Beecham Ltd
Publication of EP0859834A2 publication Critical patent/EP0859834A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the use of a novel lipase in therapy.
  • Lipoprotein Associated Phospholipase A2 (Lp-PLA2> [also known as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase)] is responsible, during the conversion of LDL to its oxidised form, 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.
  • PAF acetyl hydrolase Platelet Activating Factor Acetyl Hydrolase
  • 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.
  • the amino acid and DNA sequence of the enzyme lipoprotein associated Lp-PLA2 are disclosed in WO95/00649 (SmithKline Beecham pic).
  • WO 95/09921 suggests a range of possible therapeutic use for platelet activating factor acetylhydrolase, in regulating pathological inflammatory events such as asthma, anaphylaxis, reperfusion injury and central nervous system ischemia, antigen-induced arthritis, atherogenesis, Crohn's Disease, ischemic bowel necrosis/necrotising enterocolitis, ulcerative colitis, ischemic stroke, ischemic brain injury, systemic lupus erythematosus, acute pancreatitis, septicemia, acute post streptococcal glomerulonephritis, pulmonary edema resulting from IL-2 therapy, allergic inflammation, ischemic renal failure, preterm labour and adult respiratory distress syndrome.
  • pathological inflammatory events such as asthma, anaphylaxis, reperfusion injury and central nervous system ischemia, antigen-induced arthritis, atherogenesis, Crohn's Disease, ischemic bowel necrosis/necrotising enterocolitis,
  • Lp- PLA 2 Lp- PLA 2
  • SEQ ID NO 1 is the lower seqence and Lp-PLA2 the upper sequence. Vertical lines indicate identical residues.
  • I I I I :: I I I I I I I I I I I I I : : . I . : .. I : I I I I I . I i I .. : I I I.:.. 252 QFRCAVALI.AWMFPLERDFYPKARGPVFFINTEKFQTMESVNLMKKICAQ 301
  • inhibitors of the polypeptide may be of use in various therapeutic applications such as atherosclerosis, myocardial infarction, reperfusion injury, acute and chronic inflammation, rheumatoid arthritis, stroke, diabetes and neuropsychiatric illnesses but fails to suggest a possible therapeutic role for the polypetide per se.
  • the present invention provides for the use of a polypeptide having the amino acid sequence given in SEQ BD NO 1, and fragments, analogs or derivative thereof in therapy, in particular for the treatment of diseases associated with PAF.
  • Such diseases include those hereinbefore mentioned for PAF-acetyl hydrolase, in particular, acute respiratory distress syndrome, asthma, acute pancreatitis, inflammatory bowel disease, solid organ transplant and necrotizing entercolitis, as well as AIDS [Platlet activating factor: a candidate HIV- 1 induced nerotoxin, Gelbard et al. J.Virol, 68, 4828-4635 (1994)].
  • the term 'polypeptide(s)' refers to a polypeptide having the amino acid sequence given in SEQ ID NO 1 and fragments, analogs and derivatives thereof.
  • 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 subsutuent 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
  • 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.
  • 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.
  • compositions comprise a therapeutically effective amount of the active agent, and a pharmaceutically acceptable carrier or excipienL
  • a pharmaceutically acceptable 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.
  • 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.
  • 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.
  • 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.
  • Polypeptide for use in the present invention may be obtained from genetically modified host cells using gentic engineering techniques well known to those skilled in the art.
  • the polynucleotide for use in 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 1.
  • 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 polynucleoude or a non-naturally occurring variant of the polynucleotide.
  • 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.
  • the term "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.
  • 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 may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invenuon.
  • 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)).
  • Suitable polynucleotides also include those which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences, in particular 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.
  • Suitable 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 hosL
  • 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, lacZ, 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 TRP1 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 ⁇ (£. coli), pBR322 (£. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-£. coli gram-negative bacteria), pHV14 (£.
  • Yeast expression vectors are also known in the art. See, e.g., U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applications 103,409; 100.561; 96,491.
  • 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, are also useful, see for example, PCT applications WO 90/06358 and WO 92 06212 as well as EP 290,261-B 1.
  • 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 inco ⁇ orated 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.
  • Suitable hosts include prokaryotes for example bacterial cells, such as £. 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.
  • prokaryotes for example bacterial cells, such as £. coli, Streptomyces, Salmonella typhimurium
  • 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.
  • 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 £. 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.
  • 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.
  • a “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.
  • this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), 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 (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • 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 episomal 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 particular 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. Ausbel et al. (ed.) (1992). Protein sequences that are substantially the same can be identified by proteolytic digestion, gel electrophoresis and microsequencing.
  • the term “functionally equivalent” intends that the amino acid sequence of the subject protein is one that will exhibit enzymatic activity of the same kind as that of the lipase.
  • 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.
  • 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.
  • Oligonucleotides 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 foiming 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. DNA Sequencing
  • 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.
  • the NSDL cDNA was isolated from its host plasmid pBluescript 11 SK+ ⁇ as an approximate 1800bp EcoRl-Xhol fragment, the enzyme activity inactivated for 10 minutes at 80° C followed by blunt ending with the Klenow fragment of DNA polymerase 1, in the presence of excess dNTPs.
  • the blunt end fragment was ligated into the Sma 1 site of the baculovirus expression vector, pBacPAK9 (Clontech) with the resulting plasmid transformed into E.Coli (high efficiency JM109 -Promega) and plated on LB/1.5% w/v agar plates containing lOO ⁇ g/ml ampicillin.
  • NSDL novel serine dependent lipase
  • DNA from selected clones was diluted to lOOng/ ⁇ l with water and co-transfected with BakPAK ⁇ DNA into BSU 361 digested baculovirus DNA (Clontech), using lipid mediated transfer (Lipofectin-Life Technologies).
  • the viral DNA was used to infect 1.5xl0 6 Spodopterajrugiperda cells (sf-9; ATCC CRL 171 1) seeded in 2ml of IPL- 41 medium plus lipid and yeast supplements (complete medium). 72 hours post- transfection, the virus particles released into the medium were harvested and used to create a plaque assay (Summers, M.D. and Smith G.E.; Texas Agricultural Experiment Station Bulletin No 1555, 1987).
  • Ten plaques were chosen at random and each used to infect 5x10 ⁇ fresh sf-9 cells in 2ml complete medium. Four days post-infection 1.8ml medium from each plaque was used to infect a further 5x10 ⁇ fresh sf-9 cells. Four days post infection the sf-9 cells were lysed in 200 ⁇ l 50mM HEPES buffer, lOmM CHAPS pH 7.4, plus 10 ⁇ g/ml each of the protease inhibitors pepstatin A, antipain. Leupeptin, aprotinin), the debris removed by centrifugation at 3000xg for 4 minutes and l ⁇ l of the supernatant assayed in a colourimetric assay. Virus titre was improved by further rounds of infection in sf-9 cells using a moi of 0.01 and protein expression optimised at 2 days post-infection. Purification of NSDL was carried out from a 30 litre culture of infected cells.
  • Sf9 cells were lysed by freeze thawing 3 times in liquid nitrogen after the addition of 40ml/L of lysis buffer (50mM Tris (pH 8.5), 5mM Chaps, ImM EDTA, ImM DTT, 20% glycerol,50 ⁇ g/ml Benzamidine, plus lO ⁇ g/ml protease inhibitors.
  • the sample was centrifuged at 50,000xg for 20 minutes to pellet the cell debris, following the addition of DNase to reduce the viscosity.
  • the supernatant was loaded onto a Q sepharose column (Pharmacia) which had been equilibrated with lysis buffer minus protease inhibitors (buffer A).
  • the column was washed with buffer A and the protein was eluted using a linear gradient of NaCl. (0- 1M)
  • the enzyme eluted at 300mM NaCl. Active fractions were concentrated using a YM30 membrane in an Amicon ultrafiltration cell, followed by dialysis against 50mM Mes,( pH 6.0) ,5mM Chaps, ImM DTT, ImM EDTA, 20% Glycerol (buffer B).
  • the sample was loaded onto a blue sepharose 6 fast flow column (Pharmacia), equilibrated with buffer B.
  • the column was washed with buffer B, followed by 50mM Mops, (pH 7.0), 5mM Chaps, ImM DTT, ImM EDTA, 20%- glycerol, 0.3M NaCl (buffer C) and enzyme activity was eluted using 50mM Tris (pH 8.0), 5mM Chaps, ImM DTT, ImM EDTA, 20% glycerol, IM NaCl (buffer D).
  • Active fractions were concentrated over a YM30 membrane and dialysed against lOmM Tris (pH 7.4), 5mM Chaps, ImM DTT, 20% Glycerol (buffer E).
  • the concentrated protein was applied to a hydroxyapatite column (Biorad) , equilibrated with buffer E and the enzyme was eluted with a linear gradient from buffer E alone, to buffer E containing 150mM KH2PO-*
  • the activity eluted at around The protein was concentrated over a YM30 membrane followed by a 10 fold dilution with buffer A and was then applied to a Resource Q column (Pharmacia) pre- equilibrated with buffer A.
  • the enzyme does not bind to the column and is collected by washing with buffer A, contaminants are eluted using buffer A plus IM NaCl.
  • 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 un polypeptide de SEQ ID NO 1, utile en thérapie.
EP96933431A 1995-09-29 1996-09-26 Paf-acetylhydrolase paf et son utilisation en therapie Withdrawn EP0859834A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
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
WOPCT/GB95/02320 1995-09-29
GB9617781 1996-08-24
GBGB9617781.1A GB9617781D0 (en) 1996-08-24 1996-08-24 New use
PCT/EP1996/004268 WO1997012963A2 (fr) 1995-09-29 1996-09-26 Paf-acetylhydrolase paf et son utilisation en therapie

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Publication number Priority date Publication date Assignee Title
AU751594B2 (en) * 1997-08-13 2002-08-22 Icos Corporation Truncated platelet-activating factor acetylhydrolase
KR100781425B1 (ko) 2000-02-16 2007-12-03 스미스클라인비이참피이엘시이 Ｌｄｌ-ｐｌａ2 억제제로서 피리미딘-4-온 유도체
EA023796B1 (ru) 2010-12-06 2016-07-29 Глэксо Груп Лимитед ПИРИМИДИНОНОВЫЕ СОЕДИНЕНИЯ ДЛЯ ПРИМЕНЕНИЯ В ЛЕЧЕНИИ ЗАБОЛЕВАНИЙ ИЛИ СОСТОЯНИЙ, ОПОСРЕДОВАННЫХ Lp-PLA
US20130267544A1 (en) 2010-12-17 2013-10-10 Peter Adamson Use of LP-PLA2 Inhibitors in the Treatment and Prevention of Eye Diseases
RU2014107486A (ru) 2011-07-27 2015-09-10 Глэксо Груп Лимитед Бициклические пиримидоновые соединения
US8975400B2 (en) 2011-07-27 2015-03-10 Glaxo Group Limited 2,3-dihydroimidazo[1, 2-c] pyrimidin-5(1 H)-one compounds use as LP-PLA2 inhibitors
WO2014114694A1 (fr) 2013-01-25 2014-07-31 Glaxosmithkline Intellectual Property Development Limited Inhibiteurs de la phospholipase associée aux lipoprotéines a2 (lp-pla2) à base de 2,3-dihydro-imidazol[1,2-c]pyrimidin-5(1 h)-one
KR20150108896A (ko) 2013-01-25 2015-09-30 글락소스미스클라인 인털렉츄얼 프로퍼티 디벨로프먼트 리미티드 Lp-pla2의 억제제로서의 비시클릭 피리미돈 화합물
AU2014210259B2 (en) 2013-01-25 2016-11-03 Glaxosmithkline Intellectual Property Development Limited Compounds
WO2016012916A1 (fr) 2014-07-22 2016-01-28 Glaxosmithkline Intellectual Property Development Limited Dérivés 1,2,3,5-tétrahydro-imidazo [1,2-c]pyrimidine utiles pour le traitement de maladies et de troubles médiés par la lp-pla2
WO2016012917A1 (fr) 2014-07-22 2016-01-28 Glaxosmithkline Intellectual Property Development Limited Dérivés 1,2,3,5-tétrahydro-imidazo [1,2-c]pyrimidine utiles pour le traitement de maladies et de troubles médiés par la lp-pla2
PH12022551104A1 (en) 2019-11-09 2023-11-13 Shanghai Simr Biotechnology Co Ltd Tricyclic dihydroimidazopyrimidone derivative, preparation method therefor, pharmaceutical composition and use thereof
CN115304620A (zh) 2021-05-07 2022-11-08 上海赛默罗生物科技有限公司 嘧啶酮衍生物、其制备方法、药物组合物和用途

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EP0974663A1 (fr) * 1993-06-25 2000-01-26 Smithkline Beecham Plc Phospholipase A2 associée à une lipoprotéine, inhibiteurs de cette phospholipase et son utilisation à des fins diagnostiques et thérapeutiques
EP1096016B1 (fr) * 1993-10-06 2006-01-11 Icos Corporation Facteur d'activation des plaquettes-acetylhydrolase

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Title
See references of WO9712963A2 *

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JP2002515728A (ja) 2002-05-28
WO1997012963A2 (fr) 1997-04-10

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