WO2002013845A2 - Inflammation associee au recepteur couple a la proteine g - Google Patents

Inflammation associee au recepteur couple a la proteine g Download PDF

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Publication number
WO2002013845A2
WO2002013845A2 PCT/EP2001/009466 EP0109466W WO0213845A2 WO 2002013845 A2 WO2002013845 A2 WO 2002013845A2 EP 0109466 W EP0109466 W EP 0109466W WO 0213845 A2 WO0213845 A2 WO 0213845A2
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Prior art keywords
polynucleotide
seq
polypeptide
specified
cells
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PCT/EP2001/009466
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WO2002013845A3 (fr
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Gabor Jarai
Shida Yousefi
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Novartis Ag
Novartis Pharma Gmbh
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Priority to JP2002518985A priority Critical patent/JP2004505648A/ja
Priority to EP01980246A priority patent/EP1364017A2/fr
Priority to AU2002212140A priority patent/AU2002212140A1/en
Publication of WO2002013845A2 publication Critical patent/WO2002013845A2/fr
Publication of WO2002013845A3 publication Critical patent/WO2002013845A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to the use of an inflammation-associated G-protein coupled receptor gene designated EX20, the protein molecule encoded by EX20 and related molecules in therapeutic and diagnostic applications.
  • Cells that are attracted into tissues during inflammation include various inflammatory phagocytes such as neutrophilic and eosinophilic granulocytes and monocytes. These cells have been associated with inflammation and tissue destruction in several inflammatory diseases including respiratory tract inflammation in both acute and chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, asthma, adult respiratory distress syndrome (ARDS), rheumatoid arthritis, inflammatory bowel disease (IBD), ulcerative colitis, primary sclerosing cholangitis and Crohn's disease.
  • COPD chronic obstructive pulmonary disease
  • emphysema emphysema
  • asthma chronic respiratory distress syndrome
  • ARDS adult respiratory distress syndrome
  • IBD rheumatoid arthritis
  • ulcerative colitis primary sclerosing cholangitis and Crohn's disease.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Critical steps in the action of leukocytes in inflammatory conditions include the migration of these cells into the tissues, e.g. into the airways in respiratory inflammations or to the joints in rheumatoid arthritis, cell activation and the release of a range of inflammatory mediators, leukotrienes, oxygen radicals, proteases.
  • the isolation of genes and proteins whose expression is upregulated upon cytokine stimulation, for example by GM-CSF, can identify molecular targets that can be exploited to offer therapeutic benefits.
  • differential display Liang, P., and Pardee, A.B., Science 257:967-971
  • SAGE serial analyses of gene expression
  • Velculescu, V.E., Zhang, L., Vogelstein, B., and Kinzler, K. . Science, 270:484- 487 differerential hybridization of complex cDNA probes high density cDNA or oligonucleotide arrays bound to solid support
  • cDNA-RDA Representational Difference Analysis of cDNA
  • cDNA-RDA is a PCR-based subtractive enrichment procedure.
  • Hubank, M., and Schatz D.G. Nucleic Acids Res. 22: 5640-5648; O'Neill, M. J., and Sinclair, A.H. Nucleic Acids Res. 25: 2681-2682.
  • This technique offers several advantages including the isolation of few false positives, the fact that unwanted difference products can be competitively eliminated and genes producing rare transcripts can also be detected and isolated.
  • Tissue distribution and disease association of the over-expression of these genes and proteins can be established using tissue samples derived from appropriately selected patients. Various techniques, for example histology methods, such as in situ hybridisation and immunohistochemistry can be applied for this purpose. Over-expression of these genes would indicate their importance in the disease condition from which a number of clinically important applications would arise.
  • GPCR transmembrane-spanning G-protein coupled receptor
  • TM-I through -VII seven transmembrane helices
  • the GPCR gene family is the largest known receptor family.
  • GPCRs are transducers of extracellular signals and they allow tissues to respond to a wide array of signalling molecules.
  • G-protein coupled receptors are important targets in therapeutic applications because they are involved in a wide variety of physiological and pathological processes. It is estimated that 60-70% of currently marketed drugs indeed act on members of the GPCR superfamily.
  • GPCRs identified may lead to the development of therapeutics (small molecule drugs, antisense molecules, antibody molecules) directly targeted to the gene or protein product of the gene, or may target the biochemical pathway at an upstream or downstream location if the development of such drugs is easier than directly targeting the gene.
  • Therapeutics small molecule drugs, antisense molecules, antibody molecules
  • Polynucleotide sequences comprising the gene and sequence variants thereof may be used to develop a clinical diagnostic test for inflammatory conditions.
  • DNA sequences of GPCRs involved in inflammatory conditions and the amino acid sequences encoded by these genes facilitates large scale production of proteins by recombinant techniques and identification of the tissues and cells naturally producing the proteins.
  • sequence information also permits the preparation of antibody substances or other novel binding molecules specifically reactive with the proteins encoded by the GPCR genes that may be used in modulating the natural ligand/antiligand binding reactions in which the proteins are involved.
  • EX20 is upregulated upon stimulation, for example by GM-CSF, of cytokines which are critical for regulating inflammatory processes in the abovementioned diseases.
  • GM-CSF cytokines which are critical for regulating inflammatory processes in the abovementioned diseases.
  • EX20 and the protein which it encodes are useful in the diagnosis of the aforementioned inflammatory diseases, and the encoded protein is useful as a therapeutic target for identification of agents suitable for treatment of those diseases.
  • the present invention provides, in one aspect, a pharmaceutical composition
  • a pharmaceutical composition comprising as active ingredient (A) a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:16 or a functionally equivalent variant of said amino acid sequence, i.e. a variant thereof which retains the biological or other functional activity thereof, e.g.
  • EX20 a polynucleotide, hereinafter alternatively referred to as EX20, comprising a nucleotide sequence encoding the polypeptide (A); or (C) an antibody which is immunoreactive with the polypeptide (A); or (D) an antisense oligonucleotide comprising a nucleot
  • isolated refers to material removed from its original environment.
  • Hybridization or “hybridizes” refers to any process by which a strand of a polynucleotide binds with a complementary strand through base pairing.
  • “Stringent conditions” refer to experimental conditions which allow up to 20% base pair mismatches, typically two 15 minute washes in 0.1 XSSC (15mM NaCl, 1.5 mM sodium citrate, pH 7.0) at 65°C.
  • “Homology” or “homologous” refers to a degree of similarity between nucleotide or amino acid sequences, which may be partial or, when sequences are identical, complete.
  • “Expression vector” refers to a linear or circular DNA molecule which comprises a segment encoding a polypeptide of interest operably linked to additional segments which provide for its transcription.
  • Antisense refers to selective inhibition of protein synthesis through hybridisation of an oligo- or polynucleotide to its complementary sequence in messenger RNA (mRNA) of the target protein.
  • mRNA messenger RNA
  • the antisense concept was first proposed by Zamecnik and Stephenson (Proc. Natl. Acad. Sci. USA 75:280-284; Proc. Natl. Acad. Sci. USA 75:285-288) and has subsequently found broad application both as an experimental tool and as a means of generating putative therapeutic molecules (Alama, A., Pharmacol. Res. 36:171-178; Dean, N.M., Biochem. Soc. Trans. 24:623-629; Bennet, C.F., J. Pharmacol. Exp. Ther. 280:988- 1000; Crooke, S.T., Antisense Research and Applications, Springer).
  • variant means, in relation to amino acid sequences, an amino acid sequence that is altered by one or more amino acids. The changes may involve amino acid substitution, deletion or insertion.
  • variant in relation to nucleotide sequences, the term “variant” as used herein means a nucleotide sequence that is altered by one or more nucleotides; the changes may involve nucleotide substitution, deletion or insertion.
  • a preferred functionally equivalent variant of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:16 is one having at least 80%, more preferably at least 90%, and especially more than 95% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO: 16.
  • amino acid sequence having x% identity to a reference sequence such as SEQ ID NO:2 or SEQ ID NO: 16 is meant a sequence which is identical to the reference sequence except that it may include up to 100-x amino acid alterations per each 100 amino acids of the reference sequence.
  • a subject amino acid sequence having at least 80% identity to a reference sequence up to 20% of the amino acid residues in the reference sequence may be substituted, deleted or inserted with another amino acid residue.
  • Percentage identity between amino acid sequences can be determined conventionally using known computer programs, for example the FASTDB program based on the algorithm of Brutlag et al (Com ⁇ .App.Biosci. (1990) 6:237-245).
  • the polynucleotide (B) may be cDNA, genomic DNA or RNA.
  • the polynucleotide (B) is cDNA comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO: 15 or a DNA comprising a nucleotide sequence which hybridises to SEQ ID NO:l or SEQ ID NO: 15 under stringent conditions. Nucleotide sequences which satisfy such hybridisation requirements include those resulting from deletions, insertions or substitutions of one or more nucleotides.
  • the invention also provides an isolated polynucleotide comprising the nucleotide sequence of SEQ TD NO:15.
  • the polynucleotide (B) comprises a portion having at least 20, e.g. at least 50, e.g. at least 100, e.g. at least 200, e.g. at least 500, e.g. at least 1000, e.g. at least 1100 contiguous bases from SEQ ID NO:l or SEQ ID NO:15.
  • the polynucleotide (B) comprises a nucleotide sequence encoding at least 10, e.g. at least 50, e.g. at least 100, e.g. at least 200, e.g. at least 300 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:16.
  • the polynucleotide (B) may be isolated by first isolating a fragment of it by PCR using degenerate primers that are designed using amino acid sequence motives conserved among members of a family or sub family of GPCRs.
  • the degenerate primers can be used to amplify a fragment from cDNA that have been prepared from RNA isolated from human cells, specifically leukocytes or especially from phagocytes e.g neutrophilic and eosinophilic granulocytes or from genomic DNA.
  • the isolated fragment is then sequenced and full lengths clones are obtained by first isolating overlapping fragments containing the 5' and 3" ends of the gene using 5' and 3' RACE (rapid amplification of cDNA ends) using gene specific primers designed using that sequence and RNA isolated from human cells, specifically from leukocytes or especially from phagocytes e.g neutrophilic and eosinophilic granulocytes or from genomic DNA and then joining those fragments together by standard methods.
  • 5' and 3' RACE rapid amplification of cDNA ends
  • the polynucleotide (B) may also be isolated by first isolating a fragment of it by PCR using degenerate primers that are designed using amino acid sequence motives conserved among members of a family or sub family of GPCRs.
  • the degenerate primers can be used to amplify a fragment from cDNA that have been prepared from RNA isolated from human cells specifically from leukocytes and especially from phagocytes e.g neutrophilic and eosinophilic granulocytes or from genomic DNA.
  • the isolated fragment is then sequenced and full lengths clones are obtained by using this fragment or a part thereof as probe for screening a human cDNA library, preferably a leukocyte or, especially, a granulocyte cDNA library or a human genomic DNA library.
  • the polynucleotide (B) for example having the sequence SEQ ID NO:l or SEQ ID NO:15, may be prepared from the nucleotides which it comprises by chemical synthesis, e.g. automated solid phase synthesis using known procedures and apparatus.
  • the polypeptide (A) may be produced by cloning a polynucleotide sequence as hereinbefore described into an expression vector containing a promoter and other appropriate regulating elements for transcription, transferring into prokaryotic or eukaryotic host cells such as bacterial, plant, insect, yeast, animal or human cells, and culturing the host cells containing the recombinant expression vector under suitable conditions.
  • prokaryotic or eukaryotic host cells such as bacterial, plant, insect, yeast, animal or human cells.
  • the polypeptide (A) comprises a portion having at least 10, e.g. at least 50, e.g. at least 100, e.g. at least 200, e.g. at least 300 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:16.
  • the invention also provides an isolated polypeptide, particularly a recombinant polypeptide, comprising the amino acid sequence of SEQ ID NO: 16.
  • the polypeptide (A) may be expressed as a recombinant fusion protein with one or more heterologous polypeptides, for example to facilitate purification.
  • it may be expressed as a recombinant fusion protein with a heterologous polypeptide such as a polyhistidine containing a cleavage site located between the polynucleotide sequence of the invention and the heterologous polypeptide sequence, so that the polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:16 may be cleaved and purified away from the heterologous moiety using well known techniques.
  • a heterologous polypeptide such as a polyhistidine containing a cleavage site located between the polynucleotide sequence of the invention and the heterologous polypeptide sequence
  • the polypeptide (A) may also be synthesised, in whole or in part, from the amino acids which it comprises using well known chemical methods, for example automated solid phase techniques.
  • the polypeptide (A) may be purified by well known standard procedures.
  • the present invention also provides an antibody (C) which is immunoreactive with a polypeptide (A) as hereinbefore described.
  • the antibody may be a polyclonal or monoclonal antibody.
  • Such antibodies may be prepared using conventional procedures. Methods for the production of polyclonal antibodies against purified antigen are well established (cf. Cooper and Paterson in Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc., Chapter 11).
  • a host animal such as a rabbit, or a mouse, is immunised with a purified polypeptide (A), or immunogenic portion thereof, as antigen and, following an appropriate time interval, the host serum is collected and tested for antibodies specific against the polypeptide.
  • the present invention provides an antisense oligonucleotide (D) comprising a nucleotide sequence complementary to that of a polynucleotide (B), in particular a nucleotide sequence complementary to SEQ ID NO:l or SEQ ID NO:15.
  • the antisense oligonucleotide may be DNA, an analogue of DNA such as a phosphorothioate or methylphosphonate analogue of DNA, RNA, an analogue of RNA, or a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the antisense oligonucleotides may be synthesised by conventional methods, for example using automated solid phase techniques.
  • the present invention also provides a polynucleotide probe comprising at least 15 contiguous nucleotides of a polynucleotide (B) as hereinbefore described or a complement thereof.
  • the probe may be cDNA, genomic DNA or RNA. It may be, for example, a synthetic oligonucleotide comprising 15 to 50 nucleotides, or may be a longer molecule comprising, for example, up to 1100, e.g. up to 1000, e.g. up to 500 contiguous nucleotides of (B).
  • the probe can be labelled, e.g. with a fluorophore or a chemiluminescent or radioactive label, to provide a detectable signal.
  • the polynucleotide probe is capable of selectively hybridising under stringent conditions to a polynucleotide fragment having a sequence SEQ ID NO:l or SEQ ID NO: 15.
  • the probe has a sequence such that under such hybridisation conditions it hybridizes only to its cognate sequence.
  • DNA probes as described above are useful in a number of screening applications including Northern and Southern blot analyses, dot blot and slot blot analyses, and fluorescence in situ hybridisation (FISH).
  • the present invention also includes a pair of oligonucleotides having nucleotide sequences useful as primers for DNA amplification of a fragment of a polynucleotide (B), i.e. of EX20, wherein each primer of said pair is at least 15 nucleotides in length and said pair have sequences such that when used in a polymerase chain reaction (PCR) with either human genomic DNA or a suitable human cDNA target they result in synthesis of a DNA fragment containing all or preferably part of the sequence of EX20.
  • the primer pair is preferably capable of amplifying the coding region of EX20 or portion thereof. Examples of such primer pairs are shown hereinafter as SEQ ID NOs 3-4 and SEQ ID NOs 5-6 respectively.
  • the role of the polypeptide (A) in inflammatory diseases characterised by neutrophilic or eosinophilic inflammation can be determined using conventional allergen driven animal models for inflammatory conditions, e.g. an ovalbumin-induced mouse or rat model.
  • Polynucleotides, polypeptides, antibodies, antisense oligonucleotides or probes as hereinbefore described, hereinafter alternatively referred to collectively as agents of the invention, may be used in the treatment (prophylactic or symptomatic) or diagnosis of inflammatory diseases such as those hereinbefore described.
  • upregulation of EX20 may induce anti-inflammatory events, so that treatment which enhances this upregulation may be appropriate.
  • a polypeptide (A) may be used to treat a mammal, particularly a human, deficient in or otherwise in need of that polypeptide; a polynucleotide (B) may be used in gene therapy where it is desired to increase EX20 activity, for instance where a subject has a mutated or missing EX20 gene; an antibody (C) or an antisense oligonucleotide (D) may be used to inhibit EX20 activity, where this is desired; an antibody (C) may be used to detect, or determine the level of expression of, a polypeptide (A) or to inhibit ligand/antiligand binding activities of a polypeptide (A); and a probe of the invention may be used to detect the presence or absence of the EX20 gene, i.e. to detect genetic abnormality, or to determine the level of expression of EX20 in a cell sample, e.g. in diagnosis of inflammatory disease.
  • Gene therapy refers to an approach to the treatment of human disease based upon the transfer of genetic material into somatic cells of an individual. Gene transfer can be achieved directly in vivo by administartion of gene-bearing viral or non-viral vectors into blood or tissues, or indirectly ex vivo through the introduction of genetic material into cells manipulated in the laboratory followed by delivery of the gene-containing cells back to the individual. By altering the genetic material within a cell, gene therapy may correct underlying disease pathophysiology. Suitable vectors, and procedures, for gene delivery to specific tissues and organ systems in animals are described in Dracopoli, N.C. et al., Current Protocols in Human Genetics. John Wiley and Sons Inc., Chapters 12 and 13 respectively.
  • gene therapy may involve delivery of a viral or non-viral gene therapy vector containing an expression cassette of the EX20 gene under suitable control elements to the lungs of diseased individuals (eg. asthmatics) so that the underlying disease pathophysiology is corrected or ameliorated.
  • a viral or non-viral gene therapy vector containing an expression cassette of the EX20 gene under suitable control elements to the lungs of diseased individuals (eg. asthmatics) so that the underlying disease pathophysiology is corrected or ameliorated.
  • the present invention provides:
  • a method of treating an inflammatory disease which comprises administering to a subject in need thereof an effective amount of a polypeptide (A), a polynucleotide (B), an antibody (C) or an antisense oligonucleotide (D) as hereinbefore described;
  • polypeptide A
  • polynucleotide B
  • an antibody C
  • antisense oligonucleotide D
  • a method of detecting genetic abnormality or a predisposition to disease in a subject which comprises incubating a genetic sample from the subject with a polynucleotide probe of the invention as hereinbefore defined, under conditions where the probe hybridises to complementary polynucleotide sequence, to produce a first reaction product, and comparing the first reaction product to a control reaction product obtained with a normal genetic sample, where a difference between the first reaction product and the control reaction product indicates a genetic abnormality in the subject or a predisposition to disease;
  • a method of detecting the presence of a polynucleotide (B), e.g. comprising SEQ ID NO:l or SEQ ID NO:15, in cells or tissues which comprises contacting DNA from the cell or tissue with a polynucleotide probe as hereinbefore defined under conditions where the probe is specifically hybridizable with a polynucleotide (B), and detecting whether hybridization occurs; and
  • a polynucleotide e.g. comprising SEQ ID NO:l or SEQ ID NO:15
  • a method of detecting an abnormality in the nucleotide sequence of a polynucleotide (B) in a patient which comprises amplifying a target nucleotide sequence in DNA isolated from the patient by a polymerase chain reaction using a pair of primers as hereinbefore described which target the sequence to be amplified and analysing the amplified sequence to determine any polymorphism present therein.
  • polymorphism means any sequence difference as compared with the sequence of a polynucleotide (B) as hereinbefore described.
  • Hybridisation of a polynucleotide probe of the invention with complementary polynucleotide sequence may be detected using in situ (eg. HSH) hybridization, Northern or Southern blot analyses, dot blot or slot blot analyses.
  • the abnormality may also be detected for example by conformation sensitive gel electrophoresis (CSGE) and DNA sequencing.
  • CSGE conformation sensitive gel electrophoresis
  • the genetic abnormality may result in a change in the amino acid sequence of the individual's EX20 protein relative to the the amino acid sequence of a normal EX20 protein, or loss of protein.
  • the change may not alter the amino acid sequence but may instead alter expression of the EX20 gene by altering the sequence of controlling elements either at the 5'-, or 3'-end of the gene, or altering the sequence of control elements within intronic regions of the gene. Changes may also affect the way the gene transcript is processed or translated.
  • the invention also includes kits for the detection of an abnormality in the polynucleotide sequence of an individual's EX20 gene or for determining the level of expression of EX20 in an individual's cells.
  • Hybridisation kits for such use comprise a probe of the invention as hereinbefore described, which probe may be modified by incorporation of a detectable, e.g.
  • chemiluminescent, radioactive or fluorescent, label therein may include other reagents such as labelling reagents, i.e. reagents to incorporate a detectable label such as a radioactive isotope, chemiluminescent or fluorescent group into a hybridised product, and buffers.
  • PCR amplification kits comprise primer pairs such as those described above together with a DNA polymerase such as Taq polymerase, and may include additional reagents, such as an amplification buffer and the like. Specific embodiments of the PCR amplification kits can include additional reagents specific for a number of techniques that detect polynucleotide changes, including CSGE and DNA sequencing.
  • Determination of the level of expression of a polynucleotide (B) can be used in diagnosis of inflammatory diseases such as those abovementioned. Accordingly, the invention includes a method of determining whether a subject has an inflammatory disease, for example an inflammatory disease associated with increased GM-CSF levels, comprising determining, in a cell sample from the subject, the level of expression of a polynucleotide (B) as hereinbefore described, particularly a polynucleotide (B) comprising the nucleotide sequence of SEQ 3D NO:l or SEQ ID NO:15, or a nucleotide sequence which hybridises thereto under stringent conditions, and comparing said level with the level of expression of the polynucleotide in a cell sample from a healthy subject.
  • an inflammatory disease for example an inflammatory disease associated with increased GM-CSF levels
  • An increased level of expression of a polynucleotide (B) indicates an inflammatory disease.
  • the measured level indicates the nature of the inflammatory disease.
  • the level of expression of a polynucleotide (B) may be determined, for example, by Nothern blot analysis, reverse transcription-polymerase chain reaction (RT- PCR), in situ hybridization, immunoprecipitation, Western blot hybridization or immunohistochemistry.
  • mRNA is isolated from cells of interest, for example from BAL fluid cells or peripheral blood cells.
  • Two primers are designed, one with identical sequence to the region between nucleotides 765 and 783 of SEQ ID NO: 1, the other with a nucleotide sequence that is the reverse complement of nucleotides 815-835 of SEQ ID NO: 1.
  • An appropriately modified and labelled probe is then made corresponding to the reverse complement of the sequence between nucleotides 787-813 of SEQ ID NO: 1.
  • these two primers and probe are used in TaqMan (PE Applied Biosystems) equipment to determine the mRNA quantity of EX20 in samples obtained from patients under investigation and healthy subjects.
  • a probe of approximately 200 to 1200 bp corresponding to any part of the sequence shown in SEQ ID NO: 1 can be used.
  • Several overlapping probes of different length can be used in such a test, for example the one described in Example 3.
  • Samples can be collected from patients and embedded in paraffin. Such samples can be, for example, peripheral blood cells, bronchoalveolar lavage (BAL) fluid cells, or various tissue sections. Labelled riboprobes prepared as described in Example 3 can then be used to determine the expression level of EX20 in those samples and thereby identify the inflammatory condition.
  • BAL bronchoalveolar lavage
  • the invention also includes a method of monitoring treatment of a subject having an inflammatory disease such as hereinbefore described, e.g. such a disease associated with increased GM-CSF levels, with a drug such as those hereinbefore described as agents of the invention, which comprises determining the level of expression of a polynucleotide (B) or a polypeptide (A) as hereinbefore described or the level of an activity of said polypeptide in a cell sample from the subject following the treatment and comparing said level with the respective level before the treatment. Where the comparison indicates it to be desirable, administration of the drug to the subject may be altered accordingly.
  • a method of monitoring treatment of a subject having an inflammatory disease such as hereinbefore described, e.g. such a disease associated with increased GM-CSF levels
  • a drug such as those hereinbefore described as agents of the invention
  • an agent of the invention in inhibiting or reversing inflammatory conditions, for example in inflammatory airways diseases, may be demonstrated in an animal model, e.g. a mouse or rat model, of airways inflammation or other inflammatory conditions, for example as described by Szarka et al, J. Immunol. Methods (1997) 202:49- 57; Renzi et al, Am. Rev. Respir. Dis. (1993) 148:932-939; Tsuyuki et al., J. Clin. Invest. (1995) 96:2924-2931; Cernadas et al (1999) Am. J. Respir. Cell Mol. Biol. 20:1-8; Durie et al., Clin. Immunol. Immunopathol.(1 94) 73: 11-18; and Williams et al., Proc. Natl. Acad. Sci. USA (1992) 89:9784-9788.
  • an animal model e.g. a mouse or rat model
  • Inflammatory diseases to which the present invention is applicable include inflammatory or obstructive airways diseases such as asthma of whatever type or genesis, including both intrinsic (non-allergic) asthma and extrinsic (allergic) asthma.
  • Treatment of asthma is also to be understood as embracing treatment of subjects, e.g. of less than 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed or diagnosable as "whez infants", an established patient category of major medical concern and now often identified as incipient or early-phase asthmatics. (For convenience this particular asthmatic condition is referred to as "whez-infant syndrome”.)
  • inflammatory or obstructive airways diseases and conditions to which the present invention is applicable include adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary or airways disease (COPD or CO AD), including chronic bronchitis, or dyspnea associated therewith, emphysema, as well as exacerbation of airways hyperreactivity consequent to other drug therapy, in particular other inhaled drug therapy.
  • ARDS adult respiratory distress syndrome
  • COAD or CO AD chronic obstructive pulmonary or airways disease
  • chronic bronchitis or dyspnea associated therewith
  • emphysema emphysema
  • exacerbation of airways hyperreactivity consequent to other drug therapy, in particular other inhaled drug therapy in particular other inhaled drug therapy.
  • the invention is also applicable to the treatment of bronchitis of whatever type or genesis including, e.g., acute, arachidic, catarrhal, croupus, chronic or ph
  • pneumoconiosis an inflammatory, commonly occupational, disease of the lungs, frequently accompanied by airways obstruction, whether chronic or acute, and occasioned by repeated inhalation of dusts
  • pneumoconiosis an inflammatory, commonly occupational, disease of the lungs, frequently accompanied by airways obstruction, whether chronic or acute, and occasioned by repeated inhalation of dusts
  • aluminosis an inflammatory, commonly occupational, disease of the lungs, frequently accompanied by airways obstruction, whether chronic or acute, and occasioned by repeated inhalation of dusts
  • aluminosis anthracosis
  • asbestosis chalicosis
  • ptilosis ptilosis
  • siderosis silicosis
  • tabacosis tabacosis and byssinosis.
  • agents of the invention are also useful in the treatment of neutrophil or eosinophil related disorders, e.g. neutrophilia or eosinophilia, in particular neutrophil or eosinophil related disorders of the airways (e.g.
  • eosinophilic infiltration of pulmonary tissues including hypereosinophilia as it effects the airways and/or lungs as well as, for example, eosinophil-related disorders of the airways consequential or concomitant to L ⁇ ffler's syndrome, eosinophilic pneumonia, parasitic (in particular metazoan) infestation (including tropical eosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa (including Churg-Strauss syndrome), eosinophilic granuloma and eosinophil-related disorders affecting the airways occasioned by drug-reaction; and neutrophil-related disorders such as acute and chronic bronchitis, COPD, ARDS, emphysema, rheumatoid arthritis, inflammatory bowel disease (IBD), ulcerative colitis, primary sclerosing cholangitis and Crohn's disease.
  • Agents of the invention are also
  • the agents of the invention may be administered by any appropriate route, e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; topically, e.g. in an ointment or cream; transdermally, e.g. in a patch; by inhalation; or intranasally.
  • any appropriate route e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; topically, e.g. in an ointment or cream; transdermally, e.g. in a patch; by inhalation; or intranasally.
  • compositions containing agents of the invention may be prepared using conventional diluents or excipients and techniques known in the galenic art.
  • oral dosage forms may include tablets and capsules, and compositions for inhalation may comprise aerosol or other atomizable formulations or dry powder formulations.
  • the invention includes (i) an agent (A), (B), (C) or (D) of the invention in inhalable form, e.g. in an aerosol or other atomizable composition or in inhalable particulate, e.g.
  • an inhalable medicament comprising an agent (A), (B), (C) or (D) of the invention in inhalable form
  • a pharmaceutical product comprising an agent (A), (B), (C) or (D) of the invention in inhalable form in association with an inhalation device
  • an inhalation device containing an agent (A), (B), (C) or (D) of the invention in inhalable form.
  • Dosages of agents of the invention employed in practising the present invention may of course vary depending, for example, on the particular condition to be treated, the effect desired and the mode of administration.
  • suitable daily dosages for administration by inhalation are of the order of l ⁇ g to 10 mg/kg while for oral administration suitable daily doses are of the order of O.lmg to 1000 mg/kg.
  • a polypeptide (A) as hereinbefore described can be used to identify enhancers (agonists) or inhibitors (antagonists) of its activity, i.e. to identify compounds useful in the treatment of inflammatory diseases, particularly inflammatory or obstructive airways diseases. Accordingly, the invention also provides a method of identifying a substance suitable for use in the treatment of inflammatory diseases comprising combining a candidate substance with a polypeptide (A) as hereinbefore described and measuring the effect of the candidate substance on the activity of said polypeptide (A).
  • the activity of the polypeptide (A) may be measured, for example, by measuring intracellular Ca 2+ or cAMP (cyclic AMP) levels or by a change in shape or by an appropriate reporter gene assay.
  • the invention also includes a method of identifying a substance suitable for use in the treatment of inflammatory diseases which binds to a polypeptide (A) as hereinbefore described comprising mixing a candidate substance with said polypeptide (A) and determining whether binding has occurred.
  • BSA bovine serum albumin
  • cAMP cyclic adenosine monophosphate
  • FCS fetal calf serum
  • GM-CSF granulocyte macrophage colony stimulating factor
  • IPTG isopropyl-b-D-thiogalactopiranoside
  • PBS phosphate buffered saline
  • PBMC peripheral blood mononuclear cells
  • TEV tobacco etch virus
  • Blood (200 ml) is collected in tubes containing sodium citrate under sterile conditions from normal donors with no history of respiratory diseases. Neutrophils are purified by well established methods. PBMC are separated from peripheral blood cells by Ficoll Hypaque (Pharmacia) centrifugation. The remaining cell population, mainly granulocytes and erythrocytes, are treated with erythrocyte lysis solution (155 mM NF ⁇ Cl, 10 mM KHCO 3 , 0.1 mM EDTA, PH 7.3). To determine the purity, granulocytes are stained with Hansel stain (Difco Laboratories Ltd) and are differentiated by light microscopy at high power magnification.
  • Hansel stain Hansel stain
  • RNA 300 ng of mRNA is used to synthesize cDNA using the Superscript Choice System (Gibco/BRL) and oligo(dT) primer for first strand synthesis.
  • Double stranded cDNA is extracted once with an equal volume of phenol:chloroform:isoamyl alcohol and precipitated with 0.5 volume 7.5 M ammonium acetate and 2.5 volumes of ethanol. Following centrifugation the resultant pellet is washed with 70% ethanol and resuspended in 16 ⁇ l of sterile water.
  • the resulting cDNA samples are used in the cDNA subtraction and selective enrichment procedures for the isolation of genes preferentially expressed upon GM-CSF stimulation in neutrophils.
  • This Example describes the generation of a subtracted library by cDNA-RDA and the isolation of an EX20 cDNA clone.
  • cDNA-RDA is performed essentially as described by Hubank, M., and D.G. Schatz. Nucleic Acids Res. 22: 5640-5648; and O'Neill, M. J., and A. H. Sinclair. Nucleic Acids Res. 25: 2681-2682.
  • Double stranded cDNA samples prepared from mRNA isolated from unstimulated and stimulated neutrophils are digested with DpnJL restriction enzyme and ligated to R-adaptors obtained by annealing a 24mer (SEQ ID NO: 7) to a 12mer (SEQ ID NO: 8) oligonucleotide.
  • Amplicons for both 'tester' (cDNA of stimulated neutrophils) and 'driver' (cDNA of unstimulated neutrophils) are generated using Expand Long Template PCR System and Expand Buffer 1 (Roche Diagnostics). Five 100 ⁇ l PCRs for each tester and ten for each driver are performed. Dpnlt digestion is used to remove the R-adaptors from both driver and tester amplicons. J-adaptors, obtained by annealing a 24mer (SEQ ID NO: 9) to a 12mer (SEQ ID NO:10) are then ligated to the tester population. Subtractive hybridizations are performed in 5 ⁇ l reactions at 67°C for 20 hours.
  • DPI difference product one
  • 250 ng of tester cDNA is mixed with 25 ⁇ g of driver cDNA at a ratio of 100:1.
  • DPI cDNA is then digested with Dp ⁇ . to remove J-adaptors prior to ligation of N- adaptors, that are obtained by annealing a 24mer (SEQ ID NO:ll) to a 12mer (SEQ ID NO:12) oligonucleotide.
  • SEQ ID NO:ll 24mer
  • SEQ ID NO:12 12mer
  • DP2 difference product two
  • 31.25 ng of tester is mixed with 25 ⁇ g of driver cDNA at a ratio of 800:1.
  • Digested and excess adaptors are removed by washing the cDNA on Microcon 30 filters (Amicon).
  • the subtracted library is fractionated by agarose gel electrophoresis.
  • the libraries are plated onto agar plates containing 50 ⁇ g/ml carbenicillin, and lOOmM IPTG and 50 ⁇ g/ml X-Gal. Plates are incubated at 37°C overnight and then briefly at 4°C to allow blue white staining to be clearly distinguishable and plasmids are purified from 3 ml cultures of the white colonies. Inserts of individual clones are analysed by determining their nucleotide sequence on an automated ABI310 sequencer (Perkin-Elmer) using M13 reverse and forward primers. The resulting sequences are analysed in sequence similarity searches using the BLAST algorithm and sequence alignments are done using the GCG software package (Wisconsin Package Version 9.1).
  • EX20 A clone called EX20 is identified as containing an insert with significant sequence similarity to the database entry of the human orphan GPCR HM74 (GenBank accession number D10923;Nomura et al.: Molecular cloning of cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide receptors. Int. Immunol. 5(10):1239- 1249, 1993). The full length coding region of the EX20 gene is then isolated by PCR using the known sequence in the public database. Amplification products are analysed by determining their nucleotide sequences on both strands on an automated ABI310 sequencer (Perkin-Elmer) using Ml 3 reverse and forward primers as well as gene specific primers.
  • sequence contig is obtained using the GCG software package (Wisconsin Package Version 9.1).
  • the obtained cDNA sequence contig shows 99% sequence identity with HM74 in the studied region.
  • Translation of this nucleotide sequence results in a protein of 387 amino acid residues as shown in SEQ ID NO:2.
  • Other sequence variants of the EX20 gene are also isolated.
  • One of these variants shown in SEQ ID NO:15 is characterised by several single nucleotide differences along the coding region and a 5 bp-long deletion between nucleotide positions 1086-1090 of SEQ ID NO:l. Translation of this nucleotide sequence results in an open reading frame of 363 amino acids as shown in SEQ ID NO:16.
  • This version of the EX20 protein is characterised by 18 single amino acid substitutions along the protein sequence and a truncation resulting in a protein that has an intracellular C-terminus which is shorter by 24 amino acid residues when compared to the protein sequence shown in SEQ ID NO:2. These amino acid sequence differences may have functional consequences for example in ligand binding, receptor activation or downstream signalling events coupled to the EX20 receptor.
  • Example 3 This example describes the analysis of the expression and tissue distribution of the EX20 gene by in situ hybridisation.
  • a subclone is constructed containing an 1161 bp insert between the EcoRI and Xbal sites of the pSPORTl plasmid (Life Technologies).
  • the insert of this subclone is identical to the insert described below in Example 4 and is generated in an identical manner.
  • the SP6 promoter in the vector transcribes the antisense strand of the insert that can be used as probe in the in situ hybridisation experiments.
  • the T7 promoter in the vector is used to transcribe the sense strand, which is used as a negative control.
  • Serial tissue sections from paraffin-embedded samples are hybridized with radiolabeled cRNA probes that are synthesized from the 1.2kb insert.
  • Riboprobes are transcribed in vitro in the presence of 33 P-uridine 5'-triphosphate with SP6 (antisense) and T7 (sense) RNA polymerases. The probes are then column-purified and then subjected to electrophoresis on a 5% TBE-urea acrylamide gel to confirm size and purity. Tissue sections are digested with Proteinase and then hybridized with the probes at approximately 8.0 x 10 8 dpm/ml at 65°C for 18 hours. Slides are treated with RNAse A and washed stringently for 2 hours in O.lx SSC at 65°C.
  • the slides are then coated with Kodak NTB-2 emulsion, exposed for 7 days at 4°C, and developed using Kodak D-19 Developer and Fixer. Slides are stained with hematoxylin and eosin and imaged using a Sony Digital Photo Camera attached to a Nikon microscope.
  • alternate sections are hybridized with two types of control probes. All tissues are initially screened with a probe for beta-actin mRNA to ensure that RNA has been preserved within the samples. Adjacent serial sections are also hybridized with a sense control riboprobe derived from the same region of the gene as the antisense probe.
  • the images are evaluated and it is found that the EX20 gene is preferentially overexpressed in tissues affected by various inflammatory diseases in leukocyte subsets. Strong signals are observed in macrophages and neutrophils in respiratory inflammations in COPD, emphysema, ARDS and asthma, in synovial histio-monocytes in rheumatoid arthritis, in neutrophils and epithelioid histiocytes in Crohn's disease. A subset of lyphocytes also shows intensive signals in the airways in asthma and COPD, in the synovium in rheumatoid arthritis and in the inflammatory infiltrates in eczematous dermatitis. The intensity of the signals in the inflammatory cells is consistently greater in inflamed tissues compared to the intensity of signal in these same cell types in non-inflamed or normal appearing tissues.
  • This example relates to the expression of the full length functional EX20 in a mammalian expression system using stable transfection and the use of the transfected cells for the identification of natural ligands or artificial agonists of the EX20 protein.
  • a unique EcoRI site .GAATTC is incorporated 5' to the EX20 start codon (ATG) by PCR amplification using the following primer (SEQ ID NO: 13): 5'- TCACTAGAATTCATCATGAATCGGCA-3'
  • Another primer (SEQ ID NO: 14) is used to introduce a unique Xbal (TCTAGA) site 3' to the EX20 stop codon (TAA, reverse complement: TTA) : 5'- GTCTAGAAGCTTACTCGATGCAAC -3'
  • the recombinant amplified product is digested with EcoRI and Xbal restricion enzymes and ligated as a 1176 bp fragment into EcoRI Xbal digested pcDNA3.1(+) (Invitrogen) mammalian expression vector and transformed into E. coli DH5cc cells.
  • Transformants are selected using the ampicillin resistance gene present on pcDNA3.1(+) and recombinant vectors containing the EX20 insert are identified by isolating plasmids from randomly selected colonies and analysing the plasmids by restriction digestion and agarose gel electrophoresis using standard methods.
  • the recombinant EX20 insert containing plasmid vector is then transfected into CHO-K1 cells.
  • a confluent flask of CHO cells grown in Dulbecco's Modified Eagle's Medium/Ham's fl2 (50:50) with 10% FCS and 2mM glutamine is trypsinized and plated at a dilution of 1:20 into 2 wells of a 6-well plate in 2 ml/well of the same medium . Cells are then incubated for 24 hours at 37°C with 5% CO 2 . The next day the transfection mix is prepared. 1 ⁇ g plasmid DNA is mixed into 100 ⁇ l OptiMEM serum free medium (Life Technologies) for each well.
  • Lipofectamine is diluted in 100 ⁇ l OptiMEM in a separate tube. The two solutions are mixed and incubated for 15 minutes at room temperature. During incubation, cells are washed with OptiMEM to remove serum. 0.8 ml/transfection of serum-free OptiMEM is then added to the DNA-liposome transfection mix and 1 ml of that solution is then added to each well. Control cells are treated in an identical manner but omitting plasmid DNA from the transfection mix. Cells are then incubated for 5 hours at 37°C with 5% CO 2 and then the transfection mix is replaced with 2 ml normal growth medium.
  • transfectants are selected by washing cells in PBS, trypsinizing and re-plating them into T75 flasks with lmg/ml G418 (Life Technologies). Cells are then incubated at 37°C with 5% CO 2 , regularly changing the medium every 2 days until cells in the control flask have died. Cells are then dilution cloned by placing them at a density of one cell per well into individual wells of a 96-well plate and growing them to confluence. After further expansion of the cells individual colonies are screened for the expresssion of EX20 by RT-PCR and by using poly- or monoclonal antibodies raised against the EX20 protein.
  • the transfectant cell line stably expressing the EX20 receptor protein and non transfected controls are grown to confluence in T162 flasks, trypsinized and resuspeneded in an appropriate volume, approximately 50 ml of growth medium with no antibiotic. Cells are seeded at 30,000 cells/well in 100 ⁇ l/well into 96-well plates that will allow the formation of confluent mono-layers at the time of assay the next day. After 24 hours cells are incubated with cytoplasmic calcium indicator Fluo-3-AM (4 mM) in 100ml cell culture medium containing 20mM HEPES and 2.5 mM probenecid at 37°C for 60 min.
  • Cells are washed 4 times with PBS containing 20 mm HEPES and 2.5 mM probenecid and 100ml of that solution is then added to each well.
  • the test compounds from collections of natural ligands and synthetic compound libraries are added to the cells and the fluorescent signal is read every second for the first 60 seconds and every 5 seconds for the next 30 seconds.
  • Natural or synthetic agonists are identified by comparing the level of signal generated by the same compound in EX20 expressing and non-expressing control cells.
  • This Example relates to the expression of full length EX20 with a 6 histidine tag after the ATG start codon using the Baculovirus system in Spodoptera fmgiperda Sf9 cells, and to the purification of the resulting polypeptide.
  • a unique EcoRI site (GAATTC) is incorporated 5' to the EX20 start codon (ATG) by PCR amplification using the following primer (SEQ ID NO-.13)
  • the recombinant amplified product is digested with EcoRI and Xbal restricion enzymes and ligated as a 1176 bp fragment into EcoRI Xbal digested pFastbacTMHTa baculovirus transfer vector (Life Technologies).
  • the EX20 gene is expressed as a fusion potein as the EX20 coding region is placed after a 6x His affinity tag followed by a spacer region, a recognition site for TEV protease and an additional 7 amino acid linker region. Expression of the EX20 fusion protein containing the 6x His tag aids affinity purification and the TEV protease cleavage site is used to remove the 6x His tag.
  • EX20 sequence is transposed into Bacmid DNA carried by DHlOBac cells (Life Technologies; Bac to Bac Baculovirus expression system). EX20 recombinant Bacmids are isolated from DHlOBac cells and successful transposition is confirmed by PCR analyses.
  • Recombinant EX20 Bacmid DNA is transfected into Sf9 cells using published protocols (Bac to Bac baculovirus expression system manual; Life Technologies). Recombinant baculoviruses are harvested from the culture medium after 3-day incubation at 27°C. The cell supernatants are clarified by centrifugation for 5 minutes at 500xg and kept at 4°C. The recombinant Baculovirus is amplified by infecting Sf9 cells (SF900 SFMII medium; Life Technologies) at a cell density of lxl 0 6 cells/ml and a multiplicity of infection (MOI) of 0.01 for 48 hours. Sf9 cells are then centrifuged at lOOOx g for 5 minutes. The supernatants containing high titre virus are stored at 4°C.
  • Sf9 cells maintained at densities of between 2xl0 5 and 3xl0 6 cells/ml in SF900 SFMH medium; Life Technologies) in either shaker flasks (rotated at 90 RPM) or spinner flasks (stirring at 75 RPM) are infected with the amplified recombinant Baculovirus at a cell density of 1.5x 10 ⁇ at an MOI of 2.0 for 60 hours. Following infection Sf9 cells are centrifuged at lOOOx g for 5 minutes, the supernatants poured off and the cell pellets frozen at -80°C.
  • the cells (lxlO 9 ) are resuspended in 100 ml lysis buffer (20 mM Hepes pH 7.9, 100 mM NaCl, 5% glycerol, 2 mM E-mercaptoethanol, 0.5 mM imidazole, 0.1% Nonidet P-40, 40 pg/ml AEBSF, 0.5 pg/ml leupeptin, 1 pg/ml aprotinin and 0.7 pg/ml pepstatin A). Cells are incubated on ice for 15 minutes then centrifuged at 39,000x g for 30 minutes at 4°C.
  • Metal chelate affinity chromatography is carried out at room temperature with a column attached to a BioCAD chromatography workstation.
  • a 20 ml Poros MC/M (16mmDxl00mmL) column is charged with Ni 2+ prior to use and after each injection.
  • the column is washed with 10 column volumes (CV) 50 mM EDTA pH 8, 1 M NaCl followed by 10CV water.
  • the column is charged with 500 ml 0.1 M NiSO4 pH 4.5-5, washed with 10CV water, then any unbound Ni 2+ removed by washing with 5CV 0.3 M NaCl. All steps are performed with a flow rate of 20 ml/min.
  • the charged MC/M column is equilibrated with 5CV Buffer B (20 mM Hepes pH 7.9, 100 mM NaCl, 5% glycerol, 2 mM E-mercaptoethanol, 1 mM PMSF, 100 mM imidazole) to saturate the sites followed by 10CV Buffer A (as Buffer B except 0.5 mM imidazole).
  • 5CV Buffer B 20 mM Hepes pH 7.9, 100 mM NaCl, 5% glycerol, 2 mM E-mercaptoethanol, 1 mM PMSF, 100 mM imidazole
  • 10CV Buffer A as Buffer B except 0.5 mM imidazole
  • any unbound material is removed by washing with 12 CV buffer A and EX20 eluted by applying a 0-50% Buffer B gradient over 10 CV. Fractions (8 ml) are collected over the gradient. EX20 containing fractions are combined and protease inhibitors added to the final concentrations described for the lysis buffer above. DTT is also added to a final concentration of 1 mM. The combined fractions are dialysed overnight against 4 litres 20 mM Hepes pH 7.9, 1 mM DTT, 0.2 mM PMSF at 4°C. The protein concentartion is determined and, if needed, samples are concentrated using a Millipore Ultrafree-15 centrifugation device (MW cut-off 50 kDa) at 4°C.
  • the device is pre-rinsed with water prior to use.
  • the final storage buffer used for long term storage at -80°C is 20 mM Hepes pH 7.9, 1 mM DTT, -100 mM NaCl, 5% glycerol. Glycerol can be omitted from the sample for storage at 4°C.
  • This example relates to the generation of polyclonal antibodies against the EX20 protein.
  • Rabbits are immunised at 4 subcutaneous sites with 500 ⁇ g purified EX20 protein according to the following schedule:
  • Test bleeds 500 ⁇ l are taken and the serum assessed for antibody titre. Serum is collected when a maximum titre is reached. This is done by collecting blood (10 ml) and allowing it to clot for 2 hours at 4°C. The blood is centrifuged at lOOOx g for 5 minutes to separate the serum. The serum is removed and stored at -20°C until assayed.
  • Nunc-Immuno Plate Maxisorp 96 well plates (Nunc, Basle, CH) are used as a solid support and coated with the purified EX20 protein (100 ng/well) overnight at 4°C. The plates are blocked for 3 hours at 37°C with PBS containing 2% BSA (Sigma) and 0.02% NaN 3 (Sigma). After blocking, plates are incubated overnight at room temperature with plasma in different dilutions of PBS. The presence of polyclonal antibodies is checked with both biotin labelled IgG-antibodies to rabbit (Goat anti-rabbit IgG antiserum, 1:25000 dilution), with an incubation time of 40 minutes.
  • Alkaline phosphatase conjugated streptavidin (Immununo Research, Dianova, CH) is then added at a dilution of 1:10000. Development of the reaction is carried out by adding phosphate substrate (Sigma, f.c. 1 mg/ml) dissolved in diethanolamine. After 45 minutes, absorbance is read at 405 nm with a reference of 490 nm with an ELISA plate reader (Biorad).
  • This example relates to the generation of monoclonal antibodies against the EX20 protein.
  • mice Female Balb/c mice are immunised intraperitoneally (ip) with 100 ⁇ g of EX20 protein according to the schedule given below:
  • Serum is assessed for antibody titre by ELISA (Example 6) after the animal is sacrificed for the preparation of spleen cells for fusion. If antibody titre is sufficient, (1/1000 to 1/100,000), the hybridomas are screened, otherwise discarded.
  • Sp2/0 murine myeloma cells (ATCC #CRL 1581; maintained in culture medium containing 20 ⁇ g/ml 8-azaguanine) are cultivated for one week before fusion in RPMI 1640 (8- azaguanine is not included), 10% (v/v) FCS and 1% penicillin-streptomycin (50IU/ml and 50 ⁇ g/ml, respectively).
  • the cells are harvested by centrifugation (200 xg for 5 minutes) and washed three times in cold RPMI 1640. Approximately 2.5xl0 6 cells are used per 96 well microtitre plate.
  • the mouse is killed by an overdose of anesthetic (Forene), the spleen dissected and pressed through a cell strainer (70 ⁇ m mesh cell strainer; Becton & Dickinson, Oxford, UK, Cat. No 2350).
  • the cell suspension is washed three times in RPMI 1640 (as above) and counted: 5.10 6 cells 196 well plate are necessary.
  • the spleen and myeloma cells are mixed (2:1), centrifuged (200 xg for 5 min) and the pellet warmed in a 37°C water bath.
  • Prewarmed polyethylene glycol 4000 1 ml per 10 8 cells
  • 20 ml of prewarmed wash medium over two minutes.
  • After centrifugation the pellet is carefully resuspended in selection medium (RPMI 1640, 10% FCS, 1% penicillin-streptomycin, 10% BM condimed HI (feeder cell replacement from Boehringer Mannheim, Lewes, UK; Cat. No.
  • HAT-media supplement hypoxanthine, aminopterin and thymidine to select against unfused myeloma cells; Boehringer Mannheim, Lewes, UK; Cat. No. 644 579
  • plated 200 ⁇ l/well of a 96 well microtitre plate.
  • the expanded clones which are still positive are cloned by limiting dilution.
  • Cells are diluted serially in four dilutions steps in a 96 well microtitre plate; 5, 2, 1 and 0.5 cells/well.
  • HAT- media supplement is replaced with HT-media supplement (Boehringer Mannheim, Lewes, UK; Cat. No. 623 091).
  • HT-media supplement Boehringer Mannheim, Lewes, UK; Cat. No. 623 091
  • After approximately one week the cells are screened by ELISA (Example 5).
  • the cells of those wells containing a single positive clone are expanded.
  • the cells are grown in culture flasks in standard medium (RPMI 1640, 10% (v/v) FCS and 1 % penicillin-streptomycin) until the hybridomas overgrow and die.
  • standard medium RPMI 1640, 10% (v/v) FCS and 1 % penicillin-streptomycin
  • the debris is removed by centrifugation and the supernatant containing the antibodies is titred using ELISA (Example 5) before storing under sterile conditions at 4°C, -20°C or -70°C.

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Abstract

L'invention concerne une composition pharmaceutique comprenant comme ingrédient actif: A) un polypeptide comprenant la séquence d'acide aminé de SEQ ID NO:2 ou SEQ ID NO:16 ou un variant fonctionnellement équivalent à ladite séquence d'acide aminé; ou B) un polynucléotide comprenant une séquence nucléotidique codant pour le polypeptide A); ou C) un anticorps immunoréactif avec le polypeptide A); ou D) un oligonucléotide antisens comprenant une séquence nucléotidique complémentaire de celle du polynucléotide B). L'invention concerne également l'utilisation de A), B), C) et D) dans des applications diagnostiques et thérapeutiques.
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EP1315963A1 (fr) * 2000-09-01 2003-06-04 Boehringer Ingelheim Pharma GmbH & Co.KG Procede d'identification de substances influen ant de maniere positive des etats inflammatoires de maladies inflammatoires chroniques des voies aeriennes
WO2004071378A2 (fr) * 2003-02-17 2004-08-26 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques relatives a des etats pathologiques lies au recepteur hm74a couple a la proteine g
WO2004083388A2 (fr) * 2003-03-14 2004-09-30 Bristol-Myers Squibb Company Polynucleotide codant un nouveau variant de recepteur de hm74, hgprbmy74 couple a une proteine g humaine
US6902902B2 (en) 2001-11-27 2005-06-07 Arena Pharmaceuticals, Inc. Human G protein-coupled receptors and modulators thereof for the treatment of metabolic-related disorders
WO2006021892A2 (fr) * 2004-08-27 2006-03-02 Endocube S.A.S. Hm74 et hm74a associes a l'inflammation dans les cellules endotheliales cuboides
US7189524B1 (en) 2002-11-25 2007-03-13 Amgen, Inc. Receptor ligands and methods of modulating receptors

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WO1998056820A1 (fr) * 1997-06-12 1998-12-17 Smithkline Beecham Corporation Recepteur hm74a
WO2002018938A1 (fr) * 2000-09-01 2002-03-07 Boehringer Ingelheim Pharma Gmbh & Co. Kg Procede d'identification de substances influençant de maniere positive des etats inflammatoires de maladies inflammatoires chroniques des voies aeriennes

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EP1315963A1 (fr) * 2000-09-01 2003-06-04 Boehringer Ingelheim Pharma GmbH & Co.KG Procede d'identification de substances influen ant de maniere positive des etats inflammatoires de maladies inflammatoires chroniques des voies aeriennes
US6902902B2 (en) 2001-11-27 2005-06-07 Arena Pharmaceuticals, Inc. Human G protein-coupled receptors and modulators thereof for the treatment of metabolic-related disorders
US7829298B2 (en) 2001-11-27 2010-11-09 Arena Pharmaceuticals, Inc. Human G protein-coupled receptors for metabolic-related disorders
US7189524B1 (en) 2002-11-25 2007-03-13 Amgen, Inc. Receptor ligands and methods of modulating receptors
WO2004071378A3 (fr) * 2003-02-17 2005-01-06 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques relatives a des etats pathologiques lies au recepteur hm74a couple a la proteine g
US7465548B2 (en) 2003-02-17 2008-12-16 Bayer Healthcare Ag Methods of screening for potential therapeutics for diseases associated with G-protein coupled receptor HM74a
WO2004071378A2 (fr) * 2003-02-17 2004-08-26 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques relatives a des etats pathologiques lies au recepteur hm74a couple a la proteine g
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US7094572B2 (en) 2003-03-14 2006-08-22 Bristol-Myers Squibb Polynucleotide encoding a novel human G-protein coupled receptor variant of HM74, HGPRBMY74
WO2004083388A2 (fr) * 2003-03-14 2004-09-30 Bristol-Myers Squibb Company Polynucleotide codant un nouveau variant de recepteur de hm74, hgprbmy74 couple a une proteine g humaine
US7371822B2 (en) 2003-03-14 2008-05-13 Bristol-Myers Squibb Company Human G-protein coupled receptor variant of HM74, HGPRBMY74
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WO2006021892A3 (fr) * 2004-08-27 2006-08-17 Endocube S A S Hm74 et hm74a associes a l'inflammation dans les cellules endotheliales cuboides

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