+

WO1997012041A1 - FORMES COURTES DE LA CHIMIOKINE β-8 - Google Patents

FORMES COURTES DE LA CHIMIOKINE β-8 Download PDF

Info

Publication number
WO1997012041A1
WO1997012041A1 PCT/US1996/015592 US9615592W WO9712041A1 WO 1997012041 A1 WO1997012041 A1 WO 1997012041A1 US 9615592 W US9615592 W US 9615592W WO 9712041 A1 WO9712041 A1 WO 9712041A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
ckβ
cells
polypeptides
seq
Prior art date
Application number
PCT/US1996/015592
Other languages
English (en)
Inventor
John White
Edward Appelbaum
Daniel O'shannessy
James Allan Fornwald
Kevin O'donnell
Original Assignee
Smithkline Beecham Corporation
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 to EP96936051A priority Critical patent/EP0859842A4/fr
Priority to PL96326080A priority patent/PL326080A1/xx
Priority to AU73790/96A priority patent/AU711573B2/en
Priority to NZ320932A priority patent/NZ320932A/xx
Priority to BR9610671A priority patent/BR9610671A/pt
Priority to JP9513722A priority patent/JPH11512610A/ja
Priority to EA199800352A priority patent/EA199800352A1/ru
Priority to KR1019980702304A priority patent/KR19990063834A/ko
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to US08/722,719 priority patent/US6001606A/en
Publication of WO1997012041A1 publication Critical patent/WO1997012041A1/fr
Priority to NO981387A priority patent/NO981387L/no
Priority to MXPA/A/1998/002380A priority patent/MXPA98002380A/xx
Priority to BG102413A priority patent/BG102413A/bg
Priority to US09/334,923 priority patent/US6488925B2/en

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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • 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

  • This invention relates to polypeptide and polynucleotide sequences encoding a short form of Chemokine Beta-8 (Ck ⁇ -8).
  • the present invention relates to the use of the polynucleotide and polypeptide sequences, the production of the polynucleotide and polypeptide sequences, and inhibiting the action of the polypeptide.
  • Chemokines also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are small, inducible, proinflammatory, proteins. In general, chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds.
  • chemokines Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and are therefore referred to as the "C-X-C” subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are thus referred to as the "C-C” subfamily. The first cysteine forms a disulfide bond with the third cysteine, and the second with the fourth, resulting in a similar tertiary structure for many of the chemokines. Structurally, many chemokines undergo proteolytic processing to yield a functional, "mature" protein. This usually involves cleavage of a short leader sequence on the N-terminal portion of the protein. At least 14 distinct ⁇ -chemokines and 12 ⁇ -chemokines have been described at the protein and/or cDNA level.
  • the intercrine cytokines exhibit a wide variety of functions. One important feature is their ability to stimulate chemotactic migration of distinct cell types such as monocytes, neutrophils, T lymphocytes, basophils, and fibroblasts.
  • the ⁇ and ⁇ subfamilies differ somewhat in their cell target selectivity. Most of the ⁇ chemokines attract neutrophils, fibroblasts, T cells, and NK cells, ⁇ -chemokines primarily attract monocytes and T lymphocytes. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction.
  • MIP-1 macrophage inflammatory protein
  • PF-4 platelet factor-4
  • IL-8 Interleukin-8
  • GRO is an autocrine growth factor for melanoma cells.
  • Chemokines have been implicated in a number of physiological and disease conditions, particularly those that have an inflammatory component. These include but are not limited to: lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis. Many chemokines were initially isolated as products of an inflammatory response. For example, MIP-1 was originally isolated as an endotoxin-induced proinflammatory cytokine produced by macrophages. Other members of this subfamily have similarly been identified, based on inflammation induction as well as amino acid sequence homology. This includes the full length as well as the shortened forms of the Ck ⁇ -8 polypeptide of the present invention.
  • a mixture of novel polypeptides being truncated forms of human Ck ⁇ -8 as well as biologically active and diagnosticaliy or therapeutically useful fragments, analogs and derivatives thereof.
  • isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnosticaliy or therapeutically useful fragments, analogs and derivatives thereof.
  • a process for producing such polypeptides by recombinant techniques which comprise culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences, under conditions promoting expression of said proteins and subsequent recovery of said proteins.
  • antibodies against such a polypeptide there are provided antibodies against such a polypeptide.
  • antagonists to such a polypeptide which may be used to inhibit the action of such a polypeptide, for example, to treat aplastic anemia, myelodysplastic syndrome, asthma and arthritis.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the Ck ⁇ -8 nucleic acid sequence.
  • diagnostic assays for detecting diseases related to the underexpression and overexpression of the polypeptide and for detecting mutations in the nucleic acid sequences encoding such a polypeptide.
  • CXC chemokines
  • any DNA coding sequence which encodes the same mature polypeptides shall be considered within the scope of this invention. Any variants of this sequence, allelic or non-naturally occurring shall also be included.
  • the present invention also includes polynucleotides in which the coding sequence is fused in the same reading frame to another DNA sequence which aids in expression or secretion of the protein from a cell or serves as a marker to identify the protein in cells (e.g., HA tag).
  • This invention further relates to polynucleotide sequences which hybridize to the described sequence with at least 50% and preferably 70% sequence identity. These polynucleotides encode polypeptides which preferably retain the same biological function or activity as the mature polypeptide.
  • the sequence may be polynucleotides which have, preferably, 50 bases that have identity to, and hybridize to the sequences of the present invention, but do not retain activity. Such sequences may be used as a probe or PCR primer.
  • polypeptides of the present invention may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. Derivatives of these polypeptides may be one in which one or more amino acid substitutions are present; one in which one or more amino acids contain substituent groups; one in which the mature protein is fused with another compound; or one in which additional amino acids are fused to mature proteins.
  • the polypeptides of this invention are preferably provided in isolated form, and preferably purified to homogeneity.
  • the present invention also relates to vectors which include polynucleotides of this invention, host cells which are genetically engineered with this vector, and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered with the vectors of this invention (cloning or expression). The culture conditions for these cells in order to activate promoters, select transformants, or amplify the truncated Ck ⁇ -8 gene will be apparent to those skilled in the art.
  • This polynucleotide sequence may be included in any one of a variety of expression vectors so long as they are replicable and viable in the host.
  • the polynucleotide sequence inserted into the expression vector is under the control of an appropriate promoter to direct mRNA synthesis (e.g., LTR promoter).
  • the expression vector also contains a ribosome binding site for translation initiation, a transcription terminator, enhancer elements which will increase transcription (e.g., SV40 late enhancer), and appropriate selectable markers to ensure maintenance of the vector in the host (e.g., ampicillin resistance gene).
  • Host cells can be higher eukaryotic cells such as mammalian or insect cells, lower eukaryotic cells such as yeast, or prokaryotic cells such as bacteria.
  • Proteins can be synthesized in the host cells under the control of an appropriate promoter.
  • Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • the polypeptide of this invention can be synthetically produced by conventional peptide synthesizers. Following transformation or transfection of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Polypeptides of the invention may also include an initial methionine residue.
  • Ck ⁇ -8 is a chemoattractant for leukocytes and therefore may be employed in a variety of immunoregulatory and inflammatory functions as well as a number of disease conditions. This protein is expressed predominantly in tissues of hemopoietic origin.
  • One important biological effect of the Ck ⁇ -8 polypeptide on leukocytes is stimulation of the mobilization of Ca++ reserves. This mobilization pertains to functional activation of the cell. It has been found that stimulation of differentiated EOL-3 cells with a mixture containing four shortened forms of Ck ⁇ -8 (SEQ ID NOS: 1-4) results in an immediate increase in intracellular Ca++ which is dose dependent. When tested individually, the short form having SEQ ID NO: 4 was the most active.
  • the long form of Ck ⁇ -8 was approximately 1000 times less potent than the short forms at mobilizing Ca++.
  • the potency of the short forms of the present invention may have implications for the effectiveness of therapeutic applications of this chemokine including, but not limited to, protection of bone marrow stem cells from chemotherapeutic agents during chemotherapy, removal of leukemic cells, stimulation of an immune response, regulation of hematopoiesis and lymphocyte trafficking, treatment of psoriasis, solid tumors, enhancing host defenses against resistant chronic and acute infection, and stimulation of wound healing.
  • the polynucleotides and polypeptides of the present invention may be used as a research reagents, in synthesis of DNA and manufacture of DNA vectors, and for the purpose of developing therapeutics and diagnostics for the treatment of human disease (e.g., in the expansion of immature hematopoietic progenitor cells). Fragments of this truncated Ck ⁇ -8 polynucleotide sequence may also be used as a hybridization probe to isolate other genes which have a high sequence similarity. Techniques for these kinds of experiments are known to those skilled in the art.
  • This invention is also related to the use of these sequences as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences.
  • diseases are related to under-expression of the chemokine polypeptides.
  • individuals carrying mutations in the Ck ⁇ -8 gene may be detected at the DNA level by a variety of techniques such as PCR.
  • Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents.
  • the detection of specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, RFLP analysis, and Southern blotting of genomic DNA. Mutations can also be detected by in situ analysis.
  • the present invention also relates to a diagnostic assay for detecting altered levels of Ck ⁇ -8 protein in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of a disease or susceptibility to a disease, for example, a tumor.
  • Assays used to detect levels of Ck ⁇ -8 protein in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western blot analysis, ELISA assays and "sandwich" assays.
  • This invention provides a method for identification of the receptors for the chemokine polypeptides.
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art including ligand panning and FACS sorting.
  • An alternative approach for receptor identification involves photoaffinity linking the labeled polypeptides with the cell membrane or extract preparations that express the receptor molecule.
  • the labeled complex can be isolated and subjected to protein microsequencing.
  • the amino acid sequence obtained would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
  • This invention provides a method of screening compounds to identify agonists and antagonists to the chemokine polypeptide of the present invention.
  • Chemotaxis may be assayed by placing cells chemoattracted by these polypeptides on top of a filter with pores large enough to admit the cells (5 mm). Solutions of potential agonists or antagonists are placed in a bottom chamber so that cells migrate or are prevented from migrating through the membrane over a period of time. Alternatively, the receptors of Ck ⁇ -8 would be incubated with a labeled polypeptide in the presence of a compound. The ability of the compound to either block this interaction or interact with the receptor in the absence of polypeptide could be measured.
  • Examples of potential truncated Ck ⁇ -8 antagonists include antibodies, oligonucleotides which bind to the polypeptides or polypeptides which bind to the receptor of the wild-type polypeptide, but fail to retain biological activity.
  • Antisense technology used to control gene expression can also be a potential antagonist. The availability of the aforementioned technologies should be obvious to those skilled in the art.
  • Antagonists may be employed to treat infectious diseases such as silicosis, sarcoidosis, idiopathic pulmonary fibrosis, idiopathic hyper-eosinophilic syndrome, and endotoxic shock- all by preventing production of the polypeptide of the present invention.
  • the antagonists may also be employed for treating atherosclerosis, by preventing monocyte infiltration in the artery wall.
  • Antagonists may be employed to treat histamine-mediated allergic reactions and various immunological disorders involving the dermis.
  • the antagonists may also be used to treat chronic and acute inflammation by preventing the attraction of monocytes to the wound area. They may also be used to treat inflammatory pulmonary disease, general inflammation, and rheumatoid arthritis by preventing monocyte influx into affected areas.
  • Antagonists may also be employed to treat cases of bone marrow failure in aplastic anemia or myelodysplastic syndrome. They may also be used to treat asthma and allergy as well as subepithelial basement membrane fibrosis which is a feature of the asthmatic lung.
  • the chemokine polypeptide and agonists and antagonists may be employed in combination with a suitable pharmaceutical carrier such as saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of this invention.
  • the polypeptides, agonists, and antagonists may be employed in conjunction with other therapeutic compounds.
  • compositions may be administered in a convenient manner such as by topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intranasal, or intradermal routes in an amount which is effective for treating the specific indication.
  • chemokine polypeptides, agonists, or antagonists which are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in vivo.
  • This type of gene therapy is well known in the art.
  • cells from a patient may be engineered with DNA or RNA encoding the short form of Ck ⁇ -8 polypeptide ex vivo and the engineered cells expressing these polypeptides provided to the patient.
  • cells may be engineered in vivo for expression of a polypeptide in vivo.
  • Retroviral plasmid vectors are used to transduce packaging cell lines (e.g., PE501) by various means (e.g., electroporation) to form producer cell lines.
  • the retroviral expression vector containing the polynucleotide sequences is under the control of a promoter such as LTR and contains a selectable drug resistance marker (e.g., neo).
  • a promoter such as LTR
  • a selectable drug resistance marker e.g., neo
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed to transduce eukaryotic cells, for example, fibroblasts or endothelial cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will then express the nucleic acid sequence(s) encoding the protein.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequences are specifically targeted to and can hybridize with a particular location on an individual chromosome.
  • the mapping of DNA to chromosomes is an important step in correlating genes associated with disease.
  • One technique known to those of skill in the art involves preparing primers used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping strategies include, but are not limited to: in situ hybridization, prescreening with labeled flow-sorted chromosomes, sublocalization to fragments of specific chromosomes using the same PCR primers, and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • the physical position of the sequence can be correlated with genetic map data.
  • the relationship between genes and disease can be identified through linkage analysis. Any differences in the cDNA or genomic sequences between affected and unaffected individuals can be indicative of disease. For example, a mutation in the DNA found only in the affected individuals may be the causative agent of the disease.
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library.
  • Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Polyclonal antibodies prepared by standard procedures can be used to isolate the polypeptides from tissue expressing that polypeptide.
  • monoclonal antibodies any technique which provides antibodies produced by continuous cell lines cultures can be used. Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to immunogenic polypeptides of this invention.
  • transgenic mice may be used to express humanized antibodies to immunogenic polypeptides of this invention.
  • 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. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • the various restriction enzymes used herein for "digestion" of DNA are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. Size separation of the cleaved fragments is performed using 8% polyacrylamide gel.
  • 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. Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology 1973, 52, 456-457.
  • the DNA sequence encoding Ck ⁇ -8, ATCC# 75676 was initially amplified using PCR primers corresponding to the 5' and 3' end sequences of the processed Ck ⁇ -8 protein (minus the signal peptide sequence) and the vector sequences 3' to the Ck ⁇ -8 gene.
  • the 5' oligonucleotide primer has the sequence 5' TCAGGATCCGTCACAAAAGATGCAGA 3' (SEQ ID NO: 5) and the 3' primer has the sequence 5'CGCTCTAGAGTAAAACGACGGCCAGT 3" (SEQ ID NO: 6).
  • primers respectively contain BamHI and Xbal restriction sites used for cloning into the polylinker region of the ampicillin resistant bacterial expression vector PQE-9 (Qiagen, Inc., Chatsworth, CA).
  • the amplified sequences were ligated into PQE-9 in frame with the sequence encoding for the histidine tag and the ribosome binding site (RBS).
  • This ligation was used to transform E. coli strain M15/rep 4 (Qiagen) which contains multiple copies of plasmid pREP4.
  • pREP4 expresses the lad repressor and also confers kanamycin resistance. Transformants were selected by their ability to grow on LB plates supplemented with ampicillin/kanamycin.
  • Clones containing the desired constructs were grown overnight in liquid culture in LB media supplemented with both Amp (100 ⁇ g/ml) and Kan (25 ⁇ g/ml). This culture was used to inoculate a large culture at a ratio of 1: 100 to 1:250. The cells were grown to an optical density 600 of between 0.4 and 0.6. IPTG (isopropyl-B-D-thiogalacto-pyranoside) was then added to a final concentration of 1 mM. Cells were then grown an extra 3 to 4 hours, then harvested by centrifugation.
  • IPTG isopropyl-B-D-thiogalacto-pyranoside
  • the cell pellet was solubilized in the chaotropic agent 6 M Guanidine HCl, clarified, and subjected to chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al. J. Chromatography 1984, 411, 177-184).
  • Truncated Ck ⁇ -8 (95% pure) is eluted from the column in 6 M guanidine HCl pH 5 and for the purposes of renaturation adjusted to 3 M guanidine HCl, 100 mM sodium phosphate, 10 mM glutathione (reduced) and 2 mM glutathione (oxidized). After incubation in this solution for 12 hours, the protein was dialyzed to 10 mM sodium phosphate.
  • Example 2 Expression of recombinant Ck ⁇ -8 in COS cells
  • plasmid, CMV-Ck ⁇ -8 HA is derived from a vector pcDNAI Amp (Invitrogen) containing the ampicillin resistance gene and the CMV promoter followed by a polylinker region, SV40 intron, and polyadenylation site.
  • a DNA fragment encoding the truncated Ck ⁇ -8 sequences and an HA tag fused in frame to the 3' end is cloned into the polylinker region of the vector; therefore, the recombinant protein expression is directed under the CMV promoter.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson et al.. Cell 1984, 37:767).
  • the infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • COS cells are transfected with the expression vector by DEAE- Dextran method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1989)). Cells are labeled for 8 hours with 35S- cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., ID. 1984,37:767). Both cell lysate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.
  • RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5
  • SF9 cells were infected with a recombinant baculovirus designed to express the Ck ⁇ -8 cDNA.
  • Cells were infected in a 10 liter culture at an MOI of 2 and cultured under low serum conditions at 28°C for 72-96 hours. Cellular debris from the infected culture was removed by low speed centrifugation. Protease inhibitor cocktail was added to the supernatant at a final concentration of 20 ⁇ g/ml ( 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml E-64 and 1 mM EDTA).
  • These particular culture conditions i.e., low serum
  • the level of the Ck ⁇ -8 in the supernatant was monitored by loading 20-30 ⁇ l of supernatant on a 15% SDS-PAGE gel.
  • the shortened forms of Ck ⁇ -8 were detected as visible bands, corresponding to an expression level of several mg per liter.
  • Truncated Ck ⁇ -8 was further purified through a three-step purification procedure: 1) Heparin binding affinity chromatography. Supernatant of baculovirus culture was mixed with 1/3 volume of buffer containing 100 mM HEPES/MEM/NaOAc pH 6 and filtered through 0.22 ⁇ m membrane. The sample was then applied to a heparin binding column (HE1 poros 20, BlO-Perceptive System Inc.).
  • Ck ⁇ -8 The short forms of Ck ⁇ -8 was eluted at approximately 300 mM NaCl in a linear gradient of 50 to 500 mM NaCl in 50 mM HEPES/MES/NaOac at pH 6; 2) Cation exchange chromatography.
  • the protein enriched from heparin chromatography was subjected to a 5-fold dilution with a buffer containing 50 mM HEPES/MES/NaOAc pH 6.
  • the resultant mixture was then applied to a cation exchange column (S/M poros 20, BlO-Perceptive System Inc.).
  • Truncated Ck ⁇ -8 was eluted at 250 mM NaCl in a linear gradient of 25 to 300 mM NaCl in 50 mM HEPES/MES/NaOAc at pH 6; 3) Size exclusion chromatography. Following the cation exchange chromatography, truncated Ck ⁇ -8 was further purified by applying to a size exclusion column (HW50, TOSO HAAS, 1.4 x 45 cm.). Amino Acid sequencing of the purified sample showed a mixture of at least four dominant sequences corresponding to the four truncated forms of the protein (SEQ ID NOs: 1 , 2, 3, and 4).
  • Example 4 Stimulation of EOL-3 cells and PBLs with a mixture comprising short forms of Ck ⁇ -8
  • EOL-3 cells were grown under standard growth conditions and differentiated for two weeks with 1 ⁇ M Na Butyrate.
  • PBLs pe ⁇ pheral blood leukocytes
  • PBLs were obtained from human donors by vena puncture.
  • PBLs were purified by standard techniques. Both cell preparations were separately loaded with FURA-2 ( 1 ⁇ M) for 45 minutes. Cells at lxl06/ml (2 ml total) were transferred to plastic cuvettes.
  • the fluorescent spectrophotometer was adjusted to give a baseline recording before the short forms (871a, 871b. 871c, 871RP) or long form (889) of Ck ⁇ -8 was added at a concentration of 0.33 to 33 nM. The increase in fluorescence was monitored with the spectrophotometer.
  • the change in free Ca++ inside the cell was calculated by first lysing the cells with Triton xlOO in the presence of excess Ca++ (to obtain Fmax) and then adding 5 mM EGTA, to obtain Fmin.
  • the ratio of free Ca++ can be calculated from the min and max values.
  • MCP-1 monocyte chemotactic protein- 1

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Diabetes (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On décrit de nouveaux polypeptides relatifs à la forme tronquée de la Ckβ-8 humaine, ainsi que des procédés d'utilisation de ces polypeptides.
PCT/US1996/015592 1993-12-22 1996-09-27 FORMES COURTES DE LA CHIMIOKINE β-8 WO1997012041A1 (fr)

Priority Applications (13)

Application Number Priority Date Filing Date Title
EA199800352A EA199800352A1 (ru) 1995-09-29 1996-09-27 ПОЛИПЕПТИД ХЕМОКИНА β-8 (ВАРИАНТЫ) И СПОСОБ ЕГО ПОЛУЧЕНИЯ, КОМПОЗИЦИИ ПОЛИПЕПТИДОВ, ВЕКТОР, КЛЕТКА-ХОЗЯИН, АГОНИСТ И АНТАГОНИСТ, ВСЕ - УПОМЯНУТОГО ПОЛИПЕПТИДА, И СПОСОБ ИДЕНТИФИКАЦИИ ПОСЛЕДНЕГО, СПОСОБ ЛЕЧЕНИЯ БОЛЬНОГО, ИСПЫТЫВАЮЩЕГО ПОТРЕБНОСТЬ В Хк-β-8 (ВАРИАНТЫ), СПОСОБ ЛЕЧЕНИЯ БОЛЬНОГО, ИСПЫТЫВАЮЩЕГО ПОТРЕБНОСТЬ В ПОДАВЛЕНИИ Хк-β-8, СПОСОБ ДИАГНОСТИКИ ЗАБОЛЕВАНИЯ ИЛИ ПРЕДРАСПОЛОЖЕННОСТИ К ЗАБОЛЕВАНИЮ, ОБУСЛОВЛЕННОМУ НЕДОСТАТОЧНОЙ ЭКСПРЕССИЕЙ ПОЛИПЕПТИДА
AU73790/96A AU711573B2 (en) 1995-09-29 1996-09-27 Short forms of chemokine beta-8
NZ320932A NZ320932A (en) 1995-09-29 1996-09-27 Short forms of chemokine b-8
BR9610671A BR9610671A (pt) 1995-09-29 1996-09-27 Formas curtas de quimiocina beta-8
JP9513722A JPH11512610A (ja) 1995-09-29 1996-09-27 短縮形ケモカインβ−８
EP96936051A EP0859842A4 (fr) 1995-09-29 1996-09-27 FORMES COURTES DE LA CHIMIOKINE -g(b)-8
PL96326080A PL326080A1 (en) 1995-09-29 1996-09-27 Short forms of chemokin beta-8
KR1019980702304A KR19990063834A (ko) 1995-09-29 1996-09-27 단형 케모킨 베타-8
US08/722,719 US6001606A (en) 1994-03-08 1996-09-30 Polynucleotides encoding myeloid progenitor inhibitory factor-1 (MPIF-1) and polypeptides encoded thereby
NO981387A NO981387L (no) 1995-09-29 1998-03-26 Korte former av chemokine <beta>-8
MXPA/A/1998/002380A MXPA98002380A (en) 1995-09-29 1998-03-26 Short shapes of bet chemiscino
BG102413A BG102413A (bg) 1995-09-29 1998-04-29 Скъсени форми на хемокин вета-8
US09/334,923 US6488925B2 (en) 1993-12-22 1999-06-17 Macrophage inflammatory protein-4 (MIP-4) polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US451795P 1995-09-29 1995-09-29
US60/004,517 1995-09-29

Related Child Applications (4)

Application Number Title Priority Date Filing Date
US08/173,209 Continuation-In-Part US5556767A (en) 1993-12-22 1993-12-22 Polynucleotide encoding macrophage inflammatory protein γ
US46568295A Continuation-In-Part 1993-12-22 1995-06-06
US4381196A A-371-Of-International 1994-03-08 1998-10-21
US33851099A Continuation 1995-09-29 1999-06-23

Publications (1)

Publication Number Publication Date
WO1997012041A1 true WO1997012041A1 (fr) 1997-04-03

Family

ID=21711157

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/015592 WO1997012041A1 (fr) 1993-12-22 1996-09-27 FORMES COURTES DE LA CHIMIOKINE β-8

Country Status (16)

Country Link
EP (1) EP0859842A4 (fr)
JP (1) JPH11512610A (fr)
KR (2) KR20030096447A (fr)
CN (2) CN1198186A (fr)
AU (1) AU711573B2 (fr)
BG (1) BG102413A (fr)
BR (1) BR9610671A (fr)
CZ (1) CZ92198A3 (fr)
EA (1) EA199800352A1 (fr)
HU (1) HUP9802699A3 (fr)
NO (1) NO981387L (fr)
NZ (1) NZ320932A (fr)
PL (1) PL326080A1 (fr)
TR (1) TR199800575T1 (fr)
WO (1) WO1997012041A1 (fr)
ZA (1) ZA968204B (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998014582A1 (fr) * 1996-09-30 1998-04-09 Human Genome Sciences, Inc. Compositions therapeutiques et procedes de traitement de maladies utilisant le facteur-1 inhibiteur des precurseurs myeloides (mpif-1), le facteur inhibiteur des colonies de monocytes (m-cif) et le facteur-4 inhibiteur de la migration des macrophages (mip-4)
WO1999028473A1 (fr) * 1997-11-27 1999-06-10 Korea Green Cross Corporation ADNc CODANT UNE C6 β-CHEMOKINE LEUKOTACTINE-1 (Lkn-1) ISOLEE D'UN ORGANISME HUMAIN
US6001606A (en) * 1994-03-08 1999-12-14 Human Genome Sciences, Inc. Polynucleotides encoding myeloid progenitor inhibitory factor-1 (MPIF-1) and polypeptides encoded thereby
WO2001036459A3 (fr) * 1999-11-15 2001-11-15 Forssmann Wolf Georg Nouvelle utilisation de hcc-2
US6451562B1 (en) 1993-12-22 2002-09-17 Human Genome Sciences, Inc. Polypeptides encoding myeloid progenitor inhibitory factor-1 (MPIF-1) polynucleotides
US6488925B2 (en) 1993-12-22 2002-12-03 Human Genome Sciences, Inc. Macrophage inflammatory protein-4 (MIP-4) polypeptides
US6495129B1 (en) 1994-03-08 2002-12-17 Human Genome Sciences, Inc. Methods of inhibiting hematopoietic stem cells using human myeloid progenitor inhibitory factor-1 (MPIF-1) (Ckbeta-8/MIP-3)
US6811773B1 (en) 1993-12-22 2004-11-02 Human Genome Sciences, Inc. Human monocyte colony inhibitory factor (M-CIF) polypeptides
CN1639569B (zh) * 2002-05-07 2012-09-05 坎莫森特里克斯公司 诱导免疫应答的方法和组合物

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0735818B1 (fr) * 1993-12-22 2004-03-31 Human Genome Sciences, Inc. PROTEINES INFLAMMATOIRES MACROPHAGES MIP-3, MIP-4 ET MIP-1gamma
JPH11505417A (ja) * 1995-05-05 1999-05-21 ヒューマン・ジェノム・サイエンシズ・インコーポレイテッド ヒトケモカインベータ−８、ケモカインベータ−１、およびマクロファージ炎症性タンパク質−４
JP2001519757A (ja) * 1995-10-24 2001-10-23 スミスクライン・ビーチャム・コーポレイション 幹細胞動員のための新規ケモカイン
US6290948B1 (en) * 1996-05-14 2001-09-18 Smithkline Beecham Corporation Method of treating sepsis and ARDS using chamohine beta-10

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CYTOKINE, January 1992, Vol. 4, No. 1, CLEMENTS et al., "Biological and Structural Properties of MIP-1 alpha Expressed in Yeast", pages 76-82. *
JOURNAL OF EXPERIMENTAL MEDICINE, March 1994, Vol. 179, JOSE et al., "Eotaxin: a Potent Eosinophil Chemoattractant Cytokine Detected in a Guinea Pig Model of Allergic Airways Inflammation", pages 881-887. *
See also references of EP0859842A4 *
THE FASEB JOURNAL, December 1989, Vol. 3, WOLPE et al., "Macrophage Inflammatory Proteins 1 and 2: Members of a Novel Superfamily of Cytokines", pages 2565-2573. *
THE JOURNAL OF EXPERIMENTAL MEDICINE, February 1992, Vol. 175, KUNA et al., "Monocyte Chemotactic and Activating Factor is a Potent Histamine-Releasing Factor for Human Basophils", pages 489-493. *
THE JOURNAL OF EXPERIMENTAL MEDICINE, Volume 169, April 1989, MATSUSHIMA et al., "Purification and Characterization of a Novel Monocyte Chemotactic and Activating Factor Produced by a Human Myelomonocytic Cell Line", pages 1485- 1490. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6811773B1 (en) 1993-12-22 2004-11-02 Human Genome Sciences, Inc. Human monocyte colony inhibitory factor (M-CIF) polypeptides
US7851596B2 (en) 1993-12-22 2010-12-14 Human Genome Sciences, Inc. Myeloid progenitor inhibitory factor-1 (MPIF-1) polypeptides
US7364865B2 (en) 1993-12-22 2008-04-29 Human Genome Sciences, Inc. Antibodies to myeloid progenitor inhibitory factor-1 (MPIF-1)
US7041460B2 (en) 1993-12-22 2006-05-09 Human Genome Sciences, Inc. Antibodies to monocyte-colony inhibitory factor (M-CIF)
US6451562B1 (en) 1993-12-22 2002-09-17 Human Genome Sciences, Inc. Polypeptides encoding myeloid progenitor inhibitory factor-1 (MPIF-1) polynucleotides
US6488925B2 (en) 1993-12-22 2002-12-03 Human Genome Sciences, Inc. Macrophage inflammatory protein-4 (MIP-4) polypeptides
US6495129B1 (en) 1994-03-08 2002-12-17 Human Genome Sciences, Inc. Methods of inhibiting hematopoietic stem cells using human myeloid progenitor inhibitory factor-1 (MPIF-1) (Ckbeta-8/MIP-3)
US6623942B2 (en) 1994-03-08 2003-09-23 Human Genome Sciences, Inc. Macrophage inflammatory protein-4 (MIP-4) polynucleotides
US6001606A (en) * 1994-03-08 1999-12-14 Human Genome Sciences, Inc. Polynucleotides encoding myeloid progenitor inhibitory factor-1 (MPIF-1) and polypeptides encoded thereby
WO1998014582A1 (fr) * 1996-09-30 1998-04-09 Human Genome Sciences, Inc. Compositions therapeutiques et procedes de traitement de maladies utilisant le facteur-1 inhibiteur des precurseurs myeloides (mpif-1), le facteur inhibiteur des colonies de monocytes (m-cif) et le facteur-4 inhibiteur de la migration des macrophages (mip-4)
WO1999028473A1 (fr) * 1997-11-27 1999-06-10 Korea Green Cross Corporation ADNc CODANT UNE C6 β-CHEMOKINE LEUKOTACTINE-1 (Lkn-1) ISOLEE D'UN ORGANISME HUMAIN
WO2001036459A3 (fr) * 1999-11-15 2001-11-15 Forssmann Wolf Georg Nouvelle utilisation de hcc-2
CN1639569B (zh) * 2002-05-07 2012-09-05 坎莫森特里克斯公司 诱导免疫应答的方法和组合物

Also Published As

Publication number Publication date
CN1405180A (zh) 2003-03-26
AU7379096A (en) 1997-04-17
EA199800352A1 (ru) 1998-12-24
TR199800575T1 (en) 1998-06-22
NZ320932A (en) 2000-01-28
EP0859842A1 (fr) 1998-08-26
KR20030096447A (ko) 2003-12-31
EP0859842A4 (fr) 1999-02-24
HUP9802699A3 (en) 2000-09-28
AU711573B2 (en) 1999-10-14
MX9802380A (es) 1998-08-30
BG102413A (bg) 1999-04-30
CN1198186A (zh) 1998-11-04
CZ92198A3 (cs) 1998-07-15
ZA968204B (en) 1997-04-09
BR9610671A (pt) 1999-07-06
HUP9802699A2 (hu) 1999-03-29
PL326080A1 (en) 1998-08-17
JPH11512610A (ja) 1999-11-02
KR19990063834A (ko) 1999-07-26
NO981387L (no) 1998-05-29
NO981387D0 (no) 1998-03-26

Similar Documents

Publication Publication Date Title
EP0735818B1 (fr) PROTEINES INFLAMMATOIRES MACROPHAGES MIP-3, MIP-4 ET MIP-1gamma
US5504003A (en) Macrophage inflammatory protein-3 and -4
AU708903B2 (en) Human chemokine polypeptides
AU723891B2 (en) Human chemokine beta-11 and human chemokine alpha-1
US6458349B1 (en) Chemokine β-4 polypeptides
EP0832233B1 (fr) Chemokine beta-13 humaine
EP0799311B1 (fr) Chemokine beta 9 humaine
AU711573B2 (en) Short forms of chemokine beta-8
EP0885293A1 (fr) Polypeptides de chemokine humaine
US20030138400A1 (en) Short forms of chemokine beta-8
CN1198186U (zh) 趋化因子β－8的短形式
MXPA98002380A (en) Short shapes of bet chemiscino
JPH08140683A (ja) 新規な白血球活性化因子
CA2233367A1 (fr) Formes courtes de la chimiokine .beta.-8
KR19990042713A (ko) 사람에서 분리한 c 6 베타-케모카인 lkn-1의 cdna 및 재조합 lkn-1의 제조방법
EP1006188B1 (fr) Polypeptides de type chemokine humaine
EP0777494B1 (fr) Polypeptides de type chemokine humaine
KR100250091B1 (ko) 사람에서 분리한 c 6 베타-케모카인 lkn-3의 cdna 및재조합 lkn-3의 제조방법
KR100250090B1 (ko) 사람에서 분리한 c 6 베타-케모카인 lkn-2의 cdna 및재조합 lkn-2의 제조방법
AU753088B2 (en) Human chemokine polypeptides
AU753730B2 (en) Human chemokine beta-13
JP2002320492A (ja) ヒトケモカインβ−１１およびヒトケモカインα−１
AU4507900A (en) Human chemokine beta-11 and human chemokine alpha-1
KR19990087164A (ko) 사람 케모킨 폴리펩티드
JP2002136294A (ja) ヒトケモカインポリペプチド

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AU BB BG BR CA CN CZ EE GE HU IL IS JP KG KP KR LK LR LT LV MD MG MK MN MX NO NZ PL RO SG SI SK TR TT UA US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 1997 513722

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: PA/a/1998/002380

Country of ref document: MX

Ref document number: PV1998-921

Country of ref document: CZ

ENP Entry into the national phase

Ref document number: 2233367

Country of ref document: CA

Ref document number: 2233367

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1998/00575

Country of ref document: TR

Ref document number: 98-00794

Country of ref document: RO

Ref document number: 96197297.1

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 1019980702304

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 320932

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 1996936051

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 199800352

Country of ref document: EA

WWP Wipo information: published in national office

Ref document number: PV1998-921

Country of ref document: CZ

WWP Wipo information: published in national office

Ref document number: 1996936051

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1019980702304

Country of ref document: KR

WWR Wipo information: refused in national office

Ref document number: PV1998-921

Country of ref document: CZ

WWR Wipo information: refused in national office

Ref document number: 1019980702304

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1996936051

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载