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WO1993018783A1 - Use of interleukin-10 to induce the production of interleukin-1 receptor antagonist - Google Patents

Use of interleukin-10 to induce the production of interleukin-1 receptor antagonist Download PDF

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Publication number
WO1993018783A1
WO1993018783A1 PCT/US1993/002173 US9302173W WO9318783A1 WO 1993018783 A1 WO1993018783 A1 WO 1993018783A1 US 9302173 W US9302173 W US 9302173W WO 9318783 A1 WO9318783 A1 WO 9318783A1
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seq
leu
information
interleukin
glu
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PCT/US1993/002173
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French (fr)
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Rene De Waal Malefyt
Jan De Vries
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Schering Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • 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
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates generally to a method for inducing the 'production of interleukin-1 receptor antagonist (IL-1 ra) by administering an effective amount of interleukin-10.
  • IL-1 ra interleukin-1 receptor antagonist
  • the invention relates to the use of interleukin-10 (IL-10) to treat diseases and conditions associated with elevated levels of interleukin-1.
  • the invention relates to the treatment of a wide variety of diseases and conditions associated with undesirable inflammatory reactions, for example, septic shock, rheumatoid arthritis, and the like, e.g. Gallin et al., editors, Inflammation (Raven Press, New York, 1988); Evans et al., Circulatory Shock, Vol. 29, pgs. 279-290 (1989); Waage et al., J. Exp. Med., Vol. 169, pgs. 333-338 (1989), and the like.
  • the invention is based in part on the discovery that IL-10 induces the production of IL-1 ra, which serves to block the biological activity of IL-1.
  • the invention includes pharmaceutical compositions comprising interleukin-10.
  • the interleukin-10 of the invention is selected from the group consisting of the mature polypeptides having the open reading frames that are defined by the amino acid sequences given in SEQ. ID. NOS. 1 and 2 herein (all SEQ. IDs. are given immediately before the Claims), wherein the standard three-letter abbreviation is used to indicate L-amino acids, starting from the N-terminus.
  • IL-10 are sometimes referred to as human IL-10 (or human cytokine synthesis inhibitory factor) and viral IL-10 (or BCRF1), respectively; e.g. Moore et al., Science, Vol. 248, pgs. 1230-1234 (1990); Vieira et al., Proc. Natl. Acad. Sci., Vol. 88, pgs. 1172-1176 (1991); Fiorentino et al., J. Exp. Med, Vol. 170, pgs. 2081-2095 (1989); Hsu et al., Science, Vol. 250, pgs. 830-832 (1990). More preferably, the mature IL-10 used in the method of the invention is selected from the group consisting of the mature polypeptides having the open reading frames that are defined by the amino acid sequences given in SEQ. ID. NOS. 3 and 4 herein.
  • Figure 1 is a diagram of the vector pcD(SR ⁇ ) used for expressing IL-10 in mammalian cells.
  • Figure 2 is a diagram of the vector TRP-C11 used for expressing IL-10 in bacteria.
  • Figure 3 shows plasmid pGSRG carrying the open reading frame
  • the invention is directed to a method of using IL-10 to ameliorate the deleterious effects of IL-1 in individuals suffering from inflammatory conditions or diseases.
  • the invention includes pharmaceutical compositions comprising IL-10 for carrying out the method.
  • IL-10 for use in the invention is selected from the group of mature polypeptides encoded by the open reading frames defined by cDNA inserts of pH5C, pH15C, and pBCRFI (SR ⁇ ), which are deposited with the American Type Culture Collection (ATCC), Rockville, Maryland, under accession numbers 68191 , 68192, and 68193, respectively.
  • ATCC American Type Culture Collection
  • i Assays for lnterle ⁇ kin-ip
  • IL-10s exhibit several biological activities which could form the basis of assays and units.
  • IL-10s have the property of inhibiting the synthesis of at least one cytokine in the group consisting of IFN- ⁇ , lymphotoxin, IL-2, IL-3, and GM-CSF in a population of T helper cells induced to synthesize one or more of these cytokines by exposure to syngeneic antigen-presenting cells (APCs) and antigen.
  • APCs syngeneic antigen-presenting cells
  • the APCs are treated so that they are incapable of replication but their antigen- processing machinery remains functional. This is conveniently accomplished by irradiating the APCs, e.g. with about 1500-3000 R (gamma or X-radiation) before mixing with the T cells.
  • cytokine inhibition may be assayed in primary or, preferably, secondary mixed lymphocyte reactions (MLR), in which case syngeneic APCs need not be used.
  • MLRs are well known in the art, e.g. Bradley, pgs. 162-166, in Mishell et al., eds. Selected Methods in Cellular Immunology (Freeman, San Francisco, 1980); and Battisto et al., Meth. in Enzymol., Vol. 150, pgs. 83-91 (1987). Briefly, two populations of allogenic lymphoid cells are mixed, one of the populations having been treated prior to mixing to prevent proliferation, e.g. by irradiation.
  • the cell populations are prepared at a concentration of about 2 x 10 6 cells/ml in supplemented medium, e.g. RPM1 1640 with 10% fetal calf serum.
  • supplemented medium e.g. RPM1 1640 with 10% fetal calf serum.
  • the cells remaining after 7 days in the primary MLR are re-stimulated by freshly prepared, irradiated stimulator cells.
  • the sample suspected of containing IL-10 may be added to the test cultures at the time of mixing, and both controls and test cultures may be assayed for cytokine production from 1 to 3 days after mixing.
  • T cell populations and/or APC populations for IL-10 assays employs techniques well known in the art which are fully described in DiSabato et al., eds., Meth. in Enzymol., Vol. 108 (1984).
  • APCs for the preferred IL-10 assay are peripheral blood monocytes. These are obtained using standard techniques, e.g. as described by Boyum, Meth. in Enzymol., Vol. 108, pgs. 88-102 (1984); Mage, Meth. in Enzymol., Vol. 108, pgs. 118-132 (1984); Litvin et al., Meth. in Enzymol., Vol. 108, pgs.
  • helper T cells are used in the IL-10 assays, which are obtained by first separating lymphocytes from the peripheral blood and then selecting, e.g. by panning or flow cytometry, helper cells using a commercially available anti-CD4 antibody, e.g. OKT4 described in U.S. patent 4,381 ,295 and available from Ortho Pharmaceutical Corp. The requisite techniques are fully disclosed by Boyum in Scand. J. Clin.
  • PBLs are obtained from fresh blood by Ficoll-Hypaque density gradient centrifugation.
  • antigens can be employed in the assay, e.g. Keyhole limpet hemocyanin (KLH), fowl ⁇ -globulin, or the like. More preferably, in place of antigen, helper T cells are stimulated with anti-CD3 monoclonal antibody, e.g. OKT3 disclosed in U.S. patent 4,361 ,549, in the assay.
  • KLH Keyhole limpet hemocyanin
  • helper T cells are stimulated with anti-CD3 monoclonal antibody, e.g. OKT3 disclosed in U.S. patent 4,361 ,549, in the assay.
  • Cytokine concentrations in control and test samples are measured by standard biological and/or immunochemical assays. Construction of immunochemical assays for specific cytokines is well known in the art when the purified cytokine is available: e.g. Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam, 1984); Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and U.S. patent 4,486,530 are exemplary of the extensive literature on the subject.
  • ELISA kits for human IL-2, human IL-3, and human GM-CSF are commercially available from Genzyme Corp. (Boston, MA); and an ELISA kit for human IFN- ⁇ is commercially available from Endogen, Inc.
  • Biological assays of the cytokines listed above can also be used to determine IL-10 activity.
  • a biological assay for human lymphotoxin is disclosed by Aggarwal in Meth. in Enzymol., Vol. 116, pgs. 441-447 (1985), and by Matthews et al., pgs. 221-225, in Clemens et al., eds,, Lymphokines and Interferons: A Practical Approach (IRL Press, Washington, D.C., 1987).
  • Human IL-2 and GM-CSF can be assayed with factor dependent cell lines CTLL-2 and KG-1 , available from the ATCC under accession numbers TIB 214 and CCL 246, respectively.
  • Human IL-3 can be assayed by it ability to stimulate the formation of a wide range of hematopoietic cell colonies in soft agar cultures, e.g. as described by Metcalf, The Hemopoietic Colony Stimulating Factors (Elsevier, Amsterdam, 1984). IFN- ⁇ can be quantified with anti-viral assays, e.g. Meager, pgs. 129-147, in Clemens et a!., eds. (cited above). Cytokine production can also be determined by mRNA analysis. Cytokine mRNAs can be measured by cytoplasmic dot hybridization as described by White et al., J. Biol. Chem., Vol.
  • Units of IL-10 activity can be defined in a variety of ways. Preferably, units are based on the ability of IL-10 to inhibit the production of IFN- ⁇ in phytohemaglutinin-stimulated peripheral blood mononuclear cells. IFN- ⁇ levels in samples and standards are conveniently measured immunometricaily. Units of IL-10 activity can also be defined in terms of IL-10's ability to augment the IL-4-induced proliferation of MC/9 cells, which are described in U.S. patent 4,559,310 and available from the ATCC under accession number CRL 8306.
  • 1 unit/ml is defined as the concentration of IL-10 which gives 50% of maximum stimulation of MC/9 proliferation above the level of IL-4 in the following assay.
  • Add IL-4 25 ⁇ l/well of 1600 U/ml (400 U/ml final) diluted in medium and incubate overnight, e.g. 20-24 hours.
  • Add 3 H-thymidine e.g. 50 ⁇ Ci/ml in medium
  • 0.5-1.0 ⁇ Ci/well 0.5-1.0 ⁇ Ci/well and again incubate the cells overnight; thereafter harvest the cells and measure the incorporated radioactivity.
  • polypeptides of the present invention When polypeptides of the present invention are expressed in soluble form, for example as a secreted product of transformed yeast or mammalian cells, they can be purified according to standard procedures of the art, including steps of ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and/or the like; e.g. "Enzyme Purification and Related Techniques," Methods in Enzymology, 22:233-577 (1977; and Scopes, R., Protein Purification: Principles and Practice (Springer- Verlag, New York, 1982) provide guidance in such purifications.
  • polypeptides of the invention when expressed in insoluble form, for example as aggregates, inclusion bodies, or the like, they can be purified by standard procedures in the art, including separating the inclusion bodies from disrupted host cells by centrifugation, solubilizing the inclusion bodies with chaotropic and reducing agents, diluting the solubilized mixture, and lowering the concentration of chaotropic agent and reducing agent so that the poly- peptide takes on a biologically active conformation.
  • IL-10 is administered as a pharmaceutical composition comprising an effective amount of IL-10 and a pharmaceutical carrier.
  • a pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient.
  • compositions useful for parenteral administration of such drugs are well known, e.g. Remington's Pharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, PA 1980).
  • compositions of the invention may be introduced into a patient's body by implantable or injectable drug delivery system, e.g. Urquhart et al., Ann. Rev. Pharmacol. Toxicol., Vol. 24, pgs. 199-236 (1984); Lewis, ed. Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, New York, 1981); U.S. patent 3,773,919; U.S. patent 3,270,960; and the like.
  • the IL-10 is formulated in a unit dosage injectable form (e.g., solution, suspension or emulsion) in association with a pharmaceutical carrier.
  • IL-10 is preferably formulated in purified form substantially free of aggregates and other proteins at a concentration in the range of about 5 to 20 ⁇ g/ml.
  • IL-10 is administered by continuous infusion so that an amount in the range of about 50-800 ⁇ g is delivered per day (i.e. about 1-16 ⁇ g/kg/day). The daily infusion rate may be varied based on monitoring of side effects and blood cell counts.
  • Example 1 Expression of human CSIF in a bacterial host
  • a synthetic human CSIF gene is assembled from a plurality of chemically synthesized double-stranded DNA fragments to form an expression vector designated TAC-RBS-hCSIF.
  • Cloning and expression are carried out in a standard bacterial system, for example E. coli K-12 strain JM101 , JM103, or the like, described by Viera and Messing, in Gene, Vol. 19, pgs. 259-268 (1982). Restriction endonuclease digestions and ligase reactions are performed using standard protocols, e.g. Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1982).
  • the alkaline method (Maniatis et al., cited above) is used for small scale plasmid preparations.
  • a modification of the alkaline method is used in which an equal volume of isopropanol is used to precipitate nucleic acids from the cleared lysate.
  • Precipitation with cold 2.5 M ammonium acetate is used to remove RNA prior to cesium chloride equilibrium density centrifugation and detection with ethidium bromide.
  • DNA is synthesized by phosphoramidite chemistry using Applied Biosystems 380A synthesizers. Synthesis, deprotection, cleavage and purification (7M urea PAGE, elution, DEAE-cellulose chromatography) are done as described in the 380A synthesizer manual.
  • Complementary strands of synthetic DNAs to be cloned (400 ng each) are mixed and phosphorylated with polynucleotide kinase in a reaction volume of 50 ml.
  • This DNA is ligated with 1 mg of vector DNA digested with appropriate restriction enzymes, and ligations are in a volume of 50 ml at room temperature for 4 to 12 hours.
  • Conditions for phosphorylation, restriction enzyme digestions, polymerase reactions, and ligation have been described (Maniatis et al., cited above).
  • Colonies are scored for lacZ + (when desired) by plating on L agar supplemented with ampiciliin, isopropyl-1-thio-beta-D-galactoside (IPTG) (0.4 mM) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (x-gal) (40 mg/ml).
  • the TAC-RBS vector is constructed by filling-in with DNA polymerase the single Bam HI site of the tacP-bearing plasmid pDR540 (Pharmacia).
  • the synthetic IL-10 gene is assembled into a pUC19 plasmid in eight steps. At each step inserts free of deletions and/or inserts can be detected after cloning by maintaining the lacZ( ⁇ ) gene of pUC19 in frame with the ATG start codon inserted in step 1. Clones containing deletion and/or insertion changes can be filtered out by scoring for blue colonies on L-ampicillin plates containing x-gal and IPTG. Alternatively, at each step sequences of inserts can be readily confirmed using a universal sequencing primer on small scale plasmid DNA preparations, e.g. available from Boehringer Mannheim.
  • the TAC-RBS vector is digested with Sst I, treated with T4 DNA polymerase (whose 3'-exonuclease activity digests the 3'-protruding strands of the Sstl cuts to form blunt-end fragments), and after deactivation of T4 DNA polymerase, treated with Eco RI to form a 173 bp fragment containing the TAC-RBS region and having a blunt end at the ATG start codon and the Eco RI cut at the opposite end. Finally, the 173 bp TAC- RBS fragment is isolated.
  • step 2 the isolated TAC-RBS fragment of step 1 is mixed with Eco FQjKpn I-digested plasmid pUC19 and synthetic fragment 1A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 6 and 7 herein, which has a blunt end at its upstream terminus and a staggered end corresponding to a Kpnl cut at its downstream terminus. This Kpnl end is adjacent to and downstream of a BstEU site. The fragments are ligated to form the pUC19 of step 2.
  • step 3 synthetic fragments 2A/B and 3A/B are mixed with Bst EU/Sma I-digested pUC19 of step 2 (after amplification and purification) and ligated to form pUC19 of step 3.
  • the nucleic acid sequences of synthetic fragment 2A/B are shown in SEQ. ID. NOs. 8 and 9 herein and the nucleic acid sequences of synthetic fragment 3A/B are shown in SEQ. ID. NOs. 10 and 11 herein. Note that the downstream terminus of fragment 3A/B contains extra bases which form the Smal blunt-end. These extra bases are cleaved in step 4.
  • fragments 2A/B and 3A/B have complementary 9-residue single-stranded ends which anneal upon admixture, leaving the upstream Bst En cut of 2A/B and the downstream blunt end of 3A/B to ligate to the pUC19.
  • step 4 the pUC19 of step 3 is digested with AflU/Xbal, amplified, purified, repurified, mixed with synthetic fragment 4A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 12 and 13 herein, and ligated to form pUC19 of step 4.
  • step 5 the pUC19 of step 4 is digested with Xba ⁇ /Sall, amplified and purified, and mixed with synthetic fragment 5A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 14 and 15 herein and ligated to form the pUC19 of step 5. Note that the Sa/I-staggered end of fragment 5A/B is eliminated by digestion with Hpa I in step 6.
  • step 6 the pUC19 of step 5 is digested with HpaVPstl, amplified and purified, and mixed with synthetic fragment 6A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 16 and 17 herein and ligated to form the pUC19 of step 6.
  • step 7 the pUC19 of step 6 is digested with Clal/Sphl, amplified and purified, and mixed with synthetic fragment 7A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 18 and 19 herein and ligated to form the pUC19 of step 7.
  • step 8 the pUC19 of step 7 is digested with Mlul/Hin ⁇ m, amplified and purified, and mixed with synthetic fragments 8A/B and 9A/B and ligated to form the final construction, which is then inserted into E. coll K-12 strain JM101 , e.g. available from the ATCC under accession number 33876, by standard techniques.
  • the nucleic acid sequences of synthetic fragment 8A/B are shown in SEQ. ID. NOs. 20 and 21 herein and the nucleic acid sequences of synthetic fragment 9A/B are shown in SEQ. ID. NOs. 22 and 23 herein.
  • a gene encoding the open reading frame of vlL-10 was amplified by polymerase chain reaction using primers that allowed later insertion of the amplified fragment into an Eco Rl-digested pcD(SR ⁇ ) vector ( Figure 1).
  • the coding strand of the inserted fragment is shown in SEQ. ID. NO. 15 herein.
  • Clones carrying the insert in the proper orientation were identified by expression of vlL-10 and/or the electrophoretic pattern of restriction digests.
  • One such vector carrying the vlL-10 gene was designated pBCRFI (SR ⁇ ) and was deposited with the ATCC under accession number 68193.
  • pBCRFI(SR ⁇ ) was amplified in E. coli MC1061 , isolated by standard techniques, and used to transfect COS 7 monkey cells as follows: One day prior to transfection, approximately 1.5 x 10 6 COS 7 monkey cells were seeded onto individual 100 mm plates in Dulbecco's modified Eagle medium (DME) containing 5% fetal calf serum (FCS) and 2 mM glutamine.
  • DME Dulbecco's modified Eagle medium
  • FCS 5% fetal calf serum
  • COS 7 cells were removed from the dishes by incubation with trypsin, washed twice in serum-free DME, and suspended to 10 7 cells/ml in serum-free DME. A 0.75 ml aliquot was mixed with 20 ⁇ g DNA and transferred to a sterile 0.4 cm electroporation cuvette. After 10 minutes, the cells were pulsed at 200 volts, 960 ⁇ F in a BioRad Gene Pulser unit. After another 10 minutes, the cells were removed from the cuvette and added to 20 ml of DME containing 5% FCS, 2mM glutamine, penicillin, streptomycin, and gentamycin. The mixture was aliquoted to four 100 mm tissue culture dishes.
  • a gene encoding the mature vlL-10 shown in SEQ. ID. NO. 4 herein may be expressed in E. coli.
  • the cDNA insert of pBCRFI (SR ⁇ ) is recloned into an M13 plasmid where it is altered twice by site-directed mutagenesis: first to form a Clal site at the 5'-end of the coding region for the mature vlL-10 polypeptide, and second to form a Bam HI site at the 3'-end of the coding region for the mature vlL-10 polypeptide.
  • the mutated sequence is then readily inserted into the TRPC11 expression vector described below.
  • the TRPC11 vector was constructed by ligating a synthetic consensus RBS fragment to C/a I linkers (ATGCAT) and by cloning the resulting fragments into Cla I-restricted pMT11 hc (which had been previously modified to contain the Cla I site).
  • pMT11hc is a small (2.3 kilobase) high copy, AMP R , TET S derivative of pBR322 that bears the ⁇ VX plasmid Eco Hl-Hin ⁇ WL polylinker region. ( ⁇ VX is described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982).
  • One transformant from the TRPC11 construction had a tandem RBS sequence flanked by Cla I sites.
  • One of the Cla I sites and part of the second copy of the RBS sequence were removed by digesting this plasmid with Pstl, treating with Bal31 nuclease, restricting with Eco RI and treating with T4 DNA polymerase in the presence of all four deoxynucleotide triphosphates.
  • TRPC11 is employed as a vector for vlL-10 by first digesting it with Cla I and Bam HI, purifying it, and then mixing it in a standard ligation solution with the Clal-Bam HI fragment of the M13 containing the nucleotide sequence coding for the mature BCRF1.
  • the insert-containing TRPC11 referred to as TRPC11-BCRF1 , is propagated in E. coli K12 strain JM101 , e.g. available from the ATCC under accession number 33876.
  • Example 4 Induction of IL-1 Receptor Antagonist bv IL-10 Human peripheral blood monocytes were isolated from 500 mL blood of normal donors by standard techniques: e.g. Figdor et al., Blood, Vol. 60, pg. 46 (1982); Figdor et al., J. Immunol. Meth., Vol. 68, pg. 73 (1984). Briefly, mononuclear cells were isolated by density centrifugation in a blood component separator, followed by fractionation into lymphocytes and monocytes by centrifugal elutnation. The monocyte preparation was >95% pure, as judged by nonspecific esterase staining and contained more than 98% viable cells.
  • Monocytes were cultured in Yssel's medium (Yssel et al., J. Immunol. Meth., Vol. 72, pg.219) containing human serum albumin (HSA) supplemented with 1% pooled heat-inactivated human AB+ serum.
  • HSA human serum albumin
  • This culture medium was endotoxin.-free as determined by the Limulus amebocyte lysate assay ( ⁇ 0.2 ng/mL of endotoxin).
  • the monocytes were cultured at 37°C at a concentration of 4X 10 6 cells/mL in Teflon' bags (Jansen MNL, St. Niklaas, Belgium), which prevented adhesion of these cells.
  • T and GGTAC at positions 51 and 56- 60 differ from those of the native sequence; together with SEQ ID NO: 7, SEQ ID NO: 6 forms double-stranded Fragment 1A/B of synthetic CSIF gene with 4-base sticky end at positions 57-60.
  • T position 2 differs from that of the native sequence; together with SEQ ID NO: 9, SEQ ID NO: 8 forms double- stranded Fragment 2A/B of synthetic CSIF gene with 5- and 9-base sticky ends at positions 1 -5 and 54-62.
  • T at position 69 differs from that of the native sequence; together with SEQ ID NO: 13, SEQ ID NO: 12 forms double-stranded Fragment 4A/B of synthetic CSIF gene.
  • CTAGATAAAA CTGGATCATC TCAGACAAGG CTTGGCAACC CAGGTAACCC 50 TTAAAGTCCT CCAGCAAGGA CTC 73
  • TCGAC, A and T at positions 1 -5, 9 and 63 differ from those of the native sequence; together with SEQ ID NO: 14, SEQ ID NO: 15 forms double-stranded Fragment 5A/B of synthetic CSIF gene with 4-base sticky end at positions 1-4.
  • CTGCA at positions 58-63 differ from those of the native sequence; together with SEQ ID NO: 17, SEQ ID NO: 16 forms double-stranded Fragment 6A/B of synthetic CSIF gene with 4- base sticky end at positions 59-63.
  • CACGCGTTCT TCACCTGCTC CACGGCCTTG CTCTTGTTTT GACAGGGAAG 5 AAAT 5

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Abstract

A method is provided for treating inflammatory conditions which comprises administering to a patient an effective amount of interleukin-10.

Description

USE OF INTERLEUKIN-10 TO INDUCE THE PRODUCTION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST
Field of the Invention
The invention relates generally to a method for inducing the 'production of interleukin-1 receptor antagonist (IL-1 ra) by administering an effective amount of interleukin-10.
SUMMARY OF THE INVENTION
The invention relates to the use of interleukin-10 (IL-10) to treat diseases and conditions associated with elevated levels of interleukin-1. In particular, the invention relates to the treatment of a wide variety of diseases and conditions associated with undesirable inflammatory reactions, for example, septic shock, rheumatoid arthritis, and the like, e.g. Gallin et al., editors, Inflammation (Raven Press, New York, 1988); Evans et al., Circulatory Shock, Vol. 29, pgs. 279-290 (1989); Waage et al., J. Exp. Med., Vol. 169, pgs. 333-338 (1989), and the like.
The invention is based in part on the discovery that IL-10 induces the production of IL-1 ra, which serves to block the biological activity of IL-1. The invention includes pharmaceutical compositions comprising interleukin-10. Preferably, the interleukin-10 of the invention is selected from the group consisting of the mature polypeptides having the open reading frames that are defined by the amino acid sequences given in SEQ. ID. NOS. 1 and 2 herein (all SEQ. IDs. are given immediately before the Claims), wherein the standard three-letter abbreviation is used to indicate L-amino acids, starting from the N-terminus. These two forms of IL-10 are sometimes referred to as human IL-10 (or human cytokine synthesis inhibitory factor) and viral IL-10 (or BCRF1), respectively; e.g. Moore et al., Science, Vol. 248, pgs. 1230-1234 (1990); Vieira et al., Proc. Natl. Acad. Sci., Vol. 88, pgs. 1172-1176 (1991); Fiorentino et al., J. Exp. Med, Vol. 170, pgs. 2081-2095 (1989); Hsu et al., Science, Vol. 250, pgs. 830-832 (1990). More preferably, the mature IL-10 used in the method of the invention is selected from the group consisting of the mature polypeptides having the open reading frames that are defined by the amino acid sequences given in SEQ. ID. NOS. 3 and 4 herein.
Brief Description of the Drawings
Figure 1 is a diagram of the vector pcD(SRα) used for expressing IL-10 in mammalian cells.
Figure 2 is a diagram of the vector TRP-C11 used for expressing IL-10 in bacteria. Figure 3 shows plasmid pGSRG carrying the open reading frame
(ORF) of mouse IL-10, viral IL-10r or human IL-10 inserted into its Xhol restriction site; it also shows the sequence of the RBS-ATG-polylinker regions of the final construction (called TAC-RBS).
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to a method of using IL-10 to ameliorate the deleterious effects of IL-1 in individuals suffering from inflammatory conditions or diseases. The invention includes pharmaceutical compositions comprising IL-10 for carrying out the method. IL-10 for use in the invention is selected from the group of mature polypeptides encoded by the open reading frames defined by cDNA inserts of pH5C, pH15C, and pBCRFI (SRα), which are deposited with the American Type Culture Collection (ATCC), Rockville, Maryland, under accession numbers 68191 , 68192, and 68193, respectively. i. Assays for lnterleυkin-ip, IL-10s exhibit several biological activities which could form the basis of assays and units. In particular, IL-10s have the property of inhibiting the synthesis of at least one cytokine in the group consisting of IFN-γ, lymphotoxin, IL-2, IL-3, and GM-CSF in a population of T helper cells induced to synthesize one or more of these cytokines by exposure to syngeneic antigen-presenting cells (APCs) and antigen. In this activity, the APCs are treated so that they are incapable of replication but their antigen- processing machinery remains functional. This is conveniently accomplished by irradiating the APCs, e.g. with about 1500-3000 R (gamma or X-radiation) before mixing with the T cells.
Alternatively, cytokine inhibition may be assayed in primary or, preferably, secondary mixed lymphocyte reactions (MLR), in which case syngeneic APCs need not be used. MLRs are well known in the art, e.g. Bradley, pgs. 162-166, in Mishell et al., eds. Selected Methods in Cellular Immunology (Freeman, San Francisco, 1980); and Battisto et al., Meth. in Enzymol., Vol. 150, pgs. 83-91 (1987). Briefly, two populations of allogenic lymphoid cells are mixed, one of the populations having been treated prior to mixing to prevent proliferation, e.g. by irradiation. Preferably, the cell populations are prepared at a concentration of about 2 x 106 cells/ml in supplemented medium, e.g. RPM1 1640 with 10% fetal calf serum. For both controls and test cultures, mix 0.5 ml of each population for the assay. For a secondary MLR, the cells remaining after 7 days in the primary MLR are re-stimulated by freshly prepared, irradiated stimulator cells. The sample suspected of containing IL-10 may be added to the test cultures at the time of mixing, and both controls and test cultures may be assayed for cytokine production from 1 to 3 days after mixing.
Obtaining T cell populations and/or APC populations for IL-10 assays employs techniques well known in the art which are fully described in DiSabato et al., eds., Meth. in Enzymol., Vol. 108 (1984). APCs for the preferred IL-10 assay are peripheral blood monocytes. These are obtained using standard techniques, e.g. as described by Boyum, Meth. in Enzymol., Vol. 108, pgs. 88-102 (1984); Mage, Meth. in Enzymol., Vol. 108, pgs. 118-132 (1984); Litvin et al., Meth. in Enzymol., Vol. 108, pgs. 298-302 (1984); Stevenson, Meth. in Enzymol., Vol. 108, pgs. 242-249 (1989); and Romain et al., Meth. in Enzymol., Vol. 108, pgs. 148-153 (1984), which references are incorporated by reference. Preferably, helper T cells are used in the IL-10 assays, which are obtained by first separating lymphocytes from the peripheral blood and then selecting, e.g. by panning or flow cytometry, helper cells using a commercially available anti-CD4 antibody, e.g. OKT4 described in U.S. patent 4,381 ,295 and available from Ortho Pharmaceutical Corp. The requisite techniques are fully disclosed by Boyum in Scand. J. Clin. Lab. Invest, Vol. 21 (Suppl. 97), pg. 77 (1968) and in Meth. in Enzymol., Vol. 108 (cited above), and by Bram et al. in Meth. in Enzymol., Vol. 121 , pgs. 737-748 (1986). Generally, PBLs are obtained from fresh blood by Ficoll-Hypaque density gradient centrifugation.
A variety of antigens can be employed in the assay, e.g. Keyhole limpet hemocyanin (KLH), fowl γ-globulin, or the like. More preferably, in place of antigen, helper T cells are stimulated with anti-CD3 monoclonal antibody, e.g. OKT3 disclosed in U.S. patent 4,361 ,549, in the assay.
Cytokine concentrations in control and test samples are measured by standard biological and/or immunochemical assays. Construction of immunochemical assays for specific cytokines is well known in the art when the purified cytokine is available: e.g. Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam, 1984); Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and U.S. patent 4,486,530 are exemplary of the extensive literature on the subject. ELISA kits for human IL-2, human IL-3, and human GM-CSF are commercially available from Genzyme Corp. (Boston, MA); and an ELISA kit for human IFN-γ is commercially available from Endogen, Inc. (Boston, MA). Polyclonal antibodies specific for human lymphotoxin are available from Genzyme Corp. which can be used in a radioimmunoassay for human lymphotoxin, e.g. Chard, An Introduction to Radioimmunoassay and Related Techniques (Elsevier, Amsterdam, 1982).
Biological assays of the cytokines listed above can also be used to determine IL-10 activity. A biological assay for human lymphotoxin is disclosed by Aggarwal in Meth. in Enzymol., Vol. 116, pgs. 441-447 (1985), and by Matthews et al., pgs. 221-225, in Clemens et al., eds,, Lymphokines and Interferons: A Practical Approach (IRL Press, Washington, D.C., 1987). Human IL-2 and GM-CSF can be assayed with factor dependent cell lines CTLL-2 and KG-1 , available from the ATCC under accession numbers TIB 214 and CCL 246, respectively. Human IL-3 can be assayed by it ability to stimulate the formation of a wide range of hematopoietic cell colonies in soft agar cultures, e.g. as described by Metcalf, The Hemopoietic Colony Stimulating Factors (Elsevier, Amsterdam, 1984). IFN-γ can be quantified with anti-viral assays, e.g. Meager, pgs. 129-147, in Clemens et a!., eds. (cited above). Cytokine production can also be determined by mRNA analysis. Cytokine mRNAs can be measured by cytoplasmic dot hybridization as described by White et al., J. Biol. Chem., Vol. 257, pgs. 8569-8572 (1982), and Gillespie et al., U.S. patent 4,483,920. Accordingly, these references are incorporated by reference. Other approaches include dot blotting using purified RNA, e.g. chapter 6, in Hames et al., eds., Nucleic Acid Hybridization A Practical Approach (IRL Press, Washington, D.C., 1985). Some samples to be tested for IL-10 activity must be pretreated to remove predetermined cytokines that might interfere with the assay. For example, IL-2 increases the production of IFN-γ in some cells. Thus depending on the helper T cells used in the assay, IL-2 may have to be removed from the sample being tested. Such removals are conveniently accomplished by passing the sample over a standard anti-cytokine affinity column. Units of IL-10 activity can be defined in a variety of ways. Preferably, units are based on the ability of IL-10 to inhibit the production of IFN-γ in phytohemaglutinin-stimulated peripheral blood mononuclear cells. IFN-γ levels in samples and standards are conveniently measured immunometricaily. Units of IL-10 activity can also be defined in terms of IL-10's ability to augment the IL-4-induced proliferation of MC/9 cells, which are described in U.S. patent 4,559,310 and available from the ATCC under accession number CRL 8306. 1 unit/ml is defined as the concentration of IL-10 which gives 50% of maximum stimulation of MC/9 proliferation above the level of IL-4 in the following assay. Prepare duplicate or triplicate dilutions of IL-4 and IL-10 in 50 μl of medium per well in a standard microtiter plate. Medium consists of RPM1 1640, 10% fetal calf serum, 50 μM 2-mercaptoethanol, 2 mM glutamine, penicillin (100 U/L) and streptomycin (100 μg/L). Add IL-4, 25 μl/well of 1600 U/ml (400 U/ml final) diluted in medium and incubate overnight, e.g. 20-24 hours. Add 3H-thymidine (e.g. 50 μCi/ml in medium) at 0.5-1.0 μCi/well and again incubate the cells overnight; thereafter harvest the cells and measure the incorporated radioactivity.
π. Purification and Pharmaceutical Compositions
When polypeptides of the present invention are expressed in soluble form, for example as a secreted product of transformed yeast or mammalian cells, they can be purified according to standard procedures of the art, including steps of ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, and/or the like; e.g. "Enzyme Purification and Related Techniques," Methods in Enzymology, 22:233-577 (1977; and Scopes, R., Protein Purification: Principles and Practice (Springer- Verlag, New York, 1982) provide guidance in such purifications. Likewise, when polypeptides of the invention are expressed in insoluble form, for example as aggregates, inclusion bodies, or the like, they can be purified by standard procedures in the art, including separating the inclusion bodies from disrupted host cells by centrifugation, solubilizing the inclusion bodies with chaotropic and reducing agents, diluting the solubilized mixture, and lowering the concentration of chaotropic agent and reducing agent so that the poly- peptide takes on a biologically active conformation. The latter procedures are disclosed in the following references, which are incorporated by reference: Winkler et al., Biochemistry, 25: 4041-4045 (1986); Winkler et al., Biotechnology, 3: 992-998 (1985); Koths et al., U.S. patent 4,569,790; and European patent applications 86306917.5 and 86306353.3.
As used herein "effective amount" means an amount sufficient to reduce or prevent inflammation. The effective amount for a particular patient may vary depending on such factors as the state, type, and extent of inflammation, the overall health of the patient, method of administration, the severity of side-effects, and the like. Generally, IL-10 is administered as a pharmaceutical composition comprising an effective amount of IL-10 and a pharmaceutical carrier. A pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient. Generally, compositions useful for parenteral administration of such drugs are well known, e.g. Remington's Pharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, PA 1980). Alternatively, compositions of the invention may be introduced into a patient's body by implantable or injectable drug delivery system, e.g. Urquhart et al., Ann. Rev. Pharmacol. Toxicol., Vol. 24, pgs. 199-236 (1984); Lewis, ed. Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, New York, 1981); U.S. patent 3,773,919; U.S. patent 3,270,960; and the like. When administered parenterally, the IL-10 is formulated in a unit dosage injectable form (e.g., solution, suspension or emulsion) in association with a pharmaceutical carrier. Examples of such carriers are normal saline, Ringer's solution, dextrose solution, and Hank's solution. Nonaqueous carriers such as fixed oils and ethyl oleate may also be used. A preferred carrier is 5% dextrose/saline. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The IL-10 is preferably formulated in purified form substantially free of aggregates and other proteins at a concentration in the range of about 5 to 20 μg/ml. Preferably, IL-10 is administered by continuous infusion so that an amount in the range of about 50-800 μg is delivered per day (i.e. about 1-16 μg/kg/day). The daily infusion rate may be varied based on monitoring of side effects and blood cell counts.
EXAMPLES
The following examples serve to illustrate the present invention. Selected vectors and hosts, the concentration of reagents, temperatures, and the values of other variables are only to exemplify the application of the present invention and are not to be considered limitations thereof.
Example 1. Expression of human CSIF in a bacterial host
A synthetic human CSIF gene is assembled from a plurality of chemically synthesized double-stranded DNA fragments to form an expression vector designated TAC-RBS-hCSIF. Cloning and expression are carried out in a standard bacterial system, for example E. coli K-12 strain JM101 , JM103, or the like, described by Viera and Messing, in Gene, Vol. 19, pgs. 259-268 (1982). Restriction endonuclease digestions and ligase reactions are performed using standard protocols, e.g. Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1982). The alkaline method (Maniatis et al., cited above) is used for small scale plasmid preparations. For large scale preparations a modification of the alkaline method is used in which an equal volume of isopropanol is used to precipitate nucleic acids from the cleared lysate. Precipitation with cold 2.5 M ammonium acetate is used to remove RNA prior to cesium chloride equilibrium density centrifugation and detection with ethidium bromide.
For filter hybridizations Whatman 540 filter circles are used to lift colonies which are then lysed and fixed by successive treatments with 0.5M NaOH, 1.5M NaCI; 1 M Tris.HCI pH8.0, 1.5M NaCI (2 min each); and heating at 80°C (30 min). Hybridizations are in 6xSSPE, 20% formamide, 0.1% sodium dodecylsulphate (SDS) and 100 μg/ml E. coli tRNA at 42°C for 6 hours using 32P-labelled (kinased) synthetic DNAs. (20xSSPE is prepared by dissolving 174 g of NaCI, 27.6 g of NaH2Pθ4-9H2θ, and 7.4 g of EDTA in 800 ml of H2O. pH is adjusted to 7.4 with NaOH, volume is adjusted to 1 liter, and the whole is sterilized by autoclaving). Filters are washed twice (15 min, room temperature) with IxSSPE, 0.1 % SDS. After autoradiography (Fuji RX film), positive colonies are located by aligning the regrown colonies with the blue-stained colonies on the filters. DNA is sequenced by the dideoxy method of Sanger et al. Proc. Natl. Acad. Sci., Vol. 74, pg. 5463 (1977). Templates for the dideoxy reactions are either single-stranded DNAs of relevant regions recloned into M13mp vectors — e.g. Messing et al. Nucleic Acids Res., Vol. 9, pg. 309 (1981 ) — or double- stranded DNA prepared by the minialkaline method and denatured with 0.2M NaOH (5 min, room temperature) and precipitated from 0.2M NaOH, 1.43M ammonium acetate by the addition of 2 volumes of ethanol. DNA is synthesized by phosphoramidite chemistry using Applied Biosystems 380A synthesizers. Synthesis, deprotection, cleavage and purification (7M urea PAGE, elution, DEAE-cellulose chromatography) are done as described in the 380A synthesizer manual.
Complementary strands of synthetic DNAs to be cloned (400 ng each) are mixed and phosphorylated with polynucleotide kinase in a reaction volume of 50 ml. This DNA is ligated with 1 mg of vector DNA digested with appropriate restriction enzymes, and ligations are in a volume of 50 ml at room temperature for 4 to 12 hours. Conditions for phosphorylation, restriction enzyme digestions, polymerase reactions, and ligation have been described (Maniatis et al., cited above). Colonies are scored for lacZ+ (when desired) by plating on L agar supplemented with ampiciliin, isopropyl-1-thio-beta-D-galactoside (IPTG) (0.4 mM) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (x-gal) (40 mg/ml). The TAC-RBS vector is constructed by filling-in with DNA polymerase the single Bam HI site of the tacP-bearing plasmid pDR540 (Pharmacia). This is then ligated to unphosphorylated synthetic oligo- nucleotides (Pharmacia) which form a double-stranded fragment encoding a consensus ribosome binding site as given in SEQ. ID. NO. 5 herein and designated RBS. After ligation, the mixture is phosphoryiated and religated with the Sst I linker ATGAGCTCAT. This complex is then cleaved with Sstl and Eco RI, and the 173 base pair (bp) fragment isolated by polyacrylamide gel electrophoresis (PAGE) and cloned into Eco RI-SsH- restricted pUC19 (Pharmacia) (as described below). The sequence of the RBS-ATG-polylinker regions of the final construction (called TAC-RBS) is shown in Figure 3.
The synthetic IL-10 gene is assembled into a pUC19 plasmid in eight steps. At each step inserts free of deletions and/or inserts can be detected after cloning by maintaining the lacZ(α) gene of pUC19 in frame with the ATG start codon inserted in step 1. Clones containing deletion and/or insertion changes can be filtered out by scoring for blue colonies on L-ampicillin plates containing x-gal and IPTG. Alternatively, at each step sequences of inserts can be readily confirmed using a universal sequencing primer on small scale plasmid DNA preparations, e.g. available from Boehringer Mannheim.
In step 1 , the TAC-RBS vector is digested with Sst I, treated with T4 DNA polymerase (whose 3'-exonuclease activity digests the 3'-protruding strands of the Sstl cuts to form blunt-end fragments), and after deactivation of T4 DNA polymerase, treated with Eco RI to form a 173 bp fragment containing the TAC-RBS region and having a blunt end at the ATG start codon and the Eco RI cut at the opposite end. Finally, the 173 bp TAC- RBS fragment is isolated.
In step 2, the isolated TAC-RBS fragment of step 1 is mixed with Eco FQjKpn I-digested plasmid pUC19 and synthetic fragment 1A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 6 and 7 herein, which has a blunt end at its upstream terminus and a staggered end corresponding to a Kpnl cut at its downstream terminus. This Kpnl end is adjacent to and downstream of a BstEU site. The fragments are ligated to form the pUC19 of step 2. In step 3, synthetic fragments 2A/B and 3A/B are mixed with Bst EU/Sma I-digested pUC19 of step 2 (after amplification and purification) and ligated to form pUC19 of step 3. The nucleic acid sequences of synthetic fragment 2A/B are shown in SEQ. ID. NOs. 8 and 9 herein and the nucleic acid sequences of synthetic fragment 3A/B are shown in SEQ. ID. NOs. 10 and 11 herein. Note that the downstream terminus of fragment 3A/B contains extra bases which form the Smal blunt-end. These extra bases are cleaved in step 4. Also, fragments 2A/B and 3A/B have complementary 9-residue single-stranded ends which anneal upon admixture, leaving the upstream Bst En cut of 2A/B and the downstream blunt end of 3A/B to ligate to the pUC19.
In step 4, the pUC19 of step 3 is digested with AflU/Xbal, amplified, purified, repurified, mixed with synthetic fragment 4A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 12 and 13 herein, and ligated to form pUC19 of step 4.
In step 5, the pUC19 of step 4 is digested with Xbaϊ/Sall, amplified and purified, and mixed with synthetic fragment 5A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 14 and 15 herein and ligated to form the pUC19 of step 5. Note that the Sa/I-staggered end of fragment 5A/B is eliminated by digestion with Hpa I in step 6.
In step 6, the pUC19 of step 5 is digested with HpaVPstl, amplified and purified, and mixed with synthetic fragment 6A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 16 and 17 herein and ligated to form the pUC19 of step 6. In step 7, the pUC19 of step 6 is digested with Clal/Sphl, amplified and purified, and mixed with synthetic fragment 7A/B whose nucleic acid sequences are shown in SEQ. ID. NOs. 18 and 19 herein and ligated to form the pUC19 of step 7.
In step 8, the pUC19 of step 7 is digested with Mlul/Hinόm, amplified and purified, and mixed with synthetic fragments 8A/B and 9A/B and ligated to form the final construction, which is then inserted into E. coll K-12 strain JM101 , e.g. available from the ATCC under accession number 33876, by standard techniques. The nucleic acid sequences of synthetic fragment 8A/B are shown in SEQ. ID. NOs. 20 and 21 herein and the nucleic acid sequences of synthetic fragment 9A/B are shown in SEQ. ID. NOs. 22 and 23 herein. After cultivation, protein is extracted from the JM101 cells and dilutions of the extracts are tested for biological activity.
Example 2. Expression of ylL-10 in COS 7 Monkey cells
A gene encoding the open reading frame of vlL-10 was amplified by polymerase chain reaction using primers that allowed later insertion of the amplified fragment into an Eco Rl-digested pcD(SRα) vector (Figure 1). The coding strand of the inserted fragment is shown in SEQ. ID. NO. 15 herein.
Clones carrying the insert in the proper orientation were identified by expression of vlL-10 and/or the electrophoretic pattern of restriction digests. One such vector carrying the vlL-10 gene was designated pBCRFI (SRα) and was deposited with the ATCC under accession number 68193. pBCRFI(SRα) was amplified in E. coli MC1061 , isolated by standard techniques, and used to transfect COS 7 monkey cells as follows: One day prior to transfection, approximately 1.5 x 106 COS 7 monkey cells were seeded onto individual 100 mm plates in Dulbecco's modified Eagle medium (DME) containing 5% fetal calf serum (FCS) and 2 mM glutamine. To perform the transfection, COS 7 cells were removed from the dishes by incubation with trypsin, washed twice in serum-free DME, and suspended to 107 cells/ml in serum-free DME. A 0.75 ml aliquot was mixed with 20 μg DNA and transferred to a sterile 0.4 cm electroporation cuvette. After 10 minutes, the cells were pulsed at 200 volts, 960 μF in a BioRad Gene Pulser unit. After another 10 minutes, the cells were removed from the cuvette and added to 20 ml of DME containing 5% FCS, 2mM glutamine, penicillin, streptomycin, and gentamycin. The mixture was aliquoted to four 100 mm tissue culture dishes. After 12-24 hours at 37°C, 5% CO2, the medium was replaced with similar medium containing only 1% FCS and the incubation continued for an additional 72 hours at 37°C, 5% CO2, after which the medium was collected and assayed for its ability to inhibit IFN-γ synthesis.
10 ml aliquots of freshly isolated PBLs (about 2x106 cells/ml) were incubated at 37°C with PHA (100 ng/ml) in medium consisting of (i) 90% DME supplemented with 5% FCS and 2 mM glutamine, and (ii) 10% supernatant from COS 7 cells previously transfected with pBCRFI(SRα). After 24 hours the cells and supematants were harvested to assay for the presence of either IFN-γ mRNA or IFN-γ protein, respectively. Controls were treated identically, except that the 10% supernatant was from COS 7 cultures previously transfected with a plasmid carrying an unrelated cDNA insert. The vlL-10-treated samples exhibited about a 50% inhibition of IFN-γ synthesis relative to the controls.
Example 3. Expression of ylL-10 in Escherichia coli
A gene encoding the mature vlL-10 shown in SEQ. ID. NO. 4 herein may be expressed in E. coli. The cDNA insert of pBCRFI (SRα) is recloned into an M13 plasmid where it is altered twice by site-directed mutagenesis: first to form a Clal site at the 5'-end of the coding region for the mature vlL-10 polypeptide, and second to form a Bam HI site at the 3'-end of the coding region for the mature vlL-10 polypeptide. The mutated sequence is then readily inserted into the TRPC11 expression vector described below.
The TRPC11 vector was constructed by ligating a synthetic consensus RBS fragment to C/a I linkers (ATGCAT) and by cloning the resulting fragments into Cla I-restricted pMT11 hc (which had been previously modified to contain the Cla I site). pMT11hc is a small (2.3 kilobase) high copy, AMPR, TETS derivative of pBR322 that bears the πVX plasmid Eco Hl-HinόWL polylinker region. (πVX is described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982). This was modified to contain the Cla I site by restricting pMT11 he with Eco RI and Bam HI, filling in the resulting sticky ends and ligating with Cla I linker (CATCGATG), thereby restoring the Eco RI and Bam HI sites and replacing the Sma I site with a Cla I site. One transformant from the TRPC11 construction had a tandem RBS sequence flanked by Cla I sites. One of the Cla I sites and part of the second copy of the RBS sequence were removed by digesting this plasmid with Pstl, treating with Bal31 nuclease, restricting with Eco RI and treating with T4 DNA polymerase in the presence of all four deoxynucleotide triphosphates. The resulting 30-40 bp fragments were recovered via PAGE and cloned into Sma I-restricted pUC12. A 248 bp E. coli trpP-bearing Eco RI fragment derived from pKC101 (described by Nichols et al. in Methods in Enzymology, Vol. 101 , pg. 155 (Academic Press, N.Y. 1983)) was then cloned into the Eco RI site to complete the TRPC11 construction, which is illustrated in Figure 2. TRPC11 is employed as a vector for vlL-10 by first digesting it with Cla I and Bam HI, purifying it, and then mixing it in a standard ligation solution with the Clal-Bam HI fragment of the M13 containing the nucleotide sequence coding for the mature BCRF1. The insert-containing TRPC11 , referred to as TRPC11-BCRF1 , is propagated in E. coli K12 strain JM101 , e.g. available from the ATCC under accession number 33876.
Example 4. Induction of IL-1 Receptor Antagonist bv IL-10 Human peripheral blood monocytes were isolated from 500 mL blood of normal donors by standard techniques: e.g. Figdor et al., Blood, Vol. 60, pg. 46 (1982); Figdor et al., J. Immunol. Meth., Vol. 68, pg. 73 (1984). Briefly, mononuclear cells were isolated by density centrifugation in a blood component separator, followed by fractionation into lymphocytes and monocytes by centrifugal elutnation. The monocyte preparation was >95% pure, as judged by nonspecific esterase staining and contained more than 98% viable cells. Monocytes were cultured in Yssel's medium (Yssel et al., J. Immunol. Meth., Vol. 72, pg.219) containing human serum albumin (HSA) supplemented with 1% pooled heat-inactivated human AB+ serum. This culture medium was endotoxin.-free as determined by the Limulus amebocyte lysate assay (<0.2 ng/mL of endotoxin). The monocytes were cultured at 37°C at a concentration of 4X 106 cells/mL in Teflon' bags (Jansen MNL, St. Niklaas, Belgium), which prevented adhesion of these cells. To test IL-1 ra induction, the monocytes in culture were activated by exposure to lipopolysaccharide (LPS, Difco Laboratories, Detroit, Ml, E. coli 0127:B8) (1 μg/mL) for 24 hours. Supernatants were then harvested and assayed for IL-1β and IL-1 ra production. Monoclonal antibody 19F1 was used at 10 μg/mL to neutralize IL-10. Results are listed in the Table below.
Figure imgf000016_0001
The descriptions of the foregoing embodiments of the invention have been presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.
On December 20th 1989, Applicants deposited separate cultures of E. coli MC 1061 carrying pH5C, pH15C, and pBCRFI (SRα) with the American Type Culture Collection, Rockville, MD, USA (ATCC), under accession numbers 68191 , 68192, and 68193, respectively. These deposits were made under conditions as provided under ATCC's agreement for Culture Deposit for Patent Purposes, which assures that the deposit will be made available to the US Commissioner of Patents and Trademarks pursuant to 35 USC 122 and 37 CFR 1.14, and will be made available to the public upon issue of a U.S. patent, which requires that the deposit be maintained. Availability of the deposited strain is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The Deposits have been modified to satisfy the requirements of the
Budapest Convention. SEQUENCE LISTING
(2) INFORMATION FOR SEQ ID NO: 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE:
(D) OTHER INFORMATION: Human IL-10 (human cytokine synthesis inhibitory factor, human CSIF)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gl
5 10 1
Val Arg Ala Ser Pro Gly Gin Gly Thr Gin Ser Glu Asn Ser Cy
20 25 3
Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Ar 35 40 4
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gin Met Lys Asp Gi
50 55 6
Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Ly
65 70 7 Gly Tyr Leu Gly Cys Gin Ala Leu Ser Glu Met lie Gin Phe Ty
80 85 9 Leu Glu Glu Val Met Pro Gin Ala Glu Asn Gin Asp Pro Asp lie
95 100 105
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg
110 115 120
Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys
125 130 135
Ser Lys Ala Val Glu Gin Val Lys Asn Ala Phe Asn Lys Leu Gin
140 145 150
Glu Lys Gly lie Tyr Lys Ala Met Ser Glu Phe Asp lie Phe lie
155 160 165
Asn Tyr lie Glu Ala Tyr Met Thr Met Lys lie Arg Asn
170 175
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 170 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: B95-8 Epstein-Barr Virus
(ix) FEATURE:
(D) OTHER INFORMATION: Viral IL-10 (BCRF1 )
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Glu Arg Arg Leu Val Val Thr Leu Gin Cys Leu Val Leu Leu
5 10 15
Figure imgf000019_0001
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(D) OTHER INFORMATION: Mature human IL-10 (human cytokine synthesis inhibitory factor, human CSIF) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Pro Gly Gin Gly Thr Gin Ser Glu Asn Ser Cys Thr His Phe
5 10 15 Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe
20 25 30
Ser Arg Val Lys Thr Phe Phe Gin Met Lys Asp Gin Leu Asp Asn
35 40 45
Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu 50 55 60
Gly Cys Gin Ala Leu Ser Glu Met He Gin Phe Tyr Leu Glu Glu
65 70 75
Val Met Pro Gin Ala Glu Asn Gin Asp Pro Asp He Lys Ala His
80 85 90 Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu
95 100 105
Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala
110 115 120
Val Glu Gin Val Lys Asn Ala Phe Asn Lys Leu Gin Glu Lys Gly 125 130 135
He Tyr Lys Ala Met Ser Glu Phe Asp He Phe He Asn Tyr He
140 145 150
Glu Ala Tyr Met Thr Met Lys He Arg Asn
155 160
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 147 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE: (D) OTHER INFORMATION: Mature viral IL-10 (BCRF1 )
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Thr Asp Gin Cys Asp Asn Phe Pro Gin Met Leu Arg Asp Leu Arg
5 10 15
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gin Thr Lys Asp Glu
20 25 30
Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys 35 40 45
Gly Tyr Leu Gly Cys Gin Ala Leu Ser Glu Met He Gin Phe Tyr
50 55 60
Leu Glu Glu Val Met Pro Gin Ala Glu Asn Gin Asp Pro Glu Ala
65 70 75 Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg
80 85 90
Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys
95 100 105
Ser Lys Ala Val Glu Gin He Lys Asn Ala Phe Asn Lys Leu Gin 110 115 120
Glu Lys Gly He Tyr Lys Ala Met Ser Glu Phe Asp He Phe He
125 130 135
Asn Tyr He Glu Ala Tyr Met Thr He Lys Ala Arg
140 145
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE: (D) OTHER INFORMATION: As double-stranded fragment, encodes a consensus ribosome binding site.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GTAAGGAGGT TTAAC 15
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: T and GGTAC at positions 51 and 56- 60 differ from those of the native sequence; together with SEQ ID NO: 7, SEQ ID NO: 6 forms double-stranded Fragment 1A/B of synthetic CSIF gene with 4-base sticky end at positions 57-60.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
AGCCCAGGCC AGGGCACCCA GTCTGAGAAC AGCTGCACCC ACTTCCCAGG 50 TAACCGGTAC 60
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 56 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: C and A at positions 1 and 6 differ from those of the native sequence; together with SEQ ID NO: 6, SEQ ID NO: 7 forms double-stranded Fragment 1A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CGGTTACCTG GGAAGTGGGT GCAGCTGTTC TCAGACTGGG TGCCCTGGCC 50 TGGGCT 56
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: T position 2 differs from that of the native sequence; together with SEQ ID NO: 9, SEQ ID NO: 8 forms double- stranded Fragment 2A/B of synthetic CSIF gene with 5- and 9-base sticky ends at positions 1 -5 and 54-62.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GTAACCTGCC TAACATGCTT CGAGATCTCC GAGATGCCTT CAGCAGAGTG 50 AAGACTTTCT TT 62
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: Together with SEQ ID NO: 8, SEQ ID NO: 9 forms double-stranded Fragment 2A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CTTCACTCTG CTGAAGGCAT CTCGGAGATC TCGAAGCATG TTAGGCAG 48
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: C and T at positions 30 and 32 differ from those of the native sequence; together with SEQ ID NO: 11 , SEQ ID NO: 10 forms double-stranded Fragment 3A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CAAATGAAGG ATCAGCTGGA CAACTTGTTC TTAAG 35
(2) INFORMATION FOR SEQ ID NO: 11 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: A and G at positions 4 and 6 differ from those of the native sequence; together with SEQ ID NO: 10, SEQ ID NO:
11 forms double-stranded Fragment 3A/B of synthetic CSIF gene with 9- base sticky end at positions 36-44.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 :
CTTAAGAACA AGTTGTCCAG CTGATCCTTC ATTTGAAAGA AAGT 44 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: T at position 69 differs from that of the native sequence; together with SEQ ID NO: 13, SEQ ID NO: 12 forms double-stranded Fragment 4A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GAGTCCTTGC TGGAGGACTT TAAGGGTTAC CTGGGTTGCC AAGCCTTGTC 50 TGAGATGATC CAGTTTTAT 69
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ix) FEATURE:
(D) OTHER INFORMATION: T and A at positions 2 and 5 differ from those of the native sequence; together with SEQ ID NO: 12, SEQ ID NO: 13 forms double-stranded Fragment 4A/B of synthetic CSIF gene with 4- base sticky end at positions 1 -4.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CTAGATAAAA CTGGATCATC TCAGACAAGG CTTGGCAACC CAGGTAACCC 50 TTAAAGTCCT CCAGCAAGGA CTC 73
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: A, T and G at positions 3, 57 and 61 differ from those of the native sequence; together with SEQ ID NO: 15, SEQ ID NO: 14 forms double-stranded Fragment 5A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CTAGAGGAGG TGATGCCCCA AGCTGAGAAC CAAGACCCAG ACATCAAGGC 50 GCATGTTAAC G 61 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: TCGAC, A and T at positions 1 -5, 9 and 63 differ from those of the native sequence; together with SEQ ID NO: 14, SEQ ID NO: 15 forms double-stranded Fragment 5A/B of synthetic CSIF gene with 4-base sticky end at positions 1-4.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
TCGACGTTAA CATGCGCCTT GATGTCTGGG TCTTGGTTCT CAGCTTGGGG 50 CATCACCTCC TCTAG 65
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ix) FEATURE:
(D) OTHER INFORMATION: CTGCA at positions 58-63 differ from those of the native sequence; together with SEQ ID NO: 17, SEQ ID NO: 16 forms double-stranded Fragment 6A/B of synthetic CSIF gene with 4- base sticky end at positions 59-63.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
AACTCCCTGG GGGAGAACCT GAAGACCCTC AGGCTGAGGC TACGGCGCTG 5 TCATCGATCT GCA 6
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: G at position 1 differs from that of the native sequence; together with SEQ ID NO: 16, SEQ ID NO: 17 forms double-stranded Fragment 6A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GATCGATGAC AGCGCCGTAG CCTCAGCCTG AGGGTCTTCA GGTTCTCCCC 50 CAGGGAGTT 59
(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: C, G and GCATG at positions 51 , 54 and 56-60 differ from those of the native sequence; together with SEQ ID NO: 19, SEQ ID NO: 18 forms double-stranded Fragment 7A/B of synthetic CSIF gene with 2- and 4-base sticky ends at positions 1-2 and 57-60.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CGATTTCTTC CCTGTCAAAA CAAGAGCAAG GCCGTGGAGC AGGTGAAGAA 50 CGCGTGCATG 60
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE: (D) OTHER INFORMATION: C, C and G at positions 1 , 3 and 6 differ from those of the native sequence; together with SEQ ID NO: 18, SEQ ID NO: 19 forms double-stranded Fragment 7A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
CACGCGTTCT TCACCTGCTC CACGGCCTTG CTCTTGTTTT GACAGGGAAG 5 AAAT 5
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: Together with SEQ ID NO: 21 , SEQ ID NO: 20 forms double-stranded Fragment 8A/B of synthetic CSIF gene with
4- and 9-base sticky ends at positions 1 -4 and 50-58.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CGCGTTTAAT AATAAGCTCC AAGACAAAGG CATCTACAAA GCCATGAGTG 5 AGTTTGAC 58
(2) INFORMATION FOR SEQ ID NO: 21 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: Together with SEQ ID NO: 20, SEQ ID NO: 21 forms double-stranded Fragment 8A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 :
ACTCATGGCT TTGTAGATGC CTTTGTCTTG GAGCTTATTA TTAAA 45
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: Together with SEQ ID NO: 23, SEQ ID NO: 22 forms double-stranded Fragment 9A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ATCTTCATCA ACTACATAGA AGCCTACATG ACAATGAAGA TACGAAACTG A
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: AGCT at positions 1 -4 differ from those of the native sequence; together with SEQ ID NO: 22, SEQ ID NO: 23 forms double-stranded Fragment 9A/B of synthetic CSIF gene with 4- and 9-base sticky ends at positions 1-4 and 56-64.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
AGCTTCAGTT TCGTATCTTC ATTGTCATGT AGGCTTCTAT GTAGTTGATG AAGATGTCAA ACTC
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: Encodes viral IL-10.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
AATTC ATG GAG CGA AGG TTA GTG GTC ACT CTG CAG TGC CTG GTG 44
CTG CTT TAC CTG GCA CCT GAG TGT GGA GGT ACA GAC CAA TGT 86 GAC AAT TTT CCC CAA ATG TTG AGG GAC CTA AGA GAT GCC TTC 128
AGT CGT GTT AAA ACC TTT TTC CAG ACA AAG GAC GAG GTA GAT 170
AAC CTT TTG CTC AAG GAG TCT CTG CTA GAG GAC TTT AAG GGC 212
TAC CTT GGA TGC CAG GCC CTG TCA GAA ATG ATC CAA TTC TAC 254
CTG GAG GAA GTC ATG CCA CAG GCT GAA AAC CAG GAC CCG GAG 296 GCT AAG GAC CAT GTC AAT TCT TTG GGT GAA AAT CTA AAG ACC 338
CTA CGG CTC CGC CTG CGC AGG TGC CAC AGG TTC CTG CCG TGT 380
GAG AAC AAG AGT AAA GCT GTG GAA CAG ATA AAA AAT GCC TTT 422
AAC AAG CTG CAG GAA AAA GGA ATT TAC AAA GCC ATG AGT GAA 464
TTT GAC ATT TTT ATT AAC TAC ATA GAA GCA TAC ATG ACA ATT 506 AAA GCC AGG TGA G 519

Claims

WE CLAIM:
1. A method of ameliorating an inflammatory reaction in a patient, the method comprising the step of administering an effective amount of interleukin-10 to the patient.
2. The method of claim 1 wherein said interleukin-10 is selected from the group consisting of viral interleukin-10 and human interleukin-10.
3. The method of claim 1 wherein the inflammatory condition is septic shock or rheumatoid arthritis.
4. The method of claim 1 wherein the IL-10 is formulated in purified form substantially free of aggregates and other proteins at a concentration in the range of 5 to 20 μg/ml.
5. The method of claim 1 wherein the IL-10 is administered by continuous infusion so that an amount in the range of 1-16 μg/kg is delivered per day.
PCT/US1993/002173 1992-03-20 1993-03-16 Use of interleukin-10 to induce the production of interleukin-1 receptor antagonist WO1993018783A1 (en)

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US07/854,553 1992-03-20

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017773A3 (en) * 1993-02-01 1994-12-22 Michel Goldman Use of a pharmaceutical composition comprising an effective amount of interleukin-10, an analog and/or an agonist of interleukin-10
WO1998010792A1 (en) * 1996-09-11 1998-03-19 Prendergast Patrick T Immune direction therapy
US6159460A (en) * 1988-05-27 2000-12-12 Amgen Inc. Method for treating interleukin-1 mediated diseases
WO2001008696A3 (en) * 1999-07-28 2001-08-30 Schering Corp Combined use of interleukin 10 and methotrexate for immuno-modulatory therapy
WO2002066069A1 (en) * 2001-02-20 2002-08-29 Kansai Technology Licensing Organization Co., Ltd. Remedies for inflammatory/tumor diseases
US6544504B1 (en) 1999-07-28 2003-04-08 Schering Corporation Combined use of interleukin 10 and methotrexate for immuno-modulatory therapy
US6599873B1 (en) 1988-05-27 2003-07-29 Amgen Inc. Interleukin-1 inhibitors, compositions, and methods of treatment
US6733753B2 (en) 1997-02-10 2004-05-11 Amgen Inc. Composition and method for treating inflammatory diseases
US6858409B1 (en) 1988-05-27 2005-02-22 Amgen Inc. Nucleic acids encoding interleukin-1 inhibitors and processes for preparing interleukin-1 inhibitors

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000349A1 (en) * 1989-06-28 1991-01-10 Schering Corporation Cytokine synthesis inhibitory factor, antagonists thereof, and methods of using same
WO1991009127A1 (en) * 1989-12-20 1991-06-27 Schering Corporation BCRF1 PROTEINS AS INHIBITORS OF INTERFERON-$g(g)
WO1993002693A2 (en) * 1991-08-06 1993-02-18 Schering Corporation Use of interleukin-10 analogs or antagonists to treat endotoxin- or superantigen induced toxicity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000349A1 (en) * 1989-06-28 1991-01-10 Schering Corporation Cytokine synthesis inhibitory factor, antagonists thereof, and methods of using same
WO1991009127A1 (en) * 1989-12-20 1991-06-27 Schering Corporation BCRF1 PROTEINS AS INHIBITORS OF INTERFERON-$g(g)
WO1993002693A2 (en) * 1991-08-06 1993-02-18 Schering Corporation Use of interleukin-10 analogs or antagonists to treat endotoxin- or superantigen induced toxicity

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CURRENT OPINION IN IMMUNOLOGY vol. 4, June 1992, pages 314 - 320 R. DE WAAAL MALEFYT ET AL 'INTERLEUKIN-10' *
JOURNAL OF CLINICAL IMMUNOLOGY vol. 12, no. 4, July 1992, pages 239 - 247 M. HOWARD ET AL 'Biological properties of Interleukin 10' *
JOURNAL OF EXPERIMENTAL MEDICINE vol. 177, February 1993, pages 547 - 550 C. GERARD ET AL 'Interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia' *
JOURNAL OF EXPWERIMENTAL MEDICINE vol. 174, November 1991, pages 1209 - 1220 R. DE WAAL MALEFYT ET AL 'Interleukin-10 inhibits cytokine synthesis by human monocytes : An autoregulatory role of IL-10 produced by monocytes' *
JOURNAL OF IMMUNOLOGY. vol. 148, no. 3, 1 February 1992, BALTIMORE US pages 808 - 814 PETER RALPH ET AL 'IL-10, T lymphocyte inhibitor of human blood cell production of IL-1 and tumor necrosis factor' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 88, February 1991, WASHINGTON US pages 1172 - 1176 P. VIEIRA ET AL 'Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones : homology to Epstein-Barr virus open reading frame BCRFI' cited in the application *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6159460A (en) * 1988-05-27 2000-12-12 Amgen Inc. Method for treating interleukin-1 mediated diseases
US6599873B1 (en) 1988-05-27 2003-07-29 Amgen Inc. Interleukin-1 inhibitors, compositions, and methods of treatment
US6858409B1 (en) 1988-05-27 2005-02-22 Amgen Inc. Nucleic acids encoding interleukin-1 inhibitors and processes for preparing interleukin-1 inhibitors
WO1994017773A3 (en) * 1993-02-01 1994-12-22 Michel Goldman Use of a pharmaceutical composition comprising an effective amount of interleukin-10, an analog and/or an agonist of interleukin-10
WO1998010792A1 (en) * 1996-09-11 1998-03-19 Prendergast Patrick T Immune direction therapy
US6733753B2 (en) 1997-02-10 2004-05-11 Amgen Inc. Composition and method for treating inflammatory diseases
WO2001008696A3 (en) * 1999-07-28 2001-08-30 Schering Corp Combined use of interleukin 10 and methotrexate for immuno-modulatory therapy
US6544504B1 (en) 1999-07-28 2003-04-08 Schering Corporation Combined use of interleukin 10 and methotrexate for immuno-modulatory therapy
WO2002066069A1 (en) * 2001-02-20 2002-08-29 Kansai Technology Licensing Organization Co., Ltd. Remedies for inflammatory/tumor diseases

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IL105101A0 (en) 1993-07-08
CN1081378A (en) 1994-02-02
ZA931942B (en) 1993-09-29

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