WO1991017184A1 - Inhibiteurs ameliores d'interleukine-1 - Google Patents

Inhibiteurs ameliores d'interleukine-1 Download PDF

Info

Publication number
WO1991017184A1
WO1991017184A1 PCT/US1991/002127 US9102127W WO9117184A1 WO 1991017184 A1 WO1991017184 A1 WO 1991017184A1 US 9102127 W US9102127 W US 9102127W WO 9117184 A1 WO9117184 A1 WO 9117184A1
Authority
WO
WIPO (PCT)
Prior art keywords
irap
amino acid
mirap
inhibitor
protein
Prior art date
Application number
PCT/US1991/002127
Other languages
English (en)
Inventor
Donald B. Carter
Original Assignee
The Upjohn Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Upjohn Company filed Critical The Upjohn Company
Publication of WO1991017184A1 publication Critical patent/WO1991017184A1/fr

Links

Classifications

    • 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]
    • C07K14/545—IL-1
    • 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]
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the present invention relates to novel compounds which inhibit the activity oflnterleukin-1 (IL-1).
  • Interleukin-1 is a polypeptide that is produced after infection, injury, or antigenic challenge.
  • the macrophage is a primary source of IL-1; epidermal, epithelial, lymphoid, and vascular tissues also synthesize IL-1.
  • IL-1 When IL-1 circulates, it acts like a hormone and induces a broad spectrum of systemic changes in neurological, metabolic, hematologic, and endochronologic systems. Some of the IL-1 that is synthesized also remains associated with the plasma membrane and induces changes in local tissue without producing systemic responses.
  • IL-1 affects mesenchymal tissue remodeling where it contributes to both destructive and repair processes.
  • IL-1 activates lymphocytes and plays an important role in the initiation of the immune response. A few IL-1 receptors have been identified, but their affinities often do not match the potency of the biological response. The most consistent property of IL-1 is up-regulation of cellular metabolism and increased expression of several genes that encode biologically active molecules.
  • IL-1 is a highly inflammatory molecule that stimulates the production of arachidonic acid metabolites. IL-1 also acts synergistically with other cytokines, particularly tumor necrosis factor. The multitude of biological responses to IL-1 is an example of the rapid, adaptive changes that take place to increase the host defensive mechanisms. IL-1 mediates several components of the acute phase response to infection and injury. The most dramatic biological property is its ability to increase arachidonate metabolites in a variety of cells including PGE in the brain,
  • IL-1 reduces lipoxygenase products in lymphocytes and other cells.
  • IL-1 has been cloned and there are two forms of the molecule.
  • Precursor IL-1 is cleaved into a 17.5 kdal peptide, which is the dominant
  • IL-1 induces prostaglandins and lymphocyte activation as well as many other biological activities. These include PGE production, protease release from synovial cells and chrondrocytes, bone resorption, acute phase protein synthesis, and other effects.
  • IL-1 ⁇ contains an active region which is responsible for the immunostimulatory, immuno-restorative and antitumoral properties of IL-1.
  • This region corresponds to residues 163-171 of the IL-1 ⁇ molecule and although it is believed to be an active site, it is not associated with any of the inflammatory or pyrogenic properties of IL-1 ⁇ . It is believed that an active region other than the 163-171 sequence is responsible for the role IL-1 plays in the inflammation process.
  • the identification of the 163-171 sequence as an active region was first reported by scientists working at the Sclavo Research Center in Siena, Italy. Accordingly, the nine residue sequence is often referred to as the "Sclavo" region.
  • Sclavo region As used herein, the terms “Sclavo region”, “Sclavo fragment” and “Sclavo peptide” are meant to more broadly define peptides including the 163-171 peptide and those modeled after the nine amino acid sequences of IL-1 ⁇ .
  • Interleukin-1 activity is suppressed when IL-1 inhibitors are present.
  • IL-1 inhibitors exist and are useful tools in understanding the mechanisms which regulate the action of IL-1. Since the cytokine Interleukin-1 is one of the major mediators of tissue destruction and chronic inflammation, IL-1 inhibition can be used in treating inflammatory diseases. IL-1 inhibitors can also be used as immuno- suppressant molecules since IL-1 plays a central role in the immune response by stimulating proliferation and differentiation of T and B lymphocytes.
  • the present invention provides an improved Interleukin-1 inhibitor.
  • a gene for a naturally occurring IL-1 inhibitor has been isolated and sequenced and the DNA sequence encoding the "Sclavo" region has been inserted into the IL-1 inhibitor gene.
  • the resulting modified IL-1 inhibitor has a greater affinity for the IL-1 receptor and the desirable activities of IL-1 ⁇ derived from the Sclavo region active site.
  • Nencioni, L. et al., J. Immunol. Vol. 139, 3:800-804 (1987) disclose that the nine residue fragment derived from human IL-1 ⁇ residues 163-171 enhances antibody responses to both T helper-dependent and T helper-independent antigens. It was shown that the nine residue fragment enhances T helper-dependent antibody response as evaluated in the hemolytic plaque assay of spleen cells from mice immunized with sheep red blood cells. It was also shown that the nine residue fragment was able to enhance the in vivo immune response to a T helper- independent antigen, the poorly immunogenic polysaccharidic antigen from
  • Peppoloni S. et al., Nat. Immuno. Cell Growth Regul. 8:10-19 (1989) disclose that the nine residue 163-171 peptide increases the natural killer activity against K562 leukemia cells while the presence of human Interleukin-1 ⁇ does not confer a similar increase in natural killer activity.
  • the increase in tumor cell lysis could not be ascribed to the cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines.
  • the peptide enhanced a natural killer cytotoxicity of peripheral blood mononuclear cells, highly purified large granular lymphocytes were not susceptible to its inhibitory effect.
  • the addition to the cultures of antibodies to human Interleukin-2 completely blocked the 163-171 peptide-induced boost of natural killer cytotoxicity.
  • Liao et al. J. Exp. Med. 159:126-136 (1984) teach the identification of an IL-1 inhibitor found in urine from febrile patients.
  • the IL-1 inhibitor disclosed by Liao et al. is between 20-40 kdal.
  • Liao et al. note that the evidence suggests that the molecule is a protein or a glycoprotein but that the evidence is insufficient to support such a statement without additional information.
  • Scala et al. J. Exp. Med. 159:1637-1652 (1984) disclose a soluble factor derived from the ROHA-9 cell line (a human Epstein-Barr virus-transformed lymphoblastoid cell line) which resembles monocyte-derived human EL-1 inhibitor. Additionally, Scala et al. disclose the presence of a co-existent inhibitory factor. This inhibitory factor has a molecular weight of 95 kdal.
  • Arend et al., J. Immunol. 134:6 (1985) teach an inhibitor of IL-1 interaction with chondrocytes or thymocytes which is produced by human monocytes cultured on adherent human complexes or antibodies. The molecular weight of this factor is approximately 22 kdal.
  • IL-1 inhibitors include those having molecular weights of 30-35 kdal, 85 kdal, and 18-25 kdal.
  • the IL inhibitor of molecular weight 18-25 kdal is an immuno-suppressant glycoprotein isolated from urine of pregnant women.
  • Other IL-1 inhibitors disclosed in Seckinger and Dayer are the 22 kdal molecule reported by Arend, et al. which is derived from human monocytes stimulated by adherent immune complexes. A 95 kdal molecule derived from human monocytes stimulated by cytomegalovirus is also disclosed.
  • an IL-1 inhibitor with a molecular weight of about 95 kdal derived from human macrophages exposed to influenza and syncytial virus and from human virus-infected B cells is reported.
  • the cDNA disclosed is cloned to E. coli where it is expressed to yield Interleukin-1 inhibitor.
  • the present invention provides an improved IL-1 inhibitor.
  • an Interleukin-1 Receptor Antagonist Protein which functions as an IL-1 inhibitor is modified by the addition of a "Sclavo peptide".
  • the present invention relates to a DNA molecule which encodes a biologically active Interleukin-1 inhibitor containing a Sclavo region. Furthermore, the present invention relates to such a DNA molecule being incorporated in an expression vector and to a suitable host transformed with an expression vector that contains the DNA molecule encoding IL-1 inhibitor.
  • the present invention relates to a method of producing an improved biologically active IL-1 inhibitor by transforming a suitable host with an expression vector which contains a DNA molecule encoding an improved IL-1 inhibitor and culturing the transformed host cells in conditions promoting expression.
  • the present invention comprises a polypeptide which contains the amino acid sequence of IRAP augmented with the addition of a "Sclavo peptide".
  • the amino acid sequence for naturally occurring IRAP derived from U937 cells is extremely similar to the amino acid sequence for Interleukin-1 ⁇ (IL-1 ⁇ ). This similarity is demonstrated by the presence of identical residues or conservative substitutions of residues at corresponding regions of the two molecules throughout the two sequences. Accordingly, it is thought that the similarities of the sequences are responsible for IRAP's ability to bind to the IL-1 ⁇ receptor but the differences between the two sequences are responsible for the fact that IL-1 ⁇ is an agonist while IRAP is an antagonist.
  • Chart 1 shows a comparison between the IL-1 ⁇ amino acid sequence and the IRAP amino acid sequence. Asterisks (*) beneath the sequence represent identical residues. Periods (.) beneath the sequence represent residues where a conservative substitution has occurred.
  • IL-1 ⁇ differs from IRAP in that the IRAP molecule is missing residues corresponding to residues 163-171 of IL-1 ⁇ . This region of the IL-1 ⁇ molecule is believed to be responsible for the immmunostimulatory,
  • IL-1 ⁇ immunorestorative, antitumoral and radio-protective properties of IL-1 ⁇ . It is also believed that this region is not responsible for any of the inflammatory-related, pyrogenic or toxic effect characteristics of the naturally occurring agonist. Thus, the 163-171 region is thought to be an active region of IL-1 ⁇ but not one responsible for the molecule's role in the inflammation process.
  • Chart 1 shows a comparison between the IL-1 ⁇ amino acid sequence and the IRAP amino acid sequence.
  • Chart 2 shows the nucleotide and amino acid sequence of IRAP and Chart 3 shows the amino acid sequence of IRAP.
  • the present invention uses the following formulae as a basis for constructing molecules:
  • an improved IRAP is provided by inserting a peptide sequence modeled after amino acid residues 163-171 of IL-1 ⁇ into the corresponding region of the IRAP molecule.
  • the region of the IRAP molecule where the Sclavo peptide is inserted is at the position occupied by amino acid residue 76-79 as shown above and in Chart 3.
  • IRAP residues 76-80 correspond to the 163-171 region of IL-1 ⁇ (Scalvo 1-9). Substitutions, deletions and insertions of amino acids may be made throughout the molecule following Dayhoff s rule
  • substitutions may be made within the Sclavo peptide and, in particular, residue 3 may be substituted with any amino acid residue.
  • proline may not be substituted for Sclavo peptide amino acid residues 6, 7 or 8.
  • tenth and eleventh residues may be added to the Sclavo peptide.
  • any of IRAP amino acid residues 76-85 may be deleted.
  • IRAP amino acid residues 76-79 may be deleted and the Sclavo peptide inserted.
  • IRAP amino acid residue 80 may be substituted by a conservative replacement.
  • Sclavo amino acid 3 may be substituted with any amino acid residue.
  • Three improved IRAP molecules are disclosed: MIRAP-1; MIRAP-2; and, MIRAP-3.
  • MIRAP-1 represents the preferred embodiment.
  • amino acid sequences can be written which assign the designation "IRAP residue 76" or “76” and "IRAP residue 85” or “85” although more than 9 residues may lie between the two marked residues. Rather, 76 and 85 serve as reference points to the natural IRAP sequence which is shown in Chart 3.
  • improved IRAP molecules may be depicted as short sequences starting with a residue labelled 76 and ending with a residue labelled 85. According to this representation, residues 26- 75 are present in the molecule adjacent to the residue designated 76. Residues 86- 177 of natural IRAP are present in the molecule adjacent to the residue designated 85.
  • residues 26-75 of IRAP precede the residue labelled 76 and residues 86-177 of IRAP follow the residue labelled 85.
  • This improved IRAP possesses an enhanced ability to bind to the IL-1 receptor.
  • the desirable traits of IL-1 ⁇ which are derived from the active site at the Sclavo region are combined with IRAP's ability to block IL-1 binding to the IL-1 receptor.
  • an improved IRAP is produced.
  • the improved IRAP has IL-1 blocking ability and immunostimulatory activity.
  • Sclavo peptide or “Sclavo region” include those peptides which comprise conservative substitutions of the 163-171 peptide of IL-1 ⁇ .
  • the 163-171 peptide of IL-1 ⁇ consists of the amino acid sequence VQGEESNDK. Each amino acid residue may be substituted with a conservative equivalent except proline may not be used in Sclavo positions 7, 8 or 9 and the third residue, G, may be substituted with any amino acid.
  • the gene for IRAP is isolated and the cloned into an appropriate vector so that genetic manipulations may be performed.
  • a series of oligonucleotides are constructed which encode peptides modeled after the IL-1 ⁇ 163-171 peptide. These oligonucleotides were inserted into the portion of the IRAP gene which encodes residues 76-80 of IRAP.
  • the gene encoding IRAP was isolated by screening a cDNA library made from the RNA of U937 cells.
  • the cDNA library was screened with oligonucleotide probes which hybridize to nucleotide sequences that can encode an amino acid sequence known to exist within the IRAP protein.
  • the known amino acid sequence upon which the probes were modeled was discovered by sequencing a portion of the IRAP protein isolated from the cultured cells.
  • the nucleotide sequence which encodes IRAP including the sequence that encodes the 25 amino acid signal sequence is shown in Chart 2 and the amino acid sequence of the IRAP protein including the 25 amino acid signal sequence is shown in Charts 2 and 3.
  • the protein is 177 amino acids. Residues 1-25, the signal sequence, are cleaved off and the mature protein is produced.
  • the gene may be used to produce IRAP using techniques well known to those having ordinary skill in the art.
  • the gene may be inserted into an expression vector and the expression vector may be used to transform a suitable host.
  • the transformed host may be cultured in conditions which promote expression of the IRAP gene and, thus, production of the IRAP protein.
  • Improved IL-1 inhibitors contain peptides modeled after the 163-171 peptide.
  • the IL-1 ⁇ -derived Sclavo sequences is VQGEESNDK.
  • Each of the residues may be replaced with a conservative substitution except proline may not be used in Sclavo positions 7, 8 or 9.
  • the amino acid at position 3, G may be replaced by any amino acid.
  • additional amino acid residues may be added except proline.
  • the nucleotide sequence encoding the peptide sequence is synthesized as an
  • oligonucleotide This oligonucleotide is inserted into the gene encoding IRAP. The recombinant gene is then placed in an expression vector and the improved IRAP will be produced.
  • the established cell line U937 (ATCC) cells was grown in RPMI 1640 medium containing 10% fetal calf serum (FCS; Sterile Systems, Logan, UT), 100 ⁇ g/ml streptomycin (GIBCO, Grand Island, NY), and 100 units/ml penicillin
  • the cell line U937 was originally described by Sundstrom and Nilsson, Int. J. Cancer, 17:565-577 (1976). Cells were harvested and washed in phosphate buffered saline (PBS) and reseeded in the same medium containing 100 nM PMA at 2 x 10 5 cells/cm 2 . The cells were kept in this medium at 37°C for forty-eight hours, at which point most of the cells adhere to the plate surface. The medium was removed, the cells were washed with PBS and refed with RPMI 1640 containing 1% FCS and 100 units/ml GM-CSF (Amgen, Thousand Oaks, CA). The cells were incubated in this medium for 24-120 hr and then harvested. Cell debris was removed by high speed centrifugation. Supematants thus obtained were assayed for inhibiting activity.
  • PBS phosphate buffered saline
  • IL-4 human Interleukin-4
  • RPMI 1640 medium containing 1% Hyclone serum, Penn/Strep and glutamine
  • Measurement of IRAP messenger RNA and assay for the IRAP protein indicated that the kinetics of induction were roughly twice as fast as that seen with GM-CSF. That is, the 1/2 maximal levels of both protein and RNA were reached in less than 10 hours as compared to 24 hours being the norm for GM-CSF. At 24 hours, protein secreted in the supernatant after IL-4 treatment was nearly double that measured in GM-CSF supematants at 48 hours.
  • the assay used to determine the presence of an IL-1 inhibitor has been described in Tracey et al., Immunopharmacology, 15:47-62 (1988).
  • This assay the 1A5/HT2-T-Cell IL-1 Antagonist Assay, confirms the presence of a product capable of inhibiting IL-1 activity.
  • supematants recovered from harvesting the treated cells were added with IL-1 and phytohemaglutin (PHA) to a murine T-cell line which produces IL-2 when exposed to IL-1 and PHA.
  • PHA phytohemaglutin
  • the production of IL-2 can be determined by adding the medium recovered from the treated murine T-cells to an IL-2 dependent murine T-cell line. If IL-2 has been produced by the stimulated cells, it will be present and available for the IL-2 dependent cells to use. If the production of IL-2 has been blocked by the presence of the IL-1 inhibitor, no IL-2 will be present and the IL-2 dependent cells will die.
  • radiolabelled thymidine is added to medium. If the IL-2 dependent cells are alive and growing, the radio- nucleotide will be incorporated into the cells. If the cells are dead, all the radiolabel will be washed away when the medium is removed. Thus, when the cells are harvested and placed in a scintillation counter, the presence of the radiolabel can be detected. Accordingly, the presence of IL-2 can be established and, therefore, the presence of IRAP can be determined.
  • Murine T cell line LBRM-33-1A5 (ATCC) cells were maintained at 37°C in 5% CO 2 atmosphere in RPMI 1640 with 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 10% FCS.
  • IL-2 dependent murine T cells HT-2 as described in Watson, J., J. Exp. Med., 150:1510-1519, (1979) were maintained in continuous culture in culture medium further supplemented with 10 mM HEPES buffer (GIBCO), 5 x 10 -5 M 2- mercaptoethanol (Eastman Kodak, Rochester, NY) and 15% supernatant from concavalin A-stimulated rat spleen cells.
  • This supernatant used as a source of IL-2, was generated by stimulation of Wistar-Sprague rat spleen cells (Charles River, Cambridge, MA) at 5 x 10 6 cells/ml in culture medium with 5 ⁇ g/ml concavalin A (Pharmacia, Piscataway, NJ).
  • IRAP Receptor Binding Assay
  • the whole cell IL-1 competition assay using YT-NC1 cells and [ 125 I]-labelled BL-1 ⁇ was used to follow purification of the U937 IL-1 inhibitor.
  • Receptor binding assays were conducted in 96-well microtiter plates and contained 1 million YT-NCl cells, 50 pM [ 125 I]-IL-1 ⁇ , and an aliquot of "inhibitor" sample in a final volume of 200 ⁇ l. The cells were incubated at ambient temperature for one hour and unbound radioactivity was removed by filtration. Non-specific binding was determined by including 50 nM unlabelled IL-1 in the assay, and was generally less than 10%. The presence of IRAP was detected by a decrease in the amount of labelled [ 125 I]-IL-1 bound to the cells.
  • the 1A5 and Receptor binding assay are both used throughout the
  • IRAP is then purified from samples found to contain inhibitor activity and characterized using various protein chemistry techniques.
  • the sample in medium is concentrated by ultrafiltration (Amicon, YM5 membrane, molecular mass cutoff 5000 daltons).
  • IRAP produced in the culture media of U-937 cells stimulated with PMA and GM-CSF, was present in a large volume of solution with a relatively high concentration of albumin as the principle contaminant. In order for the purification to be successful, it was necessary to remove gram quantities of albumin and other serum proteins from microgram quantities of the desired protein. As a first step, the supematants were concentrated by ultrafiltration. This step removed PMA and peptides having masses less than 10,000 daltons, which might influence the inhibitor bioassays.
  • Protein solutions were concentrated in ultrafiltration cells having 2 liter and 400 ml volume capacity (Amicon Corp., Danvers, Mass.) at 4°C by nitrogen pressure. Added to stabilize the inhibitor protein during processing and to reduce proteolysis which was a likely causative factor of reduced yields was 0.2 mM phenylmethanesul- fonylfluoride (PMSF).
  • PMSF phenylmethanesul- fonylfluoride
  • a portion of the concentrated material was analyzed to determine certain physical characteristics of IRAP.
  • Analytic techniques performed included analytic gel filtration chromatography, isoelectric focusing, native polyacrylamide gel electrophoresis profiles and mono Q fractionation.
  • the remainder of the material was purified using protein preparation techniques. These included preparative gel filtration using a Superose 12 FPLC prep column, two procedures using TSK Bio-Sil 125 HPLC columns, and a final purification step using a C4 reverse phase HPLC column. Between preparative procedures, the presence of IRAP was confirmed using 1A5 and Receptor Binding assays. SDS PAGE profiles were performed on essentially pure IRAP to determine physical properties.
  • the molecular mass of the inhibitor activity on Superose 12 FPLC columns was 25,000 daltons, with a range of error of 19,000 through 32,000 daltons.
  • peaks of activity were observed.
  • the standard used to measure the distance travelled by the protein was the ratio of distance travelled by the peak to the distance travelled by the dye front.
  • the first peak travelled 53% of the distance travelled by the dye.
  • the second peak travelled 61% of the distance travelled by the dye. Both peaks represent the inhibitor, either as monomer and dimer forms under native conditions, or as two populations of proteins having common molecular masses but differing in glycosylation.
  • IRAP was shown to bind to anion exchange matrices at pH values greater than 7.5.
  • optimal results were obtained using quaternary anion exchange columns, such as Mono Q or QAE Fast Flow matrices (Pharmacia) rather than by classical DEAE Cellulose supports. Elution from the former columns was achieved by utilization of a linear salt gradient with fractions collected and assayed. In the column gradient elution procedure, the starting buffer was 25 mM Tris HC1, pH 8.5, and 10% glycerol, while the termination buffer was the same buffer including 1 M NaCl. Samples were prepared for column chromatography by dialysis against the starting buffer for at least a three hour period at 4°C in dialysis bags having a 5,000 dalton exclusion limit. The typical column was 1.6 x 50 cm (dimensions) for QAE Fast Flow and 0.5 x 5 cm for the Mono Q column (Pharmacia-LKB).
  • Inhibitory activity was associated with a protein fraction which elutes in the gradient at a position corresponding to a 125-160 mM NaCl concentration.
  • Isoelectric Focusing Native isoelectric focusing was conducted in 6% polyacrylamide gels [37.5:1.0 acrylamide:bis- acrylamide ratio] in a narrow range pH gradients established with 2.0% (w/v) Bio-Lytes, pH 4-6, supplemented with 0.5% (w/v) Bio-Lytes, pH 3-10. Gels were polymerized in 0.5 cm diameter borosilicate glass tubes (Bio-Rad) using ammonium persulfate, and allowed to stand at room temperature for at least 1 day prior to use. Samples of IRAP were mixed with 1 volume of 50% glycerol containing 0.05% methyl red, applied to the high pH end of the gradient (top), and
  • the anodic and cathodic solutions were 0.1 M phosphoric acid and 0.1 M ethanolamine, respectively.
  • Fractions of 1 ml were collected at a flow rate of 1 ml/min. Fractions from each series of runs were pooled, and analyzed by the 1A5/HT2 assay (bioassay) and the receptor binding assay described above. Protein assays of the fractions were determined by a protein assay kit (A 595; Bio-Rad Laboratories). Appropriate fractions were collected individually concentrated by Centricon-10 units (Amicon). After analysis by SDS PAGE and bioassay, appropriate fraction concentrates were pooled and further concentrated to 1 ml by Speed Vac centrifugation (Savant).
  • the Superose 12 pool concentrate was injected in multiple cycles onto a TSK Bio-Sil 125.
  • This fractionation because of the precision used in peak selection, resulted in the removal of almost all of the albumin and over a 500-fold purification for this step alone.
  • This step was repeated a second time to remove residual albumin and low molecular weight contaminants. While resulting in a 3- fold enhancement of specific activity, it is conceivable that this second mn of the column could have been avoided, since the C4 reverse phase fractionation (next step) works effectively on all preparations of the inhibitor having specific activities of at least 100 U/mg.
  • C4 reverse phase HPLC columns were used to further resolve the inhibitor protein.
  • the resolution capabilities of this column were dependent primarily on the hydrophobic interactions of the protein with the C4 matrix, and the relative affinity of the protein for either the stationary phase (C4) or the mobile phase (0.1% TFA, pH 2, variable concentrations [v/v] of acetonitrile).
  • the columns used were 1 x 25 cm dimensions with starting solutions of 0.1% TFA and 0% acetonitrile. The gradient of acetonitrile is incremented from 28-32% over a 52 minute period. Other variations can also be used in these test runs. Under all conditions, the same elution position was achieved.
  • the protein was injected onto the column in the buffer it was stored in prior to the ran with no requirement for a pre-incubation or pre-equilibration step. Fractions were collected, taken to dryness with a SpeedVac (Savant) centrifugal evaporator, and redissolved in a minimal volume of tissue culture media for biological activity measurements.
  • SpeedVac SpeedVac
  • IRAP elutes at a position corresponding to an acetonitrile concentration of between 30-31% (v/v). The bioactivity was recovered following this operation.
  • the elution position for this protein is a physical characteristic which is highly reliable (5% maximal error in elution position).
  • One dimensional SDS PAGE was performed using 14% - 20%, preferably 18%, gels in a Protean II system or in a mini Protean II system (Bio-Rad), according to the method of Laemmli.
  • samples were diluted 1:1 with denaturation buffer (2% SDS, 12.5% glycerol, 0.125 M Tris HCl, pH 6.8, and 3% ⁇ - mercaptoethanol), and heated in a boiling water bath for 2 minutes.
  • Electrophoresis was conducted at constant power (5 watts/gel) for 1 hour (mini gels) or 6 hours (regular gels) at room temperature and terminated when the dye front (bromophenol blue) is 0.5 cm from the bottom.
  • the completed gels were fixed in 50% ethanol and 10% acetic acid, and stained with Coomassie Brilliant Blue G-250 or with a commercially available silver staining kit (Bio-Rad). Photos of gels were made using Type 665 film (Polaroid).
  • Two dimensional SDS PAGE was also done. Samples to be analyzed were desalted by microdialysis (Enprotech) against deionized water. Two dimensional electrophoresis was conducted using 1 mm diameter capillary-tube gels (7% polyacrylamide) in the first dimension (4-8 °C; 1200 V, constant voltage) for 10-12 hours. Ampholines having a pH range of 4-6 were used (Bio-Lytes, 2% w/v, Bio-Rad Laboratories). Focused sample gels were electrophoresed onto a second dimension of 18% polyacrylamide (9 cm x 7 cm) (0.1% SDS) according to the method of Laemmli.
  • the protein (both pi forms, 4.9 and 5.1) was subjected to incubation with N- glycanase to determine if it is glycosylated.
  • To 10 ⁇ l of inhibitor protein were added 10 ⁇ l of 1% (w/v) SDS. This solution was heated for 2 minutes in boiling water.
  • To the resulting solution was added NaP04 (0.14 M final), pH 8.0, and NP-40 (1.0%). As a result the NP-40 to SDS ratio was at least 7:1.
  • N-glycanase (excess) was added and incubation allowed to proceed for overnight at 37°C. Controls were ran using ovalbumin, RNAse B and a2-macroglobulin to determine the amount of N-Glycanase needed for complete conversion of a specific amount of total glycoprotein.
  • a protein band migrates with a mass of 22 kdal, indicating that at least 3 kdal of the mass of the apoprotein are carbohydrate.
  • N-Terminal Sequence-Automated Edman degradation chemistry was performed in an Applied Biosystems, Inc., Model 470A gas phase protein sequenator fitted with an on-line Model 120A PTH amino acid analyzer. Data were collected and yields calculated on a Nelson Analytical 3000 Series chromatography system. Results suggested that the N-terminus was blocked (a sequence APHG was observed, but at low yield; identification of protein retained on the disk was performed by amino acid composition). However, this conclusion is speculative as the N-terminus may have been artificially blocked as often observed in low protein isolation conditions.
  • Endoproteinase Lys C Digestion Three hundred ⁇ l of 10 mM Tris HCl, pH 8.7 were introduced to the above solution of S-pyridylethylated inhibitor protein, followed by the addition of 1 unit of reconstituted endoproteinase Lys C (EKC;
  • Endo Lys C generated peptide mixture was resolved on a C18 reverse phase HPLC column (4.5 X 150 mm) using a linear gradient from 0 to 36% acetonitrile in 0.1% TFA over a period of 52 minutes.
  • the column was monitored at 206 nm and peptides were collected manually during peak elution. Solvent was removed by vacuum centrifugation (Savant).
  • One of the peptides sequenced has the amino acid sequence KIDVVPIE where the letters correspond to the one-letter symbol code for amino acids.
  • Degenerate oligonucleotides which contained all possibilities represented by the amino acid sequence KDDWP were synthesized using an Applied Biosystems Synthesizer.
  • degenerate oligonucleotide pools were decoded by using Northern blot analysis to determine the homology of the degenerate oligonucleotide with the U937 mRNA.
  • the degenerate oligonucleotide pool giving the best hybridization signal to the U937 mRNA was shown to be DC-166:
  • the human DNA sequence contains about 1782 nucleotides including 5' and 3' nontranslated sequences. The longest open reading frame begins with the fifty third nucleotide and extends through the 584th nucleotide.
  • the protein derived by translation of the nucleotide sequence beginning with the first Met from Nucleotide 53 is set forth in Chart 1. This sequence is comprised of 177 amino acids (including 25 amino acids of a signal peptide). Thus, the mature IRAP is 152 amino acid residue shown in Chart 2 as 26-177.
  • the coding sequence of the IRAP gene for the mature protein is nucleotides 128-584.
  • the human sequence includes one N- glycosylation site at position 377-379 of the DNA sequence (Asn 109).
  • the ⁇ 1.6 kb EcoRI/Klenow/Spel fragment was isolated from pGEM-2/P5.
  • This fragment contains part of the coding sequence for IRAP, from nucleotide 215 to 584.
  • This fragment was inserted into one expression vector pTrp2-m4 (MKOlsen, SKRockenbach, KACurry and C-SCTomich, J. of Biotechnology 9:179-190, 1989) which was treated with Hind ⁇ l/Klenow/Clal, together with two oligonucleotides.
  • the oligonucleotides contain the coding sequence for IRAP from nucleotide 128-214, the translation initiation codon nTG and the ends of Clal and Spel. In the oligonucleotide sequence, codon usage is maximized and secondary structure is minimized in order to optimize translational efficiency.
  • the resulting vector is named pTrp2-ILinh which carries the IRAP sequence under the control of the E. coli trp promoter and an AT-rich ribosome binding site, in a pBR322 background (Chart 4). DNA sequencing was carried out to confirm the sequence of the region
  • E. coli K12S carrying pTrp2-ILinh was induced for expression under tryptophan starvation conditions.
  • Cell extracts exhibit inhibitor activity and SDS- PAGE analysis shows a faint band corresponding to the expected size, demonstrating low level of expression.
  • E. coli was transformed with pUC-ILinh. Extracts from E. coli K12S (pUC- ILinh) induced for expression by tryptophan starvation shows inhibitor activity.
  • E. coli K12S (pUC-DLinh) were propagated, induced for expression by tryptophan starvation and centrifiiged.
  • the E. coli pellets were resuspended in 10 mM Tris HCl, pH 8.0. The suspension was freeze thawed; first the pellets were frozen immediately in dry ice for 10-15 minutes after which the mixtures were thawed in warm water. The pellets were freeze thawed at least once, preferably more than four times. The supernatant was then collected by centrifugation (Sorvall SS-34, 8°C at 20,000 RPM).
  • This enriched material was further refined by HPLC on a TSK DEAE 5PW (0.75 X 75 mm) column using a linear NaCl gradient (10-125 mM) in 20 mM Tris HCl, pH 9.0, over a 25 minute period.
  • the pooled material was concentrated by Centricon 10.
  • Other minor contaminants were finally removed by gel filtration on a TSK Bio-Sil 125 HPLC column (0.75 X 600 mm) in 50 mM phosphate, 1 M NaCl, pH 6.8.
  • the purified recombinant protein was stored frozen at -20°C in 20 mM Tris HCl, pH 7.2. SDS PAGE with urea indicated the molecular weight of the purified recombinant IRAP protein to be about 17,000 daltons.
  • IRAP molecules In order to construct improved IRAP molecules according to the present invention, manipulations are performed on the IRAP gene to insert oligo nucleotides that encode a Sclavo peptide into the appropriate region of the IRAP gene. The resulting recombinant gene encodes the improved IRAP which is produced when the gene is expressed.
  • oligonucleotides To construct oligonucleotides, an Applied Biosystems 380 oligonucleotide synthesizer was used. The following oligonucleotides and their complementary strands were constructed:
  • Oligo-1 encodes the amino acid sequence VQWEESNDKALFLGI.
  • Oligo-2 encodes the amino acid sequence VQGEESNDKALFLGI.
  • Oligo-3 encodes the amino acid sequence IEPHESNDKIALFLGI.
  • the IRAP gene was isolated from pUC-ILinh by digestion with Clal and BamHl and inserted into pGEM2 digested with Clal and BamHl forming
  • pGEM2IRAP To insert the complimented oligonucleotides encoding Sclavo peptides, pGEM2IRAP was digested with Kpnl and Ncol, effectively deleting the coding sequence from amino acid residue 75 to 85. The oligonucleotides were inserted into the digested pGEM2IRAP by ordinary ligation, forming pGEM2MIRAP-1,
  • Plasmid pGEM2MIRAP-1 contains oligo-1 inserted into the KpnI/Ncol digested IRAP gene construct.
  • the MIRAP-1 gene encodes the natural IRAP amino acid sequence 26-75 followed by VQWEESNDKALFLGI followed by the natural IRAP amino acid sequence 86-177.
  • Plasmid pGEM2MIRAP-2 contains oligo-2 inserted into the KpnI/Ncol digested IRAP gene construct.
  • the MIRAP-2 gene encodes the natural IRAP amino acid sequence 26-75 followed by VQGEESNDKALFLGI followed by the natural
  • Plasmid pGEM2MIRAP-3 contains oligo-3 inserted into the KpnI/Ncol digested IRAP gene construct.
  • the MIRAP-3 gene encodes the natural IRAP amino acid sequence 26-75 followed by ffiPHESNDKIALFLGI followed by the natural
  • pGEM2MIRAP-1 pGEM2MIRAP-2
  • pGEM2MIRAP-3 pGEM2MIRAP-3
  • the ⁇ 1.6 kb EcoRI/KIenow/Spel fragment was isolated from each plasmid respectively.
  • the fragment contains the coding sequence for the improved IRAP, from residue 30 to the end.
  • This fragment was inserted into one expression vector pTrp2-m4 (MKOlsen, SKRockenbach, KACurry and C-SCTomich, J. of Biotechnology
  • oligonucleotides contain the coding sequence for IL-1 inhibitor from residue 26-29, the translation initiation codon ATG and the ends of Clal and
  • pTrp2-MIRAP-1 pTrp2-MIRAP-2
  • pTrp2-MIRAP-3 pTrp2-MIRAP-3
  • pTrp2-MIRAP carries the improved IRAP sequence under the control of the E. coli trp promoter and an AT-rich ribosome binding site, in pBR322 (Chart 6). DNA sequencing was carried out to confirm the sequence of the region manipulated.
  • E. coli K12S carrying pTrp2-MIRAP was induced for expression under tryptophan starvation conditions.
  • Cell extracts exhibit inhibitor activity and SDS-
  • the 1.2 kb EcoRI-BamHI fragment containing the trp promoter, ribosome binding site and the coding sequence for BL-1 inhibitor was isolated from pTrp2-MIRAP and cloned into the EcoRI and BamHl sites of pUC19.
  • pUC-MIRAP refers to pUC-
  • MIRAP-1 MIRAP-1, pUC-MIRAP-2 or pUC-MIRAP-3) .
  • E. coli was transformed with pUC-MIRAP. Extracts from E. coli K12S (pUC-MIRAP).
  • MIRAP MIRAP induced for expression by tryptophan starvation shows inhibitor activity. SDS-PAGE analysis of the extracts shows a prominent band at the expected size, indicating high level expression. This construction strategy was followed for each of the three MIRAP gene embodiments described above (MIRAP-1, MIRAP-2 and MIRAP-3).
  • the modified IRAP genes produced improved IRAP.
  • the bioactivity of the product may be determined by in vitro transcription and translation of the gene followed by testing of the product using the 1A5/HT2 Bioassay.
  • E. coli K12S containing one of pUC-MIRAP-1, pUC-MIRAP-2, or pUC-MIRAP- 3 are propagated, induced for expression by tryptophan starvation and centrifiiged.
  • the E. coli pellets are resuspended in 10 mM Tris HCl, pH 8.0.
  • the suspension is freeze thawed; first the pellets are frozen immediately in dry ice for 10-15 minutes after which the mixtures are thawed in warm water.
  • the pellets are freeze thawed at least once, preferably more than four times.
  • the supernatant is then collected by centrifugation (Sorvall SS-34, 8°C at 20,000 RPM). This clarified solution is loaded onto a FPLC system equipped with an equilibrated 1 X 10 cm (QAE fast flow;
  • the pooled material is concentrated by Centricon 10. Other minor contaminants are finally removed by gel filtration on a TSK Bio-Sil-125 HPLC column (0.75 X 600 mm) in 50 mM phosphate, 1 M NaCl, pH 6.8.
  • the purified recombinant protein is stored frozen at -20 C in 20 mM Tris HCl, pH 7.2. SDS PAGE with urea indicates the molecular weight of any of the purified recombinant MIRAP protein to be about 17,000 daltons.

Abstract

L'invention concerne des molécules d'ADN qui codent des inhibiteurs améliorés d'interleukine-1 biologiquement active, ainsi que des molécules d'ADN qui codent des inhibiteurs améliorés d'interleukine-1 incorporés dans des vecteurs d'expression. En outre, des cellules-hôtes transformées avec des vecteurs d'expression qui renferment des molécules d'ADN codant des inhibiteurs améliorés d'IL-1 et un procédé de production d'inhibiteurs améliorés d'IL-1 biologiquement active, sont également présentés. L'invention concerne également des inhibiteurs améliorés et essentiellement purs de IL-1 biologiquement active.
PCT/US1991/002127 1990-04-27 1991-04-03 Inhibiteurs ameliores d'interleukine-1 WO1991017184A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US51546890A 1990-04-27 1990-04-27
US515,468 1990-04-27

Publications (1)

Publication Number Publication Date
WO1991017184A1 true WO1991017184A1 (fr) 1991-11-14

Family

ID=24051476

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/002127 WO1991017184A1 (fr) 1990-04-27 1991-04-03 Inhibiteurs ameliores d'interleukine-1

Country Status (2)

Country Link
AU (1) AU7683391A (fr)
WO (1) WO1991017184A1 (fr)

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019185A1 (fr) * 1992-03-23 1993-09-30 Biocine Sclavo Spa Antigene de recombinaison contenant dans sa sequence un domaine immunostimulant heterologue- son utilisation comme vaccin
EP0690673A1 (fr) * 1993-12-14 1996-01-10 University Of Pittsburgh Of The Commonwealth System Of Higher Education Traitement systemique par genes des affections des tissus conjonctifs
AU671116B2 (en) * 1992-03-30 1996-08-15 Immunex Corporation Fusion proteins comprising tumor necrosis factor receptor
US5608035A (en) * 1994-02-02 1997-03-04 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
WO1997028828A1 (fr) 1996-02-09 1997-08-14 Amgen Boulder Inc. Composition comprenant un inhibiteur de l'interleukine 1 et un polymere a liberation controlee
US5739282A (en) * 1994-10-13 1998-04-14 Applied Research Systems Ars Holding N.V. Interleukin-1 antagonist
US5786331A (en) * 1994-02-02 1998-07-28 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5837495A (en) * 1994-10-13 1998-11-17 Applied Research Systems Ars Holding N.V. DNA encoding interleukin-1 antagonist
US5861476A (en) * 1994-02-02 1999-01-19 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5880096A (en) * 1994-02-02 1999-03-09 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5922573A (en) * 1994-09-21 1999-07-13 Dompe' S.P.A. IL-1 receptor antagonists with enhanced inhibitory activity
US5981713A (en) * 1994-10-13 1999-11-09 Applied Research Systems Ars Holding N.V. Antibodies to intereleukin-1 antagonists
US6013253A (en) * 1997-08-15 2000-01-11 Amgen, Inc. Treatment of multiple sclerosis using consensus interferon and IL-1 receptor antagonist
US6159460A (en) * 1988-05-27 2000-12-12 Amgen Inc. Method for treating interleukin-1 mediated diseases
WO2002062375A1 (fr) * 2001-02-06 2002-08-15 Merck Patent Gmbh Antagoniste de recepteur d'interleukine-1 modifie (il-1ra) presentant une antigenicite reduite
WO2003014703A2 (fr) * 2001-08-09 2003-02-20 Curagen Corporation Acides nucleiques, polypeptides, polymorphismes nucleotidiques simples et techniques d'utilisation de ceux-ci
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
WO2008054603A2 (fr) 2006-10-02 2008-05-08 Amgen Inc. Protéines de liaison à l'antigène du récepteur a de l'il-17
EP1944373A3 (fr) * 1999-11-24 2008-07-23 Danisco A/S Procédé de purification d'un antagoniste du récepteur de l'interleukine (IL-1ra)
EP1992636A2 (fr) 1999-11-12 2008-11-19 Amgen Inc. Procédé pour la correction d'un mauvais repliement de bisulfure dans les molécules Fc
EP2002846A2 (fr) 1996-12-06 2008-12-17 Amgen Inc. Thérapie combinée utilisant un inhibiteur IL-1 pour traiter les maladies liées au IL-1
EP2087908A1 (fr) 2001-06-26 2009-08-12 Amgen, Inc. Anticorps opgl
US20100120684A1 (en) * 2007-05-01 2010-05-13 Dahlen Eva Maria Mutants of interleukin- 1 receptor antagonist and uses thereof
EP2213685A1 (fr) 2002-09-06 2010-08-04 Amgen Inc. Anticorps monoclonal anti-IL-1R1 thérapeutique
EP2241328A1 (fr) 2000-05-12 2010-10-20 Immunex Corporation Inhibiteurs d'interleukine 1 dans le traitement de maladies
WO2011046958A1 (fr) 2009-10-12 2011-04-21 Amgen Inc. Utilisation des proteines se liant a un antigene du recepteur a de l'il-17
EP2366715A2 (fr) 2005-11-14 2011-09-21 Amgen Inc. Molécules chimère d'anticorps anti-RANKL et de PTH/PTHRP
US8063182B1 (en) 1989-09-12 2011-11-22 Hoffman-Laroche Inc. Human TNF receptor fusion protein
US8106098B2 (en) 1999-08-09 2012-01-31 The General Hospital Corporation Protein conjugates with a water-soluble biocompatible, biodegradable polymer
US8323635B2 (en) 2007-11-14 2012-12-04 General Regeneratives, Ltd. Methods of using interleukin-1 receptor antagonist as a myeloprotective agent
WO2013016220A1 (fr) 2011-07-22 2013-01-31 Amgen Inc. Récepteur a de il-il-17 requis pour biologie il-17c
WO2013170636A1 (fr) 2012-05-18 2013-11-21 爱德迪安（北京）生物技术有限公司 Protéine et conjugué protéique pour le traitement du diabète et applications associées
US8853150B2 (en) 2010-07-29 2014-10-07 Eleven Biotherapeutics, Inc. Chimeric IL-1 receptor type I antagonists
WO2015067791A1 (fr) 2013-11-11 2015-05-14 Ascendis Pharma Relaxin Division A/S Promédicaments à bae de relaxine
WO2015191783A2 (fr) 2014-06-10 2015-12-17 Abbvie Inc. Biomarqueurs des maladies inflammatoires et leurs procédés d'utilisation
US9339528B2 (en) 2009-10-26 2016-05-17 General Regeneratives, Ltd. Methods for treating epithelium trauma of the intestinal mucosa using interleukin-1 receptor antagonist
US10041044B2 (en) 2016-07-29 2018-08-07 Trustees Of Boston University Age-associated clonal hematopoiesis accelerates cardio-metabolic disease development
WO2020035482A1 (fr) 2018-08-13 2020-02-20 Iltoo Pharma Combinaison d'interleukine 2 et d'un inhibiteur de l'interleukine 1, conjugués et utilisations thérapeutiques de celle-ci
US10799589B2 (en) 2013-03-13 2020-10-13 Buzzard Pharmaceuticals AB Chimeric cytokine formulations for ocular delivery
WO2023222565A1 (fr) 2022-05-16 2023-11-23 Institut National de la Santé et de la Recherche Médicale Procédés d'évaluation de l'épuisement de cellules souches hématopoïétiques induites par une inflammation chronique

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2068320C (fr) * 1989-11-29 2000-10-17 Robert Hageman Production d'un inhibiteur recombinant de l'interleukine-1 humaine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0231728A2 (fr) * 1986-02-07 1987-08-12 SCLAVO S.p.A. Peptide synthétique à activité de l'interleucine 1 humaine
WO1989001946A1 (fr) * 1987-08-26 1989-03-09 Biogen, Inc. Substances biologiques et procedes de production et d'utilisation de ces substances biologiques en therapeutique
EP0343684A1 (fr) * 1988-05-27 1989-11-29 Synergen, Inc. Inhibiteurs d'interleukine-1

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0231728A2 (fr) * 1986-02-07 1987-08-12 SCLAVO S.p.A. Peptide synthétique à activité de l'interleucine 1 humaine
WO1989001946A1 (fr) * 1987-08-26 1989-03-09 Biogen, Inc. Substances biologiques et procedes de production et d'utilisation de ces substances biologiques en therapeutique
EP0343684A1 (fr) * 1988-05-27 1989-11-29 Synergen, Inc. Inhibiteurs d'interleukine-1

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
The Journal of Immunology, vol. 137, no. 10, 15 November 1986 G. Antoni et al.: "A short synthetic peptide fragment of human interleukin 1 with immunostimulatory but not inflammatory activity", pages 3201-3204 *
The Journal of Immunology, vol. 143, no. 1, 1 July 1989 D. Boraschi et al.: "A monoclonal antibody to the IL-1beta peptide 163-171 blocks adjuvanticity but not pyrogenicity of IL-1beta in vivo", pages 131-134 *

Cited By (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6858409B1 (en) 1988-05-27 2005-02-22 Amgen Inc. Nucleic acids encoding interleukin-1 inhibitors and processes for preparing interleukin-1 inhibitors
US6599873B1 (en) 1988-05-27 2003-07-29 Amgen Inc. Interleukin-1 inhibitors, compositions, and methods of treatment
US6159460A (en) * 1988-05-27 2000-12-12 Amgen Inc. Method for treating interleukin-1 mediated diseases
US8063182B1 (en) 1989-09-12 2011-11-22 Hoffman-Laroche Inc. Human TNF receptor fusion protein
US8163522B1 (en) 1989-09-12 2012-04-24 Hoffman-Laroche Inc. Human TNF receptor
WO1993019185A1 (fr) * 1992-03-23 1993-09-30 Biocine Sclavo Spa Antigene de recombinaison contenant dans sa sequence un domaine immunostimulant heterologue- son utilisation comme vaccin
AU671116B2 (en) * 1992-03-30 1996-08-15 Immunex Corporation Fusion proteins comprising tumor necrosis factor receptor
EP0690673A1 (fr) * 1993-12-14 1996-01-10 University Of Pittsburgh Of The Commonwealth System Of Higher Education Traitement systemique par genes des affections des tissus conjonctifs
EP0690673A4 (fr) * 1993-12-14 1996-05-29 Univ Pittsburgh Traitement systemique par genes des affections des tissus conjonctifs
US5861476A (en) * 1994-02-02 1999-01-19 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5880096A (en) * 1994-02-02 1999-03-09 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5786331A (en) * 1994-02-02 1998-07-28 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5767234A (en) * 1994-02-02 1998-06-16 Affymax Technologies, N.V. Peptides and compounds that bind to the IL-1 receptor
US5608035A (en) * 1994-02-02 1997-03-04 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5922573A (en) * 1994-09-21 1999-07-13 Dompe' S.P.A. IL-1 receptor antagonists with enhanced inhibitory activity
US5981713A (en) * 1994-10-13 1999-11-09 Applied Research Systems Ars Holding N.V. Antibodies to intereleukin-1 antagonists
US5837495A (en) * 1994-10-13 1998-11-17 Applied Research Systems Ars Holding N.V. DNA encoding interleukin-1 antagonist
US5739282A (en) * 1994-10-13 1998-04-14 Applied Research Systems Ars Holding N.V. Interleukin-1 antagonist
WO1997028828A1 (fr) 1996-02-09 1997-08-14 Amgen Boulder Inc. Composition comprenant un inhibiteur de l'interleukine 1 et un polymere a liberation controlee
EP2002846A2 (fr) 1996-12-06 2008-12-17 Amgen Inc. Thérapie combinée utilisant un inhibiteur IL-1 pour traiter les maladies liées au IL-1
US6733753B2 (en) 1997-02-10 2004-05-11 Amgen Inc. Composition and method for treating inflammatory diseases
US6013253A (en) * 1997-08-15 2000-01-11 Amgen, Inc. Treatment of multiple sclerosis using consensus interferon and IL-1 receptor antagonist
US8106098B2 (en) 1999-08-09 2012-01-31 The General Hospital Corporation Protein conjugates with a water-soluble biocompatible, biodegradable polymer
EP1992636A2 (fr) 1999-11-12 2008-11-19 Amgen Inc. Procédé pour la correction d'un mauvais repliement de bisulfure dans les molécules Fc
EP1944373A3 (fr) * 1999-11-24 2008-07-23 Danisco A/S Procédé de purification d'un antagoniste du récepteur de l'interleukine (IL-1ra)
EP2241328A1 (fr) 2000-05-12 2010-10-20 Immunex Corporation Inhibiteurs d'interleukine 1 dans le traitement de maladies
WO2002062375A1 (fr) * 2001-02-06 2002-08-15 Merck Patent Gmbh Antagoniste de recepteur d'interleukine-1 modifie (il-1ra) presentant une antigenicite reduite
EP3492100A1 (fr) 2001-06-26 2019-06-05 Amgen Inc. Anticorps pour opgl
EP2087908A1 (fr) 2001-06-26 2009-08-12 Amgen, Inc. Anticorps opgl
WO2003014703A3 (fr) * 2001-08-09 2005-05-19 Curagen Corp Acides nucleiques, polypeptides, polymorphismes nucleotidiques simples et techniques d'utilisation de ceux-ci
WO2003014703A2 (fr) * 2001-08-09 2003-02-20 Curagen Corporation Acides nucleiques, polypeptides, polymorphismes nucleotidiques simples et techniques d'utilisation de ceux-ci
EP2213685A1 (fr) 2002-09-06 2010-08-04 Amgen Inc. Anticorps monoclonal anti-IL-1R1 thérapeutique
EP2277543A1 (fr) 2002-09-06 2011-01-26 Amgen, Inc Anticorps monoclonal anti-IL-1R1 thérapeutique
EP3020414A1 (fr) 2002-09-06 2016-05-18 Amgen, Inc Anticorps monoclonal anti-il-1r1 thérapeutique
EP2366715A2 (fr) 2005-11-14 2011-09-21 Amgen Inc. Molécules chimère d'anticorps anti-RANKL et de PTH/PTHRP
EP2816060A1 (fr) 2005-11-14 2014-12-24 Amgen Inc. Molécules chimère d'anticorps PTH/PTHRP de rang 1
WO2008054603A2 (fr) 2006-10-02 2008-05-08 Amgen Inc. Protéines de liaison à l'antigène du récepteur a de l'il-17
EP3165539A1 (fr) 2006-10-02 2017-05-10 Kirin-Amgen, Inc. Protéines se liant à des antigènes a du récepteur il-17
US20100120684A1 (en) * 2007-05-01 2010-05-13 Dahlen Eva Maria Mutants of interleukin- 1 receptor antagonist and uses thereof
US8303945B2 (en) * 2007-05-01 2012-11-06 Alligator Bioscience Ab Mutants of interleukin-1 receptor antagonist
US9163072B2 (en) 2007-05-01 2015-10-20 Alligator Bioscience Ab Mutants of interleukin-1 receptor antagonist
US8323635B2 (en) 2007-11-14 2012-12-04 General Regeneratives, Ltd. Methods of using interleukin-1 receptor antagonist as a myeloprotective agent
WO2011046958A1 (fr) 2009-10-12 2011-04-21 Amgen Inc. Utilisation des proteines se liant a un antigene du recepteur a de l'il-17
US9339528B2 (en) 2009-10-26 2016-05-17 General Regeneratives, Ltd. Methods for treating epithelium trauma of the intestinal mucosa using interleukin-1 receptor antagonist
US9458216B2 (en) 2010-07-29 2016-10-04 Eleven Biotherapeutics, Inc. Nucleic acid encoding chimeric IL-1 receptor type I antagonists
US8853150B2 (en) 2010-07-29 2014-10-07 Eleven Biotherapeutics, Inc. Chimeric IL-1 receptor type I antagonists
WO2013016220A1 (fr) 2011-07-22 2013-01-31 Amgen Inc. Récepteur a de il-il-17 requis pour biologie il-17c
WO2013170636A1 (fr) 2012-05-18 2013-11-21 爱德迪安（北京）生物技术有限公司 Protéine et conjugué protéique pour le traitement du diabète et applications associées
US9745359B2 (en) 2012-05-18 2017-08-29 Adda Biotech Inc. Protein and protein conjugate for diabetes treatment, and applications thereof
US11208451B2 (en) 2012-05-18 2021-12-28 Adda Biotech Inc. Protein and protein conjugate for diabetes treatment, and applications thereof
US10472404B2 (en) 2012-05-18 2019-11-12 Adda Biotech Inc. Protein and protein conjugate for diabetes treatment, and applications thereof
US10799589B2 (en) 2013-03-13 2020-10-13 Buzzard Pharmaceuticals AB Chimeric cytokine formulations for ocular delivery
WO2015067791A1 (fr) 2013-11-11 2015-05-14 Ascendis Pharma Relaxin Division A/S Promédicaments à bae de relaxine
WO2015191783A2 (fr) 2014-06-10 2015-12-17 Abbvie Inc. Biomarqueurs des maladies inflammatoires et leurs procédés d'utilisation
US10041044B2 (en) 2016-07-29 2018-08-07 Trustees Of Boston University Age-associated clonal hematopoiesis accelerates cardio-metabolic disease development
WO2020035482A1 (fr) 2018-08-13 2020-02-20 Iltoo Pharma Combinaison d'interleukine 2 et d'un inhibiteur de l'interleukine 1, conjugués et utilisations thérapeutiques de celle-ci
WO2023222565A1 (fr) 2022-05-16 2023-11-23 Institut National de la Santé et de la Recherche Médicale Procédés d'évaluation de l'épuisement de cellules souches hématopoïétiques induites par une inflammation chronique

Also Published As

Publication number Publication date
AU7683391A (en) 1991-11-27

Similar Documents

Publication Publication Date Title
WO1991017184A1 (fr) Inhibiteurs ameliores d'interleukine-1
Wrann et al. T cell suppressor factor from human glioblastoma cells is a 12.5‐kd protein closely related to transforming growth factor‐beta.
Park et al. Characterization of the human B cell stimulatory factor 1 receptor.
Pennica et al. Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin
Le et al. Biology of disease
Hirano et al. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin
US20060172932A1 (en) Novel erythropoietin receptor agonists
US20090286855A1 (en) Interleukin-18 mutants, their production and use
EP1428883A1 (fr) Facteurs stimulateurs des mégacaryocytes
KR970002917B1 (ko) 인터루킨-i 억제제
AU2002324249A1 (en) Interleukin-18 mutants, their production and use
ZHANG et al. Purification and characterization of a recombinant murine interleukin‐6: Isolation of N‐and C‐terminally truncated forms
KR100270348B1 (ko) 전사인자 에이피알에프
KR20000075749A (ko) 신규의 폴리펩티드, 그 폴리펩티드를 암호하는 ｄｎａ 및 그용도
JP2863265B2 (ja) インターロイキン１インヒビター
PT667872E (pt) Interleucina-6 mutante com actividade biologica melhorada em relacao a da interleucina 6 selvagem
US6537781B1 (en) Methods and compositions concerning canine interleukin 5
JP3028847B2 (ja) マウスｇｐ１３０蛋白質
WO1987004466A1 (fr) Interleukine
EP0544719A1 (fr) Production de cytokine
JP3652365B6 (ja) ナチュラルキラー刺激因子
JP3652365B2 (ja) ナチュラルキラー刺激因子
JP3786433B2 (ja) 均一なｎ末端を有する組換え型ヒトインターロイキン６及びその製造方法
JP3993816B2 (ja) 顆粒球コロニー刺激因子レセプターをコードするｄｎａ
AU8446891A (en) Lymphokine 154

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR CA FI HU JP KP KR LK MC MG MW NO PL RO SD SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CM DE DK ES FR GA GB GR IT LU ML MR NL SE SN TD TG

NENP Non-entry into the national phase

Ref country code: CA