WO1996017869A2 - Interleukin-6 (il-6) antagonists - Google Patents
Interleukin-6 (il-6) antagonists Download PDFInfo
- Publication number
- WO1996017869A2 WO1996017869A2 PCT/IT1995/000208 IT9500208W WO9617869A2 WO 1996017869 A2 WO1996017869 A2 WO 1996017869A2 IT 9500208 W IT9500208 W IT 9500208W WO 9617869 A2 WO9617869 A2 WO 9617869A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- interleukin
- sil
- receptor
- mutant
- Prior art date
Links
- 102000004889 Interleukin-6 Human genes 0.000 title claims abstract description 55
- 108090001005 Interleukin-6 Proteins 0.000 title claims abstract description 55
- 229940100601 interleukin-6 Drugs 0.000 title claims abstract description 53
- 239000005557 antagonist Substances 0.000 title claims abstract description 20
- 230000027455 binding Effects 0.000 claims abstract description 21
- 102200104843 rs587781525 Human genes 0.000 claims abstract description 18
- 102220323198 rs371265931 Human genes 0.000 claims abstract description 17
- 230000035772 mutation Effects 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 12
- 201000010099 disease Diseases 0.000 claims abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 230000002159 abnormal effect Effects 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 3
- 229940079593 drug Drugs 0.000 claims abstract description 3
- 102200043522 c.688A>G Human genes 0.000 claims description 22
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
- 229940127448 Interleukin-6 Antagonists Drugs 0.000 claims description 2
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 abstract description 3
- 102000052611 human IL6 Human genes 0.000 abstract description 3
- 239000000556 agonist Substances 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 230000002265 prevention Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 33
- 102000005962 receptors Human genes 0.000 description 31
- 108020003175 receptors Proteins 0.000 description 31
- 150000001413 amino acids Chemical group 0.000 description 13
- 239000000499 gel Substances 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 102000004127 Cytokines Human genes 0.000 description 7
- 108090000695 Cytokines Proteins 0.000 description 7
- 102000004495 STAT3 Transcription Factor Human genes 0.000 description 7
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 102000010781 Interleukin-6 Receptors Human genes 0.000 description 6
- 108010038501 Interleukin-6 Receptors Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229920002401 polyacrylamide Polymers 0.000 description 5
- 102000004140 Oncostatin M Human genes 0.000 description 4
- 108090000630 Oncostatin M Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 238000000749 co-immunoprecipitation Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- ZKAOJVJQGVUIIU-GUBZILKMSA-N Asp-Pro-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ZKAOJVJQGVUIIU-GUBZILKMSA-N 0.000 description 3
- 241000880493 Leptailurus serval Species 0.000 description 3
- KZTLZZQTJMCGIP-ZJDVBMNYSA-N Thr-Val-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KZTLZZQTJMCGIP-ZJDVBMNYSA-N 0.000 description 3
- RWAYYYOZMHMEGD-XIRDDKMYSA-N Trp-Leu-Ser Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 RWAYYYOZMHMEGD-XIRDDKMYSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- UVDDTHLDZBMBAV-SRVKXCTJSA-N His-His-Cys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CS)C(=O)O)N UVDDTHLDZBMBAV-SRVKXCTJSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 2
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 2
- UJGDFQRPYGJBEH-AAEUAGOBSA-N Trp-Ser-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N UJGDFQRPYGJBEH-AAEUAGOBSA-N 0.000 description 2
- XQVRMLRMTAGSFJ-QXEWZRGKSA-N Val-Asp-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XQVRMLRMTAGSFJ-QXEWZRGKSA-N 0.000 description 2
- 230000001270 agonistic effect Effects 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000006451 grace's insect medium Substances 0.000 description 2
- 239000012133 immunoprecipitate Substances 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 102000011767 Acute-Phase Proteins Human genes 0.000 description 1
- 108010062271 Acute-Phase Proteins Proteins 0.000 description 1
- QTIZKMMLNUMHHU-DCAQKATOSA-N Asp-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O QTIZKMMLNUMHHU-DCAQKATOSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101100338243 Caenorhabditis elegans hil-6 gene Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- WOSRKEJQESVHGA-CIUDSAMLSA-N Glu-Arg-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O WOSRKEJQESVHGA-CIUDSAMLSA-N 0.000 description 1
- VSRCAOIHMGCIJK-SRVKXCTJSA-N Glu-Leu-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VSRCAOIHMGCIJK-SRVKXCTJSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000992170 Homo sapiens Oncostatin-M Proteins 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- YCJCEMKOZOYBEF-OEAJRASXSA-N Lys-Thr-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YCJCEMKOZOYBEF-OEAJRASXSA-N 0.000 description 1
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 1
- AXKJPUBALUNJEO-UBHSHLNASA-N Ser-Trp-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(O)=O AXKJPUBALUNJEO-UBHSHLNASA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- CSZFFQBUTMGHAH-UAXMHLISSA-N Thr-Thr-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O CSZFFQBUTMGHAH-UAXMHLISSA-N 0.000 description 1
- HSVPZJLMPLMPOX-BPNCWPANSA-N Tyr-Arg-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O HSVPZJLMPLMPOX-BPNCWPANSA-N 0.000 description 1
- ADBDQGBDNUTRDB-ULQDDVLXSA-N Tyr-Arg-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O ADBDQGBDNUTRDB-ULQDDVLXSA-N 0.000 description 1
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 1
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 102000043703 human OSM Human genes 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the field of immunology. More specifically, the subject of the present invention are mutants- of the soluble forms of receptor ⁇ of interleukin 6 that interfere in a negative manner in the formation of the dimeric receptor complex between the monomeric receptor complex IL-6/sIL-6R ⁇ and gp 130. A further subject of the invention is the use of these mutants as interleukin-6 antagonists to control, prevent and treat the diseases caused by abnormal IL-6 activity.
- Interleukin-6 elicits a variety of biological responses on different target cells. However - while the physiological production of IL-6 regulates B-cells proliferation and maturation, T-cell activation and the production of acute-phase proteins in liver during inflammatory response - disregulated production of the cytokine plays a crucial role in the pathogenesis of many inflammatory, autoimmune and neoplastic diseases.
- IL-6 the mature human IL-6 protein
- h IL-6 is a 185 a ino acid polypeptide containing two disulfide bonds (Cys 45 - Cys 51, and Cys 74 - Cys 84) .
- IL-6 functions through interaction of two binding sites (known as site I and site II) with at least two specific receptors (IL-R ⁇ and gp 130) on the surface of the target cells, creating a trimeric complex IL-6- (receptor) 2 .
- This complex is formed sequentially: a first receptor (IL-6R ⁇ ) binds with low affinity to site I of IL-6 without transmitting the signal; subsequently a second receptor (gp 130) , after binding with high affinity to site II of IL-6, transduces the signal.
- a first receptor IL-6R ⁇
- gp 130 second receptor
- hIL-6 mutants have been designed that are capable of binding a first receptor to site I, but incapable of dimerising the receptor because of mutations that sterically inhibit binding of the second receptor to site II. Mutants of this type have been described in WO 94/09138 (Cetus Oncology Corporation), and WO 94/011402 and PCT/IT 94/00095 (Istituto di Ricerche di Biologia Molecolare P. Angeletti S.p.A. ) .
- soluble forms of the receptor ⁇ of IL-6 (sIL-6R ⁇ ) containing one or more mutations in the region interfacing with gp 130 are antagonists of interleukin 6.
- Figure 5 shows resolution on gel of the complex formed by APRF with the DNA binding site (SIE, Serum Inducible Element) .
- SIE Serum Inducible Element
- Antagonists of IL- ⁇ according to the invention may be produced synthetically or using recombinant techniques.
- the cDNA coding for sIL- 6R ⁇ can be incorporated in a plasmid before being expressed in procaryotic or eukaryotic cells. Bacteria are the preferred procaryotic microorganisms.
- the cDNA coding for the sIL-6R ⁇ mutants according to the invention can be introduced into mammal cells: these mammal cells can be chosen from the group comprising CHO, COS, C127, HepG2, SK Hep.
- the 3' primer was designed in order to introduce an artificial TAG stop codon at amino acid position 324 preceded by a six- histidine coding sequence. This was done to ensure that the sIL-6R ⁇ and the mutants thereof produced by us have a six histidine "tail" at the carboxy-terminal end of the molecule, which can be useful for purification of the molecule using metal affinity chromatography.
- the fragment generated was then introduced into the COS-7 cells expression vector pcDNAI (Invitrogen) and sequenced in its entirety, thus obtaining plas id pC6FRH.
- Plasmid pC ⁇ FRH was in turn used as a template to obtain constructs coding for the following four mutants: Asn230Asp (SEQ ID NO: 1); Asn230Asp/Ala228Asp (SEQ ID NO: 2); His280Ser/Asp281Val (SEQ ID NO 3) and Ala228Asp/Asn230Asp/His280Ser/Asp281Val (SEQ ID NO: 4), by a two-step PCR as described in the past (Landt 0., Grunert H.P., and Hahn, U. (1990) Gene 96, 125-128).
- the mutants were then expressed in COS-7 cells.
- the COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS, plus glutamine and antibiotics, at 5% CO:.
- 2.5xl0 6 COS-7 cells were seeded in 100-mm tissue culture dishes and the next day transfected with 2 ⁇ g of the various shIL-6R ⁇ expression vectors using the DEAE-dextran technique as described in Seed B., Aruffo A. (1987) Proc. Natl. Acad. Sci. USA 84, 3365- 3369. 16 h after transfection cells were split, replated in 100-mm dishes and grown in complete medium at 37°C.
- Ala228Asp/Asn230Asp (SEQ ID NO : 2 ) 2 . 0 ⁇ 1 His280Ser/Asp281Val ( SEQ ID NO : 3 ) 4 . 3 ⁇ 2 Ala228Asp/Asn230Asp/ His280Ser/Asp281Val ( SEQ ID NO : 4 ) 2 . 5 ⁇ 1 wild type sIL-6R ⁇ 2 . 0 ⁇ 1
- IL-6-dependent formation of sIL-6R ⁇ /sgpl30 heterodimers can be easily monitored in vi tro by co- immunoprecipitations with the use of suitable monoclonal antibodies.
- a selection was therefore made of the mutants showing an IL-6 binding affinity in the same order of magnitude as the wild-type and their binding to sgpl30 was evaluated by co-immunoprecipitation in the presence of IL-6.
- 35 S-labelled sgp 130 was incubated with 125 I-IL-6 (as an internal standard for the immunoprecipitation) and aliquots of transfected COS-7 cell culture medium, containing either native or mutant receptors.
- the receptor mutants were also tested for their ability to interact with gpl30 on the cell surface.
- binding experiments were performed on human melanoma A375 cells which have an excess of gpl30 molecules over IL-6R ⁇ .
- binding of 125 I-IL-6 to A375 monolayers could be strongly enhanced by addition of the soluble receptor.
- This phenomenon is due to the ability of the sIL-6R ⁇ to bind 125 I-IL-6 and subsequently to interact with the gpl30 molecules present on the surface of the cells.
- the specificity of this interaction is demonstrated by the fact that the increased binding of 125 I-IL-6 in the presence of sIL- 6R ⁇ is competed by the addition of an excess of unlabeled human Oncostatin M (OM) (fig.
- OM Oncostatin M
- this mutant to be the one that, without any decrease in the affinity for IL-6, has the greatest effect on gpl30 binding.
- its production and that of the wild type soluble receptor was scaled up using the MaxBac® system (Invitrogen' s Baculovirus Expression System) .
- the expressed and purified receptors were tested in immunoprecipitation experiments.
- Sf9 cells grown in Grace's insect medium, were used for transfection of transfer vectors, isolation of recombinant virus and preparation of high-titer virus stocks.
- High Five® cells (Invitrogen) were instead used for production of proteins. Briefly, 4xl0 7 High Five® cells, grown in complete Grace's insect medium, were seeded into 750 ml flasks and infected with the appropriate recombinant virus at a MOI of 10. After 2 hours, cells were washed and SF-900 serum-free medium was added. The culture supernatants were harvested at 36 hours post-infection, dialyzed against PBS and directly loaded on a Ni 2+ -NTA-agarose column.
- both wild type shIL-6R ⁇ and mutant were eluted in PBS/80 mM imidazole. Purified protein was dialyzed against PBS and directly used or stored at 4°C for up to three weeks.
- APRF Acute Phase Response Factor
- the mutant was added to HepG2 cells induced with the IL-6- related cytokine OM, which is also known to efficiently induce APRF phosphorylation. As shown in figure 5, the mutant did not antagonize OM activity. - -
- MOLECULE TYPE protein
- HYPOTHETICAL no
- NAME Ala228Asp/Asn230Asp/His280Ser/Asp281Val
- IDENTIFICATION METHOD Electrophoresis on a denaturing SDS/polyacrylamide gel
- D OTHER INFORMATION: amino acid sequence of a mutant form of the interleukin 6 receptor from position 222 to position 287.
Abstract
These are antagonists of interleukin-6, characterised in that they consist of soluble forms of the receptor α of human IL-6 (shIL-6Rα) containing one or more mutation in the gp130 binding interface. In a preferred embodiment, the mutations are present in a position chosen from the group comprising Ala 228, Asn 230, His 280 and Asp 281. These antagonists can be used as agents capable of preventing and treating diseases caused by abnormal IL-6 activity. The figure shows the antagonist activity of the mutant Ala228Asp/Asn 230Asp/His280Ser/Asp281Val in comparison with the agonist properties of wild type shIL-6Rα. This antagonist can be used for preparation of drugs for the prevention, control and treatment of diseases caused by abnormal IL-6 bioactivity.
Description
INTERLEUKIN-6 (IL-6) ANTAGONISTS
DESCRIPTION The present invention relates to the field of immunology. More specifically, the subject of the present invention are mutants- of the soluble forms of receptor α of interleukin 6 that interfere in a negative manner in the formation of the dimeric receptor complex between the monomeric receptor complex IL-6/sIL-6Rα and gp 130. A further subject of the invention is the use of these mutants as interleukin-6 antagonists to control, prevent and treat the diseases caused by abnormal IL-6 activity. The term "interleukin 6" or "IL-6", in the context of the present invention, means IL-6 and fragments, deletions, insertions, substitutions, mutations and modifications thereof that maintain the biological characteristics of wild IL-6. Unless otherwise specified, the term refers to human IL-6.
Interleukin-6 elicits a variety of biological responses on different target cells. However - while the physiological production of IL-6 regulates B-cells proliferation and maturation, T-cell activation and the production of acute-phase proteins in liver during inflammatory response - disregulated production of the cytokine plays a crucial role in the pathogenesis of many inflammatory, autoimmune and neoplastic diseases.
As is known from the state of the art, various attempts have been made to perfect IL-6 bioactivity inhibitors, both in order to study the role played by this cytokine in the genesis of single diseases, and in order to define drugs for treatment.
It is also known that the mature human IL-6 protein (h IL-6) is a 185 a ino acid polypeptide containing two disulfide bonds (Cys 45 - Cys 51, and Cys 74 - Cys 84) . IL-6 functions through interaction of two binding sites
(known as site I and site II) with at least two specific receptors (IL-Rα and gp 130) on the surface of the target cells, creating a trimeric complex IL-6- (receptor)2. This complex is formed sequentially: a first receptor (IL-6Rα) binds with low affinity to site I of IL-6 without transmitting the signal; subsequently a second receptor (gp 130) , after binding with high affinity to site II of IL-6, transduces the signal.
Based on this mechanism, hIL-6 mutants have been designed that are capable of binding a first receptor to site I, but incapable of dimerising the receptor because of mutations that sterically inhibit binding of the second receptor to site II. Mutants of this type have been described in WO 94/09138 (Cetus Oncology Corporation), and WO 94/011402 and PCT/IT 94/00095 (Istituto di Ricerche di Biologia Molecolare P. Angeletti S.p.A. ) .
It has now surprisingly been found, and this discovery forms the basis of the present invention, that soluble forms of the receptor α of IL-6 (sIL-6Rα) containing one or more mutations in the region interfacing with gp 130 are antagonists of interleukin 6.
In one embodiment, sIL-6Rct contains at least one mutation in a position chosen from Ala 228, Asn 230, His 280 and Asp 281. The mutations may, for example, be chosen from the group comprising: Asn230Asp (SEQ ID NO:l); Ala228Asp/Asn230Asp (SEQ ID NO:2); His280Ser/Asp281Val (SEQ ID NO:3).
In a preferred embodiment, sIL-6Rα contains multiple mutations in positions Ala 228, Asn 230, His 280 and Asp 281. A multiple mutation that has given good results is Ala228Asp/Asn230Asp/His280Ser/Asp281Val (SEQ ID NO:4).
The antagonists of IL-6, which according to the invention consist of soluble forms of the receptor α of IL-6, mutated in the interface that binds to gp 130, can be administered at concentrations therapeutically effective for the treatment and prevention of diseases
related to abnormal IL-6 activity. For this purpose, the antagonists according to the invention are administered preferably by endovenous and/or subcutaneous injection. Experts in the field are well acquainted with these methods of administration.
Up to this point a general description of the invention has been given. A more detailed description of the invention will now be provided, with the aid of the following examples, in order to give a clearer understanding of the objects, characteristics, advantages and operating methods thereof.
Figure 1A shows resolution on SDS/PAGE gel (12%), in vitro, of the co-immunoprecipitates of mutants according to the invention with sgp 130 (soluble form of gp 130) marked using 35S. Figure IB shows the position on the gel corresponding to the migrated 125I-IL-6.
Figure 2 shows, in the form of a histogram, the different capacities of the wild type sIL-6Rα receptor and the sIL-6Rα receptors mutated to interact with gp 130 on the membrane of A 375 cells.
Figure 3 shows resolution on SDS/PAGE gel (12%), in vitro, of the co-immunoprecipates of sgp 130 marked with 35S or with 2.0 μg of wild receptor or with 2.0 μg of mutant receptor (SEQ ID NO: 4) . Figure 4 shows the antagonist activity of the mutant SEQ ID NO: 4 on HepG2 cells in comparison with the agonistic properties of wild type shIL-6Rα.
Figure 5 shows resolution on gel of the complex formed by APRF with the DNA binding site (SIE, Serum Inducible Element) . (The mutant SEQ ID NO: 4 does not inhibit activation of OM-dependent APRF on HepG2 cells) .
Antagonists of IL-β according to the invention may be produced synthetically or using recombinant techniques. In the latter case the cDNA coding for sIL- 6Rα can be incorporated in a plasmid before being expressed in procaryotic or eukaryotic cells. Bacteria are the preferred procaryotic microorganisms.
Alternatively, the cDNA coding for the sIL-6Rα mutants according to the invention can be introduced into mammal cells: these mammal cells can be chosen from the group comprising CHO, COS, C127, HepG2, SK Hep. Furthermore, the protein can also be expressed in insect cells (Sf9 or HighFive) using the well known recombinant Baculovirus technology (AcNPV strain) . Example 1 Preparation of sIL-6Rα mutants sIL-6Rα cDNA was obtained as a 5'EcoRI-3'XbaI fragment by PCR using the IL-6Rα complete cDNA as template (Sporeno E., Paonessa G., Salvati A.L., Graziani R., Delmastro P., Ciliberto G. and Toniatti C. (1994) J. Biol. Chem. 269, 10991-10995). The 3' primer was designed in order to introduce an artificial TAG stop codon at amino acid position 324 preceded by a six- histidine coding sequence. This was done to ensure that the sIL-6Rα and the mutants thereof produced by us have a six histidine "tail" at the carboxy-terminal end of the molecule, which can be useful for purification of the molecule using metal affinity chromatography. The fragment generated was then introduced into the COS-7 cells expression vector pcDNAI (Invitrogen) and sequenced in its entirety, thus obtaining plas id pC6FRH. Plasmid pCβFRH was in turn used as a template to obtain constructs coding for the following four mutants: Asn230Asp (SEQ ID NO: 1); Asn230Asp/Ala228Asp (SEQ ID NO: 2); His280Ser/Asp281Val (SEQ ID NO 3) and Ala228Asp/Asn230Asp/His280Ser/Asp281Val (SEQ ID NO: 4), by a two-step PCR as described in the past (Landt 0., Grunert H.P., and Hahn, U. (1990) Gene 96, 125-128).
The mutants were then expressed in COS-7 cells. For this purpose, the COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS, plus glutamine and antibiotics, at 5% CO:. For protein expression, 2.5xl06 COS-7 cells were seeded in 100-mm tissue culture dishes and the next day transfected
with 2 μg of the various shIL-6Rα expression vectors using the DEAE-dextran technique as described in Seed B., Aruffo A. (1987) Proc. Natl. Acad. Sci. USA 84, 3365- 3369. 16 h after transfection cells were split, replated in 100-mm dishes and grown in complete medium at 37°C. After 72-96 h the medium was collected, centrifuged and used for co-immunoprecipitation experiments and binding analysis. To monitor the expression level of each mutant, 2.5xl05 transfected COS-7 cells were replated in 35-mm dishes and, 48 h after transfection, metabolically labeled with [35S]methionine for 4h. The supernatants were i munoprecipitated with anti-human IL-6Rα monoclonal antibody I6R1/9.G11. The immunoprecipitated material was then analyzed by electrophoresis through a polyacrylamide gel containing SDS.
IL-6 binding analysis of mutants
To determine the affinity for IL-6 of the sIL-6Rα mutants contained in the conditioned COS cells culture medium, the six-histidine "tail" in the -proteins produced by us was used.
Appropriate amounts of transfected COS cells supernatant, previously determined in titration experiments and to which imidazole was added up to a final concentration of 5 M, were mixed with 20-40 pM 125I-IL-6 and increasing concentrations of unlabeled cytokine. At equilibrium conditions, 40 μl of Ni:*-NTA- Agarose (Quiagen) , a resin capable of selectively binding proteins containing histidine "tails", was added and incubation prolonged for one additional hour. The ligand bound by the receptor and, therefore, indirectly resin-associated, was separated from free ligand
(supernatant) by centrifugation through a cushion of 30* sucrose in PBS. All steps were performed at 4°C. The values of free and bound cp (radioactivity count per minute) allowed drawing of competition displacement curves. The apparent Kd of the various soluble IL-6 receptors was determined after Scatchard transformation
of the results (see the article by Sporeno, Paonessa,
Salviati, Graziani, Delmastro, Ciliberto and Toniatti
(1994), J. Biol. Chem. 269, 10991-10995) . Analysis of binding data and curve fitting was done using the UltraFit software (Biosoft®) on a Macintosh computer.
As can be seen from Table 1, the binding affinity:
1) remains substantially unprejudiced for mutants Asn230Asp (SEQ ID NO: 1), Asn230Asp/Ala228Asp (SEQ ID NO:
2) ; b) is reduced in a slight but reproducible manner (between 2.5 nM and 6 nM) for His280Ser/Asp281Val (SEQ ID
NO: 3) ; and c) the activity of the wild type form is fully maintained for the mutant Ala228Asp/Asn230Asp/His280Ser/Asp281Val (SEQ ID NO: 4) .
TABLE 1
IL-6 binding affinity of soluble interleukin 6 receptor (wild and mutated) , labeled with histidine and expressed in COS cells
Mutant IL-6 binding affinity
nM
Asn230Asp ( SEQ ID NO : 1 ) 1 . 5 ± 1
Ala228Asp/Asn230Asp (SEQ ID NO : 2 ) 2 . 0 ± 1 His280Ser/Asp281Val ( SEQ ID NO : 3 ) 4 . 3 ± 2 Ala228Asp/Asn230Asp/ His280Ser/Asp281Val ( SEQ ID NO : 4 ) 2 . 5 ± 1 wild type sIL-6Rα 2 . 0 ± 1
Binding of mutants to gp!30
The IL-6-dependent formation of sIL-6Rα/sgpl30 heterodimers can be easily monitored in vi tro by co- immunoprecipitations with the use of suitable monoclonal antibodies. A selection was therefore made of the mutants showing an IL-6 binding affinity in the same order of magnitude as the wild-type and their binding to sgpl30 was evaluated by co-immunoprecipitation in the presence of IL-6.
35S-labelled sgp 130 was incubated with 125I-IL-6 (as an internal standard for the immunoprecipitation) and aliquots of transfected COS-7 cell culture medium, containing either native or mutant receptors. After in vitro binding, anti IL-6Rα mAb I6/R19.G11 was added to the mixtures and the immunoprecipitates resolved on SDS/PAGE gels. The results are shown in figure 1. As can be seen, the mutant His280Ser/Asp281Val (SEQ ID NO: 3) strongly reduced the association of the IL-6/ sIL-6Rα complex sgp 130 molecule (lane 4) . Also the single substitution mutant Asn230Asp (SEQ ID NO: 1) had decreased interaction with sgpl30 (lane 2) . The effect of this mutant on interaction with sgp 130 is of the same magnitude as that of mutant Ala228Asp/Asn230Asp (SEQ ID NO: 2) . Finally, the quadruple mutant Ala228Asp/Asn230Asp/His280Ser/Asp281Val (SEQ ID NO: 4) displayed the lowest ability to co-i munoprecipitate 35S- gp 130 (lane 5) .
The receptor mutants were also tested for their ability to interact with gpl30 on the cell surface. For this purpose, binding experiments were performed on human melanoma A375 cells which have an excess of gpl30 molecules over IL-6Rα. In fact, binding of 125I-IL-6 to A375 monolayers could be strongly enhanced by addition of the soluble receptor. This phenomenon is due to the ability of the sIL-6Rα to bind 125I-IL-6 and subsequently to interact with the gpl30 molecules present on the surface of the cells. The specificity of this interaction is demonstrated by the fact that the increased binding of 125I-IL-6 in the presence of sIL- 6Rα is competed by the addition of an excess of unlabeled human Oncostatin M (OM) (fig. 2) . This cytokine is able to bind directly to gpl30 and to compete the binding of the IL-6/IL-6Rα complex with gpl30. Unlike wild type shIL-6Rα, only a minor increase in specific binding was detected when cells were challenged with 125I-IL-6 plus the soluble mutant receptors acting as antagonists
(figure 2) . The greatest disturbance to interaction with gpl30 is shown by the mutants His280Ser/Asp281Val (SEQ ID NO: 3) and Ala228Asp/Asn230Asp/His280Ser/Asp281Val (SEQ ID NO: 4) . Demonstration that the mutant Ala228Asp/Asn230Asp/ His280Ser/Asp281Val (SEQ ID NO: 4) is an IL-6 antagonist
The results shown above indicate this mutant to be the one that, without any decrease in the affinity for IL-6, has the greatest effect on gpl30 binding. In order to study the biological activity of this mutant, its production and that of the wild type soluble receptor was scaled up using the MaxBac® system (Invitrogen' s Baculovirus Expression System) . The expressed and purified receptors were tested in immunoprecipitation experiments.
Sf9 cells, grown in Grace's insect medium, were used for transfection of transfer vectors, isolation of recombinant virus and preparation of high-titer virus stocks. High Five® cells (Invitrogen) were instead used for production of proteins. Briefly, 4xl07 High Five® cells, grown in complete Grace's insect medium, were seeded into 750 ml flasks and infected with the appropriate recombinant virus at a MOI of 10. After 2 hours, cells were washed and SF-900 serum-free medium was added. The culture supernatants were harvested at 36 hours post-infection, dialyzed against PBS and directly loaded on a Ni2+-NTA-agarose column. After washing steps with PBS/8 mM imidazole, both wild type shIL-6Rα and mutant were eluted in PBS/80 mM imidazole. Purified protein was dialyzed against PBS and directly used or stored at 4°C for up to three weeks.
The use of purified proteins made it possible to quantify the amount of receptor and to compare the ability of wild type receptor and mutant receptor to interact with sgpl30 in the presence of a wide range of IL-6 concentrations. The results (figure 3) were in good agreement with those previously obtained with COS cell-
derived receptors. Interestingly at each point of the curve the amount of co-immunoprecipitated gpl30 is lower for the mutant receptor than for the wild type receptor and this is true even at the highest concentrations of the cytokine tested (100 nM) . These findings confirm that the interaction between IL-6 and mutant sIL-6Rα generates a complex with lower affinity for gpl30.
To test the potential antagonism of the mutant for IL-6 activity, we chose the IL-6 dependent activation of transcription factor APRF (Acute Phase Response Factor) /STAT 3 in human hepatoma cells. Activation of APRF, which is dependent on tyrosine phosphorylation, is a rapid cytoplasmic event which takes place within minutes of stimulation with all cytokines of the IL-6 family.
Human HepG2 cells were stimulated with increasing concentrations of IL-6 together with a fixed amount of wild type and mutant receptors (100 nM) . As shown in figure 4, while shIL-6Rα potentiated APRF induction by IL-6 (compare lanes 2-4 and 5-7 in figure 4), the soluble mutant receptor not only lacked any agonistic activity but instead down-modulated the activity of the cytokine
(figure 4, lanes 8-10) . As predicted from the co- immunoprecipitation experiments, the inhibition was complete when cells were treated with up to 20 pM IL-β
(lanes 8 and 9, figure 4) and only a weak induction was detected at the highest concentration of IL-6 (200 pM, lane 10 of figure 4) .
To test if antagonism was specific for IL-6, the mutant was added to HepG2 cells induced with the IL-6- related cytokine OM, which is also known to efficiently induce APRF phosphorylation. As shown in figure 5, the mutant did not antagonize OM activity.
- -
SEQUENCE LISTING GENERAL INFORMATION (i) APPLICANT: ISTITUTO DI RICERCHE DI BIOLOGIA MOLECOLARE P. ANGELETTI S.p.A. (ii) TITLE OF INVENTION:
(iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Societa Italiana Brevetti
(B) STREET: Piazza di Pietra, 39 (C) CITY: Rome
(D) COUNTRY: Italy
(E) POSTAL CODE: 1-00186 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk 3.5" 1.44 MBYTES (B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS Rev. 6.22
(D) SOFTWARE: Microsoft Word 6.0 (viii) ATTORNEY INFORMATION
(A) NAME: DI CERBO, Mario (Dr.)- (C) REFERENCE: RM/X88470/PC-DC
(ix) TELECOMMUNICATION INFORMATION
(A) TELEPHONE: 06/6785941
(B) TELEFAX: 06/6794692
(C) TELEX: 612287 ROPAT (1) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: no (v) FRAGMENT TYPE: internal (vii) IMMEDIATE SOURCE:
(A) SYNTHESIS: production as recombinant protein in eucariotic cells (ix) FEATURE:
(A) NAME: Asn230Asp
- -
fC) IDENTIFICATION METHOD: Electrophoresis on a denaturing SDS/polyacrylamide gel
(D) OTHER INFORMATION: amino acid sequence of a mutant form of the interleukin 6 receptor from position 222 to position 236.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: lie Thr Val Thr Ala Val Ala Arg Asp Pro Arg Trp Leu Ser Val 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no
(v) FRAGMENT TYPE: internal (vii) IMMEDIATE SOURCE:
(A) SYNTHESIS: production as recombinant protein in eucariotic cells (ix) FEATURE:
(A) NAME: Ala228Asp/Asn230Asp
(C) IDENTIFICATION METHOD: Electrophoresis on a denaturing SDS/polyacrylamide gel
(D) OTHER INFORMATION: amino acid sequence of a mutant form of the interleukin 6 receptor from position 222 to position 236.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: lie Thr Val Thr Ala Val Asp Arg Asp Pro Arg Trp Leu Ser Val 1 5 10 15 (3) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: no (v) FRAGMENT TYPE: internal
(vii) IMMEDIATE SOURCE:
(A) SYNTHESIS: production as recombinant protein in eucariotic cells (ix) FEATURE: (A) NAME: His280Ser/Asp281Val
(C) IDENTIFICATION METHOD:Electrophoresis on a denaturing SDS/polyacrylamide gel
(D) OTHER INFORMATION: amino acid sequence of a mutant form of the interleukin 6 receptor from position 273 to position 287.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Leu Gin His His Cys Val He Ser Val Ala Trp Ser Gly Leu Arg 1 5 10 15
(4) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 66 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no
(v) FRAGMENT TYPE: internal (vii) IMMEDIATE SOURCE:
(A) SYNTHESIS: production as recombinant protein in eucariotic cells (ix) FEATURE:
(A) NAME: Ala228Asp/Asn230Asp/His280Ser/Asp281Val
(C) IDENTIFICATION METHOD:Electrophoresis on a denaturing SDS/polyacrylamide gel (D) OTHER INFORMATION: amino acid sequence of a mutant form of the interleukin 6 receptor from position 222 to position 287.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: He Thr Val Thr Ala Val Asp Arg Asp Pro Arg Trp Leu Ser Val T 1 5 10 15
Trp Gin Asp Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg P 20 25 30
Glu Leu Arg Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met
35 40 45
Val Lys Asp Leu Gin His His Cys Val He Ser Val Ala Trp Ser Gly 50 55 60 Leu Arg 65
Claims
1. Antagonist of interleukin-6 (IL-6), characterized in that it consists in soluble forms of the α receptor of IL-6 (sIL-6Rα) containing one or more mutations in the interface binding with gp 130.
2. Antagonist of interleukin-6 according to claim 1, in which sIL-6Rα contains at least one mutation in a position chosen from the group comprising Ala 228, Asn 230, His 280 and Asp 281.
3. Antagonist of interleukin 6 according to claim 1 or 2, in which sIL-6Rα contains the mutation Asn230Asp (SEQ ID NO: 1) .
4. Antagonist of interleukin 6 according to claim 1 or 2, in which sIL-6Rα contains the mutation Ala228Asp/Asn230/Asp (SEQ ID NO: 2) .
5. Antagonist of interleukin 6 according to claim 1 or 2, in which sIL-6Rα contains the mutations His280Ser/Asp281Val (SEQ ID NO: 3) .
6. Antagonist of interleukin 6 according to claim 1 or 2, in which sIL-6Rα contains the mutations
Ala228Asp/Asn230Asp/His280Ser/Asp281Val (SEQ ID NO: 4) .
7. Use of interleukin 6 antagonists according to claims 1 to 6, for the study and preparation of drugs capable of controlling, preventing and treating diseases caused by abnormal IL-6 activity.
8. Soluble receptors acting as IL-6 antagonists and their use, as described, illustrated and claimed above.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8517466A JPH09503232A (en) | 1994-12-06 | 1995-12-05 | An IL-6 antagonist that is a soluble form of interleukin-6 (IL-6) receptor alpha mutated at the GP130 binding interface |
| EP95940401A EP0742794A1 (en) | 1994-12-06 | 1995-12-05 | Interleukin-6 (il-6) antagonists |
| AU41866/96A AU4186696A (en) | 1994-12-06 | 1995-12-05 | Interleukin-6 (il-6) antagonists |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITRM94A000794 | 1994-12-06 | ||
| ITRM940794A IT1274350B (en) | 1994-12-06 | 1994-12-06 | INTERLEUCHINA-6 (IL-6) ANTAGONISTS, WHICH CONSIST OF SOLUBLE FORMS OF THE ALFA RECEPTOR OF IL-6, CHANGED IN THE INTERFACE THAT LINKS TO GP 130 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1996017869A2 true WO1996017869A2 (en) | 1996-06-13 |
| WO1996017869A3 WO1996017869A3 (en) | 1996-08-29 |
Family
ID=11402851
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IT1995/000208 WO1996017869A2 (en) | 1994-12-06 | 1995-12-05 | Interleukin-6 (il-6) antagonists |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0742794A1 (en) |
| JP (1) | JPH09503232A (en) |
| CN (1) | CN1139933A (en) |
| AU (1) | AU4186696A (en) |
| CA (1) | CA2177837A1 (en) |
| IT (1) | IT1274350B (en) |
| WO (1) | WO1996017869A2 (en) |
Cited By (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997013781A2 (en) * | 1995-09-28 | 1997-04-17 | Yeda Research And Development Co. Ltd. | Synthetic peptides that inhibit il-6 activity |
| WO1998035694A2 (en) * | 1997-02-11 | 1998-08-20 | Hadasit Medical Research Services & Development Company Ltd. | A pharmaceutical composition for treating hepatitis b virus (hbv) infection |
| WO2000025745A2 (en) * | 1998-11-05 | 2000-05-11 | Omeros Medical Systems, Inc. | Irrigation solution and method for inhibition of pain and inflammation |
| US6384193B1 (en) * | 1996-10-09 | 2002-05-07 | Imperial College Of Science Technology And Medicine | Agents for the regulation of oestrogen synthesis |
| WO2003025017A1 (en) * | 2001-09-14 | 2003-03-27 | Commonwealth Scientific And Industrial Research Organisation | Cytokine receptor |
| US6664374B1 (en) | 1999-08-27 | 2003-12-16 | The United States Of America As Represented By The Department Of Health & Human Services | Polypeptides comprising IL-6 ligand-binding receptor domains |
| WO2004073741A1 (en) | 2003-02-24 | 2004-09-02 | Chugai Seiyaku Kabushiki Kaisha | Remedy for spinal injury containing interleukin-6 antagonist |
| WO2005037315A1 (en) | 2003-10-17 | 2005-04-28 | Chugai Seiyaku Kabushiki Kaisha | Therapeutic agent for mesothelioma |
| WO2005061000A1 (en) | 2003-12-19 | 2005-07-07 | Chugai Seiyaku Kabushiki Kaisha | Remedy for angitis |
| US7091181B2 (en) | 1994-12-12 | 2006-08-15 | Omeros Corporation | Method of inhibition of pain and inflammation during surgery comprising administration of soluble TNF receptors |
| WO2007043641A1 (en) | 2005-10-14 | 2007-04-19 | Fukuoka University | Inhibitor of transplanted islet dysfunction in islet transplantation |
| WO2007046489A1 (en) | 2005-10-21 | 2007-04-26 | Chugai Seiyaku Kabushiki Kaisha | Therapeutic agent for heart disease |
| WO2007058194A1 (en) | 2005-11-15 | 2007-05-24 | National Hospital Organization | Inhibitor of cytotoxic t cell induction |
| WO2007061029A1 (en) | 2005-11-25 | 2007-05-31 | Keio University | Therapeutic agent for prostate cancer |
| WO2007086490A1 (en) | 2006-01-27 | 2007-08-02 | Keio University | Remedy for disease associated with choroidal angiogenesis |
| WO2007116962A1 (en) | 2006-04-07 | 2007-10-18 | Osaka University | Muscle regeneration promoter |
| WO2008090901A1 (en) | 2007-01-23 | 2008-07-31 | Shinshu University | Chronic rejection inhibitor |
| EP1972638A1 (en) | 2001-04-02 | 2008-09-24 | Chugai Seiyaku Kabushiki Kaisha | Remedies for juvenile chronic arthritis and related diseases |
| EP2011514A1 (en) | 1997-03-21 | 2009-01-07 | Chugai Seiyaku Kabushiki Kaisha | A preventive or therapeutic agent for sensitized T cell-mediated diseases comprising IL-6 antagonist as an active ingredient |
| WO2009148148A1 (en) | 2008-06-05 | 2009-12-10 | 国立がんセンター総長が代表する日本国 | Neuroinvasion inhibitor |
| EP2368577A2 (en) | 2003-04-28 | 2011-09-28 | Chugai Seiyaku Kabushiki Kaisha | Methods for treating interleukin-6 related diseases |
| WO2011149046A1 (en) | 2010-05-28 | 2011-12-01 | 独立行政法人国立がん研究センター | Therapeutic agent for pancreatic cancer |
| WO2011149051A1 (en) | 2010-05-28 | 2011-12-01 | 中外製薬株式会社 | Antitumor t cell response enhancer |
| US8440196B1 (en) | 1998-08-24 | 2013-05-14 | Chugai Seiyaku Kabushiki Kaisha | Treatment for pancreatitis using IL-6 receptor antagonist antibodies |
| WO2014200018A1 (en) | 2013-06-11 | 2014-12-18 | 独立行政法人 国立精神・神経医療研究センター | Method for predicting post-therapy prognosis of relapsing-remitting multiple sclerosis (rrms) patient, and method for determining applicability of novel therapy |
| EP2898896A1 (en) | 2014-01-22 | 2015-07-29 | Université Pierre et Marie Curie (Paris 6) | Agents for use in the treatment of retinal inflammation |
| WO2018203545A1 (en) | 2017-05-02 | 2018-11-08 | 国立研究開発法人国立精神・神経医療研究センター | Method for predicting and evaluating therapeutic effect in diseases related to il-6 and neutrophils |
| WO2019151418A1 (en) | 2018-01-31 | 2019-08-08 | 元一 加藤 | Therapeutic agent for asthma containing il-6 inhibitor |
| WO2020213665A1 (en) | 2019-04-17 | 2020-10-22 | 国立大学法人広島大学 | Therapeutic agent for urological cancer which is characterized by being administered with il-6 inhibitor and ccr2 inhibitor in combination |
| EP4269440A2 (en) | 2015-02-27 | 2023-11-01 | Chugai Seiyaku Kabushiki Kaisha | Composition for treating il-6-related diseases |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7227837B1 (en) | 1998-04-30 | 2007-06-05 | At&T Labs, Inc. | Fault tolerant virtual tandem switch |
| CA2237915A1 (en) | 1998-05-19 | 1999-11-19 | Stephen Shaughnessy | Osteoporosis treatment |
| JP4799516B2 (en) * | 1998-08-24 | 2011-10-26 | 中外製薬株式会社 | A prophylactic or therapeutic agent for pancreatitis comprising an IL-6 antagonist as an active ingredient |
| KR20210025054A (en) * | 2018-06-21 | 2021-03-08 | 샤턱 랩스 인코포레이티드 | Heterodimeric proteins and uses thereof |
| CN110133241B (en) * | 2019-05-21 | 2022-05-27 | 中国食品药品检定研究院 | Novel method for measuring biological activity of recombinant human soluble gp130-Fc fusion protein |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0591892A (en) * | 1991-10-02 | 1993-04-16 | Chuzo Kishimoto | Il-6 receptor derivative |
-
1994
- 1994-12-06 IT ITRM940794A patent/IT1274350B/en active IP Right Grant
-
1995
- 1995-12-05 CN CN95191457A patent/CN1139933A/en active Pending
- 1995-12-05 WO PCT/IT1995/000208 patent/WO1996017869A2/en not_active Application Discontinuation
- 1995-12-05 CA CA002177837A patent/CA2177837A1/en not_active Abandoned
- 1995-12-05 JP JP8517466A patent/JPH09503232A/en active Pending
- 1995-12-05 EP EP95940401A patent/EP0742794A1/en not_active Withdrawn
- 1995-12-05 AU AU41866/96A patent/AU4186696A/en not_active Abandoned
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0591892A (en) * | 1991-10-02 | 1993-04-16 | Chuzo Kishimoto | Il-6 receptor derivative |
Non-Patent Citations (5)
| Title |
|---|
| COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, vol. 54, 1989, pages 713-722, XP002003860 TAGA ET AL.: "Interleukin-6 receptor and a unique mechanism of its signal transduction" * |
| DATABASE WPI Week 9320 Derwent Publications Ltd., London, GB; AN 93-161739 XP002003861 & JP,A,05 091 892 (CHUGAI PHARM CO LTD & AL) , 16 April 1993 & DATABASE STRAND TPSD AN: E52907, * |
| EMBO J., vol. 12, no. 4, 1993, pages 1705-1712, XP002003859 YAWATA ET AL.: "Structure-function analysis of human IL-6 receptor : dissociation of amino acid residues required for IL-6-binding and IL-6 signal transduction through gp130" * |
| EUR. CYTOKINE NETWORK, vol. 5, no. 3, May 1994 - June 1994, pages 293-300, XP000571448 LIAUTARD ET AL.: "Epitope analysis of human IL-6 receptor gp80 molecule with monoclonal antibodies" * |
| JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 20, 19 May 1995, pages 12242-12249, XP000566420 SALVATI ET AL.: "Interleukin-6 (IL-6) antagonism by soluble IL-6 receptor alpha mutated in the predicted gp130-binding interface" * |
Cited By (44)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7091181B2 (en) | 1994-12-12 | 2006-08-15 | Omeros Corporation | Method of inhibition of pain and inflammation during surgery comprising administration of soluble TNF receptors |
| WO1997013781A2 (en) * | 1995-09-28 | 1997-04-17 | Yeda Research And Development Co. Ltd. | Synthetic peptides that inhibit il-6 activity |
| WO1997013781A3 (en) * | 1995-09-28 | 1997-05-15 | Yeda Res & Dev | Synthetic peptides that inhibit il-6 activity |
| US6384193B1 (en) * | 1996-10-09 | 2002-05-07 | Imperial College Of Science Technology And Medicine | Agents for the regulation of oestrogen synthesis |
| WO1998035694A2 (en) * | 1997-02-11 | 1998-08-20 | Hadasit Medical Research Services & Development Company Ltd. | A pharmaceutical composition for treating hepatitis b virus (hbv) infection |
| WO1998035694A3 (en) * | 1997-02-11 | 1998-11-19 | Hadasit Med Res Service | A pharmaceutical composition for treating hepatitis b virus (hbv) infection |
| US6217858B1 (en) | 1997-02-11 | 2001-04-17 | Hadasit & Medical Research Services & Development Company, Ltd. | Pharmaceutical composition for treating hepatitis B virus (HBV) infection |
| US6410009B1 (en) | 1997-02-11 | 2002-06-25 | Hadasit Medical Research Services & Development Co., Ltd. | Pharmaceutical composition for treating hepatitis B virus (HBV) infection |
| EP2011514A1 (en) | 1997-03-21 | 2009-01-07 | Chugai Seiyaku Kabushiki Kaisha | A preventive or therapeutic agent for sensitized T cell-mediated diseases comprising IL-6 antagonist as an active ingredient |
| EP2322216A1 (en) | 1997-03-21 | 2011-05-18 | Chugai Seiyaku Kabushiki Kaisha | A preventive or therapeutic agent for sensitized T cell-mediated diseases comprising IL-6 antagonist as an active ingredient |
| US8440196B1 (en) | 1998-08-24 | 2013-05-14 | Chugai Seiyaku Kabushiki Kaisha | Treatment for pancreatitis using IL-6 receptor antagonist antibodies |
| WO2000025745A2 (en) * | 1998-11-05 | 2000-05-11 | Omeros Medical Systems, Inc. | Irrigation solution and method for inhibition of pain and inflammation |
| WO2000025745A3 (en) * | 1998-11-05 | 2000-08-24 | Omeros Med Sys Inc | Irrigation solution and method for inhibition of pain and inflammation |
| US6664374B1 (en) | 1999-08-27 | 2003-12-16 | The United States Of America As Represented By The Department Of Health & Human Services | Polypeptides comprising IL-6 ligand-binding receptor domains |
| EP3640261A1 (en) | 2001-04-02 | 2020-04-22 | Chugai Seiyaku Kabushiki Kaisha | Therapeutic agent for chronic arthritides diseases of childhood-related diseases |
| EP2298812A2 (en) | 2001-04-02 | 2011-03-23 | Chugai Seiyaku Kabushiki Kaisha | Remedies for juvenile chronic arthritis and related diseases |
| EP1972638A1 (en) | 2001-04-02 | 2008-09-24 | Chugai Seiyaku Kabushiki Kaisha | Remedies for juvenile chronic arthritis and related diseases |
| WO2003025017A1 (en) * | 2001-09-14 | 2003-03-27 | Commonwealth Scientific And Industrial Research Organisation | Cytokine receptor |
| WO2004073741A1 (en) | 2003-02-24 | 2004-09-02 | Chugai Seiyaku Kabushiki Kaisha | Remedy for spinal injury containing interleukin-6 antagonist |
| EP2368577A2 (en) | 2003-04-28 | 2011-09-28 | Chugai Seiyaku Kabushiki Kaisha | Methods for treating interleukin-6 related diseases |
| EP3903819A1 (en) | 2003-04-28 | 2021-11-03 | Chugai Seiyaku Kabushiki Kaisha | Methods for treating interleukin-6 related diseases |
| EP4098279A1 (en) | 2003-04-28 | 2022-12-07 | Chugai Seiyaku Kabushiki Kaisha | Methods for treating interleukin-6 related diseases |
| EP3167901A1 (en) | 2003-04-28 | 2017-05-17 | Chugai Seiyaku Kabushiki Kaisha | Methods for treating interleukin-6 related diseases |
| WO2005037315A1 (en) | 2003-10-17 | 2005-04-28 | Chugai Seiyaku Kabushiki Kaisha | Therapeutic agent for mesothelioma |
| WO2005061000A1 (en) | 2003-12-19 | 2005-07-07 | Chugai Seiyaku Kabushiki Kaisha | Remedy for angitis |
| WO2007043641A1 (en) | 2005-10-14 | 2007-04-19 | Fukuoka University | Inhibitor of transplanted islet dysfunction in islet transplantation |
| WO2007046489A1 (en) | 2005-10-21 | 2007-04-26 | Chugai Seiyaku Kabushiki Kaisha | Therapeutic agent for heart disease |
| WO2007058194A1 (en) | 2005-11-15 | 2007-05-24 | National Hospital Organization | Inhibitor of cytotoxic t cell induction |
| WO2007061029A1 (en) | 2005-11-25 | 2007-05-31 | Keio University | Therapeutic agent for prostate cancer |
| WO2007086490A1 (en) | 2006-01-27 | 2007-08-02 | Keio University | Remedy for disease associated with choroidal angiogenesis |
| EP3135298A1 (en) | 2006-01-27 | 2017-03-01 | Keio University | Remedy for disease associated with choroidal angiogenesis |
| WO2007116962A1 (en) | 2006-04-07 | 2007-10-18 | Osaka University | Muscle regeneration promoter |
| WO2008090901A1 (en) | 2007-01-23 | 2008-07-31 | Shinshu University | Chronic rejection inhibitor |
| WO2009148148A1 (en) | 2008-06-05 | 2009-12-10 | 国立がんセンター総長が代表する日本国 | Neuroinvasion inhibitor |
| WO2011149046A1 (en) | 2010-05-28 | 2011-12-01 | 独立行政法人国立がん研究センター | Therapeutic agent for pancreatic cancer |
| WO2011149051A1 (en) | 2010-05-28 | 2011-12-01 | 中外製薬株式会社 | Antitumor t cell response enhancer |
| EP4115906A1 (en) | 2010-05-28 | 2023-01-11 | Chugai Seiyaku Kabushiki Kaisha | Antitumor t cell response enhancer |
| WO2014200018A1 (en) | 2013-06-11 | 2014-12-18 | 独立行政法人 国立精神・神経医療研究センター | Method for predicting post-therapy prognosis of relapsing-remitting multiple sclerosis (rrms) patient, and method for determining applicability of novel therapy |
| US10519232B2 (en) | 2014-01-22 | 2019-12-31 | Sorbonne Universite | Agents for use in the treatment of retinal inflammation |
| EP2898896A1 (en) | 2014-01-22 | 2015-07-29 | Université Pierre et Marie Curie (Paris 6) | Agents for use in the treatment of retinal inflammation |
| EP4269440A2 (en) | 2015-02-27 | 2023-11-01 | Chugai Seiyaku Kabushiki Kaisha | Composition for treating il-6-related diseases |
| WO2018203545A1 (en) | 2017-05-02 | 2018-11-08 | 国立研究開発法人国立精神・神経医療研究センター | Method for predicting and evaluating therapeutic effect in diseases related to il-6 and neutrophils |
| WO2019151418A1 (en) | 2018-01-31 | 2019-08-08 | 元一 加藤 | Therapeutic agent for asthma containing il-6 inhibitor |
| WO2020213665A1 (en) | 2019-04-17 | 2020-10-22 | 国立大学法人広島大学 | Therapeutic agent for urological cancer which is characterized by being administered with il-6 inhibitor and ccr2 inhibitor in combination |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2177837A1 (en) | 1996-06-07 |
| AU4186696A (en) | 1996-06-26 |
| ITRM940794A1 (en) | 1996-06-06 |
| IT1274350B (en) | 1997-07-17 |
| ITRM940794A0 (en) | 1994-12-06 |
| CN1139933A (en) | 1997-01-08 |
| WO1996017869A3 (en) | 1996-08-29 |
| JPH09503232A (en) | 1997-03-31 |
| EP0742794A1 (en) | 1996-11-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO1996017869A2 (en) | Interleukin-6 (il-6) antagonists | |
| RU2135584C1 (en) | Homodimer p40 of interleukin-12 | |
| Hammacher et al. | Structure‐function analysis of human IL‐6: identification of two distinct regions that are important for receptor binding | |
| JP2657221B2 (en) | Human Fcγ receptor III | |
| EP0673420B1 (en) | Mammalian receptors for interleukin-10 (il-10) | |
| US6232446B1 (en) | TNF ligands | |
| JP2007267750A (en) | Isolated nucleic acid molecules which encode soluble il-tif/il-22 receptor or binding protein which binds to il-tif/il-22, and uses thereof | |
| CA2062975A1 (en) | Antagonists of gm-csf derived from the carboxyl terminus | |
| EP2292760A2 (en) | Mammalian cytokines, receptors, related reagents and methods | |
| EP0790305B1 (en) | Mutant human growth hormones and their uses | |
| JP3907661B2 (en) | INTERFERON-α / β BINDING PROTEIN, PROCESS FOR PRODUCING THE SAME AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME | |
| US6171824B1 (en) | Hybrid cytokines | |
| JP2002512782A (en) | G-CSF receptor agonist antibody and screening method thereof | |
| Moritz et al. | The N-terminus of gp130 is critical for the formation of the high-affinity interleukin-6 receptor complex | |
| US6280975B1 (en) | IL-6 mutein and DNA encoding thereto | |
| EP0769055A1 (en) | Variants of leukemia inhibitory factor | |
| CA2124338A1 (en) | Region of cytoplasmic domain of the human interleukin-4 receptor, as antagonists of il-4 | |
| WO2004028555A1 (en) | Resistin binding proteins, their preparation and use | |
| IL126484A (en) | Il-6 mutein, its preparation and pharmaceutical compositions containing it |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WWE | Wipo information: entry into national phase |
Ref document number: 95191457.X Country of ref document: CN |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 1995940401 Country of ref document: EP |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2177837 Country of ref document: CA |
|
| AK | Designated states |
Kind code of ref document: A2 Designated state(s): AU CA CN JP US |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| ENP | Entry into the national phase |
Ref document number: 1996 687490 Country of ref document: US Date of ref document: 19961016 Kind code of ref document: A |
|
| WWP | Wipo information: published in national office |
Ref document number: 1995940401 Country of ref document: EP |
|
| WWW | Wipo information: withdrawn in national office |
Ref document number: 1995940401 Country of ref document: EP |