EP4153613A1 - Il1-r1 derived inhibitor of il-1b and use thereof - Google Patents
Il1-r1 derived inhibitor of il-1b and use thereofInfo
- Publication number
- EP4153613A1 EP4153613A1 EP20936359.7A EP20936359A EP4153613A1 EP 4153613 A1 EP4153613 A1 EP 4153613A1 EP 20936359 A EP20936359 A EP 20936359A EP 4153613 A1 EP4153613 A1 EP 4153613A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- disease
- composition
- seq
- human
- polypeptide
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 239000003112 inhibitor Substances 0.000 title description 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 71
- 239000000203 mixture Substances 0.000 claims abstract description 70
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 65
- 101001033249 Homo sapiens Interleukin-1 beta Proteins 0.000 claims abstract description 20
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 16
- 230000000694 effects Effects 0.000 claims abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 84
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 79
- 229920001184 polypeptide Polymers 0.000 claims description 77
- 238000000034 method Methods 0.000 claims description 48
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 44
- 201000010099 disease Diseases 0.000 claims description 42
- 208000032672 Histiocytosis haematophagic Diseases 0.000 claims description 24
- 150000001413 amino acids Chemical group 0.000 claims description 19
- 208000022993 cryopyrin-associated periodic syndrome Diseases 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 206010016207 Familial Mediterranean fever Diseases 0.000 claims description 15
- 208000036066 Hemophagocytic Lymphohistiocytosis Diseases 0.000 claims description 12
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 12
- 208000004987 Macrophage activation syndrome Diseases 0.000 claims description 12
- 208000006117 ST-elevation myocardial infarction Diseases 0.000 claims description 12
- 208000020832 chronic kidney disease Diseases 0.000 claims description 12
- 208000014752 hemophagocytic syndrome Diseases 0.000 claims description 12
- 102000004127 Cytokines Human genes 0.000 claims description 10
- 108090000695 Cytokines Proteins 0.000 claims description 10
- 201000005569 Gout Diseases 0.000 claims description 9
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 9
- 206010012601 diabetes mellitus Diseases 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 208000027496 Behcet disease Diseases 0.000 claims description 7
- 208000009137 Behcet syndrome Diseases 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 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 claims description 7
- 206010003246 arthritis Diseases 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 208000002874 Acne Vulgaris Diseases 0.000 claims description 6
- 102100033887 Arylsulfatase D Human genes 0.000 claims description 6
- 201000001320 Atherosclerosis Diseases 0.000 claims description 6
- 206010012438 Dermatitis atopic Diseases 0.000 claims description 6
- 208000003556 Dry Eye Syndromes Diseases 0.000 claims description 6
- 206010013774 Dry eye Diseases 0.000 claims description 6
- 101000925559 Homo sapiens Arylsulfatase D Proteins 0.000 claims description 6
- 206010020751 Hypersensitivity Diseases 0.000 claims description 6
- 208000011200 Kawasaki disease Diseases 0.000 claims description 6
- 201000007100 Pharyngitis Diseases 0.000 claims description 6
- 206010037660 Pyrexia Diseases 0.000 claims description 6
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 claims description 6
- 201000010848 Schnitzler Syndrome Diseases 0.000 claims description 6
- 206010039710 Scleroderma Diseases 0.000 claims description 6
- 206010046851 Uveitis Diseases 0.000 claims description 6
- 206010000496 acne Diseases 0.000 claims description 6
- 208000026935 allergic disease Diseases 0.000 claims description 6
- 230000007815 allergy Effects 0.000 claims description 6
- 208000002399 aphthous stomatitis Diseases 0.000 claims description 6
- 201000008937 atopic dermatitis Diseases 0.000 claims description 6
- 229960002885 histidine Drugs 0.000 claims description 6
- 201000003265 lymphadenitis Diseases 0.000 claims description 6
- 208000001725 mucocutaneous lymph node syndrome Diseases 0.000 claims description 6
- 208000008494 pericarditis Diseases 0.000 claims description 6
- 230000002980 postoperative effect Effects 0.000 claims description 6
- 208000009954 pyoderma gangrenosum Diseases 0.000 claims description 6
- 230000000306 recurrent effect Effects 0.000 claims description 6
- 208000011580 syndromic disease Diseases 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 230000001154 acute effect Effects 0.000 claims description 5
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 5
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 claims 1
- 201000000028 adult respiratory distress syndrome Diseases 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 19
- 230000027455 binding Effects 0.000 abstract description 16
- 239000012634 fragment Substances 0.000 abstract description 8
- 101001076418 Homo sapiens Interleukin-1 receptor type 1 Proteins 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 241000282412 Homo Species 0.000 abstract description 3
- 239000000539 dimer Substances 0.000 abstract description 3
- 101710180389 Interleukin-1 receptor accessory protein Proteins 0.000 abstract description 2
- 101000960952 Homo sapiens Interleukin-1 receptor accessory protein Proteins 0.000 abstract 1
- 102000044594 Interleukin-1 Receptor Accessory Human genes 0.000 abstract 1
- 230000006806 disease prevention Effects 0.000 abstract 1
- 102000046828 human IL1RAP Human genes 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 55
- 239000000833 heterodimer Substances 0.000 description 48
- 125000003275 alpha amino acid group Chemical group 0.000 description 29
- 102000000589 Interleukin-1 Human genes 0.000 description 24
- 108010002352 Interleukin-1 Proteins 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 19
- 229940024606 amino acid Drugs 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- 108090001005 Interleukin-6 Proteins 0.000 description 11
- 102000004889 Interleukin-6 Human genes 0.000 description 11
- 238000007920 subcutaneous administration Methods 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 230000004044 response Effects 0.000 description 8
- 230000028327 secretion Effects 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 241000282567 Macaca fascicularis Species 0.000 description 6
- 238000004364 calculation method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 238000000954 titration curve Methods 0.000 description 6
- 101100394073 Caenorhabditis elegans hil-1 gene Proteins 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000011664 signaling Effects 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 4
- 108050006617 Interleukin-1 receptor Proteins 0.000 description 4
- 102000019223 Interleukin-1 receptor Human genes 0.000 description 4
- 101001033286 Mus musculus Interleukin-1 beta Proteins 0.000 description 4
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 4
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 4
- 208000006011 Stroke Diseases 0.000 description 4
- 102000002689 Toll-like receptor Human genes 0.000 description 4
- 108020000411 Toll-like receptor Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 102000052611 human IL6 Human genes 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000012510 peptide mapping method Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 4
- 238000004885 tandem mass spectrometry Methods 0.000 description 4
- 108010039224 Amidophosphoribosyltransferase Proteins 0.000 description 3
- 108090000317 Chymotrypsin Proteins 0.000 description 3
- 101001076407 Homo sapiens Interleukin-1 receptor antagonist protein Proteins 0.000 description 3
- 101000977771 Homo sapiens Interleukin-1 receptor-associated kinase 4 Proteins 0.000 description 3
- 229940119178 Interleukin 1 receptor antagonist Drugs 0.000 description 3
- 102000051628 Interleukin-1 receptor antagonist Human genes 0.000 description 3
- 102100023533 Interleukin-1 receptor-associated kinase 4 Human genes 0.000 description 3
- 102100033500 Interleukin-33 Human genes 0.000 description 3
- 239000012901 Milli-Q water Substances 0.000 description 3
- 101100340743 Mus musculus Il1b gene Proteins 0.000 description 3
- 101001076414 Mus musculus Interleukin-6 Proteins 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- 102000003945 NF-kappa B Human genes 0.000 description 3
- 108010057466 NF-kappa B Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000005571 anion exchange chromatography Methods 0.000 description 3
- 238000011953 bioanalysis Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 229960002376 chymotrypsin Drugs 0.000 description 3
- 230000021615 conjugation Effects 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 239000003407 interleukin 1 receptor blocking agent Substances 0.000 description 3
- 230000017306 interleukin-6 production Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 108010046141 rilonacept Proteins 0.000 description 3
- 229960001886 rilonacept Drugs 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001839 systemic circulation Effects 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- JTTIOYHBNXDJOD-UHFFFAOYSA-N 2,4,6-triaminopyrimidine Chemical compound NC1=CC(N)=NC(N)=N1 JTTIOYHBNXDJOD-UHFFFAOYSA-N 0.000 description 2
- PMUNIMVZCACZBB-UHFFFAOYSA-N 2-hydroxyethylazanium;chloride Chemical compound Cl.NCCO PMUNIMVZCACZBB-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 208000011594 Autoinflammatory disease Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 208000035690 Familial cold urticaria Diseases 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 101000852255 Homo sapiens Interleukin-1 receptor-associated kinase-like 2 Proteins 0.000 description 2
- 101000724418 Homo sapiens Neutral amino acid transporter B(0) Proteins 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- -1 IL-1β Proteins 0.000 description 2
- 102100039065 Interleukin-1 beta Human genes 0.000 description 2
- 102100036433 Interleukin-1 receptor-associated kinase-like 2 Human genes 0.000 description 2
- 108010067003 Interleukin-33 Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 201000002795 Muckle-Wells syndrome Diseases 0.000 description 2
- 102100028267 Neutral amino acid transporter B(0) Human genes 0.000 description 2
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 108010018242 Transcription Factor AP-1 Proteins 0.000 description 2
- 102100023132 Transcription factor Jun Human genes 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000000429 assembly Methods 0.000 description 2
- 230000000712 assembly Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 238000012754 cardiac puncture Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229940000406 drug candidate Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 206010064570 familial cold autoinflammatory syndrome Diseases 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000007115 recruitment Effects 0.000 description 2
- 239000012146 running buffer Substances 0.000 description 2
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- KWTQSFXGGICVPE-UHFFFAOYSA-N 2-amino-5-(diaminomethylideneamino)pentanoic acid;hydron;chloride Chemical compound Cl.OC(=O)C(N)CCCN=C(N)N KWTQSFXGGICVPE-UHFFFAOYSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 1
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 102100034428 Dual specificity protein phosphatase 1 Human genes 0.000 description 1
- 101710132784 Dual specificity protein phosphatase 1 Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101100001678 Emericella variicolor andM gene Proteins 0.000 description 1
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000998137 Homo sapiens Interleukin-33 Proteins 0.000 description 1
- 102000039996 IL-1 family Human genes 0.000 description 1
- 108091069196 IL-1 family Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102100020881 Interleukin-1 alpha Human genes 0.000 description 1
- 102100039880 Interleukin-1 receptor accessory protein Human genes 0.000 description 1
- 101710144554 Interleukin-1 receptor antagonist protein Proteins 0.000 description 1
- 102100026018 Interleukin-1 receptor antagonist protein Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 102000004890 Interleukin-8 Human genes 0.000 description 1
- 102000001702 Intracellular Signaling Peptides and Proteins Human genes 0.000 description 1
- 108010068964 Intracellular Signaling Peptides and Proteins Proteins 0.000 description 1
- 101150102972 Irak4 gene Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 102000002569 MAP Kinase Kinase 4 Human genes 0.000 description 1
- 108010068304 MAP Kinase Kinase 4 Proteins 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 101001033288 Macaca mulatta Interleukin-1 beta Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 1
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 description 1
- 108010025832 RANK Ligand Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 102000003714 TNF receptor-associated factor 6 Human genes 0.000 description 1
- 108090000009 TNF receptor-associated factor 6 Proteins 0.000 description 1
- 102000002933 Thioredoxin Human genes 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100024568 Tumor necrosis factor ligand superfamily member 11 Human genes 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 229940094361 arcalyst Drugs 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000007623 carbamidomethylation reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000022811 deglycosylation Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000447 dimerizing effect Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000008622 extracellular signaling Effects 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000011990 functional testing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 102000005396 glutamine synthetase Human genes 0.000 description 1
- 108020002326 glutamine synthetase Proteins 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 238000012531 mass spectrometric analysis of intact mass Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 102000002574 p38 Mitogen-Activated Protein Kinases Human genes 0.000 description 1
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 108700015048 receptor decoy activity proteins Proteins 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010846 tandem mass spectrometry analysis Methods 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 239000003643 water by type Substances 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
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- the invention relates to the field of biological pharmaceuticals as well as their use in conditions associated with inflammatory disorders (e.g. rheumatoid arthritis, Crohn’s disease, etc.), diabetes, cardiovascular disease and gout. More specifically, the invention relates to a heterodimeric IL-lRl/lL-lRAcP -derived composition that is capable of inhibiting IL-1 ⁇ cytokine.
- the interleukin-1 (IL-1) family of cytokines comprises 11 proteins (IL-1F1 to IL-1F11) encoded by 11 distinct genes in humans and mice.
- IL-l-type cytokines are major mediators of innate immune reactions, and blockade of the founding members IL-1 or IL-1 ⁇ by the interleukin- 1 receptor antagonist (IL-1RA) has demonstrated a central role of IL-1 in a number of human autoinflammatory diseases.
- IL-1 or IL-1 ⁇ rapidly increase messenger RNA expression of hundreds of genes in multiple different cell types.
- the potent proinflammatory activities of IL-1 and IL-1 ⁇ are restricted at three major levels: (i) synthesis and release, (ii) membrane receptors, and (iii) intracellular signal transduction.
- This pathway summarizes extracellular and intracellular signaling of IL-1 or IL-1 ⁇ , including positive- and negative-feedback mechanisms that amplify or terminate the IL-1 response.
- a complex sequence of combinatorial phosphorylation and ubiquitination events results in activation of nuclear factor kappa-B signaling and the JNK and p38 mitogen-activated protein kinase pathways, which, cooperatively, induce the expression of canonical IL-1 target genes (such as IL-6, IL-8, MCP-1, COX-2, IB, IL-1, IL-1 ⁇ , MKP-1) by transcriptional and posttranscriptional mechanisms.
- canonical IL-1 target genes such as IL-6, IL-8, MCP-1, COX-2, IB, IL-1, IL-1 ⁇ , MKP-1
- IL-1 interleukin-1
- IL-18 and IL-33 cytokines
- TLRs Toll-like-receptors
- cytotoxic stresses see Weber A, et ah, Sci Signal., 2010 Jan 19;3(105), the entire teachings of which are incorporated by reference herein).
- IL-1 and IL-1 ⁇ independently bind the type I IL-1 receptor (IL-lRl), which is ubiquitously expressed.
- a third specific ligand, the IL-1 receptor antagonist (IL-IRA) binds the IL-1RI with similar specificity and affinity but does not activate the receptor and trigger downstream signaling.
- the IL-1 receptor accessory protein (IL-lRAcP) serves as a co-receptor that is required for signal transduction of IL-l/IL-lRI complexes, and this co-receptor is also necessary for activation of IL- 1R1 by other IL-1 family members, in particular IL-18 and IL-33.
- the type II IL-1 receptor (IL- 1R2) binds IL-1 and IL-1 ⁇ but lacks a signaling-competent cytosolic part and thus serves as a decoy receptor.
- the IL-1RA, the plasma membrane-anchored IL-1R2, and the naturally occurring "shed" domains of each of the extracellular IL-1 receptor chains (termed sIL-lRI, sIL-lRII, and sIL-lRAcP, where "s” stands for soluble) provide inducible negative regulators of IL-1 signaling in the extracellular space whose abundance, which is regulated by a combination of increased transcription and controlled release, can limit or terminate IL-1 effects.
- the initial step in IL-1 signal transduction is a ligand-induced conformational change in the first extracellular domain of the IL-IRI that facilitates recruitment of IL-lRacP.
- TIR Toll- and IL-lR-like domains
- the trimeric complex rapidly assembles two intracellular signaling proteins, myeloid differentiation primary response gene 88 (MYD88) and interleukin- 1 receptor-activated protein kinase (IRAK) 4.
- MYD88 myeloid differentiation primary response gene 88
- IRAK interleukin- 1 receptor-activated protein kinase
- Mice lacking MYD88 or IRAK4 show severe defects in IL-1 signaling.
- humans with mutations in the IRAK4 gene have defects in IL-IRI and Toll-like receptor (TLR) signaling.
- IL-1, IL-IRI, IL-RAcP, MYD88, and IRAK4 form a stable IL-l-induced first signaling module.
- This is paralleled by the (auto)phosphorylation of IRAK4, which subsequently phosphorylates IRAKI and IRAK2, and then this is followed by the recruitment and oligomerization of tumor necrosis factor-associated factor (TRAF) 6.
- IRAKI and 2 function as both adaptors and protein kinases to transmit downstream signals.
- Complexes of IRAKI, IRAK2, and TRAF6 dissociate from the initial receptor complex, and cells lacking these proteins have impaired activation of the transcription factors nuclear factor kappa-B (NF-kappa-B) and activator protein 1 (AP-1).
- IL-1 has been linked to the pathology of diabetes, cardiovascular disease, gout, certain types of arthritis (e.g. rheumatoid arthritis (RA)), as well as a number of less prevalent autoimmune diseases, such as familial Mediterranean fever (FMF), Behcet disease, etc. (Ozen S, Bilginer Y. “A clinical guide to autoinflammatory diseases: familial Mediterranean fever and next-of-kin”, Nat. Rev.
- Rilonacept is an IL-1 antagonist which includes an IL-l-specific fusion protein which comprises an IL-1 binding portion of the extracellular domain of human ILl-RAcP, an IL-1 binding portion of the extracellular domain of human IL-IRI, and a multimerizing component.
- This IL-l-specific fusion protein is described in U.S. Pat. No. 6,472,179, U.S. patent publication No. 2003/0143697, published 31 Jul. 2003, U.S. Pat. No. 7,361,350, and U.S. patent publication No. 2005/0197293, published 8 Sep. 2005 (all of which are incorporated by reference herein in their entirety).
- Rilonacept under the trade name ARCALYST was approved by U.S.
- FDA Food and Drug Administration
- Cryopyrin-Associated Periodic Syndromes including Familial Cold Auto-inflammatory Syndrome (FCAS) and Muckle-Wells Syndrome (MWS) in adults and children 12 and older.
- FCAS Familial Cold Auto-inflammatory Syndrome
- MFS Muckle-Wells Syndrome
- the present invention provides for a heterodimeric protein composition capable of binding human IL-1 ⁇ (GenBank: AAH08678.1).
- the protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 18 through 333 of human IL1-R1 (GenBank: AAM88423.1), and a second amino acid sequence which contains a first mutant of a Fc portion of human immunoglobulin gamma- 1 Fc (GenBank: J00228.1).
- the protein composition also comprises a second polypeptide which includes another first amino acid sequence containing amino acids 21 through 358 of human ILl-RAcP (GenBank: BAA25421.1), and another second amino acid sequence which contains a second mutant of the Fc portion of human immunoglobulin gamma-1 Fc.
- the first and second mutants are selected as to favor heterodimeric assembly between the first and second mutants over any homodimeric assembly.
- the protein composition may be capable of exhibiting human IL- 1 ⁇ /IL- l F2 binding activity with a Kd valus of no more than about 10 U M.
- the first polypeptide of the protein composition may contain amino acid sequence of SEQ ID NO. 1, while the second polypeptide may contain amino acid sequence of SEQ ID NO. 2.
- the present invention provides for a heterodimeric protein composition, containing a first polypeptide including amino acid sequence of SEQ ID NO. 8 and a second polypeptide including amino acid sequence of SEQ ID NO. 9.
- the present invention provides for a therapeutic composition which contains a heterodimeric protein composition, including a first polypeptide containing amino acid sequence of SEQ ID NO. 8 and a second polypeptide containing amino acid sequence of SEQ ID NO. 9.
- the therapeutic composition may also contain about 6% (m/v) sucrose, about 3% (m/v) polyethylene glycol having an average molecular weight of 3350 Da, about 50 mM sodium chloride, and about 20 mM L-Histidine pH from about 4.5 to about 7.0.
- the pH value may be about 6.5.
- the present invention provides for a therapeutic composition.
- the therapeutic composition comprises a heterodimeric protein composition capable of binding human IL-1 ⁇ .
- the protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 18 through 333 of human IL1-R1, and a second amino acid sequence which contains a first mutant of the Fc portion of human immunoglobulin gamma-1 Fc.
- the protein composition also comprises a second polypeptide which includes another first amino acid sequence containing amino acids 21 through 358 of human ILl-RAcP, and another second amino acid sequence which contains a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc.
- the first and second mutants are selected as to favor heterodimeric assembly between the first and second mutants over any homodimeric assembly.
- the protein composition may be capable of exhibiting human IL-1 ⁇ /IL- l F2 binding activity with a Kd values of no more than about 10 -11 M.
- the therapeutic composition may exhibit a half- life of the heterodimeric protein composition in systemic circulation in mice after a subcutaneous administration at a dose of 5 mg/kg of at least about 97 hours, as assayed by human Fc ELISA.
- the therapeutic composition may exhibit a half-life of the heterodimeric protein composition in systemic circulation in Cynomolgus monkeys after a subcutaneous administration at a dose of 10 mg/kg of at least about 3 days, as assayed by human Fc ELISA.
- the therapeutic composition may comprise a heterodimeric protein comprised of a first polypeptide containing amino acid sequence of SEQ ID NO. 1 and a second polypeptide containing amino acid sequence of SEQ ID NO. 2.
- the therapeutic composition may also contain about 6% (m/v) sucrose, about 3% (m/v) polyethylene glycol with an average molecular weight of about 3350 Da, about 50 mM sodium chloride, and about 20 mM L-Histidine pH 6.5.
- the present invention provides for a therapeutic composition which contains a heterodimeric protein composition, including a first polypeptide containing amino acid sequence of SEQ ID NO. 8 and a second polypeptide containing amino acid sequence of SEQ ID NO. 9.
- the therapeutic composition may also contain about 6% (m/v) sucrose, about 3% (m/v) polyethylene glycol having an average molecular weight of 3350 Da, about 50 mM sodium chloride, and about 20 mM L-Histidine pH from about 4.5 to about 7.0.
- the pH value may be about 6.5.
- the present teachings provide for a substance or a composition containing a heterodimeric protein assembly including a polypeptide of SEQ ID NO. 8 and another polypeptide of SEQ ID NO. 9 for use in the treatment of certain disorders or diseases associated with IL-1 ⁇ modulation, including, but not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST- elevation myocardial infarction (STEMI), acute respiratory distress syndrom e/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytos
- the present teachings provide for a method of treating or preventing a disease or condition associated with modulation of activity of human IL-1 ⁇ .
- the method includes administering to a patient in need for treating or preventing a disease associated with modulation of activity of human IL-1 ⁇ a therapeutically effective amount of a pharmaceutical composition including a heterodimeric protein containing a first polypeptide including amino acid sequence of SEQ ID NO. 8 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 9.
- Diseases associated with IL-1 ⁇ modulation include, but are not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-elevation myocardial infarction (STEMI), acute respiratory distress syndrom e/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet disease, breast cancer, non-small cell lung cancer,
- Figure 1 illustratively shows a heterodimeric protein assembly of the present teachings comprising an extracellular portion of IL1-R1 fused with an IgG-Fc domain (Fc-II) via a flexible linker and an extracellular portion of ILl-RAcP fused with another IgG-Fc domain (Fc-V) via another flexible linker;
- Fc-II IgG-Fc domain
- Fc-V IgG-Fc domain
- Figure 2 shows a representative series of buffer-normalized sensograms at various concentrations of IL-1 ⁇ /IL-lF2, the lowest curve represents IL-1 ⁇ /IL- l F2 concentration of 0.919 nM and each subsequent curve represents 1.838, 3.676, 7.35, 14.7 and 29.4 nM respectively;
- Figure 3 shows a representative IL1 binding data, relative response was calculated by subtraction of ‘buffer only’ background, error bars reflect standard deviation values calculated by Bioacore T200 Evaluation Software package;
- Figure 4 shows representative ‘Response vs. Concentration’ curve, concentration of IL- 1 ⁇ /IL- l F2 is shown on the X-axis in Mol and Response in RU (Req) is shown on the Y-axis;
- Figure 5 shows concentration of ILlR-FcV-RAcP-FcII heterodimer (in ng/ml) in the serum of the initial set of three Cynomolgus Monkey after a single subcutaneous administration at a dose of 10 mg/kg (vertical bars represent standard deviation values at various time points);
- Figure 6 shows concentration of ILlR-FcV-RAcP-FcII heterodimer (in ng/ml) in the serum of the follow-up set of three Cynomolgus Monkey after a single subcutaneous administration at a dose of 10 mg/kg, the three curves shown represent measurements taken from three individual animals designated FI 290, F1269 and F 1254;
- Figure 7 shows ILlR-FcV-RAcP-FcII heterodimer titration curve of mouse IL6 secretion induced by mouse IL-1B /IL-lF2in MEFs, the insert table shows curve fitting results using 4- parameter algorithm and curve interpolation for determination of the IC50 value;
- Figure 8 shows ILlR-FcV-RAcP-FcII heterodimer titration curve of human IL6 secretion induced by human IL-IB /IL-1F2 in MRC5 cells, the insert table shows curve fitting results using 4-parameter algorithm and curve interpolation for determination of the IC50 value;
- Figure 9 shows ILlR-FcV-RAcP-FcII heterodimer titration curve of human IL6 secretion induced by M. Rhesus IL-IB /IL-lF2in MRC5 cells, the insert table shows curve fitting results using 4-parameter algorithm and curve interpolation for determination of the IC50 value.
- the teachings disclosed herein are based, in part, upon engineering of a heterodimeric protein assembly that is capable of binding to human IL-1 ⁇ and attenuating its function.
- the heterodimeric protein assembly of the present teachings comprises extracellular portions of ILl-Rl (GenBank: AAM88423.1) and of IL-lRAcP (GenBank: BAA25421.1), or functional fragments thereof. Each, the ILl-Rl portion and the IL-lRAcP portion, is fused to a distinct mutant of Fc portion of the human Ig Gamma-1 (GenBank: J00228.1).
- the two distinct Fc mutants in the heterodimeric protein assembly are engineered as to favor the heteromeric dimer formation between the two Fc mutants over any homomeric assembly.
- a DNA expression vector has been constructed for overproducing the heterodimeric protein assembly in a heterologous protein expression system, and mammalian cells have been prepared stably expressing the heterodimeric protein assembly to a high expression level.
- a protein purification procedure has been devised allowing obtaining a physiologically relevant substantially pure preparation of the heterodimeric protein assembly of the present teachings.
- purified protein molecule demonstrates a high degree of specific activity in an in vitro Enzyme-Linked Immunosorbent Assay (ELISA) using human IL- 1b (GenBank: AAH08678.1).
- the protein molecule exhibits an acceptable pharmacokinetics profile upon subcutaneous animal administration, while not resulting in any body weight loss or adverse clinical events.
- Design, preparation and preliminary characterization of composition of matter of the present teachings are disclosed, in part, in an International Patent Application Publication No. WO/2014/035361, published on March 6, 2014, and International Patent Application Serial No. PCT/US/2013/026349, filed on February 15, 2013, both of which are incorporated herein by reference in their entirety.
- the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
- the methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants).
- Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
- sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
- sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
- the methods of the invention may include statistical calculations, e.g. determination of IC50 or EC50 values, etc..
- the skilled artisan can readily appreciate that such can be performed using a variety of commercially available software, e.g. PRISM (GraphPad Software Inc, La Jolla, CA, USA) or similar.
- homologous in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from super families in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.
- homologous when modified with an adverb such as "highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
- sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
- polypeptides described herein may be comprised of more than one contiguous amino acid chain, thus forming dimers or other oligomeric formations.
- the polypeptides of the present teachings for use in mammals are expressed in mammalian cells that allow for proper post-translational modifications, such as CHO or HEK293 cell lines, although other mammalian expression cell lines are expected to be useful as well. It is therefore anticipated that the polypeptides of the present teachings may be post-translationally modified without substantially effecting its biological function.
- fusion proteins having at least a biologically active portion of the human IL1-R1 or IL-lRAcP or a functional fragment thereof, and one or more fusion domains.
- fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an Fc), maltose binding protein (MBP), or human serum albumin.
- a fusion domain may be selected so as to confer a desired property.
- the ILl-Rl or IL- lRAcP polypeptide portions may be fused with a domain that stabilizes the ILl-Rl or IL-lRAcP polypeptides in vivo (a "stabilizer” domain), optionally via a suitable peptide linker.
- a stabilizer means anything that increases the half life of a polypeptide in systemic circulation, regardless of whether this is because of decreased destruction, decreased clearance, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on certain proteins. Likewise, fusions to human serum albumin can confer desirable properties.
- fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains that confer an additional biological function, e.g. promoting accumulation at the targeted site of action in vivo.
- multimerizing e.g., dimerizing, tetramerizing
- functional domains that confer an additional biological function, e.g. promoting accumulation at the targeted site of action in vivo.
- the heterodimeric protein assemblies of the present teachings comprise an extracellular portion of IL1-R1, or a functional fragment thereof, fused with a IgG-Fc domain, and an extracellular portion IL-lRAcP, or a functional fragment thereof, fused with another IgG-Fc domain.
- the IgG-Fc domain and the another IgG-Fc domain are chosen as to favor a heterodimeric protein assembly over any homodimeric protein assembly.
- the extracellular portion of IL1-R1 may be fused with the IgG-Fc domain via a flexible linker, while IL-lRAcP, or a functional fragment thereof, may be fused with the another IgG-Fc domain via the flexible linker of the same amino acid sequence or via another flexible linker.
- the extracellular portion of ILl-Rl fused with IgG-Fc domain (Fc-II) via a flexible linker may comprise the amino acid sequence of SEQ ID NO. 1
- IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker may comprise the amino acid sequence of SEQ. ID NO. 2.
- hlLl-Rl-hlgGl-Fc polypeptide SEQ ID NO. 1
- the present teachings provides for a recombinant DNA molecule having an open reading frame coding for a polypeptide comprising the leading 333 amino acids of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker, and for another recombinant DNA molecule having an open reading frame coding for another polypeptide comprising the leading 358 amino acids of the human IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker.
- the polypeptide comprising the leading 333 amino acids of the human ILl-Rl fused with IgG-Fc domain (Fc-II) via a flexible linker comprises the amino acid sequence of SEQ. ID NO. 3.
- the corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO. 4.
- the another polypeptide comprises the leading 358 amino acids of the human IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker may comprise the amino acid sequence of SEQ. ID NO. 5.
- the corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO. 6.
- hlLl-Rl-hlgGl-Fc polypeptide SEQ ID NO. 3
- FPPKPKDTLM ISRTPEVTCV W DVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV 420 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSRDELTKNQ 480 VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFKLVSKLTV DKSRWQQGNV 540 FSCSV HEAL HNHYTQKSLS LSPGK 565 hlLl-Rl-hlgGl-Fc DNA (SEQ ID NO. 4)
- CTGTCTCCGG GTAAA 1695 hIL- 1 RAcP-hlgG 1 -F c polypeptide (SEQ ID NO. 5) TLLWCW SL YFYGILQSDA SERCDDWGLD T RQIQVFED EPARIKCPLF EHFLKFNYST 60 AHSAGLTLIW YWTRQDRDLE EPINFRLPEN RISKEKDVLW FRPTLLNDTG NYTC LRNTT 120 YCSKVAFPLE VVQKDSCFNS P KLPVHKLY IEYGIQRITC PNVDGYFPSS VKPTITWY G 180 CYKIQNFN V IPEG NLSFL IALISNNGNY TCVVTYPENG RTFHLTRTLT VKVVGSPKNA 240 VPPVIHSPND HW YEKEPGE ELLIPCTVYF SFL DSRNEV WWTIDGKKPD DITIDVTINE 300 SISHSRTEDE TRTQILSIKK VTSEDLKRSY VCHAR
- the present invention provides for a recombinant mammalian expression plasmid for high expression of a polypeptide comprising the leading 333 amino acids of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker, and for another recombinant DNA molecule having an open reading frame coding for another polypeptide comprising the leading 358 amino acids of the human IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker.
- This plasmid comprises two cytomegalovirus (CMV) promoters to drive transcription of the two genes coding for said polypeptide and said another polypeptide, each followed by a transcription termination sequence and a polyadenylation sequence.
- CMV cytomegalovirus
- the plasmid also contains an origin of replication and a gene conferring ampicillin resistance, for supporting plasmid propagation and selection in bacteria.
- the plasmid further contains a gene for Glutamine synthetase, a selectable marker widely used for establishing stable CHOK1 and NSO cell lines.
- the mammalian expression plasmid of the present teachings comprises the nucleotide sequence of SEQ ID NO. 7.
- hIL 1 -R 1 -hlgG 1 -F c-II/ IL-lRAcP- hlgGl-Fc-V expression plasmid (SEQ ID NO. 7)
- the present teachings provide for a mammalian expression system for production of a heterodimeric protein assembly comprising a polypeptide comprising amino acid residues 18 through 333 of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker, and another polypeptide comprising amino acid residues 21 through 358 of the human IL- lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker.
- the mammalian expression system of the present teachings comprises Chinese hamster ovary cells (CHO-K1) harboring a plasmid comprising nucleotide sequence of SEQ ID NO. 7.
- the mammalian expression system of the present teachings yields a heterodimeric protein assembly comprising a polypeptide of SEQ ID NO. 8 and another polypeptide of SEQ ID NO. 9.
- hlLl-Rl-hlgGl-Fc polypeptide SEQ ID NO. 8.
- the present teachings provide for a substance or a composition, comprising a heterodimeric protein assembly comprising a polypeptide of SEQ ID NO. 8 and another polypeptide of SEQ ID NO. 9, for use in the treatment of certain disorders or diseases associated with IL-1 ⁇ modulation, including, but not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST- elevation myocardial infarction (STEMI), acute respiratory distress syndrom e/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistio
- the present teachings provide for a method of treating or preventing a disease or condition associated with modulation of activity of human IL-1 ⁇ .
- the method includes administering to a patient in need for treating or preventing a disease associated with modulation of activity of human IL-1 ⁇ a therapeutically effective amount of a pharmaceutical composition including a heterodimeric protein including a first polypeptide including amino acid sequence of SEQ ID NO. 8 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 9.
- Diseases associated with IL-1 ⁇ modulation include, but are not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-elevation myocardial infarction (STEMI), acute respiratory distress syndrom e/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet disease, breast cancer, non-small cell lung cancer,
- Example 1 Preparation of polypeptides of the present invention.
- hlLl-Rl-hlgGl-Fc polypeptide of SEQ ID NO. 1 and hlL-lRAcP-hlgGl-Fc polypeptide of SEQ ID NO. 2 were co-expressed in CHO-K1 using molecular biology, cell culture and protein biochemistry techniques known in the art and described in PCT Publication WO/2014/035361, and PCT Application Serial No. PCT/US/2013/026349.
- CHO-K1 cells expressing the polypeptides were harvested and lysed utilizing well established protocols.
- the supernatant containing expressed polypeptides was first applied to a Protein A affinity column.
- the pH adjusted Protein A column eluate was further purified by anion-exchange chromatography (AIEX) utilizing Q Sepharose resin.
- AIEX flowthrough was analyzed by size-exclusion HPLC (SEC-HPLC), SDS-PAGE and other analytical techniques, as appropriate.
- a therapeutic composition comprising hlLl-Rl-hlgGl-Fc and hlL- lRAcP-hlgGl-Fc polypeptides was formulated to contain 40 mg/ml of the polypeptides, 6% (m/v) sucrose, 3% (m/v) polyethylene (PEG) 3350, 50 mM sodium chloride, and 20 mM L-Histidine pH from about 4.5 to about 7.0, preferably about 6.5 .
- polypeptides contained in the final product were analyzed as outlined in the following example. Unexpectedly, the polypeptides in the final product predominantly contained hlLl-Rl-hlgGl-Fc polypeptide of SEQ ID NO. 8 and hlL-lRAcP-hlgGl-Fc polypeptide of SEQ ID NO. 9.
- Example 2 Peptide Mapping and Characterization of polypeptides of the present invention.
- Solvent B Acetonitrile (ACN) with 0.1% formic acid
- Tandem Mass Spectrometry Analysis Spectra were acquired using a QTOF 6550 mass spectrometer (Agilent Technologies, Santa Clara, CA). The mass spectrometer was operated in positive ion mode. Mass spectra were acquired over m/z 350-2000 at 20,000 resolution (m/z 1521) and data-dependent acquisition selected the top 10 most abundant precursor ions for tandem mass spectrometry by CID fragmentation using an isolation width of 4.0 Da, formula of (slope)*(m/z)/100 + offset was used for collision energy. Dynamic exclusion was used to minimize redundancy of MS/MS collection and maximize peptide identifications.
- a concatenated forward-reverse database was constructed to calculate the in situ false discovery rate (FDR). Cutoff scores were dynamically assigned to each data set to maintain the false discovery rate at less than 0.1% at the peptide level. Manual inspection was also applied for every uniquely identified peptides of each of the analyzed samples.
- Example 3 Evaluation of polypeptides of the present teachings affinity binding to RANKL using Surface Plasmon Resonance (SPR) assay.
- SPR Surface Plasmon Resonance
- the binding affinity of prepared polypeptides of ILlR-FcV-RAcP-FcII heterodimer to IL-1 ⁇ /IL-lF2 was measured using a specially designed Surface Plasmon Resonance (SPR) assay.
- SPR Surface Plasmon Resonance
- the assay was carried out using capturing method where anti human IgG were cross-linked to the surface of sensor chip for capturing ILlR-FcV-RAcP-FcII heterodimer via its IgG (Fc) fragments.
- Series of different concentrations of IL-1 ⁇ /IL-lF2 were used for calculation of the dissociation constant (Kd).
- BiaCore T200 Instrument # 12108, GE Healthcare, with Biacore T200 Control and Evaluation Software packages.
- ILlR-FcV-RAcP-FcII heterodimer stock solution 20 mg/ml of the polypeptides, 6% (m/v) sucrose, 3% (m/v) PEG3350, 50 mM sodium chloride, and 20 mM L-Histidine pH 6.5.
- IL-1 ⁇ /IL-lF2 Human recombinant, E.
- CM5 Sensor Chip was placed into the instrument and primed with Biacore running buffer, lx HBS-EP, for 6 min at 10 m ⁇ /min, repeated twice. All steps were carried out at 25°C. Channels 1 and 2 was used for the experiment and channels 3 and 4 were reserved as a backup;
- Anti -Human IgG from the kit 0.5 mg/ml in 0.15 M NaCl, was diluted 20-fold in Immobilization Buffer (10 mM Na-acetate pH 5.0) to a final concentration of 25 pg/ml;
- Reagents for immobilization procedure were prepared as follows: EDC (1 -ethyl-3 -(3- dimethylaminopropyl)-carbodiimide) - 0.4 M in Milli-Q water; NHS (N- hydroxysuccinimide) - 0.1 M in Milli-Q water; 1 M Ethanolamine-HCl pH 8.5 in Milli-Q water;
- Immobilization Anti -Human IgG were injected into the chip at 10 m ⁇ /min for 5 min;
- the chip was washed with lx HBS-EP 2 times at 10 m ⁇ /min for 6 min and then the “dry” working cycle without addition of any protein component was run twice.
- the working cycle consisted of Ligand (ILlR-FcV-RAcP-FcII heterodimer) Loading Step of 1 min, Wash Step of 3 min, Sample (IL-1) Loading Step of 1 min, Wash step of 16.7 min, Chip Regeneration Step, 1 min, 3 M MgCl 2 . All steps were run at 10 m ⁇ /min except Sample Loading Step that was run at 30 ⁇ l/min;
- the goal of this experiment was to measure association constant for ILlR-FcV-RAcP- FcII heterodimer and IL-1 ⁇ /IL- l F2.
- Anti-human IgG were covalently immobilized on CM5 Sensor Chip then ILlR-FcV-RAcP-FcII heterodimer was loaded and followed by various concentrations of human IL-1 ⁇ /IL- l F2 Series of sensograms were generated and used for calculation of Kd value.
- Human IL-1 ⁇ /IL- l F2 were used at the concentrations specified in Table 2 where concentration of 3.676 nM was run two time independently as an internal control for the instrument reproducibility;
- Kd Steady-State data analysis using 1 : 1 Langmuir binding model was used. According to this method, Kd is calculated from series of plots of steady-state analyte binding levels (Req) against concentration. The obtained data are summarized in Table 2.
- IL-1 ⁇ /IL- l F2 concentrations and binding (Relative Response). Standard Deviation values, %, were calculated by Biacore T200 Evaluation Software and then converted into Standard Deviation by multiplying Rmax* StDev %. The StDev values are plotted as error bars on Figure 3.
- Example 4 Pharmacokinetics (PK) of ILlR-FcV-RAcP-FcII heterodimer after subcutaneous administration in mice.
- Polypeptides of ILlR-FcV-RAcP-FcII heterodimer were co-expressed and purified essentially as described in the forgoing examples.
- the polypeptides were formulated in the following buffer: 1% w/v Sucrose, lOOmM Sodium Chloride, 20 mM L-Arginine Hydrochloride, 25 mM Sodium Bicarbonate, pH 6.3.
- the dosing stock concentration used was 0.5 mg/mL of the polypeptide.
- mice Fourteen male DBA/1 mice were randomized by body weight into seven groups of two animals on Day 0 of the study.
- a single dose of ILlR-FcV-RAcP-FcII heterodimer (5 mg/kg in 10 ml/kg) was administered subcutaneously (dorsally) on Day 0 to mice in six of the groups.
- the mice in the remaining group remained untreated and were bled via cardiac puncture for plasma preparation on Day 0 of the study.
- Plasma was prepared from blood samples collected from mice in the treated groups via the orbital sinus or terminal cardiac puncture at specified times throughout the study. Body weights were recorded for all animals on the treatment day (Day 0) and then three times per week, including the termination day of each group.
- Body weight change was not measured in groups culled for sample collection at 0 hours and within 36 hours of dose administration. Mean body weight loss between Day 0 and termination of the groups culled between 96 hours and 21 days post-dose was minimal. No mice lost body weight exceeding ethical limits.
- plasma samples were analyzed by Enzyme Linked Immunosorbent Assay (ELISA) for Hu-Fc proteins. Quantification of Hu-Fc in mouse plasma samples by ELISA was used as a read-out for circulating levels of ILlR-FcV-RAcP-FcII heterodimer. The assay was performed on samples from all mice in the study.
- ELISA Enzyme Linked Immunosorbent Assay
- the polypeptides (detected as Human-Fc protein) were detected in the plasma of animals at all time-points post-dose.
- One Phase Decay Model equation using Prism 5.0c (GraphPad Software Inc, La Jolla, CA, USA) was then used to determine pharmacokinetics of the polypeptides as detected by Hu-Fc ELISA.
- Peak circulating level of Hu-Fc (Cmax) was determined to be 1.284 ⁇ g/mL, and time to peak circulating levels (Tmax) was 24 hours post-dose.
- the half-life (Tl/2) was 97 hours, 31 minutes and the rate constant (K) was 0.0071 hr-1.
- Hu-Fc was below the level of detection in the plasma of the untreated animals. The results of the study are summarized in Table 4.
- an additional 3 male Cynomolgus monkeys received a single dose of ILlR-FcV-RAcP- FcII heterodimer by subcutaneous administration on Day 1 at a dose level of 10 mg/kg and blood samples were collected at designated time points until Day 21.
- the results of the bioanalysis from the follow-up additional set of three animals are shown in Figure 5. All the animals were observed once daily for any reactions to treatment during the study. Body weights were measured and recorded prior to dosing. Blood samples for pharmacokinetic analysis were collected at the designated time points. The collected serum samples were stored at -80 °C for bioanalysis. The determination of plasma concentrations of the polypeptides was performed using ELISA method.
- Cynomolgus monkeys (values in parenthesis are mean CV%) All animals were widely exposed to ILlR-FcV-RAcP-FcII heterodimer. The observed inter-individual variability was relatively high with a CV% of about 60%. The latter was explained by the lowest drug exposure found in animal F1290 ( Figure 6), which was at least 5-fold less exposed to ILlR-FcV-RAcP-FcII heterodimer than the remaining two animals. The maximal concentration (Cmax) was reached between 1st and 2nd days. The estimated Tl/2 was evaluated to be about 4 days.
- Example 6 Interspecies Specific Activity of of ILlR-FcV-RAcP-FcII heterodimer.
- ILlR-FcV-RAcP-FcII is a heterodimer comprised of soluble portions of human IL-IR and IL-lRAcP each linked to a unique IgGl Fc portion. Sequence alignment of the 333 amino acid portion of the human IL-IR with relevant portions from several species demonstrates only a modest sequence identity (-64%) with IL-IR portions from rodents (mouse, rat). However, the sequence identity is much higher between human IL-IR and those of other primates (e.g. 91% with marmoset monkey).
- Mouse Embryo Fibroblasts used for the experiments.
- DMEM Dulbecco’s Modification of Eagle’s Medium, high glucose (4.5 g/L), Invitrogen,
- IL-1 ⁇ IL-1F2 Human recombinant, E. coli-derived, Alai 17-Ser269, Accession # NP_000567, R&D systems, Cat # 201-LB, Lot # AD1412111
- IL-1 ⁇ IL-1F2 M. Rhesus recombinant, E. coli-derived, Alai 17-Ser269, Accession # P48090, R&D systems, Cat # 1318-RL, Lot # GUG0110111
- This assay employs the quantitative sandwich enzyme immunoassay technique.
- a monoclonal antibody specific for IL-6 has been pre-coated onto a microplate.
- Standards and samples are pipetted into the wells and any IL-6 present is bound by the immobilized antibody.
- an enzyme-linked polyclonal antibody specific for IL-6 is added to the wells.
- a substrate solution is added to the wells and color develops in proportion to the amount of IL-6 bound in the initial step. The color development is stopped and the intensity of the color is measured.
- the IL6 production data were calculated from the calibration curve shown on Figure 9.
- the insert table shows curve fitting results using 4-parameter algorithm and curve interpolation for determination of the IC50 value.
- the calculated ILlR-FcV-RAcP-FcII heterodimer IC50 value for mouse IL-1B /IL-1F2 is >210 ng/ml.
- ILlR-FcV-RAcP-FcII heterodimer is an efficient inhibitor of human IL-1 ⁇ /IL-1F2, but not mouse IL-IB /IL-1F2 signaling pathway: ILlR-FcV- RAcP-FcII heterodimer IC50 value for human IL-IB /IL-1F2 is 0.19 ng/ml and for mouse IL-IB /IL-1F2- >200 ng/ml (0.95 pM and >1000 pM respectively, assuming molecular mass of ILIR- FcV-RAcP-FcII heterodimer as 200 kDa).
- ILlR-FcV-RAcP-FcII heterodimer titration curve of human IL6 secretion induced by human IL-IB /IL-1F2 in MRC5 cells is shown in Figure 8.
- the calculated IC50 value of ILlR-FcV-RAcP-FcII heterodimer against human IL-IB /IL-1F2 (X- column in the Curve Interpolation table) is 0.22 ng/mL.
- ILlR-FcV-RAcP-FcII heterodimer titration curve of human IL6 secretion induced by M. Rhesus IL-IB /IL-1F2 in MRC5 cells is shown in Figure 9.
- the calculated ILlR-FcV-RAcP-FcII heterodimer IC50 value for human IL- IB /IL-1F2 is 0.38 ng/ml.
- IL-6 recovery from ILlR-FcV-RAcP-FcII heterodimer preparation with a final concentration of 200 ng/ml was 95%.
- ILlR-FcV-RAcP-FcII heterodimer is an efficient inhibitor of both human andM Rhesus IL-1B /IL-1F2 signaling pathway:
- ILlR-FcV-RAcP-FcII heterodimer IC50 value for human IL-1B /IL-1F2 is 0.19 ng/ml and for M.
- IL-6 production upon treatment of mouse or human cells with IL-IB /IL-1F2 was used a functional test for inhibitory properties of a novel drug candidate ILlR-FcV- RAcP-FcII heterodimer against human, mouse and M. Rhesus orthologs of IL-IB /IL-1F2.
- Suitable cell lines were identified and experimental conditions including cell density, treatment duration linear range for IL6 detection and were optimized for all three orthologs. The obtained data are summarized in Table 6.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Emergency Medicine (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Endocrinology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
Claims
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2020/034114 WO2021236091A1 (en) | 2020-05-22 | 2020-05-22 | Il1-r1 derived inhibitor of il-1b and use thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
EP4153613A1 true EP4153613A1 (en) | 2023-03-29 |
EP4153613A4 EP4153613A4 (en) | 2024-01-24 |
Family
ID=78708753
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP20936359.7A Pending EP4153613A4 (en) | 2020-05-22 | 2020-05-22 | Il1-r1 derived inhibitor of il-1b and use thereof |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP4153613A4 (en) |
JP (1) | JP2023527171A (en) |
CN (1) | CN115768787A (en) |
BR (1) | BR112022022089A2 (en) |
MX (1) | MX2022014410A (en) |
WO (1) | WO2021236091A1 (en) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3905921B2 (en) * | 1992-10-02 | 2007-04-18 | ジェネティクス インスチチュート リミテッド ライアビリティー カンパニー | COMPOSITION CONTAINING COAGULATION FACTOR VIII, METHOD FOR PRODUCING THE SAME, AND METHOD FOR USING SURFACTANT AS STABILIATOR |
JP6225197B2 (en) * | 2013-02-15 | 2017-11-01 | アール−ファーム・ジョイント・ストック・カンパニーR−Pharm,Jsc | IL-1β inhibitor composition and use thereof |
US11155600B2 (en) * | 2014-03-24 | 2021-10-26 | R-Pharm Overseas, Inc. | Human IL1-R1 derived inhibitor of IL-1β |
-
2020
- 2020-05-22 WO PCT/US2020/034114 patent/WO2021236091A1/en active Application Filing
- 2020-05-22 CN CN202080101065.9A patent/CN115768787A/en active Pending
- 2020-05-22 BR BR112022022089A patent/BR112022022089A2/en unknown
- 2020-05-22 EP EP20936359.7A patent/EP4153613A4/en active Pending
- 2020-05-22 MX MX2022014410A patent/MX2022014410A/en unknown
- 2020-05-22 JP JP2022571138A patent/JP2023527171A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
CN115768787A (en) | 2023-03-07 |
WO2021236091A1 (en) | 2021-11-25 |
BR112022022089A2 (en) | 2022-12-13 |
MX2022014410A (en) | 2022-12-06 |
EP4153613A4 (en) | 2024-01-24 |
JP2023527171A (en) | 2023-06-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2013378122B2 (en) | IL-1beta inhibitor composition and use thereof | |
CA2502385C (en) | Il-1 receptor based antagonists and methods of making and using | |
KR102424590B1 (en) | Fibronectin based scaffold domain proteins that bind to myostatin | |
CN101014617B (en) | Il-1 antagonist formulations | |
KR20180004094A (en) | Immunotherapeutics for cancer and autoimmune diseases | |
KR100511544B1 (en) | Recombinant il-5 antagonists useful in treatment of il-5 mediated disorders | |
FI119374B (en) | Recombinant IL-5 Antagonists Useful in the Treatment of IL-5 Related Diseases | |
KR20150041626A (en) | Asgpr antibodies and uses thereof | |
KR20230007555A (en) | Thrombin cleavable linker with xten and its uses thereof | |
JP2010514417A (en) | Complex of natriuretic peptide and antibody constant region | |
KR20170004967A (en) | Hybrid immunoglobulin containing non-peptidyl linkage | |
KR20160086819A (en) | Encapsulated cell therapy cartridge | |
WO1996021000A9 (en) | Recombinant il-5 antagonists useful in treatment of il-5 mediated disorders | |
US11155600B2 (en) | Human IL1-R1 derived inhibitor of IL-1β | |
WO1997048418A1 (en) | Improved method for treatment and diagnosis of il-5 mediated disorders | |
US20210403534A1 (en) | IL1-R1 DERIVED INHIBITOR OF IL-1b AND USE THEREOF | |
EP4153613A1 (en) | Il1-r1 derived inhibitor of il-1b and use thereof | |
US10584147B2 (en) | Procoagulant fusion compound | |
CN113637084A (en) | Biomacromolecule targeted specific complement inhibitor and preparation method and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20221130 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
A4 | Supplementary search report drawn up and despatched |
Effective date: 20240104 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: A61P 37/00 20060101ALI20231221BHEP Ipc: A61P 19/02 20060101ALI20231221BHEP Ipc: A61P 3/10 20060101ALI20231221BHEP Ipc: A61P 3/00 20060101ALI20231221BHEP Ipc: A61P 43/00 20060101ALI20231221BHEP Ipc: C07K 19/00 20060101ALI20231221BHEP Ipc: A61K 38/20 20060101ALI20231221BHEP Ipc: C07K 14/545 20060101AFI20231221BHEP |