NZ554481A - Fusion protein that binds to IL-1 receptor and one of IL18, IL4 or IL13 - Google Patents
Fusion protein that binds to IL-1 receptor and one of IL18, IL4 or IL13Info
- Publication number
- NZ554481A NZ554481A NZ554481A NZ55448105A NZ554481A NZ 554481 A NZ554481 A NZ 554481A NZ 554481 A NZ554481 A NZ 554481A NZ 55448105 A NZ55448105 A NZ 55448105A NZ 554481 A NZ554481 A NZ 554481A
- Authority
- NZ
- New Zealand
- Prior art keywords
- protein
- lra
- receptor
- fusion protein
- segment
- Prior art date
Links
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 69
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 69
- 108090000176 Interleukin-13 Proteins 0.000 title claims abstract description 23
- 102000003816 Interleukin-13 Human genes 0.000 title claims abstract description 23
- 102000019223 Interleukin-1 receptor Human genes 0.000 title claims description 51
- 108050006617 Interleukin-1 receptor Proteins 0.000 title claims description 51
- 102000003810 Interleukin-18 Human genes 0.000 title claims description 49
- 108090000171 Interleukin-18 Proteins 0.000 title claims description 49
- 102000004388 Interleukin-4 Human genes 0.000 title claims description 47
- 108090000978 Interleukin-4 Proteins 0.000 title claims description 46
- 230000028993 immune response Effects 0.000 claims abstract description 9
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 88
- 108090000623 proteins and genes Proteins 0.000 claims description 68
- 102000004169 proteins and genes Human genes 0.000 claims description 66
- 102000000589 Interleukin-1 Human genes 0.000 claims description 63
- 108010002352 Interleukin-1 Proteins 0.000 claims description 63
- 230000000694 effects Effects 0.000 claims description 51
- 238000000034 method Methods 0.000 claims description 36
- 150000007523 nucleic acids Chemical class 0.000 claims description 36
- 108020004707 nucleic acids Proteins 0.000 claims description 35
- 102000039446 nucleic acids Human genes 0.000 claims description 35
- 206010061218 Inflammation Diseases 0.000 claims description 33
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 32
- 230000004054 inflammatory process Effects 0.000 claims description 31
- 229920001184 polypeptide Polymers 0.000 claims description 29
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 29
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 29
- 230000001225 therapeutic effect Effects 0.000 claims description 24
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 20
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 20
- 102000005962 receptors Human genes 0.000 claims description 19
- 108020003175 receptors Proteins 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 18
- 201000010099 disease Diseases 0.000 claims description 16
- 239000003814 drug Substances 0.000 claims description 14
- 239000013598 vector Substances 0.000 claims description 14
- 239000005557 antagonist Substances 0.000 claims description 13
- 230000014509 gene expression Effects 0.000 claims description 12
- 230000002757 inflammatory effect Effects 0.000 claims description 12
- 108060003951 Immunoglobulin Proteins 0.000 claims description 9
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 claims description 9
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 claims description 9
- 102000018358 immunoglobulin Human genes 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 230000001965 increasing effect Effects 0.000 claims description 7
- 238000005304 joining Methods 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 5
- 208000026935 allergic disease Diseases 0.000 claims description 4
- 208000027866 inflammatory disease Diseases 0.000 claims description 4
- 208000023275 Autoimmune disease Diseases 0.000 claims description 3
- 230000000735 allogeneic effect Effects 0.000 claims description 3
- 102000023732 binding proteins Human genes 0.000 claims description 3
- 108091008324 binding proteins Proteins 0.000 claims description 3
- 210000004748 cultured cell Anatomy 0.000 claims description 3
- 230000000447 dimerizing effect Effects 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 55
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 55
- 238000003556 assay Methods 0.000 description 30
- 230000003472 neutralizing effect Effects 0.000 description 26
- 230000004927 fusion Effects 0.000 description 23
- 230000027455 binding Effects 0.000 description 22
- 230000033115 angiogenesis Effects 0.000 description 20
- 230000001419 dependent effect Effects 0.000 description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
- 238000006386 neutralization reaction Methods 0.000 description 18
- 210000001519 tissue Anatomy 0.000 description 17
- 102000009840 Angiopoietins Human genes 0.000 description 16
- 108010038486 Interleukin-4 Receptors Proteins 0.000 description 16
- 208000006673 asthma Diseases 0.000 description 16
- 108010009906 Angiopoietins Proteins 0.000 description 15
- 102000010787 Interleukin-4 Receptors Human genes 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 15
- 102000051628 Interleukin-1 receptor antagonist Human genes 0.000 description 14
- 208000035475 disorder Diseases 0.000 description 14
- 238000002347 injection Methods 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 102000044166 interleukin-18 binding protein Human genes 0.000 description 14
- 108010070145 interleukin-18 binding protein Proteins 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 13
- 108010008165 Etanercept Proteins 0.000 description 13
- 101100481410 Mus musculus Tek gene Proteins 0.000 description 13
- 229940073621 enbrel Drugs 0.000 description 13
- 206010028980 Neoplasm Diseases 0.000 description 12
- 230000004663 cell proliferation Effects 0.000 description 12
- 101100481408 Danio rerio tie2 gene Proteins 0.000 description 11
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 11
- 102000057041 human TNF Human genes 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- 239000011780 sodium chloride Substances 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 10
- 108090000695 Cytokines Proteins 0.000 description 10
- 101000960954 Homo sapiens Interleukin-18 Proteins 0.000 description 10
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 10
- 108700021006 Interleukin-1 receptor antagonist Proteins 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 10
- 231100000673 dose–response relationship Toxicity 0.000 description 10
- 102000043959 human IL18 Human genes 0.000 description 10
- 102000058223 human VEGFA Human genes 0.000 description 10
- 229940054136 kineret Drugs 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 230000009266 disease activity Effects 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 description 8
- 201000004681 Psoriasis Diseases 0.000 description 8
- 230000001413 cellular effect Effects 0.000 description 8
- 238000006471 dimerization reaction Methods 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 238000013207 serial dilution Methods 0.000 description 8
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 description 7
- 101710144554 Interleukin-1 receptor antagonist protein Proteins 0.000 description 7
- 102100026018 Interleukin-1 receptor antagonist protein Human genes 0.000 description 7
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 208000010247 contact dermatitis Diseases 0.000 description 7
- 239000003102 growth factor Substances 0.000 description 7
- 102000055229 human IL4 Human genes 0.000 description 7
- 206010039073 rheumatoid arthritis Diseases 0.000 description 7
- 230000028327 secretion Effects 0.000 description 7
- 229940124597 therapeutic agent Drugs 0.000 description 7
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 102000039996 IL-1 family Human genes 0.000 description 6
- 108091069196 IL-1 family Proteins 0.000 description 6
- 230000000975 bioactive effect Effects 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 238000009826 distribution Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 210000004072 lung Anatomy 0.000 description 6
- 230000037311 normal skin Effects 0.000 description 6
- 230000003287 optical effect Effects 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 201000004624 Dermatitis Diseases 0.000 description 5
- 229940119178 Interleukin 1 receptor antagonist Drugs 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 230000030833 cell death Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000010172 mouse model Methods 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 238000001525 receptor binding assay Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 230000008961 swelling Effects 0.000 description 5
- 238000002054 transplantation Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 102000000503 Collagen Type II Human genes 0.000 description 4
- 108010041390 Collagen Type II Proteins 0.000 description 4
- 101001076407 Homo sapiens Interleukin-1 receptor antagonist protein Proteins 0.000 description 4
- 101150106931 IFNG gene Proteins 0.000 description 4
- 101710115512 Nuclear receptor coactivator 5 Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- -1 e.g. Proteins 0.000 description 4
- 210000002683 foot Anatomy 0.000 description 4
- 230000013595 glycosylation Effects 0.000 description 4
- 238000006206 glycosylation reaction Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000003407 interleukin 1 receptor blocking agent Substances 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 102000004506 Blood Proteins Human genes 0.000 description 3
- 108010017384 Blood Proteins Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 102000004557 Interleukin-18 Receptors Human genes 0.000 description 3
- 108010017537 Interleukin-18 Receptors Proteins 0.000 description 3
- 108700012920 TNF Proteins 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000003527 anti-angiogenesis Effects 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 208000037976 chronic inflammation Diseases 0.000 description 3
- 230000006020 chronic inflammation Effects 0.000 description 3
- 210000005069 ears Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 229940048921 humira Drugs 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 210000001503 joint Anatomy 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000012743 protein tagging Effects 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 229940116176 remicade Drugs 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 108091008601 sVEGFR Proteins 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 238000011763 DBA/1J (JAX™ mouse strain) Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 206010014025 Ear swelling Diseases 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 206010015150 Erythema Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 2
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 2
- 206010029113 Neovascularisation Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 208000034038 Pathologic Neovascularization Diseases 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 206010038933 Retinopathy of prematurity Diseases 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 206010039710 Scleroderma Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 208000038016 acute inflammation Diseases 0.000 description 2
- 230000006022 acute inflammation Effects 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 201000009961 allergic asthma Diseases 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001088 anti-asthma Effects 0.000 description 2
- 239000000924 antiasthmatic agent Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 238000000423 cell based assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 210000003979 eosinophil Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- ZAHQPTJLOCWVPG-UHFFFAOYSA-N mitoxantrone dihydrochloride Chemical compound Cl.Cl.O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO ZAHQPTJLOCWVPG-UHFFFAOYSA-N 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000010422 painting Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 239000000546 pharmaceutical excipient 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
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 229940044551 receptor antagonist Drugs 0.000 description 2
- 239000002464 receptor antagonist Substances 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- FJQZXCPWAGYPSD-UHFFFAOYSA-N 1,3,4,6-tetrachloro-3a,6a-diphenylimidazo[4,5-d]imidazole-2,5-dione Chemical compound ClN1C(=O)N(Cl)C2(C=3C=CC=CC=3)N(Cl)C(=O)N(Cl)C12C1=CC=CC=C1 FJQZXCPWAGYPSD-UHFFFAOYSA-N 0.000 description 1
- 101150090724 3 gene Proteins 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 206010000050 Abdominal adhesions Diseases 0.000 description 1
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 1
- 208000035285 Allergic Seasonal Rhinitis Diseases 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 208000003120 Angiofibroma Diseases 0.000 description 1
- 102000009088 Angiopoietin-1 Human genes 0.000 description 1
- 108010048154 Angiopoietin-1 Proteins 0.000 description 1
- 101710131689 Angiopoietin-1 receptor Proteins 0.000 description 1
- 102000009075 Angiopoietin-2 Human genes 0.000 description 1
- 108010048036 Angiopoietin-2 Proteins 0.000 description 1
- 208000008822 Ankylosis Diseases 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 208000023328 Basedow disease Diseases 0.000 description 1
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 1
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010007710 Cartilage injury Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- PHEDXBVPIONUQT-UHFFFAOYSA-N Cocarcinogen A1 Natural products CCCCCCCCCCCCCC(=O)OC1C(C)C2(O)C3C=C(C)C(=O)C3(O)CC(CO)=CC2C2C1(OC(C)=O)C2(C)C PHEDXBVPIONUQT-UHFFFAOYSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000015943 Coeliac disease Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 206010011017 Corneal graft rejection Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 206010011891 Deafness neurosensory Diseases 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 0.000 description 1
- 208000019872 Drug Eruptions Diseases 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 208000010412 Glaucoma Diseases 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
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 206010072579 Granulomatosis with polyangiitis Diseases 0.000 description 1
- 208000015023 Graves' disease Diseases 0.000 description 1
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 1
- 206010019755 Hepatitis chronic active Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000924552 Homo sapiens Angiopoietin-1 Proteins 0.000 description 1
- 101001002634 Homo sapiens Interleukin-1 alpha Proteins 0.000 description 1
- 101000801232 Homo sapiens Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 description 1
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 206010021245 Idiopathic thrombocytopenic purpura Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 208000005726 Inflammatory Breast Neoplasms Diseases 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 206010021980 Inflammatory carcinoma of the breast Diseases 0.000 description 1
- 102100020881 Interleukin-1 alpha Human genes 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102000004559 Interleukin-13 Receptors Human genes 0.000 description 1
- 108010017511 Interleukin-13 Receptors Proteins 0.000 description 1
- 102100035017 Interleukin-18-binding protein Human genes 0.000 description 1
- 101710205006 Interleukin-18-binding protein Proteins 0.000 description 1
- 108010082786 Interleukin-1alpha Proteins 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 208000029523 Interstitial Lung disease Diseases 0.000 description 1
- 206010023198 Joint ankylosis Diseases 0.000 description 1
- 208000003456 Juvenile Arthritis Diseases 0.000 description 1
- 206010059176 Juvenile idiopathic arthritis Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101000974353 Mus musculus Nuclear receptor coactivator 5 Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 101800000135 N-terminal protein Proteins 0.000 description 1
- 201000004404 Neurofibroma Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101800001452 P1 proteinase Proteins 0.000 description 1
- 241001111421 Pannus Species 0.000 description 1
- 206010065159 Polychondritis Diseases 0.000 description 1
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 1
- 206010036774 Proctitis Diseases 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 1
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 1
- 206010037649 Pyogenic granuloma Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 1
- 208000036284 Rhinitis seasonal Diseases 0.000 description 1
- 208000009966 Sensorineural Hearing Loss Diseases 0.000 description 1
- 208000021386 Sjogren Syndrome Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 206010042033 Stevens-Johnson syndrome Diseases 0.000 description 1
- 231100000168 Stevens-Johnson syndrome Toxicity 0.000 description 1
- 208000025317 T-cell and NK-cell neoplasm Diseases 0.000 description 1
- 206010043189 Telangiectasia Diseases 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- 241000390203 Trachoma Species 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 206010070517 Type 2 lepra reaction Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 206010046851 Uveitis Diseases 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 206010046914 Vaginal infection Diseases 0.000 description 1
- 201000008100 Vaginitis Diseases 0.000 description 1
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 208000004064 acoustic neuroma Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 231100000354 acute hepatitis Toxicity 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 201000010105 allergic rhinitis Diseases 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 210000003423 ankle Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000007503 antigenic stimulation Effects 0.000 description 1
- 208000002399 aphthous stomatitis Diseases 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 201000003710 autoimmune thrombocytopenic purpura Diseases 0.000 description 1
- 201000004982 autoimmune uveitis Diseases 0.000 description 1
- 229940120638 avastin Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 208000020670 canker sore Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000019069 chronic childhood arthritis Diseases 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 208000004921 cutaneous lupus erythematosus Diseases 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000032692 embryo implantation Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 229960003699 evans blue Drugs 0.000 description 1
- 239000012526 feed medium Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 210000004744 fore-foot Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 201000011066 hemangioma Diseases 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 102000044072 human ANGPT1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 201000004653 inflammatory breast carcinoma Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000266 injurious effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- 201000004614 iritis Diseases 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 201000002215 juvenile rheumatoid arthritis Diseases 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 201000010666 keratoconjunctivitis Diseases 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 201000011486 lichen planus Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 230000002956 necrotizing effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 201000003142 neovascular glaucoma Diseases 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 229950010444 onercept Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000006201 parenteral dosage form Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 229940076372 protein antagonist Drugs 0.000 description 1
- 108010003189 recombinant human tumor necrosis factor-binding protein-1 Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 208000017022 seasonal allergic rhinitis Diseases 0.000 description 1
- 231100000879 sensorineural hearing loss Toxicity 0.000 description 1
- 208000023573 sensorineural hearing loss disease Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 208000009056 telangiectasis Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 206010044325 trachoma Diseases 0.000 description 1
- 238000013024 troubleshooting Methods 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 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
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- 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
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/08—Antibacterial agents for leprosy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- 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
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- 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
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
-
- 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
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- 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
-
- 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
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)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Diabetes (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Physical Education & Sports Medicine (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Endocrinology (AREA)
- Pulmonology (AREA)
- Rheumatology (AREA)
- Oncology (AREA)
- Dermatology (AREA)
- Communicable Diseases (AREA)
- Virology (AREA)
- Neurology (AREA)
- AIDS & HIV (AREA)
- Cardiology (AREA)
- Transplantation (AREA)
Abstract
Disclosed is a fusion protein comprising: a first segment that is located at the amino terminus of the fusion protein and specifically binds to and neutralizes ILI8, IL4, or IL13; and a second segment that is located at the carboxyl terminus of the fusion protein and specifically binds to an IL-I receptor, wherein the domains are operably linked. The fusion protein can be used to modulate an immune response in a subject.
Description
554481
CHIMERIC PROTEIN
RELATED APPLICATION
This application claims priority to US provisional Application Serial No. US 60/618,476, filed on October 12, 2004; US provisional Application Serial No. US 60/628,994, filed on November 17, 2004; and US provisional Application entitled "IL-lra as a fusion partner to target angiogenesis," filed on February 1, 2005, the content of which is incorporated by reference in its entirety.
The present invention is directed to chimeric protein therapeutic agents useful in treatment of various diseases such as inflammation, asthma and cancer.
BACKGROUND OF THE INVENTION
Inflammation is the body's defense reaction to injuries such as those caused by mechanical damage, infection or antigenic stimulation. An inflammatory reaction may be expressed pathologically when inflammation is induced by an inappropriate stimulus such as an autoantigen, expressed in an exaggerated manner or persists well after the removal of the injurious agents. Inflammation often co-exists with asthma and angiogenesis-related indications. A number of therapeutic proteins have developed for inhibiting inflammatory reactions, treating inflammation-related asthma, and reducing pathological angiogenesis. However, many of them are not satisfactory due to poor efficacy, side effects, or instability.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or at least to provide the public with a useful choice.
This invention relates to use IL-1 receptor antagonist (IL-lra) or its function equivalent as a fusion partner to bioactive or therapeutic proteins. Examples of the bioactive or therapeutic proteins include, but are not limited to, tumor necrosis factor (TNF) neutralizers, IL-18 neutralizers, IL-4/IL-13 neutralizers, VGEF neutralizer,
FIELD OF THE INVENTION
SUMMARY OF INVENTION
301158966 l.DOC
2 ? M 4Y 2009
554481
WO 2006/043972 PCT/US2005/012194
angiopoietin neutralizer, and others useful in treatment of inflammation, asthma and angiogenesis-related indications.
One aspect of this invention features a fusion protein that contains a first segment that is located at the amino terminus of the fusion protein and specifically binds to and neutralizes a first cytokine or growth factor; and a second segment that is located at the carboxyl terminus of the fusion protein and specifically binds to a receptor of a second cytokine or a growth factor, e.g., IL-1 receptors which are rich at inflammatory sites. The domains are operably linked, and the first or second cytokine is rich at an inflammatory site.
The just -described fusion protein can be glycosylated. It can further include a linker segment that joins the first segment and the second segment. The linker segment is capable of dimerizing. In one example, the linker segment contains the Fc fragment of an immunoglobulin or a functional equivalent there of. Preferably, the immunoglobulin is an IgA, IgE, IgD, IgG, or IgM. More preferably, the immunoglobulin is IgG or its Fc fragment, e.g., SEQ ID NO.: 2. The immunoglobulin chain contains SEQ ID NO: 9, 11, 12, 14, 23, or 24; or a functional equivalent thereof.
In the just-described fusion protein, the first segment can bind to and neutralizes VEGF, Ang, TNF, IL18, IL4, or IL6, or a functional equivalent thereof. For example, the first segment contains the sequence of a chain of an immunoglobulin that specifically binds to and neutralizes VEGF, Angiopoitins, TNF, IL18, IL4, IL-13 or IgE; or a functional equivalent thereof. The first segment can also contain the sequence of a receptor of VEGF, Ang, TNF, IL18, IL4, IL13 or IgE, e.g., SEQ ID NO.: 3, 6, 15, or 19.
In the just-described fusion protein, the second segment can specifically bind to a receptor of IL-1. The second segment can be an antagonist of IL-1, such as a segment containing the sequence of IL-lra (SEQ ID NO.: 1) or a functional equivalent analogue thereof. Accordingly, the above-described fusion protein can contain SEQ ID NO: 5, 8, 10, 13, 17, 18, 21, 22, 24, or 25.
In one particular aspect, the invention provides a fusion protein comprising a first segment that is located at the amino terminus of the fusion protein and specifically binds to and neutralizes VEGF, Angiopoitin, TNF, IL18, IL4, IL13 or IgE; and a
301225371 l.DOC
INTELLECTUAL PROPERTY OFFICE OF N.Z.
-7 SEP 2009
RECEIVED
554481
second segment that is located at the carboxyl terminus of the fusion protein and specifically binds to an IL-1 receptor,
wherein the domains are operably linked.
Another aspect of this invention features an isolated nucleic acid containing a sequence that encodes a fusion protein described above. It can contain a sequence encoding one of SEQ ID NOs: 1-25.
In another aspect, the invention features a vector comprising a nucleic acid as described above. Further, the invention features an isolated host cell comprising a nucleic acid or vector as described above, as well as a method for producing a polypeptide, comprising culturing such host cell in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cell or the medium of the cell.
Within the scope of this invention is a composition containing (i) the above-described fusion protein or a nucleic acid encoding it and (ii) a pharmaceutically acceptable carrier. Also within the scope of this invention is a method of modulating an immune response in a subject. The method includes identifying a subject having or being at risk of acquiring a condition characterized by an excessive inflammatory response, an immune response, and an angiogenesis response; and administering to the subject an effective amount of the above-described fusion proteins or a nucleic acid encoding the fusion protein. The subject can be one that has received or is contemplated to receive an allogeneic or xenogeneic transplant. Examples of the condition include an inflammatory disease, an autoimmune disease, an allergic disease, or a cancer. In the case, the condition is an angiogenesis-dependent cancer, a fusion protein contains SEQ ID NO: 24 is preferred.
The invention further features the use of a fusion protein as hereinbefore described or a nucleic acid encoding the fusion protein, in the manufacture of a medicament for modulating an immune response in a subject.
In another aspect, the invention features a method of increasing the half-life of a recombinant protein in a subject. The method includes joining the recombinant protein to a segment containing SEQ ID NO.: 1 or a functional equivalent there of to form a fusion
301158966 1.DOC
3
554481
WO 2006/043972 PCT/US2005/012194
protein chimera, wherein the recombinant protein binds to a cytokine or a growth factor. In one aspect, the recombinant protein binds to and neutralizes VEGF, TNF, IL18, IL4, IL13 or IgG. The method may additionally involve determining the half-life of the fusion protein in a subject.
The invention also features a method of increasing the efficacy of a recombinant protein in a subject. The method includes joining the recombinant protein to a segment containing SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera. The method may additionally involve determining the efficacy of the fusion protein in a subject. In one embodiment, the fusion protein chimera binds and neutralizes simultaneously to both IL-1 receptor and the cytokines or growth factor at inflammation site or at an IL-1 receptor-rich disease site in a subject. In another embodiment, the fusion protein chimera neutralizes or antagonizes the activities of both IL-1 and the cytokine or growth factor at inflammation site or at an IL-1 receptor-rich disease site in a subject.
In one particular aspect, the invention provides a method of increasing the efficacy of a recombinant protein in a subject, the method comprising:
joining the recombinant protein to a segment containing SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera,
wherein the recombinant protein binds to and neutralizes VEGF, TNF, IL18, IL4, IL13 or IgE.
In yet another aspect, the invention features a method of delivering a therapeutic protein to a target site in a subject, the method including joining the therapeutic protein to a segment containing SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera; and administering the fusion protein chimera to a subject in need thereof. The therapeutic protein is targeted to an inflammatory site that is rich in IL-1 receptor. In one embodiment, the segment containing SEQ ID NO: 1 or a functional equivalent thereof binds to IL-1 receptor, and the recombinant protein is a therapeutic protein that binds to and neutralizes a cytokine or a growth factor.
In another aspect, the invention features the use of a fusion protein chimera in the manufacture of a medicament for delivery of a therapeutic protein to a target site in a
301225371 l.DOC
INTELLECTUAL PROPERTY OFFICE OF N.Z.
-7 SEP 2009
RECEIVED
554481
WO 20067043972 PCT/US2005/012194
subject, wherein the fusion protein chimera comprises a therapeutic protein joined to a segment containing SEQ ID NO: 1 or a functional equivalent thereof, and wherein the therapeutic protein is targeted to an inflammatory site that is rich in IL-1 receptor, and binds to and neutralizes VEGF, TNF, IL18, IL4, IL13 or IgE.
An isolated polypeptide refers to a polypeptide substantially free from naturally associated molecules, i.e., it is at least 75% (i.e., any number between 75% and 100%, inclusive) pure by dry weight. Purity can be measured by any appropriate standard method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC.
301225371_1.DOC 4 A
INTELLECTUAL PROPERTY OFFICE OF N.Z.
-7 SEP 2009
RECEIVED
554481
An isolated polypeptide of the invention can be purified from a natural source (for wild type polypeptides), produced by recombinant DNA techniques, or by chemical methods.
A nucleic acid refers to a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated nucleic acid" refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The nucleic acid described above can be used to express the polypeptide of this invention. For this purpose, one can operatively link the nucleic acid to suitable regulatory sequences to generate an expression vector.
A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector can be capable of autonomous replication or integrate into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The vector includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. A "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be tr<
301158966_1 .DOC
554481
expression of protein or RNA desired, and the like. The expression vector can be introduced into host cells to produce a polypeptide of this invention. Also within the scope of this invention is a host cell that contains the above-described nucleic acid or vector, as described hereinbefore. Examples include E. coli cells, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. See e.g., Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA. To produce a polypeptide of this invention, one can culture a host cell in a medium under conditions permitting expression of the polypeptide encoded by a nucleic acid of this invention, and purify the polypeptide from the cultured cell or the medium of the cell. Alternatively, the nucleic acid of this invention can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase.
A "functional equivalent" of a proteinous factor refers to a polypeptide derivative of the protein e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity of the factor, e.g., an ability to bind to a cytokine, a growth factor, or a receptor thereof.
301158966 l.DOC
5A
554481
The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: 1st generation of production CHO cell clones of TNFRJI-Fc and TNFRII-Fc-IL-lra chimera: 24 well plate expression in serum-free medium; direct Coomasie blue protein staining; all recombinant proteins are visible ranging 0.5-1.0 ug; loading 10-15 microliters per lane.
Figure 2: Affinity purification of TNFRII-Fc-IL-lra chimera: SDS page reduced and non-reduced conditions; Coomasie blue protein staining.
Figure 3: An example of our trouble-shooting capability: reducing a degradation problem for TNFRII-Fc-IL-lra chimera by altering the first purification step — HPLC analysis of intact and partially degraded TNFRII-Fc-IL-lra chimera with TNFRII-Fc control.
Figure 4: Affinity purification of IL-4R-Fc, IL-4R-Fc-IL-lra and IL-18bp-Fc-IL-
lra.
Figure 5: Cell-based TNF alpha neutralization test indicates that similar to marketed TNFRII-Fc (Enbrel), TNFRII-Fc-IL-lra chimera neutralizes TNF alpha's killing activity on L979 cells.
Figure 6: Cell-based IL-1 neutralization test indicates that both marketed IL-lra (Kineret) and TNFRII-Fc-IL-lra chimera neutralize IL-1 's biological activity on D10 cell proliferation.
Figure 7: Human IL-4 neutralization assay of IL-4R-Fc-IL-lra and control IL-4R-
Fc.
Figure 8: Human IL-1 neutralization assay of IL-4R-Fc-IL-lra.
Figure 9: IL-18 neutralizing activity of IL-18bp-Fc-IL-Ira.
Figure 10: IL-1 neutralizing activity of IL-18bp-Fc-IL-lra.
Figure 11: IL-1 neutralizing activity of VEGFRl-Fc-IL-lra in D10 cells.
Figure 12: VEGF neutralizing activity of VEGFRl-Fc-IL-lra in HUVE cells.
6
554481
Figure 13: IL-1 receptor binding assay.
DETAILED DESCRIPTION OF THE INVENTION
This invention is based, as least in part, on the discovery that IL-lra or its functional equivalent, as a fusion partner, extend biological lives and efficacy of a number of bioactive proteins, e.g., anti-inflammation proteins, anti-asthma proteins, and anti-angiogenesis proteins. Examples of these proteins include tumor necrosis factor (TNF) neutralizers, IL-18 neutralizers, IL-4/IL-13 neutralizers, VEGF neutralizer, angiopoietin neutralizers.
N-terminal protein fusion to a bioactive protein often leads to complete activity loss, particularly for large-size protein fusion partners. For example, pro-enzymes and pro-hormones are not active due to the propeptide fusion at their N-terminus. These pro-digesting enzymes and pro-hormones become biologically active only until their propeptides are cleaved off. In addition, large size protein fusion often leads to low expression yield. Unexpectedly, IL-lra fused proteins can be produced at commercial production level in mammalian host cells. The fusion does not interfere with the activity IL-Ira's IL-1 receptor binding and neutralizing activities, or the binding and neutralizing activity of a bioactive protein to which it is fused. Also unexpectedly, IL-lra (e.g., mammalian made glycosylated) or its functional equivalent not only extends biological lives of the bioactive proteins, but also directs them to an IL-1 receptor-rich inflammatory site.
IL-lra
IL-1 is a cytokine produced by cells of the macrophage/monocyte lineage. It is produced in two forms: IL-1 alpha and IL-1 beta. IL-1 protein initiates its biological effects on cells by binding to specific IL-1 receptors (IL-1R). IL-1R is generally expressed on the plasma membrane of IL-1 responsive cells.
IL-1 receptor antagonist (IL-lra) is a human protein that acts as a natural inhibitor of IL-1. IL-lra has been used to suppress biological activities caused by IL-1. It binds to cell membrane bound IL-1 receptors and prevents IL-1 from binding to the same IL-1
7
554481
receptors. IL-1 receptor is mostly expressed at inflammatory sites (Deleuran et al, 1992; Laken VD et al, 1997) and lymphocyes (Dower SK et al, 1990). Thus, IL-lra may direct a therapeutic protein (e.g., a TNF neutralizing agent described below) fused thereto to an IL-1 receptor-rich inflammatory site. Due to this targeting effect, reduced effective doses of the therapeutic protein are needed, thereby reducing side effects or improved efficacy. Further, the synergy between IL-lra and the fusion partner leads to a therapeutic effect greater than that of each of the two proteins alone or in combination due to, at least in part, fusion protein going to the same location.
IL-lra and its functional equivalent can be used to practice this invention. IL-lra functional equivalent refers to a polypeptide derivative of the IL-lra (SEQ ID NO: 1) as described in the Summary section. It has substantially the activity of IL-lra, i.e., e.g., binding to IL-1 receptors and preventing IL-1 from binding to the same IL-1 receptors. IL-lra and its functional equivalent contains at least one interleukin-1 receptor antagonist domain, which refers to a domain capable of specifically binding to IL-1 receptor family members and preventing activation of cellular receptors to IL-1 and its family members. IL-1 receptor family contains several receptor members. Accordingly, there are several different IL-1 family agonists and antagonists. These IL-1 antagonists may not necessarily bind same IL-1 receptor family members. Here IL-lra is used to represent all the IL-1 antagonists that bind to IL-receptor family members or/and neutralize activities of IL-1 family members.
An IL-lra functional equivalent contains an interleukin-1 receptor antagonist domain. This domain refers to a domain capable of specifically binding to IL-1 receptor family members and preventing activation of cellular receptors to IL-1 and its family members. Examples of interleukin-1 receptor antagonists include IL-lra (U.S. Patent No. 6,096,728), IL-1 HY1 or IL-1 family member 5 (U.S. Patent No. 6,541,623), IL-lHy2 or IL-1 family member 10 (U.S. Patent No. 6,365,726), IL-lra beta (US6,399,573), other IL-1 antagonist members and their functional equivalents, i.e., polypeptides derived from IL-lra e.g., proteins having one or more point mutations, insertions, deletions,
truncations, or combination thereof. They retain substantially the activity of specifically
8
554481
binding to IL-1 receptor and preventing activation of cellular receptors to IL-1. They can contain SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. Preferably, the IL-lra is a glycosylated mammalian polypeptide. The activity of an Interleukin-1 receptor antagonist may be determined by cell-based IL-1 neutralization assay using IL-1 dependent D10 cells (see Example 3), and other IL-1 family member neutralizing assays.
Preferably, IL-lra or its functional equivalent is a glycosylated polypeptide. Native IL-lra is glycosylated with two N-link glycosylation sites (US patent number 6096728). These two N-link glycosylation sites are important for IL-lra's in vivo activity, particularly for its biological life, and its serum protein binding property.
Kineret, an E-coli produced IL-lra, lacks post-translational modification. As result, it tends to bind to human serum proteins significantly and has lower in vivo efficacy.
An IL-lra or its functional equivalent's antagonist activity can be determined by cell-based IL-1 neutralization assay using IL-1 dependent D10 cells (see Example 3), and other standard IL-1 family member neutralizing assays. IL-lra fusion to any protein agents increases molecular weight and lead to increased biological life in vivo. IL-lra fusion to other molecules through immunoglobin Fc (e.g., IgGl Fc) may further increase molecular weight. Due to the dimerizing ability of immunoglobin Fc, its presence can double the level of the fused proteins at a site of interest.
TNF
Tumor necrosis factor-alpha (TNF alpha) and Tumor necrosis factor beta (TNF-beta) are mammalian secreted proteins capable of inducing a wide variety of effects on a large number of cell types. The great similarities in the structural and functional characteristics of these two cytokines have resulted in their collective description as "TNF".
TNF initiates its biological effects on cells by binding to specific a TNF receptor (TNFR) expressed on the plasma membrane of TNF-responsive cells. Two distinct forms of TNFRs are known: Type I TNFR (TNFRI), which has a molecular weight of approximately 55 kilodaltons (led), and type II TNFR (TNFRII), which has a molecular
9
554481
weight of approximately 75 kd. TNFRI and TNFRII each bind to both TNF alpha and TNF beta.
The role of TNF in inflammatory diseases has been well established. TNFRII fused to human IgGl Fc fragment (trade name Enbrel) has been used for treating certain TNF-dependent disorders such as rheumatoid arthritis and psoriasis. Soluble TNFRI (Onercept, Serono) has been tested in clinical trial for treatment of psoriasis.
TNF antagonists have been identified. These antagonists, such as soluble TNFRII and TNFRI, bind to TNF and prevent TNF from binding to TNF receptors. Such proteins can be used to suppress biological activities caused by TNF. Protein-based TNF neutralizing agents can be fused to IL-lra or its functional equivalent. Like IL-1, TN F is an important mediator of inflammation reaction. The just mentioned TNF-neutralizing agents include TNF and its functional equivalents. Each of them includes one or more TNF neutralizer domains, a domain capable of neutralizing TNF, i.e., inhibiting the activity of TNF. A TNF neutralizer domain may include an extracellular domain of human TNFRII, an extracellular domain of TNFRI, or variable regions of anti TNF antibodies. Examples include the extracellular domain of TNF receptor type II (TNFRII), TNF binding protein 1 (rhTBP-1) or TNF receptor type I (TNFRI), humanized anti TNF antibody (e.g., Humira, Abbot Laboratories) and chimeric anti TNF antibody (e.g., Remicade of Johnsons&Johnson).
Since TNF alpha and IL-1 are two major players in inflammatory diseases, a fusion or chimeric of a TNF antagonist and an IL-lra or its functional equivalent can be used to block both TNF alpha and IL-1 pathways, and therefore can be used to treat acute and chronic inflammation-related diseases more effectively than each individually. TNF neutralizer activity of the chimeric protein can be determined using TNF dependent cells such as L979 cell (ATTC). More specifically, TNF-dependent cells can be killed by effective doses of recombinant TNF alpha. This TNF-dependent activity can be neutralized by addition of these TNF neutralizers into the reaction. The activity of these TNF neutralizers may also be determined by using TNF in vitro binding assays.
554481
Concurrent use of IL-lra and TNF receptor type I (not Type II) have been proposed for treatment of TNF alpha and IL-1 mediated diseases. However, a clinical trial of 242 patients and 24-weeks published by Immunex Inc and Amgen Inc in 2003 had concluded that concurrent use of Enbrel and Kineret with non-reduced individual dosage (Enbrel 25mg biweekly and Kineret lOmg daily with molar ratio about 1:12) did not increase the efficacy but leaded to higher incidence of infection and neutrapenia than that of Enbrel or Kineret monotherapy.
IL-18 and IL-4
The above-described IL-lra or a functional equivalent thereof can also be fused to other anti-inflammation, anti-asthma, or anti-angiogenesis proteins. Examples include: (i) IL-18 neutralizing agents such as IL-18 binding protein (IL-18bp), IL-18 receptor (IL-18R) extracellular domain and humanized anti IL-18 antibody; (ii) IL-4 neutralizing agents such as IL-4 receptor (IL-4R) extracellular domain (tradename Nuvance, Immunex) and humanized anti IL-4 antibody (Protein Design Labs); (iii) anti-VEGF antibodies and angiopoietin neutralizer soluble Tie2 extracellular domain. As discussed therein, addition of IL-lra at C-terminus ofthese proteins (1) increases their molecular weights; (2) adds two more glycosylation sites when produced in mammalian host; (3) targets them to an IL-1 receptor-rich inflammation site directed delivery; (4) blocks IL-18, IL-4, VBEGF, or angiopoietin and IL-1 simultaneously at 1:1 molar ratio.
Recombinant IL-18bp has been tested in clinical trials (Serono) for treating skin inflammatory indication psoriasis. Good safety profile of this IL-18bp has been demonstrated. IL-lra fusion at its C-terminus may significantly increase its biological life. Inflammatory site-targeting via IL-lra fusion can significantly increase its efficacy. Double-neutralizing IL-18 and IL-1 by IL-lra fusion also have synergy for treatment of inflammation-dependent diseases such as psoriasis (Yudoh K et al (2004). Most interestingly, IL-18 and IL-1 use same IL-1 receptor family and almost same signal transduction pathway. Double-blocking of IL-1 and IL-18 blocks almost completely whole IL-1 receptor family mediated inflammation processes. Double blocking of IL-1
11
554481
and IL-18 by a chimeric protein of this invention represent the most effective antiinflammatory therapeutic agent.
A functional equivalent of IL-18bp can also be used to practice this invention. IL-18bp or its functional equivalent contains a IL-18 neutralizer domain, a domain capable of neutralizing IL-18, i.e., inhibiting the activity of IL-18. For example, an IL-18 neutralizer domain may include an extracellular domain of human IL-18 receptor (US Patent 6,589,764), an IL-18bp, an anti IL-18 antibody, or an IL-18 mutant antagonist protein.
The IL-18 neutralizer activity of a chimeric protein of this invention can be determined using IL-18 dependent KG-1 cells. For example, human IL-18 induces IFN-g secretion from KG-1 cells (in the presence of TNFa) in a dose dependent manner. This IL-18 dependent IFN-g secretion can be inhibited by effective doses of IL-18 neutralizers. The activity of these IL-18 neutralizers may also be determined by IL-18/IL-18 receptor binding assays.
Recombinant soluble IL-4 receptor has been tested in clinical trials for treatment of asthma. Great safety profile has been demonstrated. However, its efficacy is not satisfactory. Interestingly, it was reported that IL-1 is required for allergen-specific Th2 cell activation and the development of airway hypersensitive response (Iwakura Y et al, 2003). In addition, co-existence or co-dependence of and interaction between asthma and chronic inflammation are very common in clinics. Blocking IL-1 has clear therapeutic effect on asthma at least in animal models. It is very possible that blocking IL-4 and IL-1 simultaneously at 1:1 molar ratio by a IL-lra-soluble IL-4 receptor fusion significantly improves the efficacy for treating severe asthma. Inflammatory site-targeting of IL-lra may further increases the therapeutic value of soluble IL-4 receptor in treating severe asthma compounded by the inflammation. In addition, IL-lra fusion may significantly increase soluble IL-4 receptor's biological life.
A soluble IL-4 receptor or its functional equivalent can be fused to IL-lra. IL-4 receptor or its functional equivalent contains a IL-4 neutralizer domain, a domain capable of neutralizing IL-4, i.e., inhibiting the activity of IL-4. For example, an IL-4 neutralizer
12
554481
domain may include an extracellular domain of human IL-4 receptor, anti IL-4 antibodies, or a IL-4 mutant protein antagonist having a double mutation R121D/Y124D (Schnarr et al. 1997). Interestingly, this IL-4R subunit not only binds IL-4 but also binds to IL-13 due to the nature of shared common subunit of IL-4 and IL-13 receptors.
The IL-4 neutralizer activity of a chimeric protein of this invention can be determined by IL-4 dependent TF-1 cell-based assays. For example, human IL-4-dependent proliferation of TF-1 cells can be inhibited by adding effective doses of IL-4 neutralizers. The activity of IL-4 neutralizers may also be determined by IL-4/IL-4 receptor binding assays.
VEGF and Angiopoietin
The above-described approaches can also be applied to antagonists of VEGF and Angiopoietin, as well functional equivalents thereof. VEGF is important for angiogenesis. Anti-VEGF antibody (trade name Avastin, Genentech Inc) has been used for treating cancer indications. Similarly, soluble VEGF receptor extracellular domain fused with IgGlFc has also been used to neutralize VEGF for angiogenesis related indications. A functional equivalent of VEGF contains a VEGF neutralizer domain, a domain capable of neutralizing VEGF, i.e., inhibiting the activity of VEGF. For example, a VEGF neutralizer domain may include an extracellular domain of human VEGF and variable region of an anti VEGF antibody.
The VEGF neutralizer activity of a chimeric protein of this invention can be determined using VEGF-dependent HUVEC cells. For example, human VEGF induces proliferation of HUVEC cells. This VEGF-dependent proliferation of HUVEC cells can be inhibited by effective doses of VEGF neutralizers. The activity of VEGF neutralizers may also be determined by using VEGF/VEGF receptor binding assays.
Angiopoietin soluble receptor Tie2 has also been suggested as an anti-angiogenesis therapeutic agent against cancer or angiogenesis-related rheumatoid arthritis. Co-existance and co-dependence of angiogenesis and inflammation have long been observed in clinics. The most common example is rheumatoid arthritis where
13
554481
angiogenesis and inflammation co-exist. Angiopoietin soluble receptor Tie2 or a functional equivalent thereof contains an angiopoietin neutralizer domain, which is a domain capable of neutralizing angiopoietin, i.e., inhibiting the activity of angiopoietin 1. For example, an angiopoietin neutralizer domain may include an extracellular domain of human Tie2 and anti Tie2 or angippoietin antibodies.
The Tie-2 neutralizer activity of a chimeric protein of this invention can be determined by Tie-2-dependent HUVEC cells. For example, human angiopoietin 1 induces intracellular phosphorylation of HUVEC cells. This Tie-2-dependent phosphorylation of HUVEC cells can be inhibited by effective doses of Tie-2 neutralizers. The activity of Tie-2 neutralizers may also be determined by using Tie-2/Angiopoietin 2 binding assays.
It is known that IL-1 is an important pathological angiogenesis stimulator. Neutralizing IL-1 by IL-lra or its functional equivalent inhibits angiogenesis and tumor growth in an animal model, suggesting inflammation enhances angiogenesis. For example, the most aggressive type of breast cancer is inflammatory breast cancer. It is most likely that use of the fusion IL-lra and an angiogenesis agent (e.g., anti-VEGF antibody, soluble VEGF receptor extracellular domain, or soluble Tie2 extracellular domain) has significantly better efficacy than the anti-angiogenesis agent alone in treating cancer or rheumatoid arthritis related indications.
Besides the above-mentioned therapeutic agents, other suitable protein therapeutic agents that can be fused to IL-lra or its functional equivalent are listed below:
1. E25 (olizumab). E25 is a humanized anti IgE antibody (Novartis) for treating allergic asthma, seasonal allergic rhinitis.
2. H5G1.1. H5G1.1 is a humanized anti-C5 antibody (Alexion Pharmaceuticals), which can be used for treating of psoriasis and autoimune diseases.
3. TP 10. TP 10 is a soluble complement receptor 1 (sCRl) for treatment of acute respiratory distress syndrome and organ transplantation (AVANT Immunotherapeutics).
4. ABX-IL8. ABX-IL8 is an anti IL-8 monoclonal antibody (Abgenix), which can be used for treating psoriasis.
14
554481
. CTLA4Ig. CTLA4Ig is a recombinant soluble receptor (Bristol-Myers Squibb), which can be used for immunosuppression.
In a fusion of one of the above-discussed agents and IL-lra/its functional equivalent partner, the two fusion partners have activities synergistic or complementary to each other. IL-lra binds to IL-1 receptors and directs the fused therapeutic agent to IL-1 receptor-rich inflammation site. It also neutralizes IL-1 activity. The fusion of IL-lra and any of these proteins can be used in treating inflammation, asthma, and angiogenesis-related disorders or endothelial cell proliferation-related disorders.
Angiogenesis-relatcd disorders refer to any disorders that require angiogenesis or exhibit abnormal angiogenesis. Examples include, but are not limited to, cancers, solid tumors, tumor metastasis, benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia and rubeosis, Osier-Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma and wound granulation. As used herein, endothelial cell proliferation-related disorders include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma and hypertrophic scars. Fusion proteins described herein can also be used to treat the just-listed disorders by preventing the neovascularization required for embryo implantation.
Preferably, a fusion protein of this invention includes a dimerization domain. A "dimerization domain" refers to a domain capable of engaging two polypeptides. For example, a dimerization domain may include an IgG Fc fragment (e.g., human IgG heavy chain constant region). An example of such a Fc fragment includes SEQ ID No:2. IgG Fc fragment dimmerizes through its cystaine residues for formation of inter-chain disulfide bonds (covalent). Sometime non-covalent dimerization also occurs without involving disulfide bond. Dimerized IgG Fc fragment is capable of presenting, e.g., two functional TNFRII or soluble IL-4R or IL-18bp or soluble Tie-2 molecules at its N-terminus and two functional IL-lra molecules at its C-terminus. This arrangement
554481
increases in vivo receptor/ligand binding chances for neutralizing both TNF alpha or IL-4 or IL-18 or angiopoietin and IL-1 receptors.
The activity of a covalent dimerization through disulfide bond may be determined by using reduced and non-reduced SDS page electroporesis. Molecular weight of the protein should be reduced in half when reduced condition is used. Non-covalent dimerization may be determined by using native and denatured conditions for electroporesis. In this case, molecular weight of the protein should be reduced in half when denatured condition is used.
In a polypeptide of the invention, the TNF neutralizer domain or IL-4/IL-13 neutralizer domain or IL-18 neutralizer domain or VEGF neutralizer domain or angiopoietin neutralizer domain, dimerization domain, and IL-1 receptor antagonist domain are operably linked. As used herein, "operably linked" refers to the structural configuration of the polypeptide that does not interfere with the activities of each domain. For example, an IL-4 neutralizer domain retains its capability of neutralizing IL-4; an interleukin-1 receptor antagonist domain retains its capability of specifically binding IL-1 receptor and preventing activation of cellular receptors to IL-1; and an dimerization domain retains its capability of engaging two polypeptides of the invention and presenting, e.g., two functional IL-4 receptor extracellular domain at its N-terminus and two functional IL-lra molecules at its C-terminus.
Fusion of IL-lra at C-terminus of one of the above-discussed TNF neutralizers, IL-18 neutralizers, IL-4 neutralizers, VEGF neutralizers, or angiopoietin neutralizers (1) increases the molecular weight; (2) adds two more glycosylation sites on IL-lra molecule when produced in mammalian host; (3) targets a neutralizer to IL-1 receptor-rich inflammation site directed delivery; and (4) blocks IL-1 and any of TNF, IL-18, IL-4, IL-13, IgE, VEGF, and angiopoietin simultaneously at 1:1 molar ratio. The resulting double-blocking has better efficacy for treatment of inflammation diseases and provides more complete blockage to inflammation disease processes. Double-blocking of IL-4/IL-13/VEGF/ angiopoietin and IL-1 simultaneously has better and more complete efficacy
16
554481
for treatment of the diseases where co-existance and co-dependence of inflammation and asthma or angiogenesis play important role in disease processes.
A polypeptide of this invention can be obtained as a synthetic or recombinant polypeptide. To prepare a recombinant polypeptide, a nucleic acid encoding it can be linked to another nucleic acid encoding a fusion partner, e.g., Glutathione-S-Transferase (GST), 6x-His epitope tag, or Ml 3 Gene 3 protein. The resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be isolated by methods known in the art. A variety of host-expression vector systems can be used. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors; yeast transformed with recombinant yeast expression vectors; and human cell lines infected with recombinant virus or plasmid expression vectors. Isolation and purification of recombinant polypeptides or its fragments can be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies. The isolated fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of this invention.
Compositions and Treatment Methods
Also within the scope of this invention is a method of treating a disorder characterized by an excessive immune response or angiogenesis-related disorders by administering to a subject in need thereof an effective amount of the fusion protein of this invention Subjects to be treated can be identified as having or being at risk for acquiring a condition characterized by an excessive or unwanted immune response, e.g., patients suffering from autoimmune diseases, transplant rejection, allergic diseases, or immune cell-derived cancers. This method can be performed alone or in conjunction with other drugs or therapy.
The term "treating" refers to administration of a composition to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, the symptom
17
554481
of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder. An "effective amount" is an amount of the composition that is capable of producing a medically desirable result in a treated subject. The medically desirable result may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). Exemplary diseases to be treated include acute and chronic inflammation, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory bowel diseases, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, interstitial lung fibrosis, graft-versus-host disease, cases of transplantation (including transplantation using allogeneic or xenogeneic tissues) such as bone marrow transplantation, liver transplantation, or the transplantation of any organ or tissue, allergies such as atopic allergy, AIDS, T cell neoplasms such as leukemias or lymphomas, acute hepatitis, angiogenesis related diseases (such as rheumatoid arthritis and cancer), and cardiovascular diseases
A subject to be treated may be identified as being in need of treatment for one or more of the disorders noted above. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).
18
554481
In one in vivo approach, a therapeutic composition (e.g., a composition containing a fusion protein of the invention) is administered to the subject. Generally, the protein is suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Variations in the needed dosage are to be expected in view of the variety of compositions available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the composition in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
Also within the scope of this invention is a pharmaceutical composition that contains a pharmaceutically acceptable carrier and an effective amount of a fusion protein of the invention. The pharmaceutical composition can be used to treat diseases described above. The pharmaceutically acceptable carrier includes a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, and an isotonic and absorption delaying agent.
The pharmaceutical composition of the invention can be formulated into dosage forms for different administration routes utilizing conventional methods. For example, it can be formulated in a capsule, a gel seal, or a tablet for oral administration. Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the composition with a solid carrier and a lubricant. Examples
19
554481
of solid carriers include starch and sugar bentonite. The composition can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent. The pharmaceutical composition can be administered via the parenteral route. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient. Cyclodextrins, or other solubilizing agents well known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent.
The efficacy of a composition of this invention can be evaluated both in vitro and in vivo. See, e.g., the examples below. Briefly, the composition can be tested for its ability to repress immune responses in vitro. For in vivo studies, the composition can be injected into an animal (e.g., a mouse model) and its therapeutic effects are then accessed. Based on the results, an appropriate dosage range and administration route can be determined.
The examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
Our results also indicate that IL-lra fused molecules made in mammalian hosts, contain glycosylated IL-lra, and have a larger molecular weight than those of non-IL-lra fused molecules. They have longer biological lives, and less frequent effective injection doses. Due to its inflammation site-directed nature and low effective dose and less dosing frequency, IL-lra fused molecules may have less side effects when comparing with that of non-IL-lra fused molecules or concurrent use of th enon-IL-lra fused molecules and IL-lra.
Example 1
554481
Various of expression vectors were generated. The vectors respectively encode the following proteins:
A) TNFRII-Fc-IL-1 ra (SEQ ID NO: 5), TNFRI-Fc-IL-lra (SEQ ID NO: 8) and control TNFRII-Fc (SEQ ID NO: 4) or TNFRI-Fc (SEQ ID NO:7);
B) Humira (D2E7)-IL-lra (SEQ ID NOs: 10 and 11), Remicade (cA2)-IL-lra (SEQ ID NOs: 13 and 14) and control dimerized Humira (D2E7) (SEQ ID NOs: 9 and 11), and Remicade (cA2) (SEQ ID NOs: 12 and 14);
C) IL-18bp (SEQ ID NO: 15), dimerized IL-18bp-Fc (SEQ ID NO: 16), and dimerized IL-18bp-Fc-IL-lra(SEQ ID NO: 17);
D) soluble IL-4R extracellular domain (SEQ ID NO: 19), IL-4R-Fc (SEQ ID NO: 19), and IL-4R-Fc-IL-Ira (SEQ ID NO:21);
E). VEGFRl-Fc-IL-lra and light chain (SEQ ID NOs: 24 and 23), and anti-VEGF heavy chain-IL-lra and light chain (SEQ ID NOs: 25 and 23).
Most constructs encoding proteins (SEQ ID NOs: 4-25) were sequenced and expressed in mammalian cell lines. SEQ ID NOs: 4-25 are expressed by using either native or optimized codons and artificial or native secretion signal sequence in suspension adapted mammalian hosts. Dimerized antibody products were detected by non-heated SDS page gel and Western blot.
Expression titers of TNFRII-Fc (SEQ ID NO:4) and TNFRII-Fc-IL-lra (SEQ ID NO:5) in serum-free medium in 24-well plate were found to be 50 mg-100 mg/L (Figure 1), respectively. Higher expression of TNFRII-Fc-IL-lra than TNFRII-Fc in suspension adapted CHOK1 cells (estimated by direct Coomasie blue protein staining to conditional medium) was found. This result indicate that IL-lra fused chimeric proteins can be produced in mammalian host at high level enough for commercial production.
Example 2
Scale up and purification of TNFRII-Fc-IL-lra, IL-4R-ECD-Fc-IL-lra and IL-18bp-Fc-IL-lra were carried out. Cell lines were cultured in a serum-free suspension adapted in CHO-CD4 medium (Irvine Scientific) and in-house feed medium, and scaled
21
554481
up in 3 liter bioreactor (Eplikon). TNFRII-Fc-IL-lra (SEQ ID No: 5), IL-4R-ECD-Fc-IL-lra (SEQ ID No: 20), and IL-18bp-Fc-IL-lra (SEQ ID No: 17) were produed at commercial levels. These proteins were purified by protein-A direct capture, followed by ion-exchange and hydrophobic chromatography (Figures 2, 3, and 4). Bulk purified proteins were formulated, lyophilized and SEC-HPLC analyzed.
Example 3
Activities of TNFRII-Fc-IL-lra, IL-4R-Fc-IL-lra, IL-18bp-Fc-IL-lra, and VEGFRl-Fc-IL-lra were tested by bioassays.
For cell-based IL-1 neutralization assay, IL-1 dependent D10 cells (ATCC) were used to test the blocking activity of IL-lra (Kineret), TNFRII-Fc-IL-lra, IL-4R-Fc-IL-lra, and IL-18bp-Fc-IL-lra against recombinant human IL-1-dependent proliferation of D10 cells.
Briefly, human IL-1 alpha induced D10 cell proliferation in a dose—dependent manner. The concentration, at which IL-la induced 50% of the total cell growth, i.e., the EC50, was determined. The normal EC50 range for hIL-la on D10 cells was 1-5 pg/ml. When cells were pre-incubated with IL-1 receptor antagonist at effective dose, IL-lra inhibited the cell proliferation through the blockage of the cell surface IL-1 receptors. This blockage effect was also dose-dependent. When the concentration of receptor antagonist was low, it did not block the cell surface receptors. Then, IL-1 induced cell proliferation restored. The concentration of the receptor antagonist, at 50% of IL-1 activity is blocked, was the EC50 of the antagonist.
The recombinant protein (TNFRII-Fc-IL-lra, IL-4R-Fc-ILlra, IL-18bp-Fc-IL-lra, or VEGFRl-Fc-IL-lra) acted like a soluble TNFRII, IL-18, IL-4, or VEGF neutralizer as well as IL-1 receptor antagonist. The cell-based bioassays confirmed the biological activity of these chimeric molecules (Figures 6, 8, 10, and 11).
For cell-based TNF neutralization assay, L929 cells (mouse connective cell line, ATCC) were used to test TNFRITs blocking activity against TNF alpha. Briefly, TNF alpha (TNF-a) was used to induce rapid cell death in a dose-dependent manner. The
22
554481
EC50 of TNF-a (a concentration at which TNF-a induced 50% of the total cell death) was found to be less than 50 pg/ml. When TNF-a molecules were pre-incubated with high concentrations of soluble TNF receptor (sTNFR), the soluble receptor bound to TNF-a and inhibited its binding to cell surface receptors. This blocked the TNF-a activity of inducing cell death. This blockage effect was also dose-dependent. When the concentration of sTNFR was diluted down to certain point, no blocking of the TNF-a activity was found and cell death restored. Accordingly, the EC50 of the sTNFR was determined (i.e., the concentration at which it blocked 50% of TNF-a activity.).
Serial dilutions of human TNF-alpha (BioSource) in duplicates were added into a 96-well assay plate pre-seeded with constant number of L929 cells in 10% equine serum, DMEM medium supplemented with L-glutamine and 1 ug/ml of actinomycine D in a total volume of 150 ul/well. The control wells (containing cells in the medium only)
were also included. The assay plate was incubated in a humidified chamber at 37°C 5% CO2 incubator for 1 day. The cells in each well were then fixed in 10% paraformaldehyde and stained with 1% crystal violet solution. The staining were solubilized with 30%
acetic acid. The optical density (O.D.) of each well of the assay plate, which is directly proportional to the total number of cells, was then read in a plate reader at 540 nm wave length. Cytotoxicity curve is plotted with O.D. vs. TNF-alpha concentrations.
Serial dilutions of TNFRII-Fc (Enbrel) and TNFRII-Fc-IL-lra in duplicates were mixed with fixed concentration of human TNF-alpha in 10% equine serum, DMEM medium supplemented with L-glutamine and 1 ug/ml of actinomycine D in a 96-well assay plate. The assay plate was pre-incubated for 1 hour at 37°C. The mix in each well of the assay plate was transferred into another 96-well plate that was pre-seeded with constant number of L929 cells. The final concentration of human TNF-alpha in each well was 500 pg/ml in a total volume of 150 ul/well. The assay plate was incubated in humidified chamber at 37°C 5% CO2 incubator for 1 day. The cells in each well were then fixed with 10% paraformaldehyde and stained by 1% crystal violet solution. The staining was solubilized with 30% acetic acid. The optical density (O.D.) of the assay plate was then read in a
23
554481
plate reader at 540 run wavelength. The neutralization curves were plotted with O.D. vs. the concentrations of TNFRII-Fc and TNFRII-Fc-IL-lra.
The results show that human TNF alpha dose-dependently induced L929 cell death. The O.D of the background containing cells with actinomycine D only was 0.5. Human TNF-alpha dose curve decreased from base level of 0.5 to the lowest level of 0.1. The O.D. did not decrease further from human TNF-alpha concentration at 100 pg/ml and higher, indicating the saturation stage of human TNF-alpha. All experiments were carried out in duplicates and the CV% at each point was < 9%. The EC50 of human TNF-alpha under this condition was determined to be 8 pg/ml.
It was found that both TNFRII-Fc (Enbrel) and TNFRII-Fc-IL-lra dose-dependently inhibited human TNF-alpha activity on L929 cells. The O.D of the base level (for cells in presence of human TNF-alpha (500 pg/ml) and actinomycine D) was 0.1. In presence of different concentrations of TNFRII-Fc-IL-lra, the O.D.s increased from 0.1 up to the basal level of 0.5, indicating a total neutralization. Both TNFRII-Fc and TNFRII-Fc-IL-lra totally neutralized human TNF-alpha activity at concentration of 50 ng/ml. All dilutions were tested in duplicates and the CV% at each point was < 10%. The EC50 of TNFRII-Fc (Enbrel) and TNFRII-Fc-IL-lra under this condition were 3—4 ng/ml, and 10 ng/ml.
For cell-based IL-4 neutralization assay, human IL-4 induced TF-1 cell proliferation was used. TF-1 cells were incubated with media containing human IL-4 of different concentrations and then were cultured a 96-well plate in 37°C, 5% C02 incubator for 3 days. MTS was added to the cultures and incubated for 5 hours. The optical density (OD) of the plate was read at 490nm in a plate reader. The cell proliferation curve was plotted (OD vs. human IL-4 concentration). For neutralization, serial dilutions of IL-4R-Fc and IL-4R-Fc-IL-lra were pre-incubated with constant concentration of human IL-4 (2 ng/ml) in culture medium in a 96-well plate in 37°C for 1 hour. TF-1 cells of the same number were added into each well of the 96-well plate at the end of incubation. The plate was incubated in a 37°C, 5% CO2 incubator for 3 days. MTS was added and incubated for 5 hours. The OD of the plate was read at 490nm in a
24
554481
plate reader. The cell growth inhibition curve was plotted with OD vs. IL-4R-Fc and IL-4R-Fc-IL-lra concentration.
The results (Figure 7), taken together with the results of IL-1 neutralizing assay (Figure 8), show that IL-4R-Fc-IL-lra was functional and had both IL-4R and IL-1 neutralizing activity.
For cell-based IL-18 neutralization assay, human IL-18 induced IFN-g secretion from KG-1 cells (in the presence of TNFa) in a dose dependent manner was used. The EC50 of human IL-18 (the concentration at which it induces 50% of the maximum IFNg secretion of KG-1 cells) is normally between 20 - 40 ng/ml. When human IL-18 binding protein (IL-18bp) was pre-incubated with human IL-18 before applying to the cell culture, IL-18bp bound to IL-18 and blocked its activity. This blockage effect was dose-dependent. The concentration of the binding protein, at which 50% of maximum IFNg secretion is blocked, is its EC50.
Serial dilutions of IL-18bp-Fc-IL-lra and control IL-18bp-Fc in duplicates were pre-incubated with constant concentration of human IL-18 (R & D System, 50 ng/ml) in culture medium in a 96-well assay plate at 37°C for 1 hour. Duplicate of serial dilutions of human IL-18 by itself was also included in the plate as positive control. Same number of KG-1 cells (ATCC, CCL246) with constant amount of human TNFa (BioSource Inc.) was added into each well of the 96-well assay plate at the end of incubation. The assay plate was further incubated in 37°C, 5% C02 incubator for 24 hours. 50 ul/well of the culture media was transferred from each well of the assay plate to ELISA plate. Human IFNg ELISA (BioSource Inc.) was tested according to kit's instruction. The optical density (OD) of the plate was read at 450nm in a plate reader. The IFNg secretion curve induced by human IL-18 was plotted with OD vs. human IL-18 concentrations. The IL-18bp neutralization curve was plotted with OD vs. IL-18bp-Fc-IL-Ira and control IL-18bp-Fc concentrations.
The result of cell-based assays is shown in Figure 9. Taken together with the result of IL-1 neutralization assay (Figure 10), functional IL-18bp-Fc-IL-lra chimera was produced successfully. It maintained both IL-18 and IL-1 neutralizing activity.
554481
Human VEGF (vascular endothelial cell growth factor) induces HTJVE (human umbilical vein endothelial) cell proliferation in a dose dependent manner. The EC50 of human VEGF, which is the concentration that will induce 50% of the maximum proliferation of HUVE cells, was normally between 2 - 6 ng/ml. When soluble human VEGF receptor-1 was pre-incubated with human VEGF before applying to the cell culture, this soluble human VEGF receptor-1 bound to human VEGF and block its activity on the cells. This blockage effect of soluble receptor was also dose-dependent. The concentration of the soluble receptor, at which 50% of maximum cell proliferation was blocked, is its EC50. The recombinant protein VEGFRl-Fc-IL-lra was constructed with both soluble VEGF receptor and IL-1 receptor antagonist on the same molecule. Therefore it could act as soluble VEGFR1, as well as IL-1 receptor antagonist.
Serial dilutions of VEGFRl-Fc-IL-lra in duplicates were pre-incubated with constant concentration of VEGF (BioSource, 10 ng/ml) in culture medium in a 96-well assay plate at 37°C for 1 hour. Duplicates of serial dilutions of human VEGF by itself was also included in the plate as positive control. Same number of HUVE cells (Cambrex, CC-2517) were added into each well of the 96-well assay plate at the end of incubation. The assay plate was further incubated in 37°C, 5% CO2 incubator for 96 hours. MTS (Promega) was added into each well of the assay plate at the last 4 hours of incubation. The optical density (O.D.) of the plate was then read in a plate reader at 490 nm wavelength. The cell proliferation curve by VEGF was plotted with OD vs. VEGF concentrations. The VEGF-R neutralization curve was plotted with OD vs. VEGFRl-Fc-IL-lra concentrations.
Human VEGF dose dependently stimulated HUVE cell to proliferate. The ED50 was 3 ng/ml. When VEGF at 10 ng/ml was pre-incubated with serial dilutions of VEGFRl-Fc-IL-lra before applying to the cells, VEGF dependent cell proliferation was inhibited in a dose-dependent manner. The EC50 of VEGFRl-Fc-IL-lra was 15 n/ml (Figure 12). Taken together with the result of IL-1 neutralization assay (Figure 11), functional VEGFRl-Fc-IL-lra chimera was produced successfully. It maintained both VEGF and IL-1 neutralizing activity.
26
554481
Example 4
Animal testing of IL-4R-Fc-IL- Ira in a mouse model of asthma was conducted. Female BALB/c mice (6-8 wk of age) were used. In brief, these mice received 40 ug OVA (Sigma) emulsified in 2.25mg aluminum hydroxide (Pierce, Rockford, IL) in a total volume of lOOul on day 0 and 14 by ip injection.
The mice were divided into 8-hour and 48-hour divisions. 8-hour division include saline control-8hr, OVA-8hr, IL-4R-Fc/OVA-8hr and IL-4R-Fc-IL-lra/OVA-8hr groups while 48-hour division include saline control-48hr, OVA-48hr, IL-4R-Fc/OVA-48hr and IL-4R-Fc-IL-lra/OVA-48hr groups.
On day 28, all the division groups received lOOug OVA in 0.05ml normal saline by the intranasal route except for saline control groups. Saline control groups received normal saline with aluminum by the ip route on days 0 and 14, and 0.05ml of normal saline by intranasal route on day 28.
On day 29, 48-hour division groups received additional lOOug OVA in 0.05ml normal saline by the intranasal route except for saline control groups. Saline control groups also received additional 0.05ml of normal saline by intranasal route on day 29.
Administration of IL-4R-Fc and IL-4R-Fc-IL-lra
The IL-4R-Fc/OVA-8hr, IL-4R-Fc-IL-lra/OVA-8hr, IL-4R-Fc/OVA-48hr and IL-4R-Fc-IL-lra/OVA-48hr groups received 200ug/mouse/day on days 28. They were administrated by ip injection 60 min before challenge with OVA on day 28. IL-4R-Fc/OVA—48hr and IL-4R-Fc-IL-lra/OVA-48hr groups received additional 200ug/mouse/day on day 29.
Determination of cell numbers in Bronchoalveolar lavage (BLA)
For 8-hour division, 8 hours after the single intranasal OVA challenge on day 28, the mice were killed for BAL fluid and histology studies. For 48-hour division, 48 hours after two intranasal OVA challenges on day 28 and 29, the mice were killed.
27
554481
After tying off the left lung at the mainstem bronchus, the right lung was lavaged via the tracheal cannula with 1.0 ml of normal saline. Total (leukocyte) number was determined using a hemocytometer. Differential cell counts were made from cytocentrifuged preparations, stained with leukostat (fisher Diagnostics, Pittsburgh, PA). Cells were identified as macrophages, eosinophils, neutraphils, and lymphocytes by standard hematological procedures and at least 200 cells counted under x400 magnification.
Lung histology
The trachea and left lung (upper and lower lobs) were collected and fixed in Carnoy's solution at 20C for 15 hours. After embedding in paraffin, the tissues were cut into 5 um sections. For each mouse, 10 airway sections randomly distributed throughout the left lung were assessed for the severity of the cellular inflammatory response and mucus occlusion. The intensity of the cellular infiltration around pulmonary blood vessels and airway was assessed on a semiquantitative scale ranging from 0-4+.
Results
1. Treatment with IL-4R-Fc-IL-lra blocks early phase pulmonary inflammation
Table-1. Differential cell counts in BAL fluid 8 hours after the single intranasal OVA challenge. Differential cell counts were assessed in saline control-8hr, OVA-8hr, IL-4R-Fc/OVA-8hr and IL-4R-Fc-IL-lra/OVA-8hr groups (n=5 in each group; Mean ± SEM are given).
Total cell count xl0-3
Neutraphils xl0-3
Saline control-8hr
51+8
±5
OVA-8hr
220+16
172+17
IL-4R-F c/ OVA-8hr
200+11
165+10
IL-4R-Fc-IL- lra/O VA-8hr
86±7
60+8
2. Treatment with IL-4R-Fc-IL-lra also blocks late phase pulmonary inflammation
28
554481
Table-2. Differential cell counts in BAL fluid 48 hours after two intranasal OVA challenges. Differential cell counts were assessed in saline control-48hr, OVA-48hr, IL-4R-Fc/OVA-48hr and IL-4R-Fc-IL-lra/OVA-48hr groups (n=5 in each group). Mean +_SEM are given. P<0.01 compared with
Total cell count xl0-3
Eosinophils xl0-3
Saline control-48hr
44+8
3+2
OVA-48hr
180+12
52+7
IL-4R-F c/O V A-48hr
102+10
+5
IL-4R-F c-IL-1 ra/OVA-48hr
68±7
+5
Lung histology studies
The intensive cellular infiltration around pulmonary blood vessels and airway was observed in both OVA-8hr and OVA-48hr groups. Significantly reduced cellular infiltration around pulmonary blood vessels and airway were observed in IL-4R-Fc-IL-lra-8hr and IL-4R-Fc-IL-lra-48hr groups when comparing with IL-4R-Fc/OVA-8hr and IL-4R-Fc/OVA-48hr groups. The result suggests that IL-4R-Fc-ILlra was the best treatment for asthma in this animal model.
Example 5
Animal testing of IL-18bp-IgGlFc-IL-lra in a mouse CIA model was performed. CIA was induced in 8- to 10-wk-old DBA/1J mice by an intradermal injection of bovine Collagen type II (CII) according to a recently described adaptation of the standard protocol (Banada et al., 2002). Each mouse received lOO-^il injections containing 200 |ig of CII and 200 |Lig of inactivated Mycobacterium tuberculosis (Difco, Detroit, MI) in IFA on days 0 and 21. The mice (n=5) were treated between days 21 and 36 with one of two therapeutic interventions given as i.p. injections every 3 days: PBS control, 3 mg/kg IL-18bp-Fc, and 3 mg/kg IL-18bp-Fc-IL-lra. The mice were sacrificed on day 36 by cervical dislocation. Three normal DBA/1 J mice (controls) were sacrificed at the same time.
29
554481
The clinical disease activity of the CIA was assessed every other day between days 21 and 36 by two blinded observers using a three-point scale for each paw: 0 = normal joint; 1 = slight inflammation and redness; 2 = severe erythema and swelling affectingthe entire paw, with inhibition of use; and 3 = deformed paw or joint, with ankylosis, joint rigidity, and loss of function. The total score for clinical disease activity was based on all four paws, with a maximum score of 12 for each animal (Banda et al., 2002).
Both forepaws and the right hind limb were surgically removed from all mice on day 36 and fixed in 10% buffered formalin, with preparation of tissue samples and histological analysis as previously described (Bendele et al., 2000). The histological findings in paws, ankles, and knees were scored by an experienced observer who was blinded to the treatment. The data were expressed as mean scores for inflammation, pannus, cartilage damage, and bone damage as well as an overall score, based on scales of 0-5 and five joint sets per animal as previously described (Bendele et al., 2000). Results
Effect of IL-18bp-Fc-IL-lra on clinical disease activity and joint histology The incidence of development of arthritis was 100% in all groups. Compared with PBS control alone, mice treated with either 3 mg/kg IL-18bp-Fc, and 3 mg/kg IL-1 Sbp-Fc-IL-lra between days 21 and 36 showed reduction in clinical disease activity score (Table-1). Histological analysis of the joints also indicated that treatment with either 3 mg/kg IL-18bp-Fc, and 3 mg/kg IL-18bp-Fc-IL- Ira prevented joint damage compared with the PBS group. Significant differences were observed between 3 mg/kg IL-18bp-Fc, and 3 mg/kg IL-18bp-Fc-IL-lra in either clinical disease activity scores or histological scores. IL-18bp-Fc-IL-lra was significantly better than IL-18bp-Fc (Table-1).
Table-3: Clinical disease activity in CIA mice treated with IL-18bp-Fc-IL-lra.
DBA/1J mice were immunized with 200 fig of CII in IF A, with 200 Lig of added M tuberculosis on days 0 and 21. The mice were treated for 3 wk with i.p. injections every 3 days of between days 21 and 36 with one of two therapeutic interventions given as ip
554481
injection every 3 days: PBS control, 3 mg/kg IL-18bp-Fc, and 3 mg/kg IL-18bp-Fc-IL-lra. The clinical disease activity of the CIA was determined every other day by two trained observers who were blinded to the treatment and to each other, using a three-point scale for each paw. The data are expressed as the clinical disease activity score (mean ± SEM) for each treatment group vs the days after the initial collagen injection.
Clinical Disease Activity at Day 36
PBS control
8.8±0.7
IL-18bp-Fc
6.5±0.6
IL-18bp-Fc-IL-Ira
3.8±0.5
Example 6
In vivo testing of IL-18bp-Fc-IL-lra in a contact hypersensitivity (CHS) mouse model was carried out.
Induction of CHS and treatment with IL-18bp chimera
C57BL/6 mice (8 and 14 wk of age) were used. DNFB, acetone, Evans blue, formamide. BSA, PMA, ionomycin, brefeldin A, and LPS (.Escherichia coli 026:B6) were purchased from Sigma-Aldrich (St. Louis, MO). DNFB was diluted in acetone/olive oil (4/1) immediately before use. The mice were sensitized with 25 jjI of 0.5% DNFB solution painted to the shaved dorsal skin or untreated (controls). Five days later, 10 |ul of 0.2% DNFB (a nonirritant dose) was applied onto both sides of the right ear, and the same amount of solvent alone onto the left ear. Ear thickness was monitored daily from day 5 before challenge onwards using a caliper. Ear swelling was calculated as ((Tn - Ts) right ear) - (Tn - T5) left ear)), where Tn and T5 represent values of ear thickness at day n of investigation and day 5 prior to challenge, respectively. To assure that the observed swelling was due to DNFB-specific inflammation rather than nonspecific irritation, a nonsensitized but challenged control group was included with each experiment. IL-18 or/and IL-1 were neutralized by daily ip injection of 250 jug of IL-18bp-Fc or IL-18bp-Fc-IL-lra per animal, starting 60 minutes before challenge at day 5. Control animals received the vehicle saline alone. Treatment during primary re-exposure was stopped at day 7.
31
554481
Results
Therapeutic treatment with IL-18bp-Fc-IL-Ira protects against CHS
To experimentally induce CHS, mice were sensitized with the hapten DNFB on their shaved backs. CHS was elicited 5 days later by painting DNFB onto the ears. Inflammation was scored as the increase in swelling of the DNFB-challenged vs the control ear painted with solvent only.
Administration of IL-18BP-Fc and IL-18bp-Fc-IL-lra during the elicitation phase at days 5-7 significantly reduced swelling of the DNFB-challenged ears for the total duration of the response (Table-1). Significant difference between IL-18bp-Fc and IL-18bp-Fc-IL-lra was observed (Table-1), suggesting that either double-blocking IL-1 and IL-18 together ast same location or IL-1 receptor-rich site-directed nature of IL-18bp-Fc-IL-lra played important role in the effectiveness. IL-18bp-Fc-IL-lra was significantly better than IL-18bp-Fc.
Table-4: Treatment with IL-18BP during elicitation protects against CHS.
C57BL/6 mice were sensitized with DNFB at day 0 and challenged 5 days later on the ears. Ear swelling was measured daily and expressed as the increase in swelling of the DNFB-challenged vs the vehicle-painted control ear. The animals were treated daily with IL-18bp chimera or the vehicle only. The data are the mean of 5 mice per group.
Day
6
7
No treatment
0±0
110±12
160±10
IL-18bp-Fc
0±0
80±9
105±8
Il-18BP-Fc-IL-lra
0±0
50±5
70±5
Example 7
IL-1 receptor binding experiments were carried out.
Briefly, recombinant human IL-1 receptor extracellular domain was first expressed and purified in house using a mammalian CHO cells. TNFRII-Fc-IL-lra, negative control TNFRII-Fc and positive control IL-lra (Kineret) had been coated to 96-
32
554481
well plate lug/well in 100 ul coating buffer (Sigma). The purified IL-1 receptor (0. lug/well) was then incubated in PBS at 37°C for 45 minutes. The receptor/ligand binding was detected by rabbit anti human IL-1 receptor extracellular domain antibodies (R&D Systems), followed by goat anti-rabbit IgG conjugated with HRP (Pierce). After washing with PBS-T, a color reaction was developed by mixing with TMB (Sigma, T8665). The optical density (OD) of the plate was read at 650nm in an EL800 universal microplate reader (Bio-Tek). OD values were plotted against dilution times. Figure 13 showed that both TNFRII-Fc-IL-lra and IL-lra (Kineret) bound to IL-1 receptor, and that TNFRII-Fc (Enbrel) did not. Interestingly, TNFRII-Fc-IL-lra (mammalian made) bound to IL-1 receptor significantly better than that of E-coli made IL-lra (Kineret). In addition, mammalian made IL-lra contains two N-linked glycosylated sites, thus having less serum protein binding and consistent different in vitro binding property from that of E-coli made IL-lra (Kineret).
Example 8
125-1 labeling and animal testing of TNFRII-Fc-IL-lra, IL-4R-Fc-IL-lra, and IL-18bp-Fc-IL-lra. as well as their non-IL-lra fused controls, were conducted.
125-1 labeled TNFRII-Fc-IL-lra, IL-4R-Fc-IL-lra, and IL-18bp-Fc-IL-lra were made by the Iodogen method and purified by size-exclusion chromatography (M Hui et al., 1989). IL-1 receptor binding assay had been established by using in-house mammalian recombinant IL-receptor extracellular domain fused (see above Example 4). IL-1 receptor's binding to 125-1 labeled TNFRI-Fc-IL-lra was compared side by side with non-radiolabelled TNFRII-Fc-IL-lra. The results indicate that 125-1 labeled TNFRII-Fc-IL-lra is functional in terms of IL-1 receptor binding.
Mice treated with 6 nmol TPA by ear painting in 200 ul acetone consistently developed skin inflammation in 2-3 days. 125-1 labeled TNFRII-Fc-IL-lra was injected into skin-inflammation mouse models (see below) together with 125-1 labeled TNFRII-Fc (Enbrel). Surprisingly, the results indicated that 125-1 labeled TNFRII-Fc was distributed more at inflammatory site than that of TNFRII-Fc (Table 1). This most probably is due to the IL-1 receptor binding affinity.
33
554481
125-1 labeled IL-4R-Fc-IL-lra and IL-18bp-Fc-IL-lra were also injected into skin-inflammation mouse models together with 125-1 labeled IL-4R-Fc and IL-18bp-Fc. Similar results were obtained (Tables 2 and 3).
Table 5: Distribution of 125-1 labeled TNFRII-Fc-IL-lra and TNFRII-Fc (Enbrel) in inflamed and non-inflamed skin tissues 4 hours after injection. The distribution is expressed as % of injected dose per gram of tissue (n=6).
Treatment
Tissue
% of injected dose per gram tissue (n=6)
TNFRII-Fc-IL-lra 125-1
Inflamed skin
3.8±0.2
TNFRII-Fc-IL-lra 125-1
Normal skin
1.5±0.1
TNFRII-Fc (Enbrel) 125-1
Inflamed skin
2.8±0.2
TNFRII-Fc (Enbrel) 125-1
Normal skin
1.4±0.2
Table 6: Distribution of 125-1 labeled IL-4R~Fc-IL-lra and IL-4R-Fc in inflamed and non-inflamed skin tissues 4 hours after injection. The distribution is expressed as % of injected dose per gram of tissue (n=6).
Treatment
Tissue
% of injected dose per gram tissue (n=6)
IL-4R-Fc-IL-lra 125-1
Inflamed skin
4.0±0.2
IL-4R-Fc-IL-lra 125-1
Normal skin
1.6±0.1
IL-4R-Fc 125-1
Inflamed skin
1.4±0.3
IL-4R-Fc 125-1
Normal skin
1.4±0.2
Table 7: Distribution of 125-1 labeled IL-18bp-Fc-IL-Ira and ILK-18bp-Fc in inflamed and non-inflamed skin tissues 4 hours after injection. The distribution is expressed as % of injected dose per gram of tissue (n=6).
Treatment
Tissue
% of injected dose per gram tissue (n=6)
IL-18bp-Fc-IL-Ira 125-1
Inflamed skin
3.9±0.3
IL-18bp-Fc-IL-Ira 125-1
Normal skin
1.4±0.5
IL-18bp-Fc 125-1
Inflamed skin
1.5±0.3
IL-18bp-Fc 125-1
Normal skin
1.4±0.3
Example 9
34
554481
Immunogenicity of IL-4R-Fc-IL-lra was estimated in two cynomolgus monkeys. 10 mg of IL-4R-Fc-IL-lra had been injected per week sc for 8 weeks. Serum samples were collected before and after the injection (on Days 1 and 56). The samples were analyzed by the neutralization assay established for the presence of anti chimeric IL-4R-Fc-IL-1 antibodies which neutralize IL-4 and IL-1 bioactivities of the chimeric protein. In order to further detect low concentration of neutralizing antibodies, serum samples were affinity-purified by protein-A and anti-human IgM antibodies. No antibodies neutralizing IL-4 and IL-1 bioactivities of chimeric protein were detected in the treated monkeys by using both undiluted serum and purified IgG and IgM. The results suggest that chimeric IL-4R-Fc-IL-lra is not immunogenic to monkey, and human.
Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to".
The reference to any prior art in the specification is not, and should not be taken as, an acknowledgement, or any form of suggestion, that the prior art forms part of the common general knowledge in New Zealand.
301158966 1.DOC
554481
RECEIVED at IPONZ on 19 March 2010
Claims (39)
1. A fusion protein comprising a first segment that is located at the amino terminus of the fusion protein and specifically binds to and neutralizes IL18, IL4, or IL13; and a second segment that is located at the carboxyl terminus of the fusion protein and specifically binds to an IL-1 receptor, wherein the domains are operably linked.
2. The protein of claim 1, further comprising a linker segment that joins the first segment and the second segment, wherein the linker segment is capable of dimerizing.
3. The protein of claim 2, wherein the linker segment contains the Fc fragment of an immunoglobulin or a functional equivalent there of.
4. The protein of claim 3, wherein the immunoglobulin is IgA, IgE, IgD, IgG, or IgM.
5. The protein of claim 4, wherein the immunoglobulin is IgG.
6. The protein of claim 5, wherein the Fc fragment contains SEQ ID NO.: 2.
7. The protein of any one of claims 1 to 6, wherein the first segment contains the sequence of a chain of an immunoglobulin that specifically binds to and neutralizes IL18, IL4, or IL-13.
8. The protein of any one of claims 1 to 6, wherein the first segment contains the sequence of a receptor or a binding protein of IL18, IL4, and IL 13.
9. The protein of claim 8, wherein the first segment contains SEQ ID NO.: 15 or 19. 301421667_1.DOC 36 554481 RECEIVED at IPONZ on 19 March 2010
10. The protein of any one of claims 1 to 9, wherein the protein is glycosylated.
11. The protein of any one of claims 1 to 10, wherein the second segment is an antagonist of IL-1.
12. The protein of claim 11, wherein the second segment contains the sequence of IL-lra (SEQ ID NO.: 1) or a functional equivalent analogue thereof.
13. The protein of claim 11, wherein the protein contains SEQ ID NO: 17, 18, or 21.
14. An isolated nucleic acid comprising a sequence that encodes the fusion protein of any one of claims 1 to 13.
15. The nucleic acid of claim 14, wherein the nucleic acid contains a sequence encoding one of SEQ ID NOs: 1, 2, and 15 to 21.
16. A vector comprising the nucleic acid of claim 14 or 15.
17. An isolated host cell comprising a nucleic acid of claim 14 or 15 or the vector of claim 16.
18. A method of producing a polypeptide, comprising culturing the host cell of claim 17 in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cell or the medium of the cell.
19. A composition comprising a fusion protein of any one of claims 1 to 13 or a nucleic acid encoding the fusion protein; and a pharmaceutically acceptable carrier. 3Q1421667_1.DOC 37 554481 RECEIVED at IPONZ on 19 March 2010
20. The use of a fusion protein of any one of claims 1 to 13 or a nucleic acid encoding the fusion protein, in the manufacture of a medicament for modulating an immune response in a subject.
21. The use of claim 20, wherein the subject has or is at risk of acquiring a condition characterized by an excessive immune response.
22. The use of claim 20 or 21, wherein the subject has received or is contemplated to receive an allogeneic or xenogeneic transplant.
23. The use of claim 21 or 22, wherein the condition is an inflammatory disease, an autoimmune disease or an allergic disease.
24. A method of increasing the half-life of a recombinant protein in a subject, the method comprising: joining the recombinant protein to a segment containing SEQ ID NO.: 1 or a functional equivalent thereof to form a fusion protein chimera, wherein the recombinant protein binds to and neutralizes IL 18, IL4, or IL 13.
25. A method of increasing the efficacy of a recombinant protein in a subject, the method comprising: joining the recombinant protein to a segment containing SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera, wherein the recombinant protein binds to and neutralizes IL18, IL4, or IL13.
26. The use of a fusion protein chimera in the manufacture of a medicament for delivery of a therapeutic protein to a target site in a subject, wherein the fusion protein chimera comprises a therapeutic protein joined to a segment containing SEQ ID NO: 1 or a functional equivalent thereof, and 3Q1421667_1.DOC 38 554481 RECEIVED at IPONZ on 19 March 2010 wherein the therapeutic protein is targeted to an inflammatory site that is rich in IL-1 receptor and binds to and neutralizes IL18, IL4, or IL13.
27. The method of claim 25 or the use of claim 26, wherein the segment containing SEQ ID NO: 1 or a functional equivalent thereof binds to IL-1 receptor, and the recombinant protein is a therapeutic protein that binds to and neutralizes IL18, IL4, or IL13.
28. The method or use of claim 27, wherein the fusion protein chimera binds and neutralizes simultaneously to both IL-1 receptor and IL18, IL4, or IL13 at an inflammation site or at IL-1 receptor-rich disease site in a subject.
29. The method or use of claim 27 or 28, wherein the fusion protein chimera neutralizes or antagonizes the activities of both IL-1 and IL18, IL4, or IL 13 at an inflammation site or at IL-1 receptor-rich disease site in a subject.
30. A fusion protein as claimed in claim 1, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
31. An isolated nucleic acid as claimed in claim 14, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
32. A vector as claimed in claim 16, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
33. A host cell as claimed in claim 17, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing. 3Q1421667_1.DOC 39 554481 RECEIVED at IPONZ on 19 March 2010
34. A method as claimed in claim 18, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
35. A composition as claimed in claim 19, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
36. The use as claimed in claim 20, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
37. A method as claimed in claim 24, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
38. A method as claimed in claim 25, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing.
39. The use as claimed in claim 26, substantially as hereinbefore described with particular reference to any one or more of the examples, Figures and/or Sequence Listing. AMPROTEIN CORPORATION By Its Attorney BALDWINS INTELLECTUAL PROPERTY 3Q1421667_1.DOC 40
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US61847604P | 2004-10-12 | 2004-10-12 | |
US62899404P | 2004-11-17 | 2004-11-17 | |
US65073405P | 2005-02-01 | 2005-02-01 | |
PCT/US2005/012194 WO2006043972A1 (en) | 2004-10-12 | 2005-04-08 | Chimeric protein |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ554481A true NZ554481A (en) | 2010-04-30 |
Family
ID=39445826
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NZ554481A NZ554481A (en) | 2004-10-12 | 2005-04-08 | Fusion protein that binds to IL-1 receptor and one of IL18, IL4 or IL13 |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP1799246A4 (en) |
JP (1) | JP2008515970A (en) |
KR (1) | KR20080022539A (en) |
AU (1) | AU2005296277A1 (en) |
BR (1) | BRPI0516350A (en) |
CA (1) | CA2583937A1 (en) |
IL (1) | IL182497A0 (en) |
NZ (1) | NZ554481A (en) |
WO (1) | WO2006043972A1 (en) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10259860B2 (en) * | 2007-02-27 | 2019-04-16 | Aprogen Inc. | Fusion proteins binding to VEGF and angiopoietin |
EP3575317A1 (en) * | 2007-12-26 | 2019-12-04 | Xencor, Inc. | Fc variants with altered binding to fcrn |
PL2259774T3 (en) | 2008-02-27 | 2013-04-30 | Biomet Biologics Llc | Methods and compositions for delivering interleukin-1 receptor antagonist |
US8753690B2 (en) | 2008-02-27 | 2014-06-17 | Biomet Biologics, Llc | Methods and compositions for delivering interleukin-1 receptor antagonist |
WO2009149205A2 (en) * | 2008-06-03 | 2009-12-10 | Neurotech Usa, Inc. | Cell lines that secrete soluble vegf receptors and uses thereof |
CN102171247A (en) * | 2008-07-02 | 2011-08-31 | 特鲁比昂药品公司 | TNF-alpha antagonist multi-target binding proteins |
KR20110044992A (en) * | 2008-07-02 | 2011-05-03 | 이머전트 프로덕트 디벨롭먼트 시애틀, 엘엘씨 | TVF-β antagonist multi-target binding protein |
US8722860B2 (en) | 2009-04-16 | 2014-05-13 | Abbvie Biotherapeutics Inc. | Anti-TNF-α antibodies and their uses |
MX346002B (en) | 2009-06-17 | 2017-03-01 | Abbvie Biotherapeutics Inc | Anti-vegf antibodies and their uses. |
CN102573790B (en) | 2009-08-27 | 2017-03-22 | 拜欧米特生物制剂有限责任公司 | Implantable device for production of interleukin-1 receptor antagonist |
KR101004363B1 (en) * | 2010-03-19 | 2010-12-28 | 가톨릭대학교 산학협력단 | TNF-α AND IL-21 COUPLED ANTAGONIST FOR THE PREVENTION AND TREATMENT OF AUTOIMMUNE DISEASES |
KR101004362B1 (en) | 2010-03-19 | 2010-12-28 | 가톨릭대학교 산학협력단 | TNF-α AND TWEAK COUPLED ANTAGONIST FOR THE PREVENTION AND TREATMENT OF AUTOIMMUNE DISEASES |
US20140154255A1 (en) | 2012-11-30 | 2014-06-05 | Abbvie Biotherapeutics Inc. | Anti-vegf antibodies and their uses |
US9878011B2 (en) * | 2013-03-15 | 2018-01-30 | Biomet Biologics, Llc | Treatment of inflammatory respiratory disease using biological solutions |
US10208095B2 (en) | 2013-03-15 | 2019-02-19 | Biomet Manufacturing, Llc | Methods for making cytokine compositions from tissues using non-centrifugal methods |
US9758806B2 (en) | 2013-03-15 | 2017-09-12 | Biomet Biologics, Llc | Acellular compositions for treating inflammatory disorders |
US10143725B2 (en) | 2013-03-15 | 2018-12-04 | Biomet Biologics, Llc | Treatment of pain using protein solutions |
US20140271589A1 (en) | 2013-03-15 | 2014-09-18 | Biomet Biologics, Llc | Treatment of collagen defects using protein solutions |
US9895418B2 (en) | 2013-03-15 | 2018-02-20 | Biomet Biologics, Llc | Treatment of peripheral vascular disease using protein solutions |
US9950035B2 (en) | 2013-03-15 | 2018-04-24 | Biomet Biologics, Llc | Methods and non-immunogenic compositions for treating inflammatory disorders |
WO2015081253A1 (en) | 2013-11-26 | 2015-06-04 | Biomet Biologics, Llc | Methods of mediating macrophage phenotypes |
US10441635B2 (en) | 2014-11-10 | 2019-10-15 | Biomet Biologics, Llc | Methods of treating pain using protein solutions |
US9763800B2 (en) | 2015-03-18 | 2017-09-19 | Biomet C. V. | Implant configured for hammertoe and small bone fixation |
CN113402583B (en) * | 2021-06-19 | 2023-03-21 | 江西农业大学 | QGK tripeptide and application thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6541610B1 (en) * | 1989-09-05 | 2003-04-01 | Immunex Corporation | Fusion proteins comprising tumor necrosis factor receptor |
JP3693671B2 (en) * | 1991-03-15 | 2005-09-07 | アムゲン インコーポレーテッド | PEGylation of polypeptides |
US6548634B1 (en) * | 1998-09-30 | 2003-04-15 | Chiron Corporation | Synthetic peptides having FGF receptor affinity |
US7700318B2 (en) * | 2004-08-25 | 2010-04-20 | Amprotein Corporation | Chimeric polypeptide and use thereof |
-
2005
- 2005-04-08 AU AU2005296277A patent/AU2005296277A1/en not_active Abandoned
- 2005-04-08 EP EP05737667A patent/EP1799246A4/en not_active Withdrawn
- 2005-04-08 KR KR1020077008321A patent/KR20080022539A/en not_active Application Discontinuation
- 2005-04-08 WO PCT/US2005/012194 patent/WO2006043972A1/en active Application Filing
- 2005-04-08 CA CA002583937A patent/CA2583937A1/en not_active Abandoned
- 2005-04-08 BR BRPI0516350-1A patent/BRPI0516350A/en not_active IP Right Cessation
- 2005-04-08 NZ NZ554481A patent/NZ554481A/en unknown
- 2005-04-08 JP JP2007536675A patent/JP2008515970A/en active Pending
-
2007
- 2007-04-12 IL IL182497A patent/IL182497A0/en unknown
Also Published As
Publication number | Publication date |
---|---|
IL182497A0 (en) | 2007-09-20 |
EP1799246A1 (en) | 2007-06-27 |
AU2005296277A1 (en) | 2006-04-27 |
CA2583937A1 (en) | 2006-04-27 |
KR20080022539A (en) | 2008-03-11 |
WO2006043972A1 (en) | 2006-04-27 |
EP1799246A4 (en) | 2009-08-12 |
WO2006043972A8 (en) | 2006-06-15 |
JP2008515970A (en) | 2008-05-15 |
BRPI0516350A (en) | 2008-09-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
NZ554481A (en) | Fusion protein that binds to IL-1 receptor and one of IL18, IL4 or IL13 | |
RU2711979C2 (en) | Interleukin 15 protein complex and use thereof | |
US20080292628A1 (en) | Chimeric Protein | |
CA2194299A1 (en) | Novel cytokine designated lerk-5 | |
JP2011079863A (en) | April receptor (bcma) and use thereof | |
JP2002500886A (en) | IL-18 receptor | |
WO2014207173A1 (en) | Interleukin 15 (il-15) antagonists and uses thereof for the treatment of autoimmune diseases and inflammatory diseases | |
WO2018166468A1 (en) | Igg-like long-acting immune fusion protein and use thereof | |
JP2001506982A (en) | How to control nitric oxide production | |
JPH09508009A (en) | Ligands that bind Fas antigen | |
TWI694084B (en) | Uti fusion proteins | |
TW201623330A (en) | IL-21 antibodies | |
US11667704B2 (en) | Anti-IL-17 antibody/TNFR ECD fusion protein and use thereof | |
WO2015172305A1 (en) | Fusion protein inhibiting taci-baff complex formation and preparation method therefor and use thereof | |
JP2023096044A (en) | Immunomodulatory fusion proteins | |
US20180028612A1 (en) | Compositions and methods of using a soluble TNF-alpha receptor modified for increased half-life | |
US20230340054A1 (en) | Interleukin-2 muteins and uses thereof | |
EP2357238A2 (en) | Compositions and methods for regulation of tumor necrosis factor-alpha | |
EP3101035B1 (en) | Bifunctional fusion protein, preparation method therefor, and use thereof | |
JP2002507127A (en) | Proteins that bind to TRAIL | |
US7700318B2 (en) | Chimeric polypeptide and use thereof | |
JP6320973B2 (en) | B cell activator antagonist, preparation method and use thereof | |
KR101273893B1 (en) | Modified human tumor necrosis factor receptor-1 polypeptides or fragments thereof and method for preparing the same | |
KR20110043485A (en) | Modified human tumor necrosis factor receptor-1 polypeptide or fragment thereofand method for preparing the same | |
WO2023060165A2 (en) | Interleukin-10 muteins and fusion proteins thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PSEA | Patent sealed |