NZ710831B2 - Anti-il-33 antibodies and uses thereof - Google Patents
Anti-il-33 antibodies and uses thereof Download PDFInfo
- Publication number
- NZ710831B2 NZ710831B2 NZ710831A NZ71083114A NZ710831B2 NZ 710831 B2 NZ710831 B2 NZ 710831B2 NZ 710831 A NZ710831 A NZ 710831A NZ 71083114 A NZ71083114 A NZ 71083114A NZ 710831 B2 NZ710831 B2 NZ 710831B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- antibody
- antibodies
- antigen
- human
- binding
- Prior art date
Links
- 102000004965 antibodies Human genes 0.000 title claims abstract description 518
- 108090001123 antibodies Proteins 0.000 title claims abstract description 518
- 102000017761 Interleukin-33 Human genes 0.000 claims abstract description 261
- 108010067003 Interleukin-33 Proteins 0.000 claims abstract description 261
- 230000011664 signaling Effects 0.000 claims abstract description 25
- 230000003993 interaction Effects 0.000 claims abstract description 15
- 230000027455 binding Effects 0.000 claims description 222
- 239000000427 antigen Substances 0.000 claims description 111
- 102000038129 antigens Human genes 0.000 claims description 111
- 108091007172 antigens Proteins 0.000 claims description 111
- 210000004027 cells Anatomy 0.000 claims description 68
- 210000004072 Lung Anatomy 0.000 claims description 64
- 238000004166 bioassay Methods 0.000 claims description 49
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 39
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 36
- 108010045030 monoclonal antibodies Proteins 0.000 claims description 36
- 102000005614 monoclonal antibodies Human genes 0.000 claims description 36
- 101700086956 IFNG Proteins 0.000 claims description 29
- 102100016020 IFNG Human genes 0.000 claims description 29
- 210000003651 Basophils Anatomy 0.000 claims description 23
- 101000408083 IL33 Proteins 0.000 claims description 20
- 102000028446 human IL33 protein Human genes 0.000 claims description 20
- 238000000338 in vitro Methods 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 19
- 206010061218 Inflammation Diseases 0.000 claims description 16
- 230000004054 inflammatory process Effects 0.000 claims description 16
- 230000002009 allergen Effects 0.000 claims description 14
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 claims description 11
- 238000010494 dissociation reaction Methods 0.000 claims description 9
- 230000005593 dissociations Effects 0.000 claims description 9
- 210000003979 Eosinophils Anatomy 0.000 claims description 7
- 230000004913 activation Effects 0.000 claims description 7
- NFGXHKASABOEEW-UHFFFAOYSA-N (+)-methoprene Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 210000001744 T-Lymphocytes Anatomy 0.000 claims description 6
- 238000010171 animal model Methods 0.000 claims description 6
- 230000036499 Half live Effects 0.000 claims description 5
- 238000000670 ligand binding assay Methods 0.000 claims description 2
- 238000001525 receptor binding assay Methods 0.000 claims description 2
- URRBLVUOXIGNQR-HXUWFJFHSA-N [(1R)-1-phenylethyl] N-(2-aminoethyl)-N-[(3-methoxy-4-phenylmethoxyphenyl)methyl]carbamate Chemical compound C1([C@@H](C)OC(=O)N(CCN)CC=2C=C(C(=CC=2)OCC=2C=CC=CC=2)OC)=CC=CC=C1 URRBLVUOXIGNQR-HXUWFJFHSA-N 0.000 claims 3
- 108090000978 Interleukin-4 Proteins 0.000 claims 2
- 108010002616 Interleukin-5 Proteins 0.000 claims 2
- 201000010099 disease Diseases 0.000 abstract description 34
- 101710039379 IL1RL1 Proteins 0.000 abstract description 31
- 200000000018 inflammatory disease Diseases 0.000 abstract description 30
- 101710017523 ST2 Proteins 0.000 abstract description 29
- 101710019687 SULT2A1 Proteins 0.000 abstract description 29
- 230000000903 blocking Effects 0.000 abstract description 26
- 230000014509 gene expression Effects 0.000 abstract description 16
- 230000001404 mediated Effects 0.000 abstract description 6
- 201000005794 allergic hypersensitivity disease Diseases 0.000 abstract description 4
- 230000001413 cellular Effects 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 description 47
- 108090000623 proteins and genes Proteins 0.000 description 47
- 241000238711 Pyroglyphidae Species 0.000 description 46
- 229940046533 house dust mites Drugs 0.000 description 46
- 235000018102 proteins Nutrition 0.000 description 46
- 235000001014 amino acid Nutrition 0.000 description 39
- 230000002401 inhibitory effect Effects 0.000 description 36
- 239000002663 humin Substances 0.000 description 34
- 239000000203 mixture Substances 0.000 description 34
- 150000001413 amino acids Chemical class 0.000 description 32
- 230000003042 antagnostic Effects 0.000 description 32
- 210000003819 Peripheral blood mononuclear cell Anatomy 0.000 description 31
- 239000002953 phosphate buffered saline Substances 0.000 description 30
- 239000005557 antagonist Substances 0.000 description 29
- 102100014181 IL1RL1 Human genes 0.000 description 28
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 25
- 210000004602 germ cell Anatomy 0.000 description 24
- 230000000694 effects Effects 0.000 description 23
- 230000004048 modification Effects 0.000 description 23
- 238000006011 modification reaction Methods 0.000 description 23
- 239000000284 extract Substances 0.000 description 22
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 20
- 229960000060 monoclonal antibodies Drugs 0.000 description 20
- 239000008194 pharmaceutical composition Substances 0.000 description 20
- 230000035772 mutation Effects 0.000 description 19
- 208000006673 Asthma Diseases 0.000 description 18
- 229920001184 polypeptide Polymers 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 17
- 230000035492 administration Effects 0.000 description 16
- 238000006467 substitution reaction Methods 0.000 description 16
- 206010016654 Fibrosis Diseases 0.000 description 15
- 230000004761 fibrosis Effects 0.000 description 15
- 230000004044 response Effects 0.000 description 15
- 230000002378 acidificating Effects 0.000 description 14
- 230000002829 reduced Effects 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 229920001850 Nucleic acid sequence Polymers 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 102000018358 Immunoglobulins Human genes 0.000 description 12
- 108060003951 Immunoglobulins Proteins 0.000 description 12
- 238000002820 assay format Methods 0.000 description 12
- 108091006028 chimera Proteins 0.000 description 12
- 230000001264 neutralization Effects 0.000 description 12
- 150000007523 nucleic acids Chemical class 0.000 description 11
- 102000005962 receptors Human genes 0.000 description 11
- 108020003175 receptors Proteins 0.000 description 11
- 239000012146 running buffer Substances 0.000 description 11
- 241000282693 Cercopithecidae Species 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 102100013077 CD4 Human genes 0.000 description 9
- 101700022938 CD4 Proteins 0.000 description 9
- 102100018956 IL1RAP Human genes 0.000 description 9
- 108010045623 Interleukin-1 Receptor Accessory Protein Proteins 0.000 description 9
- 125000000539 amino acid group Chemical group 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 8
- 230000000875 corresponding Effects 0.000 description 8
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 230000001225 therapeutic Effects 0.000 description 8
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 7
- 230000001154 acute Effects 0.000 description 7
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 101710027066 ALB Proteins 0.000 description 6
- 206010003246 Arthritis Diseases 0.000 description 6
- 210000003719 B-Lymphocytes Anatomy 0.000 description 6
- 101700027814 CDR3 Proteins 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 102100005499 PTPRC Human genes 0.000 description 6
- 101700059076 PTPRC Proteins 0.000 description 6
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 201000008937 atopic dermatitis Diseases 0.000 description 6
- 230000001684 chronic Effects 0.000 description 6
- 230000001419 dependent Effects 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- 201000004681 psoriasis Diseases 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 210000001519 tissues Anatomy 0.000 description 6
- 210000004369 Blood Anatomy 0.000 description 5
- 206010012438 Dermatitis atopic Diseases 0.000 description 5
- 238000007792 addition Methods 0.000 description 5
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000007405 data analysis Methods 0.000 description 5
- 230000001809 detectable Effects 0.000 description 5
- 229910052805 deuterium Inorganic materials 0.000 description 5
- 239000002552 dosage form Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 101700039188 hst2 Proteins 0.000 description 5
- 238000003018 immunoassay Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 201000006417 multiple sclerosis Diseases 0.000 description 5
- 230000036961 partial Effects 0.000 description 5
- -1 phosphoryl groups Chemical group 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 150000003431 steroids Chemical class 0.000 description 5
- 238000007920 subcutaneous administration Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000004094 surface-active agent Substances 0.000 description 5
- UCSJYZPVAKXKNQ-HZYVHMACSA-N 1-[(1S,2R,3R,4S,5R,6R)-3-carbamimidamido-6-{[(2R,3R,4R,5S)-3-{[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxan-2-yl]oxy}-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy}-2,4,5-trihydroxycyclohexyl]guanidine Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000009010 Bradford assay Methods 0.000 description 4
- 102100005826 CD19 Human genes 0.000 description 4
- 101700087100 CD19 Proteins 0.000 description 4
- 101700080416 CD69 Proteins 0.000 description 4
- 102100005832 CD69 Human genes 0.000 description 4
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- 206010020751 Hypersensitivity Diseases 0.000 description 4
- 101710006689 ITGAX Proteins 0.000 description 4
- 102100019437 ITGAX Human genes 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- 108060001084 Luciferase family Proteins 0.000 description 4
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 4
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 4
- 102000003714 TNF receptor-associated factor 6 Human genes 0.000 description 4
- 108090000009 TNF receptor-associated factor 6 Proteins 0.000 description 4
- 102100005288 TSLP Human genes 0.000 description 4
- 102000025417 antigen binding proteins Human genes 0.000 description 4
- 108091000829 antigen binding proteins Proteins 0.000 description 4
- 238000002838 bio layer interferometry Methods 0.000 description 4
- YZCKVEUIGOORGS-OUBTZVSYSA-N deuterium Chemical group [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000003899 glycosylation Effects 0.000 description 4
- 238000006206 glycosylation reaction Methods 0.000 description 4
- 238000003306 harvesting Methods 0.000 description 4
- 230000001976 improved Effects 0.000 description 4
- 230000002934 lysing Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000002609 media Substances 0.000 description 4
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 4
- 108091008117 polyclonal antibodies Proteins 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000002685 pulmonary Effects 0.000 description 4
- 108010029307 thymic stromal lymphopoietin Proteins 0.000 description 4
- 102200012730 ABCB1 N44S Human genes 0.000 description 3
- 102100007788 APC Human genes 0.000 description 3
- 206010064212 Eosinophilic oesophagitis Diseases 0.000 description 3
- UCTWMZQNUQWSLP-VIFPVBQESA-N Epinephrine Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 3
- 210000003743 Erythrocytes Anatomy 0.000 description 3
- 206010015719 Exsanguination Diseases 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 108010027440 Immunoconjugates Proteins 0.000 description 3
- 102000018748 Immunoconjugates Human genes 0.000 description 3
- 206010021972 Inflammatory bowel disease Diseases 0.000 description 3
- 102000013691 Interleukin-17 Human genes 0.000 description 3
- 108050003558 Interleukin-17 family Proteins 0.000 description 3
- 238000001282 Kruskal–Wallis one-way analysis of variance Methods 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- 241000282567 Macaca fascicularis Species 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 102000003945 NF-kappa B Human genes 0.000 description 3
- 108010057466 NF-kappa B Proteins 0.000 description 3
- 239000004698 Polyethylene (PE) Substances 0.000 description 3
- 208000005069 Pulmonary Fibrosis Diseases 0.000 description 3
- 210000003324 RBC Anatomy 0.000 description 3
- 102200003743 RMDN1 K52N Human genes 0.000 description 3
- 239000007759 RPMI Media 1640 Substances 0.000 description 3
- 206010039085 Rhinitis allergic Diseases 0.000 description 3
- 206010039710 Scleroderma Diseases 0.000 description 3
- 210000002966 Serum Anatomy 0.000 description 3
- 201000010105 allergic rhinitis Diseases 0.000 description 3
- 230000036783 anaphylactic response Effects 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000002860 competitive Effects 0.000 description 3
- 230000000295 complement Effects 0.000 description 3
- 239000003246 corticosteroid Substances 0.000 description 3
- 230000001808 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 231100000599 cytotoxic agent Toxicity 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000003292 diminished Effects 0.000 description 3
- 201000000708 eosinophilic esophagitis Diseases 0.000 description 3
- 230000003176 fibrotic Effects 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 238000011068 load Methods 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 230000003472 neutralizing Effects 0.000 description 3
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 3
- 238000010149 post-hoc-test Methods 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 230000035489 relative bioavailability Effects 0.000 description 3
- 102220117530 rs112626848 Human genes 0.000 description 3
- 238000009738 saturating Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- 206010048594 Allergic granulomatous angiitis Diseases 0.000 description 2
- 206010002198 Anaphylactic reaction Diseases 0.000 description 2
- 208000003455 Anaphylaxis Diseases 0.000 description 2
- 210000000612 Antigen-Presenting Cells Anatomy 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 229940090047 Auto-Injector Drugs 0.000 description 2
- 208000009137 Behcet Syndrome Diseases 0.000 description 2
- 201000008335 Behcet's disease Diseases 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N Benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- 230000036912 Bioavailability Effects 0.000 description 2
- 229940098773 Bovine Serum Albumin Drugs 0.000 description 2
- 108091003117 Bovine Serum Albumin Proteins 0.000 description 2
- 102200148758 CAV3 V57M Human genes 0.000 description 2
- 231100000023 Cell-mediated cytotoxicity Toxicity 0.000 description 2
- 206010057250 Cell-mediated cytotoxicity Diseases 0.000 description 2
- 208000006344 Churg-Strauss Syndrome Diseases 0.000 description 2
- 206010009900 Colitis ulcerative Diseases 0.000 description 2
- 206010011401 Crohn's disease Diseases 0.000 description 2
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102100014838 FCGRT Human genes 0.000 description 2
- 101710003435 FCGRT Proteins 0.000 description 2
- 230000035693 Fab Effects 0.000 description 2
- 208000007465 Giant Cell Arteritis Diseases 0.000 description 2
- 229960002743 Glutamine Drugs 0.000 description 2
- 201000004331 Henoch-Schoenlein purpura Diseases 0.000 description 2
- 206010019617 Henoch-Schonlein purpura Diseases 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 210000004408 Hybridomas Anatomy 0.000 description 2
- 101000186975 IL1RL1 Proteins 0.000 description 2
- 101710006572 ITGAM Proteins 0.000 description 2
- 102100019441 ITGAM Human genes 0.000 description 2
- 210000000987 Immune System Anatomy 0.000 description 2
- 206010027665 Immune disorder Diseases 0.000 description 2
- 206010021425 Immune system disease Diseases 0.000 description 2
- 206010022114 Injury Diseases 0.000 description 2
- 102000019223 Interleukin-1 receptor family Human genes 0.000 description 2
- 108050006617 Interleukin-1 receptor family Proteins 0.000 description 2
- 108010002386 Interleukin-3 Proteins 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 210000004185 Liver Anatomy 0.000 description 2
- 210000004698 Lymphocytes Anatomy 0.000 description 2
- 229940062713 MITE EXTRACT Drugs 0.000 description 2
- 210000002540 Macrophages Anatomy 0.000 description 2
- 210000003097 Mucus Anatomy 0.000 description 2
- 210000000440 Neutrophils Anatomy 0.000 description 2
- 206010053219 Non-alcoholic steatohepatitis Diseases 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229940049954 Penicillin Drugs 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 206010037162 Psoriatic arthropathy Diseases 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 206010039073 Rheumatoid arthritis Diseases 0.000 description 2
- 101710045184 SIGLEC5 Proteins 0.000 description 2
- 208000005511 Schoenlein-Henoch Purpura Diseases 0.000 description 2
- 206010040767 Sjogren's syndrome Diseases 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 229960005322 Streptomycin Drugs 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 206010043207 Temporal arteritis Diseases 0.000 description 2
- 206010047115 Vasculitis Diseases 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 201000009961 allergic asthma Diseases 0.000 description 2
- 230000001668 ameliorated Effects 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 230000001387 anti-histamine Effects 0.000 description 2
- 230000003110 anti-inflammatory Effects 0.000 description 2
- 239000000739 antihistaminic agent Substances 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 229960000626 benzylpenicillin Drugs 0.000 description 2
- 230000035514 bioavailability Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000019504 cigarettes Nutrition 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 239000002254 cytotoxic agent Substances 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 239000000850 decongestant Substances 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229960005139 epinephrine Drugs 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 102000030909 human IL1RL1 protein Human genes 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000002757 inflammatory Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000016507 interphase Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000000670 limiting Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 201000004044 liver cirrhosis Diseases 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 229940021182 non-steroidal anti-inflammatory drugs Drugs 0.000 description 2
- 230000036963 noncompetitive Effects 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 201000008482 osteoarthritis Diseases 0.000 description 2
- 230000001575 pathological Effects 0.000 description 2
- ORMNNUPLFAPCFD-MPQOODJTSA-M potassium;(2S,5R,6R)-3,3-dimethyl-7-oxo-6-[[(2R)-2-phenoxypropanoyl]amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate Chemical compound [K+].O([C@H](C)C(=O)N[C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C([O-])=O)=O)C1=CC=CC=C1 ORMNNUPLFAPCFD-MPQOODJTSA-M 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000000770 pro-inflamatory Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 201000001263 psoriatic arthritis Diseases 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 239000000779 smoke Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000003637 steroidlike Effects 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 102000002689 toll-like receptors Human genes 0.000 description 2
- 108020000411 toll-like receptors Proteins 0.000 description 2
- 230000001960 triggered Effects 0.000 description 2
- 201000006704 ulcerative colitis Diseases 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- BEJKOYIMCGMNRB-GRHHLOCNSA-N (2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid;(2S)-2-amino-3-phenylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1.OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 BEJKOYIMCGMNRB-GRHHLOCNSA-N 0.000 description 1
- UHZZMRAGKVHANO-UHFFFAOYSA-M 2-chloroethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CCCl UHZZMRAGKVHANO-UHFFFAOYSA-M 0.000 description 1
- 101700027111 3SA0 Proteins 0.000 description 1
- 229940035676 ANALGESICS Drugs 0.000 description 1
- 102200016408 ANKRD34A Q15R Human genes 0.000 description 1
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 206010069351 Acute lung injury Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000005875 Alternating hemiplegia of childhood Diseases 0.000 description 1
- 206010002220 Anaphylactic response Diseases 0.000 description 1
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 1
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 1
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 229960001230 Asparagine Drugs 0.000 description 1
- 229940009098 Aspartate Drugs 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- 108010071919 Bispecific Antibodies Proteins 0.000 description 1
- 229960001561 Bleomycin Drugs 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102100008191 CD8A Human genes 0.000 description 1
- 101700054655 CD8A Proteins 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000008787 Cardiovascular Disease Diseases 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 208000000350 Central Nervous System Disease Diseases 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 229920000453 Consensus sequence Polymers 0.000 description 1
- 229940064701 Corticosteroid nasal preparations for topical use Drugs 0.000 description 1
- 229960001334 Corticosteroids Drugs 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UWTATZPHSA-N D-serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N DL-aspartic acid Chemical compound OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 210000004443 Dendritic Cells Anatomy 0.000 description 1
- 230000036947 Dissociation constant Effects 0.000 description 1
- 102000033147 ERVK-25 Human genes 0.000 description 1
- 210000001163 Endosomes Anatomy 0.000 description 1
- 210000002889 Endothelial Cells Anatomy 0.000 description 1
- 229940088598 Enzyme Drugs 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010014950 Eosinophilia Diseases 0.000 description 1
- 229940015979 Epipen Drugs 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 101710044640 FCGR2A Proteins 0.000 description 1
- 101710044641 FCGR2B Proteins 0.000 description 1
- 102100015544 FCGR2C Human genes 0.000 description 1
- 101710044642 FCGR2C Proteins 0.000 description 1
- 102100015541 FCGR3A Human genes 0.000 description 1
- 101710044656 FCGR3A Proteins 0.000 description 1
- 101710044657 FCGR3B Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N Fucose Chemical group C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- 102200069956 GJA1 H95R Human genes 0.000 description 1
- 102100011343 GLB1 Human genes 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 229940062714 Humalog Mix Drugs 0.000 description 1
- 229940048921 Humira Drugs 0.000 description 1
- 206010048643 Hypereosinophilic syndrome Diseases 0.000 description 1
- 102100005026 IL21 Human genes 0.000 description 1
- 102200069439 IL21 R69K Human genes 0.000 description 1
- 229960000310 ISOLEUCINE Drugs 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 210000004969 Inflammatory Cells Anatomy 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090000177 Interleukin-11 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 210000004347 Intestinal Mucosa Anatomy 0.000 description 1
- 210000003734 Kidney Anatomy 0.000 description 1
- 210000000822 Killer Cells, Natural Anatomy 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108010052014 Liberase Proteins 0.000 description 1
- 206010025135 Lupus erythematosus Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 210000000138 Mast Cells Anatomy 0.000 description 1
- 210000000274 Microglia Anatomy 0.000 description 1
- 210000001616 Monocytes Anatomy 0.000 description 1
- 210000002200 Mouth Mucosa Anatomy 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010028537 Myelofibrosis Diseases 0.000 description 1
- 108091007229 NSP3 Papain-like protease domain Proteins 0.000 description 1
- 206010029888 Obliterative bronchiolitis Diseases 0.000 description 1
- 229920000272 Oligonucleotide Polymers 0.000 description 1
- 241000283898 Ovis Species 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 229940090048 Pen Injector Drugs 0.000 description 1
- 108091005771 Peptidases Proteins 0.000 description 1
- 229960005190 Phenylalanine Drugs 0.000 description 1
- 210000002381 Plasma Anatomy 0.000 description 1
- 206010035737 Pneumonia viral Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229940068968 Polysorbate 80 Drugs 0.000 description 1
- 208000003476 Primary Myelofibrosis Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010051739 Pulmonary sepsis Diseases 0.000 description 1
- 102200045029 RFK E79Q Human genes 0.000 description 1
- 206010038687 Respiratory distress Diseases 0.000 description 1
- 229920001785 Response element Polymers 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N Rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 108010070144 Single-Chain Antibodies Proteins 0.000 description 1
- 102000005632 Single-Chain Antibodies Human genes 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 210000003491 Skin Anatomy 0.000 description 1
- 206010050207 Skin fibrosis Diseases 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102200133401 TM9SF1 L18M Human genes 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 1
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 1
- 208000009421 Viral Pneumonia Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000001594 aberrant Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000000240 adjuvant Effects 0.000 description 1
- 230000001058 adult Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 231100000494 adverse effect Toxicity 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 230000001270 agonistic Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 230000000172 allergic Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000000202 analgesic Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000111 anti-oxidant Effects 0.000 description 1
- 230000000840 anti-viral Effects 0.000 description 1
- 230000000890 antigenic Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000010425 asbestos Substances 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 201000001320 atherosclerosis Diseases 0.000 description 1
- 201000001178 bacterial pneumonia Diseases 0.000 description 1
- 239000003855 balanced salt solution Substances 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000003115 biocidal Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 210000003008 brain-resident macrophage Anatomy 0.000 description 1
- 201000003848 bronchiolitis obliterans Diseases 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 102220349284 c.287A>T Human genes 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 201000008779 central nervous system disease Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated Effects 0.000 description 1
- 101700067609 ctx Proteins 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 239000000430 cytokine receptor antagonist Substances 0.000 description 1
- 230000001472 cytotoxic Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000001627 detrimental Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000003467 diminishing Effects 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 229940079593 drugs Drugs 0.000 description 1
- 230000001804 emulsifying Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing Effects 0.000 description 1
- 210000002919 epithelial cells Anatomy 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000001747 exhibiting Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic Effects 0.000 description 1
- 238000010358 genetic engineering technique Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 125000003372 histidine group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 201000009794 idiopathic pulmonary fibrosis Diseases 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001861 immunosuppresant Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 210000004964 innate lymphoid cells Anatomy 0.000 description 1
- 229940079866 intestinal antibiotics Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000002147 killing Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 230000003278 mimic Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000000869 mutational Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 1
- 229940005943 ophthalmologic Antivirals Drugs 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- VYNDHICBIRRPFP-UHFFFAOYSA-N pacific blue Chemical compound FC1=C(O)C(F)=C2OC(=O)C(C(=O)O)=CC2=C1 VYNDHICBIRRPFP-UHFFFAOYSA-N 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 230000000737 periodic Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000275 pharmacokinetic Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002587 phosphodiesterase IV inhibitor Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920001690 polydopamine Polymers 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002797 proteolythic Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000000268 renotropic Effects 0.000 description 1
- 200000000008 restenosis Diseases 0.000 description 1
- 229910052895 riebeckite Inorganic materials 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 102220210869 rs1057524586 Human genes 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000001235 sensitizing Effects 0.000 description 1
- 239000004017 serum-free culture media Substances 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 201000010001 silicosis Diseases 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 230000000392 somatic Effects 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 229940026754 topical Antivirals Drugs 0.000 description 1
- 229940083878 topical for treatment of hemorrhoids and anal fissures Corticosteroids Drugs 0.000 description 1
- 238000004450 types of analysis Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000003612 virological Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 230000003442 weekly Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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
- A61P11/00—Drugs for disorders of the respiratory system
-
- 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
- A61P11/02—Nasal agents, e.g. decongestants
-
- 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
- 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
- A61P17/04—Antipruritics
-
- 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
- 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
- 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
- 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
- A61P37/08—Antiallergic agents
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Abstract
The present invention provides antibodies that bind to interleukin-33 (IL-33) and methods of using the same. The invention includes antibodies that inhibit or attenuate IL-33-mediated signaling. The antibodies of the invention may function to block the interaction between IL-33 and ST2. Alternatively, certain antibodies of the invention inhibit or attenuate IL-33-mediated signaling without blocking the IL-33/ST2 interaction. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human IL-33 with high affinity. The antibodies of the invention are useful for the treatment of diseases and disorders associated with IL-33 signaling and/or IL-33 cellular expression, such as inflammatory diseases, or allergic diseases. y, certain antibodies of the invention inhibit or attenuate IL-33-mediated signaling without blocking the IL-33/ST2 interaction. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human IL-33 with high affinity. The antibodies of the invention are useful for the treatment of diseases and disorders associated with IL-33 signaling and/or IL-33 cellular expression, such as inflammatory diseases, or allergic diseases.
Description
ANTI-IL-33 ANTIBODIES AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to antibodies, and antigen-binding fragments thereof,
which are specific for human IL-33, and methods of use thereof.
BACKGROUND
Interleukin-33 (IL-33) is a ligand for ST2, a toll-like/interleukin-1 receptor super-family
member that associates with an accessory protein, IL-1RAcP (for reviews, see, e.g., Kakkar and
Lee, Nature Reviews – Drug Discovery 7(10):827-840 (2008), Schmitz et al., Immunity 23:479-
490 (2005); Liew et al., Nature Reviews – Immunology 10:103-110 (2010); US 2010/0260770;
US 2009/0041718). Upon activation of ST2/IL-1RAcP by IL-33, a signaling cascade is triggered
through downstream molecules such as MyD88 (myeloid differentiation factor 88) and TRAF6
(TNF receptor associated factor 6), leading to activation of NFκB (nuclear factor-κB), among
others. IL-33 signaling has been implicated as a factor in a variety of diseases and disorders.
(Liew et al., Nature Reviews – Immunology 10:103-110 (2010)).
[0002a] It is to be understood that if any prior art publication is referred to herein, such
reference does not constitute an admission that the publication forms a part of the common
general knowledge in the art in Australia or any other country.
BRIEF SUMMARY OF THE INVENTION
The present invention provides antibodies that bind human interleukin-33 ("IL-33").
The antibodies of the invention are useful, inter alia, for inhibiting ILmediated signaling and
for treating diseases and disorders caused by or related to IL-33 activity and/or signaling.
The antibodies of the invention can be full-length (for example, an IgG1 or IgG4
antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab’) or scFv
fragment), and may be modified to affect functionality, e.g., to eliminate residual effector
functions (Reddy et al., 2000, J. Immunol. 164:1925-1933).
In one embodiment, the antibodies that bind specifically to human interleukin-33 are
isolated fully human monoclonal antibodies.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof inhibit or attenuate ILmediated signaling.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof block the interaction of IL-33 and ST2.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof block the interaction of IL-33 and ST2 with an IC value of
less than about 10 nM, or blocks greater than about 50% of the interaction of IL-33 and ST2 as
17378269_1 (GHMatters) P40794NZ00
measured in an in vitro receptor/ligand binding assay at 25ºC.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof do not block, or only partially block the interaction of IL-33
and ST2.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof bind human IL-33 with a binding dissociation equilibrium
constant (K ) of less than about 1 nM as measured in a surface plasmon resonance assay at
37ºC.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof bind human IL-33 with a dissociative half-life (t½) of greater
than about 8 minutes as measured in a surface plasmon resonance assay at 37ºC.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof inhibit ILmediated degranulation of human basophils.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof inhibit ILmediated degranulation of human basophils with
an IC of less than about 600 pM as measured in an in vitro basophil activation test (BAT).
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof inhibit ILmediated IFN-gamma production from human
PBMCs.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof inhibit ILmediated IFN-gamma production from human
PBMCs with an IC of less than about 25 nM as measured in an in vitro PBMC IFN-gamma
production assay.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof inhibit ILmediated IFN-gamma production from human
PBMCs with an IC of less than about 3 nM as measured in an in vitro PBMC IFN-gamma
production assay.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof inhibit ILmediated IFN-gamma production from human
PBMCs with an IC of less than about 0.5 nM as measured in an in vitro PBMC IFN-gamma
production assay.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof reduce the frequency of CD4+ T cells, eosinophils and ILC2
cells in the lungs when administered to an animal model of allergen-induced lung inflammation.
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof reduces the level of IL-4 and IL-5 in the lungs when
administered to an animal model of allergen-induced lung inflammation.
17378269_1 (GHMatters) P40794NZ00
In one embodiment, the antibodies that bind specifically to human interleukin-33, or
antigen-binding fragments thereof, when administered to an animal model of allergen-induced
lung inflammation, result in at least a 4 fold reduction of IL-4 levels and/or at least a 5 fold
reduction in IL-5 levels when compared to allergen-challenged animals receiving an isotype
control antibody.
The present invention provides antibodies, or antigen-binding fragments thereof
comprising a heavy chain variable region (HCVR) having an amino acid sequence selected from
the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210,
226, 242, 258, 274, 290, and 308, or a substantially similar sequence thereof having at least
90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides an antibody or antigen-binding fragment of an
antibody comprising a light chain variable region (LCVR) having an amino acid sequence
selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154,
170, 186, 202, 218, 234, 250, 266, 282, 298, and 316, or a substantially similar sequence
thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
The present invention also provides an antibody or antigen-binding fragment thereof
comprising a HCVR and LCVR (HCVR/LCVR) sequence pair selected from the group consisting
of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154,
162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, and
308/316.
The present invention also provides an antibody or antigen-binding fragment of an
antibody comprising a heavy chain CDR3 (HCDR3) domain having an amino acid sequence
selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152,
168, 184, 200, 216, 232, 248, 264, 280, 296, and 314, or a substantially similar sequence
thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a
light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group
consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240,
256, 272, 288, 304, and 322, or a substantially similar sequence thereof having at least 90%, at
least 95%, at least 98% or at least 99% sequence identity.
In certain embodiments, the antibody or antigen-binding portion of an antibody
comprises a HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of
SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144, 152/160,
168/176, 184/192, 200/208, 216/224, 232/240, 248/256, 264/272, 280/288, 296/304 and
314/322.
The present invention also provides an antibody or fragment thereof further comprising
a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group
consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228,
17378269_1 (GHMatters) P40794NZ00
244, 260, 276, 292, and 310, or a substantially similar sequence thereof having at least 90%, at
least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 (HCDR2)
domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6,
22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, and 312, or
a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at
least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid
sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124,
140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, and 318, or a substantially similar
sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence
identity; and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from
the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206,
222, 238, 254, 270, 286, 302, and 320, or a substantially similar sequence thereof having at
least 90%, at least 95%, at least 98% or at least 99% sequence identity.
Certain non-limiting, exemplary antibodies and antigen-binding fragments of the
invention comprise HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively,
having the amino acid sequences selected from the group consisting of: SEQ ID NOs: 48
14-16 (e.g. H1M9559N); 202430-32 (e.g. H1M9566N); 364046-48 (e.g.
H1M9568N); 525662-64 (e.g. H4H9629P); 687278-80 (e.g. H4H9633P); 84
8894-96 (e.g. H4H9640P); 100104110-112 (e.g. H4H9659P); 116120
126-128 (e.g. H4H9660P); 132136142-144 (e.g. H4H9662P); 148152158-
160 (e.g., H4H9663P); 164168174-176 (e.g. H4H9664P); 180184190-192
(e.g., H4H9665P); 196200206-208 (e.g. H4H9666P); 212216222-224 (e.g.
H4H9667P); 228232238-240 (e.g. H4H9670P); 244248254-256 (e.g.
H4H9671P); 260264270-272 (e.g. H4H9672P); 276280286-288 (e.g.
H4H9675P); 292296302-304 (e.g. H4H9676P); and 310314320-322
(H1M9565N).
In a related embodiment, the invention includes an antibody or antigen-binding
fragment of an antibody which specifically binds IL-33, wherein the antibody or fragment
comprises the heavy and light chain CDR domains contained within heavy and light chain
variable region (HCVR/LCVR) sequences selected from the group consisting of SEQ ID NO:
2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186,
194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, and 308/316. Methods and
techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known
in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid
sequences disclosed herein. Exemplary conventions that can be used to identify the
boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM
definition. In general terms, the Kabat definition is based on sequence variability, the Chothia
17378269_1 (GHMatters) P40794NZ00
definition is based on the location of the structural loop regions, and the AbM definition is a
compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of
Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-
Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA
86:9268-9272 (1989). Public databases are also available for identifying CDR sequences
within an antibody.
[0028a] In a preferred embodiment, the antibodies are isolated human monoclonal antibodies or
antigen-binding fragments thereof that bind human interleukin 33 (IL-33), wherein the antibodies
or antigen-binding fragments thereof inhibit or attenuate ILmediated signaling and comprise
the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR)
having the amino acid sequence of SEQ ID NO: 274 and the CDRs of a light chain variable
region (LCVR) having the amino acid sequence of SEQ ID NO: 282, said complementarity
determining regions being identified by one or more of the Kabat method, the Chothia method,
or the AbM method.
In another aspect, the invention provides nucleic acid molecules encoding anti-IL-33
antibodies or antigen-binding fragments thereof. Recombinant expression vectors carrying the
nucleic acids of the invention, and host cells into which such vectors have been introduced, are
also encompassed by the invention, as are methods of producing the antibodies by culturing the
host cells under conditions permitting production of the antibodies, and recovering the
antibodies produced.
In one embodiment, the invention provides an antibody or fragment thereof comprising
a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:
1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, and 307,
or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least
99% homology thereof.
The present invention also provides an antibody or fragment thereof comprising a
LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9,
, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, and 315, or
a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least
99% homology thereof.
The present invention also provides an antibody or antigen-binding fragment of an
antibody comprising a HCDR3 domain encoded by a nucleotide sequence selected from the
group consisting of SEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215,
231, 247, 263, 279, 295, and 313, or a substantially identical sequence having at least 90%, at
least 95%, at least 98%, or at least 99% homology thereof; and a LCDR3 domain encoded by a
nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 31, 47, 63, 79, 95,
111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, and 321, or a substantially
17378269_1 (GHMatters) P40794NZ00
identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology
thereof.
The present invention also provides an antibody or fragment thereof which further
comprises a HCDR1 domain encoded by a nucleotide sequence selected from the group
consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243,
259, 275, 291, and 309, or a substantially identical sequence having at least 90%, at least 95%,
at least 98%, or at least 99% homology thereof; a HCDR2 domain encoded by a nucleotide
sequence selected from the group consisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117,
133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, and 311, or a substantially identical
sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; a
LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ
ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299,
and 317, or a substantially identical sequence having at least 90%, at least 95%, at least 98%,
or at least 99% homology thereof; and a LCDR2 domain encoded by a nucleotide sequence
selected from the group consisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157,
173, 189, 205, 221, 237, 253, 269, 285, 301, and 319, or a substantially identical sequence
having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.
According to certain embodiments, the antibody or fragment thereof comprises the
heavy and light chain CDR sequences encoded by the nucleic acid sequences of SEQ ID NOs:
1 and 9 (e.g. H1M9559N), 17 and 25 (e.g. H1M9566N), 33 and 41 (e.g. H1M9568N), 49 and 57
(e.g. H4H9629P), 65 and 73 (e.g. H4H9633P), 81 and 89 (e.g. H4H9640P), 97 and 105 (e.g.
H4H9659P), 113 and 121 (e.g. H4H9660P), 129 and 137 (e.g. H4H9662P), 145 and 153 (e.g.
H4H9663P), 161 and 169 (e.g. H4H9664P), 177 and 185 (e.g. H4H9665P), 193 and 201 (e.g.
H4H9666P), 209 and 217 (e.g. H4H9667P), 225 and 233 (e.g. H4H9670P), 241 and 249 (e.g.
H4H9671P), 257 and 265 (e.g. H4H9672P), 273 and 281 (e.g. H4H9675P), 289 and 297 (e.g.
H4H9676P), or 307 and 315 (H1M9565N).
The present invention includes anti-IL-33 antibodies having a modified glycosylation
pattern. In some applications, modification to remove undesirable glycosylation sites may be
useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for
example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al.
(2002) JBC 277:26733). In other applications, modification of galactosylation can be made in
order to modify complement dependent cytotoxicity (CDC).
In another aspect, the invention provides a pharmaceutical composition comprising a
recombinant human antibody or fragment thereof, which specifically binds IL-33 and a
pharmaceutically acceptable carrier. In a related aspect, the invention features a composition
which is a combination of an anti-IL-33 antibody and a second therapeutic agent. In one
17378269_1 (GHMatters) P40794NZ00
embodiment, the second therapeutic agent is any agent that is advantageously combined with
an anti-IL-33 antibody. Exemplary agents that may be advantageously combined with an anti-
IL-33 antibody include, without limitation, other agents that inhibit IL-33 activity (including other
antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists,
etc.) and/or agents, which do not directly bind IL-33 but nonetheless interfere with, block or
attenuate ILmediated signaling. In one embodiment the second therapeutic agent may be
selected from the group consisting of a non-steroidal anti-inflammatory (NSAID), a
corticosteroid, a bronchial dilator, an antihistamine, epinephrine, a decongestant, a thymic
stromal lymphopoietin (TSLP) antagonist, an IL-13 antagonist, an IL-4 antagonist, an IL-4/IL-13
dual antagonist, an IL-5 antagonist, an IL-6 antagonist, an IL-12/23 antagonist, an IL-22
antagonist, an IL-25 antagonist, an IL-17 antagonist, an IL-31 antagonist, an oral PDE4 inhibitor
and another IL-33 antagonist or a different antibody to IL-33.
In certain embodiments, the cytokine antagonist may be a small molecule inhibitor
(synthetic or naturally derived), or a protein (e.g. an antibody) that interacts with either the
cytokine itself, or to a receptor for the cytokine, or to a complex comprising both the cytokine
and its receptor(s) (e.g. an antibody to IL-4 or IL-6, or an antibody to the receptor for IL-4 or IL-
6). Additional combination therapies and co-formulations involving the anti-IL-33 antibodies of
the present invention are disclosed elsewhere herein.
In yet another aspect, the invention provides therapeutic methods for inhibiting IL-33
activity using an anti-IL-33 antibody or antigen-binding portion of an antibody of the invention,
wherein the therapeutic methods comprise administering a therapeutically effective amount of a
pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody
of the invention. The disorder treated is any disease or condition which is improved,
ameliorated, inhibited or prevented by removal, inhibition or reduction of IL-33 activity or
signaling. The anti-IL-33 antibodies or antibody fragments of the invention may function to block
the interaction between IL-33 and an IL-33 binding partner (e.g., an IL-33 receptor component),
or otherwise inhibit the signaling activity of IL-33.
In one embodiment, the invention provides a method for treating an inflammatory
disease or disorder, or at least one symptom associated with the inflammatory disease or
disorder, the method comprising administering an antibody that binds specifically to IL-33, or an
antigen-binding fragment thereof, or a pharmaceutical composition comprising an antibody that
binds specifically to IL-33, or an antigen-binding fragment thereof, to a patient in need thereof,
wherein the inflammatory disease or disorder is alleviated, or reduced in severity, duration or
frequency of occurrence, or at least one symptom associated with the inflammatory disease or
disorder is alleviated, or reduced in severity, duration, or frequency of occurrence.
In one embodiment, the inflammatory disease or condition is selected from the group
17378269_1 (GHMatters) P40794NZ00
consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD),
inflammatory bowel disease, multiple sclerosis, arthritis, allergic rhinitis, eosinophilic esophagitis
and psoriasis.
In one embodiment, the invention provides a method for treating a patient who
demonstrates a sensitivity to an allergen, the method comprising administering an effective
amount of an antibody or antigen binding fragment thereof that binds specifically to IL-33, or a
pharmaceutical composition comprising an antibody that binds specifically to IL-33, or an
antigen-binding fragment thereof, to a patient in need thereof, wherein the patient demonstrates
a reduced sensitivity to, or a diminished allergic reaction against the allergen, or does not
experience any sensitivity or allergic reaction to, or anaphylactic response to the allergen
following administration of the antibody or a composition comprising the antibody.
In one embodiment, the invention provides for administering an effective amount of a
second therapeutic agent useful for alleviating the inflammatory disease or disorder, or at least
one symptom of the inflammatory disease or disorder, or for diminishing an allergic response to
an allergen. As noted above, the the second therapeutic agent may be selected from the group
consisting of a non-steroidal anti-inflammatory (NSAID), a corticosteroid, a bronchial dilator, an
antihistamine, epinephrine, a decongestant, a thymic stromal lymphopoietin (TSLP) antagonist,
an IL-13 antagonist, an IL-4 antagonist, an IL-5 antagonist, an IL-6 antagonist, an IL-25
antagonist, an IL-17 antagonist, and another IL-33 antagonist or a different antibody to IL-33.
In a related aspect, the invention provides an anti-IL-33 antibody of the invention, or an
antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or
an-gen-binding fragment thereof for use in treating a disease or disorder related to, or caused
by IL-33 activity in a patient. In one embodiment, the disease or disorder related to, or caused
by IL-33 activity in a patient is an inflammatory disease or disorder, wherein the inflammatory
disease or disorder is selected from the group consisting of asthma, atopic dermatitis, chronic
obstructive pulmonary disease (COPD), inflammatory bowel disease, multiple sclerosis, arthritis,
allergic rhinitis, eosinophilic esophagitis and psoriasis.
The present invention also includes the use of an anti-IL-33 antibody or antigen binding
portion of an antibody of the invention in the manufacture of a medicament for the treatment of a
disease or disorder related to or caused by IL-33 activity in a patient. In one embodiment, the
disease or disorder related to, or caused by IL-33 activity in a patient is an inflammatory disease
or disorder, wherein the inflammatory disease or disorder is selected from the group consisting
of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), inflammatory
bowel disease, multiple sclerosis, arthritis, allergic rhinitis, eosinophilic esophagitis and
psoriasis..
Other embodiments will become apparent from a review of the ensuing detailed
description.
17378269_1 (GHMatters) P40794NZ00
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Cross Competition between Anti-IL-33 Antibodies for Human IL-33
Figure 2. Cross Competition between Anti-IL-33 Antibodies for Recombinant Monkey
IL-33
DETAILED DESCRIPTION
Before the present invention is described, it is to be understood that this invention is
not limited to particular methods and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended claims.
[0047a] In the claims which follow and in the proceeding description of the invention, except
where the context requires otherwise due to express language or necessary implication, the
word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense,
i.e. to specify the presence of the stated features, integers, steps or components but not to
preclude the presence or addition of further features integers, steps, components or groups
thereof in various embodiments of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. As used herein, the term "about," when used in reference to a particular recited
numerical value, means that the value may vary from the recited value by no more than 1%.
For example, as used herein, the expression "about 100" includes 99 and 101 and all values in
between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein
can be used in the practice or testing of the present invention, the preferred methods and
materials are now described.
Definitions
The expressions "interleukin-33," "IL-33," and the like, as used herein, refer to a human
IL-33 protein having the amino acid sequence of SEQ ID NO:307. All references to proteins,
polypeptides and protein fragments herein are intended to refer to the human version of the
respective protein, polypeptide or protein fragment unless explicitly specified as being from a
non-human species (e.g., "mouse IL-33," "monkey IL-33," etc.).
As used herein, "an antibody that binds IL-33" or an "anti-IL-33 antibody" includes
antibodies, and antigen-binding fragments thereof, that bind a soluble fragment of an IL-33
protein. Soluble IL-33 molecules include natural IL-33 proteins as well as recombinant IL-33
protein variants such as, e.g., monomeric and dimeric IL-33 constructs.
The term "antibody", as used herein, means any antigen-binding molecule or molecular
17378269_1 (GHMatters) P40794NZ00
complex comprising at least one complementarity determining region (CDR) that specifically
binds to or interacts with a particular antigen (e.g., IL-33). The term "antibody" includes
immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V )
and a heavy chain constant region. The heavy chain constant region comprises three domains,
C 1, C 2 and C 3. Each light chain comprises a light chain variable region (abbreviated herein
H H H
as LCVR or V ) and a light chain constant region. The light chain constant region comprises
one domain (C 1). The V and V regions can be further subdivided into regions of
L H L
hypervariability, termed complementarity determining regions (CDRs), interspersed with regions
that are more conserved, termed framework regions (FR). Each V and V is composed of
three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention,
the FRs of the anti-IL-33 antibody (or antigen-binding portion thereof) may be identical to the
human germline sequences, or may be naturally or artificially modified. An amino acid
consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
The term "antibody", as used herein, also includes antigen-binding fragments of full
antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding
fragment" of an antibody, and the like, as used herein, include any naturally occurring,
enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that
specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may
be derived, e.g., from full antibody molecules using any suitable standard techniques such as
proteolytic digestion or recombinant genetic engineering techniques involving the manipulation
and expression of DNA encoding antibody variable and optionally constant domains. Such DNA
is known and/or is readily available from, e.g., commercial sources, DNA libraries (including,
e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and
manipulated chemically or by using molecular biology techniques, for example, to arrange one
or more variable and/or constant domains into a suitable configuration, or to introduce codons,
create cysteine residues, modify, add or delete amino acids, etc.
Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii)
F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules;
(vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that
mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining
region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other
engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-
deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies,
tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.),
17378269_1 (GHMatters) P40794NZ00
small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also
encompassed within the expression "antigen-binding fragment," as used herein.
An antigen-binding fragment of an antibody will typically comprise at least one variable
domain. The variable domain may be of any size or amino acid composition and will generally
comprise at least one CDR which is adjacent to or in frame with one or more framework
sequences. In antigen-binding fragments having a V domain associated with a V domain, the
V and V domains may be situated relative to one another in any suitable arrangement. For
example, the variable region may be dimeric and contain V -V , V -V or V -V dimers.
H H H L L L
Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V or V
domain.
In certain embodiments, an antigen-binding fragment of an antibody may contain at
least one variable domain covalently linked to at least one constant domain. Non-limiting,
exemplary configurations of variable and constant domains that may be found within an antigen-
binding fragment of an antibody of the present invention include: (i) V -C 1; (ii) V -C 2; (iii) V -
H H H H H
C 3; (iv) V -C 1-C 2; (v) V -C 1-C 2-C 3; (vi) V -C 2-C 3; (vii) V -C ; (viii) V -C 1; (ix) V -C 2;
H H H H H H H H H H H H L L H L H
(x) V -C 3; (xi) V -C 1-C 2; (xii) V -C 1-C 2-C 3; (xiii) V -C 2-C 3; and (xiv) V -C . In any
L H L H H L H H H L H H L L
configuration of variable and constant domains, including any of the exemplary configurations
listed above, the variable and constant domains may be either directly linked to one another or
may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2
(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage
between adjacent variable and/or constant domains in a single polypeptide molecule.
Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a
homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain
configurations listed above in non-covalent association with one another and/or with one or
more monomeric V or V domain (e.g., by disulfide bond(s)).
As with full antibody molecules, antigen-binding fragments may be monospecific or
multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will
typically comprise at least two different variable domains, wherein each variable domain is
capable of specifically binding to a separate antigen or to a different epitope on the same
antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats
disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an
antibody of the present invention using routine techniques available in the art.
The antibodies of the present invention may function through complement-dependent
cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-
dependent cytotoxicity" (CDC) refers to lysis of antigen-expressing cells by an antibody of the
invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity"
(ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc
17378269_1 (GHMatters) P40794NZ00
receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and thereby lead to lysis of the target cell. CDC and ADCC can
be measured using assays that are well known and available in the art. (See, e.g., U.S. Patent
Nos 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-
656). The constant region of an antibody is important in the ability of an antibody to fix
complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be
selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.
In certain embodiments of the invention, the anti-IL-33 antibodies of the invention are
human antibodies. The term "human antibody", as used herein, is intended to include
antibodies having variable and constant regions derived from human germline immunoglobulin
sequences. The human antibodies of the invention may include amino acid residues not
encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random
or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs
and in particular CDR3. However, the term "human antibody", as used herein, is not intended to
include antibodies in which CDR sequences derived from the germline of another mammalian
species, such as a mouse, have been grafted onto human framework sequences.
The antibodies of the invention may, in some embodiments, be recombinant human
antibodies. The term "recombinant human antibody", as used herein, is intended to include all
human antibodies that are prepared, expressed, created or isolated by recombinant means,
such as antibodies expressed using a recombinant expression vector transfected into a host cell
(described further below), antibodies isolated from a recombinant, combinatorial human
antibody library (described further below), antibodies isolated from an animal (e.g., a mouse)
that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids
Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means
that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
Such recombinant human antibodies have variable and constant regions derived from human
germline immunoglobulin sequences. In certain embodiments, however, such recombinant
human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for
human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences
of the V and V regions of the recombinant antibodies are sequences that, while derived from
and related to human germline V and V sequences, may not naturally exist within the human
antibody germline repertoire in vivo.
Human antibodies can exist in two forms that are associated with hinge heterogeneity.
In one form, an immunoglobulin molecule comprises a stable four chain construct of
approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain
disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a
molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain
17378269_1 (GHMatters) P40794NZ00
(half-antibody). These forms have been extremely difficult to separate, even after affinity
purification.
The frequency of appearance of the second form in various intact IgG isotypes is due
to, but not limited to, structural differences associated with the hinge region isotype of the
antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can
significantly reduce the appearance of the second form (Angal et al. (1993) Molecular
Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant
invention encompasses antibodies having one or more mutations in the hinge, C 2 or C 3
region which may be desirable, for example, in production, to improve the yield of the desired
antibody form.
The antibodies of the invention may be isolated antibodies. An "isolated antibody," as
used herein, means an antibody that has been identified and separated and/or recovered from
at least one component of its natural environment. For example, an antibody that has been
separated or removed from at least one component of an organism, or from a tissue or cell in
which the antibody naturally exists or is naturally produced, is an "isolated antibody" for
purposes of the present invention. An isolated antibody also includes an antibody in situ within
a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one
purification or isolation step. According to certain embodiments, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
The present invention includes neutralizing and/or blocking anti-IL-33 antibodies. A
"neutralizing" or "blocking" antibody, as used herein, is intended to refer to an antibody whose
binding to IL-33: (i) interferes with the interaction between IL-33 or an IL-33 fragment and an IL-
33 receptor component (e.g., ST2, IL-1RAcP, etc.); and/or (ii) results in inhibition of at least one
biological function of IL-33. The inhibition caused by an IL-33 neutralizing or blocking antibody
need not be complete so long as it is detectable using an appropriate assay. Exemplary assays
for detecting IL-33 inhibition are described in the working Examples herein.
The anti-IL-33 antibodies disclosed herein may comprise one or more amino acid
substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and
light chain variable domains as compared to the corresponding germline sequences from which
the antibodies were derived. Such mutations can be readily ascertained by comparing the
amino acid sequences disclosed herein to germline sequences available from, for example,
public antibody sequence databases. The present invention includes antibodies, and antigen-
binding fragments thereof, which are derived from any of the amino acid sequences disclosed
herein, wherein one or more amino acids within one or more framework and/or CDR regions are
mutated to the corresponding residue(s) of the germline sequence from which the antibody was
derived, or to the corresponding residue(s) of another human germline sequence, or to a
conservative amino acid substitution of the corresponding germline residue(s) (such sequence
17378269_1 (GHMatters) P40794NZ00
changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in
the art, starting with the heavy and light chain variable region sequences disclosed herein, can
easily produce numerous antibodies and antigen-binding fragments which comprise one or
more individual germline mutations or combinations thereof. In certain embodiments, all of the
framework and/or CDR residues within the V and/or V domains are mutated back to the
residues found in the original germline sequence from which the antibody was derived. In other
embodiments, only certain residues are mutated back to the original germline sequence, e.g.,
only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino
acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other
embodiments, one or more of the framework and/or CDR residue(s) are mutated to the
corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is
different from the germline sequence from which the antibody was originally derived).
Furthermore, the antibodies of the present invention may contain any combination of two or
more germline mutations within the framework and/or CDR regions, e.g., wherein certain
individual residues are mutated to the corresponding residue of a particular germline sequence
while certain other residues that differ from the original germline sequence are maintained or
are mutated to the corresponding residue of a different germline sequence. Once obtained,
antibodies and antigen-binding fragments that contain one or more germline mutations can be
easily tested for one or more desired property such as, improved binding specificity, increased
binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the
case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments
obtained in this general manner are encompassed within the present invention.
The present invention also includes anti-IL-33 antibodies comprising variants of any of
the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more
conservative substitutions. For example, the present invention includes anti-IL-33 antibodies
having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or
fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR,
and/or CDR amino acid sequences disclosed herein.
The term "epitope" refers to an antigenic determinant that interacts with a specific
antigen binding site in the variable region of an antibody molecule known as a paratope. A
single antigen may have more than one epitope. Thus, different antibodies may bind to different
areas on an antigen and may have different biological effects. Epitopes may be either
conformational or linear. A conformational epitope is produced by spatially juxtaposed amino
acids from different segments of the linear polypeptide chain. A linear epitope is one produced
by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope
may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
The term "substantial identity" or "substantially identical," when referring to a nucleic
17378269_1 (GHMatters) P40794NZ00
acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide
insertions or deletions with another nucleic acid (or its complementary strand), there is
nucleotide sequence identity in at least about 95%, and more preferably at least about 96%,
97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of
sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid
molecule having substantial identity to a reference nucleic acid molecule may, in certain
instances, encode a polypeptide having the same or substantially similar amino acid sequence
as the polypeptide encoded by the reference nucleic acid molecule.
As applied to polypeptides, the term "substantial similarity" or "substantially similar"
means that two peptide sequences, when optimally aligned, such as by the programs GAP or
BESTFIT using default gap weights, share at least 95% sequence identity, even more
preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not
identical differ by conservative amino acid substitutions. A "conservative amino acid
substitution" is one in which an amino acid residue is substituted by another amino acid residue
having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
In general, a conservative amino acid substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid sequences differ from each
other by conservative substitutions, the percent sequence identity or degree of similarity may be
adjusted upwards to correct for the conservative nature of the substitution. Means for making
this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods
Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar
chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and
isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side
chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and
tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate
and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred
conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-
tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
Alternatively, a conservative replacement is any change having a positive value in the PAM250
log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately
conservative" replacement is any change having a nonnegative value in the PAM250 log-
likelihood matrix.
Sequence similarity for polypeptides, which is also referred to as sequence identity, is
typically measured using sequence analysis software. Protein analysis software matches
similar sequences using measures of similarity assigned to various substitutions, deletions and
other modifications, including conservative amino acid substitutions. For instance, GCG
software contains programs such as Gap and Bestfit which can be used with default parameters
17378269_1 (GHMatters) P40794NZ00
to determine sequence homology or sequence identity between closely related polypeptides,
such as homologous polypeptides from different species of organisms or between a wild type
protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be
compared using FASTA using default or recommended parameters, a program in GCG Version
6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of
the regions of the best overlap between the query and search sequences (Pearson (2000)
supra). Another preferred algorithm when comparing a sequence of the invention to a database
containing a large number of sequences from different organisms is the computer program
BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al.
(1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.
An "inflammatory disease or disorder", as used herein, refers to a disease, disorder or
pathological condition where the pathology results, in whole or in part, from, e.g., a change in
number, change in rate of migration, or change in activation, of cells of the immune system.
Cells of the immune system include, e.g., T cells, B cells, monocytes or macrophages, antigen
presenting cells (APCs), dendritic cells, microglia, NK cells, neutrophils, eosinophils, mast cells,
or any other cell specifically associated with the immunology, for example, cytokine-producing
endothelial or epithelial cells. As used herein, in one embodiment, the "inflammatory disease or
disorder" is an immune disorder or condition selected from the group consisting of asthma,
(including steroid resistant asthma, steroid sensitive asthma, eosinophilic asthma or non-
eosinophilic asthma, allergy, anaphylaxis, multiple sclerosis, inflammatory bowel disorder (e.g.
Crohn's disease or ulcerative colitis), chronic obstructive pulmonary disease (COPD, which may
or may not be related to, caused in part by, or resulting from, exposure to first or second hand
cigarette smoke), lupus, atopic dermatitis, psoriasis, scleroderma and other fibrotic diseases,
sjogren's syndrome, vasculitis (behcet's disease, Giant cell arteritis, Henoch-Schonlein purpura
and Churg Strauss syndrome) and arthritis. In another embodiment, the arthritis is selected from
the group consisting of rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. In another
embodiment, the "inflammatory disease or disorder" is an immune disorder or condition
comprises a TH -type response or a TH -type response.
The phrase "Inhibits or attenuates ILmediated signaling", as used herein, refers to
the degree to which IL-33 stimulates signal transduction through ST2 and IL-1RAcP, which is
diminished in the presence of an antagonist, such as an IL-33 antibody as described herein,
relative to the degree to which IL-33 stimulates signal transduction through ST2 and IL-1RAcP
in the absence of the antagonist such as an IL-33 antibody as described herein. To examine the
extent of inhibition, a sample is treated with a potential inhibitor/antagonist and is compared to a
control sample without the inhibitor/antagonist. Control samples, i.e., not treated with antagonist,
are assigned a relative activity value of 100%. Inhibition is achieved when the activity value
relative to the control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,
17378269_1 (GHMatters) P40794NZ00
%, 30%, 25%, or 20% or less. An endpoint in inhibition may comprise a predetermined
quantity or percentage of, e.g., an indicator of inflammation, or cell degranulation,secretion or
activation, such as the release of a cytokine. Inhibition of IL-33 signal transduction through ST2
and IL-1RAcP can be determined by assaying for IL-33 signal transduction in an in vitro assay,
such as that described herein in Example 6. In addition, in vivo assays can be used to
determine whether a molecule is an antagonist of IL-33. For example, an in vivo assay such as
that described in Examples 11 and 12 may be used to assess the effect of an antibody to IL-33
on lung inflammation in allergen-sensitized animals that are homozygous for expression of
human IL-33. Following sensitization of the animals with allergen, a subset of the animals is
treated with either an anti-IL-33 antibody of the invention or a negative isotype control antibody.
Afterwards, the animals are sacrificed and the lungs are harvested for assessment of cellular
infiltrates, as well as cytokine measurements (IL-4 and IL-5). An IL-33 antibody that is effective
as an antagonist should demonstrate a trend towards reduction in inflammatory cells in the lung,
as well as a trend towards reduction in cytokines such as IL-4 and IL-5.
Biological Characteristics of the Antibodies
The present invention includes anti-IL-33 antibodies and antigen-binding fragments
thereof that bind human IL-33 and inhibit or attenuate ILmediated signaling. An anti-IL-33
antibody is deemed to "inhibit or attenuate ILmediated signaling" if, e.g., the antibody
exhibits one or more properties selected from the group consisting of: (1) inhibition of IL
mediated signaling in a cell-based bioassay; (2) inhibition of ILinduced degranulation of
human basophils; (3) inhibition of ILinduced IFNγ production from human PBMCs; (4)
reduction in cytokine levels that are elevated in a mammal as a result of exposure to an
allergen, e.g. IL-4 or IL-5; and (5) inhibition of lung inflammation resulting from acute or chronic
exposure to an allergen (e.g. house dust mites (HDM)).
Inhibition of ILmediated signaling in a cell-based bioassay means that an anti-IL-33
antibody or antigen-binding fragment thereof inhibits or reduces the signal produced in cells that
express an IL-33 receptor and a reporter element that produces a detectable signal in response
to IL-33 binding, e.g., using the assay format as defined in Example 6 herein, or a substantially
similar assay. For example, the present invention includes antibodies and antigen-binding
fragments thereof that block ILmediated signaling in cells expressing human ST2, with an
IC of less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800
pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about
400 pM, less than about 350 pM, less than about 300 pM, less than about 250 pM, less than
about 200 pM, less than about 150 pM, less than about 100 pM, less than about 90 pM, less
than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less
than about 40 pM, less than about 30 pM, less than about 20 pM, or less than about 10 pM, as
17378269_1 (GHMatters) P40794NZ00
measured in a cell-based blocking bioassay, e.g., using the assay format as defined in Example
herein, or a substantially similar assay.
Inhibition of ILinduced degranulation of human basophils means that an anti-IL-33
antibody or antigen-binding fragment thereof inhibits or reduces the extent of ILinduced
basophil degranulation in vitro, e.g., as measured using the assay system of Example 7 or a
substantially similar assay. For example, the present invention includes antibodies and antigen-
binding fragments thereof that inhibit degranulation of human basophils in the presence of
human IL-33 (e.g., about 100 pM final concentration), with an IC of less than about 500 pM,
less than about 400 pM, less than about 350 pM, less than about 300 pM, less than about 250
pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about
90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about
50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, or less than about
pM, as measured in an in vitro human basophil degranulation assay, e.g., using the assay
format as defined in Example 7 herein, or a substantially similar assay.
Inhibition of ILinduced IFNγ production from human PBMCs means that an anti-IL-
33 antibody or antigen-binding fragment thereof inhibits or reduces the amount of IFNγ released
from PBMCs treated with human IL-33 in the presence of human IL-12, e.g., as measured using
the assay system of Example 8 or a substantially similar assay. For example, the present
invention includes antibodies and antigen-binding fragments thereof that inhibit ILinduced
release of IFNγ, in the presence of human IL-12, with an IC of less than about 50 nM, less
than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less
than about 5 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less
than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM or
less than about 300 pM, as measured in an ILinduced IFNγ release assay, e.g., using the
assay format as defined in Example 8 herein, or a substantially similar assay.
In certain embodiments, the anti-IL-33 antibodies and antigen-binding fragments of the
present invention block the binding of IL-33 to an IL-33 receptor (e.g., ST2). For example, the
present invention includes anti-IL-33 antibodies that block the binding of IL-33 to ST2 in vitro,
with an IC value of less than about 15 nM, as measured by an ELISA-based immunoassay,
e.g., using the assay format as defined in Example 4 herein, or a substantially similar assay. In
certain embodiments, the antibodies or antigen-binding fragments of the present invention block
the binding of IL-33 to ST2 in vitro with an IC value of less than about 10 nM, less than about
nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about
600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than
about 280 pM, less than about 260 pM, less than about 250 pM, less than about 240 pM, less
than about 230 pM, less than about 220 pM, less than about 200 pM, less than about 180 pM,
less than about 160 pM, or less than about 150 pM, as measured by an ELISA-based
17378269_1 (GHMatters) P40794NZ00
immunoassay, e.g., using the assay format as defined in Example 4 herein, or a substantially
similar assay.
In other embodiments, however, certain anti-IL-33 antibodies and antigen-binding
fragments of the present invention, despite having the ability to inhibit or attenuate IL
mediated signaling, do not block or only partially block the interaction of IL-33 and ST2. Such
antibodies and antigen-binding fragments thereof, may be referred to herein as "indirect
blockers." Without being bound by theory, it is believed that the indirect blockers of the
invention function by binding to IL-33 at an epitope that does overlap, or overlaps only partially,
with the ST2-binding domain of IL-33, but nonetheless interfere with ILmediated signaling
without blocking the IL-33/ST2 interaction directly.
The present invention includes anti-IL-33 antibodies and antigen-binding fragments
thereof that bind soluble IL-33 molecules with high affinity. For example, the present invention
includes antibodies and antigen-binding fragments of antibodies that bind IL-33 (e.g., at 25ºC or
37ºC) with a K of less than about 10 nM as measured by surface plasmon resonance, e.g.,
using the assay format as defined in Example 3 herein. In certain embodiments, the antibodies
or antigen-binding fragments of the present invention bind IL-33 with a K of less than about 5
nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 600
pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about
200 pM, less than about 180 pM, or less than about 160 pM, as measured by surface plasmon
resonance, e.g., using the assay format as defined in Example 3 herein, or a substantially
similar assay.
The present invention also includes anti-IL-33 antibodies and antigen-binding
fragments thereof that specifically bind to IL-33 with a dissociative half-life (t½) of greater than
about 10 minutes as measured by surface plasmon resonance at 25ºC or 37ºC, e.g., using the
assay format as defined in Example 3 herein, or a substantially similar assay. In certain
embodiments, the antibodies or antigen-binding fragments of the present invention bind IL-33
with a t½ of greater than about 20 minutes, greater than about 30 minutes, greater than about
40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about
70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about
100 minutes, as measured by surface plasmon resonance at 25ºC or 37ºC, e.g., using the
assay format as defined in Example 3 herein, or a substantially similar assay.
The antibodies of the present invention may possess one or more of the
aforementioned biological characteristics, or any combinations thereof. Other biological
characteristics of the antibodies of the present invention will be evident to a person of ordinary
skill in the art from a review of the present disclosure including the working Examples herein.
Anti-IL-33 Antibodies Comprising Fc Variants
17378269_1 (GHMatters) P40794NZ00
According to certain embodiments of the present invention, anti-IL-33 antibodies are
provided comprising an Fc domain comprising one or more mutations which enhance or
diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH.
For example, the present invention includes anti- IL-33 antibodies comprising a mutation in the
C 2 or a C 3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc
domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about
.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody
when administered to an animal. Non-limiting examples of such Fc modifications include, e.g.,
a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),
254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433
(e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428;
or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the
modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I
(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y)
modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L
modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).
In yet another embodiment, the modification comprises a 265A (e.g., D265A) and/or a 297A
(e.g., D297A) modification.
For example, the present invention includes anti-IL-33 antibodies comprising an Fc
domain comprising one or more pairs or groups of mutations selected from the group consisting
of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and
T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and
N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations
within the antibody variable domains disclosed herein, are contemplated within the scope of the
present invention.
The present invention also includes anti-IL-33 antibodies comprising a chimeric heavy
chain constant (C ) region, wherein the chimeric C region comprises segments derived from
the C regions of more than one immunoglobulin isotype. For example, the antibodies of the
invention may comprise a chimeric C region comprising part or all of a C 2 domain derived
from a human IgG1, human IgG2 or human IgG4 molecule, combined with part or all of a C 3
domain derived from a human IgG1, human IgG2 or human IgG4 molecule. According to
certain embodiments, the antibodies of the invention comprise a chimeric C region having a
chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino
acid sequence (amino acid residues from positions 216 to 227 according to EU numbering)
derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a
"lower hinge" sequence (amino acid residues from positions 228 to 236 according to EU
numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region.
17378269_1 (GHMatters) P40794NZ00
According to certain embodiments, the chimeric hinge region comprises amino acid residues
derived from a human IgG1 or a human IgG4 upper hinge and amino acid residues derived from
a human IgG2 lower hinge. An antibody comprising a chimeric C region as described herein
may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting
the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., U.S. Provisional Appl.
No. 61/759,578, filed February 1, 2013).
Epitope Mapping and Related Technologies
The present invention includes anti-IL-33 antibodies which interact with one or more
amino acids of IL-33. For example, the present invention includes anti-IL-33 antibodies that
interact with one or more amino acids located within the ST2-interacting domain of IL-33. The
epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more
(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of IL-33.
Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino
acid sequences) of IL-33.
Various techniques known to persons of ordinary skill in the art can be used to
determine whether an antibody "interacts with one or more amino acids" within a polypeptide or
protein. Exemplary techniques include, e.g., routine cross-blocking assay such as that
described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY),
alanine scanning mutational analysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol
248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision,
epitope extraction and chemical modification of antigens can be employed (Tomer, 2000,
Protein Science 9:487-496). Another method that can be used to identify the amino acids within
a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by
mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves
deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-
labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-
deuterium exchange to occur at all residues except for the residues protected by the antibody
(which remain deuterium-labeled). After dissociation of the antibody, the target protein is
subjected to protease cleavage and mass spectrometry analysis, thereby revealing the
deuterium-labeled residues which correspond to the specific amino acids with which the
antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and
Smith (2001) Anal. Chem. 73:256A-265A.
The present invention further includes anti-IL-33 antibodies that bind to the same
epitope as any of the specific exemplary antibodies described herein (e.g. H1M9559N,
H1M9566N, H1M9568N, H4H9629P, H4H9633P, H4H9640P, H4H9659P, H4H9660P,
H4H9662P, H4H9663P, H4H9664P, H4H9665P, H4H9666P, H4H9667P, H4H9670P,
17378269_1 (GHMatters) P40794NZ00
H4H9671P, H4H9672P, H4H9675P, H4H9676P, H1M9565N, etc.). Likewise, the present
invention also includes anti-IL-33 antibodies that compete for binding to IL-33 with any of the
specific exemplary antibodies described herein (e.g. H1M9559N, H1M9566N, H1M9568N,
H4H9629P, H4H9633P, H4H9640P, H4H9659P, H4H9660P, H4H9662P, H4H9663P,
H4H9664P, H4H9665P, H4H9666P, H4H9667P, H4H9670P, H4H9671P, H4H9672P,
H4H9675P, H4H9676P, H1M9565N, etc.).
One can easily determine whether an antibody binds to the same epitope as, or
competes for binding with, a reference anti-IL-33 antibody by using routine methods known in
the art and exemplified herein. For example, to determine if a test antibody binds to the same
epitope as a reference anti-IL-33 antibody of the invention, the reference antibody is allowed to
bind to an IL-33 protein. Next, the ability of a test antibody to bind to the IL-33 molecule is
assessed. If the test antibody is able to bind to IL-33 following saturation binding with the
reference anti-IL-33 antibody, it can be concluded that the test antibody binds to a different
epitope than the reference anti-IL-33 antibody. On the other hand, if the test antibody is not
able to bind to the IL-33 molecule following saturation binding with the reference anti-IL-33
antibody, then the test antibody may bind to the same epitope as the epitope bound by the
reference anti-IL-33 antibody of the invention. Additional routine experimentation (e.g., peptide
mutation and binding analyses) can then be carried out to confirm whether the observed lack of
binding of the test antibody is in fact due to binding to the same epitope as the reference
antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed
binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry
or any other quantitative or qualitative antibody-binding assay available in the art. In
accordance with certain embodiments of the present invention, two antibodies bind to the same
(or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits
binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a
competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).
Alternatively, two antibodies are deemed to bind to the same epitope if essentially all amino acid
mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate
binding of the other. Two antibodies are deemed to have "overlapping epitopes" if only a subset
of the amino acid mutations that reduce or eliminate binding of one antibody reduce or
eliminate binding of the other.
To determine if an antibody competes for binding (or cross-competes for binding) with
a reference anti-IL-33 antibody, the above-described binding methodology is performed in two
orientations: In a first orientation, the reference antibody is allowed to bind to an IL-33 protein
under saturating conditions followed by assessment of binding of the test antibody to the IL-33
molecule. In a second orientation, the test antibody is allowed to bind to an IL-33 molecule
under saturating conditions followed by assessment of binding of the reference antibody to the
17378269_1 (GHMatters) P40794NZ00
IL-33 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding
to the IL-33 molecule, then it is concluded that the test antibody and the reference antibody
compete for binding to IL-33. As will be appreciated by a person of ordinary skill in the art, an
antibody that competes for binding with a reference antibody may not necessarily bind to the
same epitope as the reference antibody, but may sterically block binding of the reference
antibody by binding an overlapping or adjacent epitope.
Preparation of Human Antibodies
Methods for generating monoclonal antibodies, including fully human monoclonal
antibodies are known in the art. Any such known methods can be used in the context of the
present invention to make human antibodies that specifically bind to human IL-33.
Using VELOCIMMUNE™ technology, for example, or any other known method for
generating fully human monoclonal antibodies, high affinity chimeric antibodies to IL-33 are
initially isolated having a human variable region and a mouse constant region. As in the
experimental section below, the antibodies are characterized and selected for desirable
characteristics, including affinity, selectivity, epitope, etc. If necessary, mouse constant regions
are replaced with a desired human constant region, for example wild-type or modified IgG1 or
IgG4, to generate a fully human anti-IL-33 antibody. While the constant region selected may
vary according to specific use, high affinity antigen-binding and target specificity characteristics
reside in the variable region. In certain instances, fully human anti-IL-33 antibodies are isolated
directly from antigen-positive B cells.
Bioequivalents
The anti-IL-33 antibodies and antibody fragments of the present invention encompass
proteins having amino acid sequences that vary from those of the described antibodies but that
retain the ability to bind human IL-33. Such variant antibodies and antibody fragments comprise
one or more additions, deletions, or substitutions of amino acids when compared to parent
sequence, but exhibit biological activity that is essentially equivalent to that of the described
antibodies. Likewise, the anti-IL-33 antibody-encoding DNA sequences of the present invention
encompass sequences that comprise one or more additions, deletions, or substitutions of
nucleotides when compared to the disclosed sequence, but that encode an anti-IL-33 antibody
or antibody fragment that is essentially bioequivalent to an anti-IL-33 antibody or antibody
fragment of the invention. Examples of such variant amino acid and DNA sequences are
discussed above.
Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for
example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and
extent of absorption do not show a significant difference when administered at the same molar
dose under similar experimental conditions, either single does or multiple dose. Some
17378269_1 (GHMatters) P40794NZ00
antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in
the extent of their absorption but not in their rate of absorption and yet may be considered
bioequivalent because such differences in the rate of absorption are intentional and are
reflected in the labeling, are not essential to the attainment of effective body drug concentrations
on, e.g., chronic use, and are considered medically insignificant for the particular drug product
studied.
In one embodiment, two antigen-binding proteins are bioequivalent if there are no
clinically meaningful differences in their safety, purity, and potency.
In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be
switched one or more times between the reference product and the biological product without
an expected increase in the risk of adverse effects, including a clinically significant change in
immunogenicity, or diminished effectiveness, as compared to continued therapy without such
switching.
In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a
common mechanism or mechanisms of action for the condition or conditions of use, to the
extent that such mechanisms are known.
Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence
measures include, e.g., (a) an in vivo test in humans or other mammals, in which the
concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other
biological fluid as a function of time; (b) an in vitro test that has been correlated with and is
reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other
mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is
measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety,
efficacy, or bioavailability or bioequivalence of an antibody.
Bioequivalent variants of anti-IL-33 antibodies of the invention may be constructed by,
for example, making various substitutions of residues or sequences or deleting terminal or
internal residues or sequences not needed for biological activity. For example, cysteine
residues not essential for biological activity can be deleted or replaced with other amino acids to
prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon
renaturation. In other contexts, bioequivalent antibodies may include anti-IL-33 antibody
variants comprising amino acid changes which modify the glycosylation characteristics of the
antibodies, e.g., mutations which eliminate or remove glycosylation.
Species Selectivity and Species Cross-Reactivity
The present invention, according to certain embodiments, provides anti-IL-33
antibodies that bind to human IL-33 but not to IL-33 from other species. The present invention
also includes anti-IL-33 antibodies that bind to human IL-33 and to IL-33 from one or more non-
17378269_1 (GHMatters) P40794NZ00
human species. For example, the anti-IL-33 antibodies of the invention may bind to human IL-
33 and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig,
hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset,
rhesus or chimpanzee IL-33. According to certain exemplary embodiments of the present
invention, anti-IL-33 antibodies are provided which specifically bind human IL-33 and
cynomolgus monkey (e.g., Macaca fascicularis) IL-33.
Immunoconjugates
The invention encompasses anti-IL-33 monoclonal antibodies conjugated to a
therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an
immunosuppressant or a radioisotope. Cytotoxic agents include any agent that is detrimental to
cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming
immunoconjugates are known in the art, (see for example, WO 05/103081).
Multispecific Antibodies
The antibodies of the present invention may be monospecific, bi-specific, or
multispecific. Multispecific antibodies may be specific for different epitopes of one target
polypeptide or may contain antigen-binding domains specific for more than one target
polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends
Biotechnol. 22:238-244. The anti-IL-33 antibodies of the present invention can be linked to or
co-expressed with another functional molecule, e.g., another peptide or protein. For example,
an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic
fusion, noncovalent association or otherwise) to one or more other molecular entities, such as
another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a
second binding specificity. For example, the present invention includes bi-specific antibodies
wherein one arm of an immunoglobulin is specific for human IL-33 or a fragment thereof, and
the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated
to a therapeutic moiety.
An exemplary bi-specific antibody format that can be used in the context of the present
invention involves the use of a first immunoglobulin (Ig) C 3 domain and a second Ig C 3
domain, wherein the first and second Ig C 3 domains differ from one another by at least one
amino acid, and wherein at least one amino acid difference reduces binding of the bispecific
antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference.
In one embodiment, the first Ig C 3 domain binds Protein A and the second Ig C 3 domain
contains a mutation that reduces or abolishes Protein A binding such as an H95R modification
(by IMGT exon numbering; H435R by EU numbering). The second C 3 may further comprise a
Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the
second C 3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M,
17378269_1 (GHMatters) P40794NZ00
N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and
V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R,
N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K,
E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody
format described above are contemplated within the scope of the present invention.
Other exemplary bispecific formats that can be used in the context of the present
invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv
fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g.,
common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine
zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab bispecific formats (see, e.g.,
Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing
formats). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation,
e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate
site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric
complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am.
Chem. Soc. [Epub: Dec. 4, 2012]).
pH-DEPENDENT BINDING
The present invention provides antibodies and antigen-binding fragments thereof that
bind IL-33 in a pH-dependent manner. For example, an anti-IL-33 antibody of the invention may
exhibit reduced binding to IL-33 at acidic pH as compared to neutral pH. Alternatively, an anti-
IL-33 antibody of the invention may exhibit enhanced binding to its antigen at acidic pH as
compared to neutral pH.
In certain instances, "reduced binding to IL-33 at acidic pH as compared to neutral pH"
is expressed in terms of a ratio of the K
D value of the antibody binding to IL-33 at acidic pH to
the K value of the antibody binding to IL-33 at neutral pH (or vice versa). For example, an
antibody or antigen-binding fragment thereof may be regarded as exhibiting "reduced binding to
IL-33 at acidic pH as compared to neutral pH" for purposes of the present invention if the
antibody or antigen-binding fragment thereof exhibits an acidic/neutral K ratio of about 3.0 or
greater. In certain exemplary embodiments, the acidic/neutral K ratio for an antibody or
antigen-binding fragment of the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,
6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,
.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater
Antibodies with pH-dependent binding characteristics may be obtained, e.g., by
screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at
acidic pH as compared to neutral pH. Additionally, modifications of the antigen-binding domain
at the amino acid level may yield antibodies with pH-dependent characteristics. For example,
17378269_1 (GHMatters) P40794NZ00
by substituting one or more amino acids of an antigen-binding domain (e.g., within a CDR) with
a histidine residue, an antibody with reduced antigen-binding at acidic pH relative to neutral pH
may be obtained. As used herein, the expression "acidic pH" means a pH of about 6.0 or less,
about 5.5 or less, or about 5.0 or less. The expression "acidic pH" includes pH values of about
6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1,
.05, 5.0, or less. As used herein, the expression "neutral pH" means a pH of about 7.0 to
about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2,
7.25, 7.3, 7.35, and 7.4.
Therapeutic Formulation and Administration
The invention provides pharmaceutical compositions comprising the anti-IL-33
antibodies or antigen-binding fragments thereof of the present invention. The pharmaceutical
compositions of the invention are formulated with suitable carriers, excipients, and other agents
that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate
formulations can be found in the formulary known to all pharmaceutical chemists: Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include,
for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)
containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA
conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions
carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid
mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral
formulations" PDA (1998) J Pharm Sci Technol 52:238-311.
The dose of antibody administered to a patient may vary depending upon the age and
the size of the patient, target disease, conditions, route of administration, and the like. The
preferred dose is typically calculated according to body weight or body surface area. When an
antibody of the present invention is used for treating a condition or disease associated with IL-
33 activity in an adult patient, it may be advantageous to intravenously administer the antibody
of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight,
more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg
body weight. Depending on the severity of the condition, the frequency and the duration of the
treatment can be adjusted. Effective dosages and schedules for administering anti-IL-33
antibodies may be determined empirically; for example, patient progress can be monitored by
periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of
dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991,
Pharmaceut. Res. 8:1351).
Various delivery systems are known and can be used to administer the pharmaceutical
17378269_1 (GHMatters) P40794NZ00
composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis
(see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but
are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The composition may be administered by any convenient
route, for example by infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other biologically active agents. Administration can be systemic or
local.
A pharmaceutical composition of the present invention can be delivered
subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to
subcutaneous delivery, a pen delivery device readily has applications in delivering a
pharmaceutical composition of the present invention. Such a pen delivery device can be
reusable or disposable. A reusable pen delivery device generally utilizes a replaceable
cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical
composition within the cartridge has been administered and the cartridge is empty, the empty
cartridge can readily be discarded and replaced with a new cartridge that contains the
pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen
delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device
comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once
the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
Numerous reusable pen and autoinjector delivery devices have applications in the
subcutaneous delivery of a pharmaceutical composition of the present invention. Examples
include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),
DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX
75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN),
NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo
Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ),
OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,
Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having
applications in subcutaneous delivery of a pharmaceutical composition of the present invention
include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo
Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK Autoinjector (Amgen, Thousand
Oaks, CA), the PENLET (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the
HUMIRA Pen (Abbott Labs, Abbott Park IL), to name only a few.
In certain situations, the pharmaceutical composition can be delivered in a controlled
release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987,
17378269_1 (GHMatters) P40794NZ00
CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be
used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC
Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be
placed in proximity of the composition’s target, thus requiring only a fraction of the systemic
dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138). Other controlled release systems are discussed in the review by Langer, 1990,
Science 249:1527-1533.
The injectable preparations may include dosage forms for intravenous, subcutaneous,
intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations
may be prepared by methods publicly known. For example, the injectable preparations may be
prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above
in a sterile aqueous medium or an oily medium conventionally used for injections. As the
aqueous medium for injections, there are, for example, physiological saline, an isotonic solution
containing glucose and other auxiliary agents, etc., which may be used in combination with an
appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene
glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50
(polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there
are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a
solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is
preferably filled in an appropriate ampoule.
Advantageously, the pharmaceutical compositions for oral or parenteral use described
above are prepared into dosage forms in a unit dose suited to fit a dose of the active
ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules,
injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is
generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of
injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and
in about 10 to about 250 mg for the other dosage forms.
Therapeutic Uses of the Antibodies
Experiments using mouse model systems, conducted by the present inventors, have
contributed to the identification of various diseases and conditions that can be treated,
prevented and/or ameliorated by IL-33 antagonism. For example, hydrodynamic delivery of
mouse IL-33 DNA resulted in the induction of lung mucus accumulation and increases in total
serum IgE in mice. In addition, mIL-33 DNA delivery resulted in up-regulation of ST2 and
various downstream cytokines as measured by microarray analysis. Experiments conducted by
the present inventors using IL-33 knock-out mice also revealed various potential therapeutic
benefits of IL-33 antagonism. For example, macroscopic scoring and skin infiltrates were found
17378269_1 (GHMatters) P40794NZ00
to be comparable between wild-type mice and IL-33 mice in a model of IMQ-induced psoriasis.
Moreover, IL-33 mice showed reduced eosinophilia and residual mucus accumulation in an
allergen-induced lung inflammation model.
The antibodies of the invention are useful, inter alia, for the treatment, prevention
and/or amelioration of any disease or disorder associated with or mediated by IL-33 expression,
signaling, or activity, or treatable by blocking the interaction between IL-33 and a IL-33 ligand
(e.g., ST2) or otherwise inhibiting IL-33 activity and/or signaling. For example, the present
invention provides methods for treating, asthma (e.g., allergic asthma, non-allergic asthma,
severe refractory asthma, asthma exacerbations, steroid resistant asthma, steroid sensitive
asthma, eosinophilic asthma or non-eosinophilic asthma, etc.), atopic dermatitis, psoriasis, other
inflammatory disorders, allergy, anaphylaxis, cardiovascular disease, central nervous system
disease, pain, arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis, etc.), giant cell
arteritis, vasculitis (behcet's disease and Churg Strauss syndrome), Henoch-Schonlein purpura.,
multiple sclerosis, inflammatory bowel disorder (e.g. Crohn's disease or ulcerative colitis), lupus,
and sjogren's syndrome.
The antibodies of the present invention are also useful for the treatment, prevention
and/or amelioration of one or more fibrotic diseases. Exemplary fibrotic diseases that are
treatable by administering the anti-IL-33 antibodies of the invention include pulmonary fibrosis
(e.g., idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, asbestos-induced
pulmonary fibrosis, and bronchiolitis obliterans syndrome), chronic asthma, fibrosis associated
with acute lung injury and acute respiratory distress (e.g., bacterial pneumonia induced fibrosis,
trauma induced fibrosis, viral pneumonia induced fibrosis, ventilator induced fibrosis, non-
pulmonary sepsis induced fibrosis and aspiration induced fibrosis), silicosis, radiation-induced
fibrosis, chronic obstructive pulmonary disease (COPD, which may or may not be related to,
caused in part by, or resulting from, exposure to first or second hand cigarette smoke),
scleroderma, ocular fibrosis, skin fibrosis (e.g., scleroderma), hepatic fibrosis (e.g., cirrhosis,
alcohol-induced liver fibrosis, non-alcoholic steatohepatitis (NASH), bilary duct injury, primary
bilary cirrhosis, infection- or viral-induced liver fibrosis, autoimmune hepatitis, kidney (renal)
fibrosis, cardiac fibrosis, atherosclerosis, stent restenosis, and myelofibrosis.
In the context of the methods of treatment described herein, the anti-IL-33 antibody
may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination
with one or more additional therapeutic agents (examples of which are described elsewhere
herein).
Combination Therapies and Formulations
The present invention includes compositions and therapeutic formulations comprising
17378269_1 (GHMatters) P40794NZ00
any of the anti-IL-33 antibodies described herein in combination with one or more additional
therapeutically active components, and methods of treatment comprising administering such
combinations to subjects in need thereof.
The anti-IL-33 antibodies of the present invention may be co-formulated with and/or
administered in combination with, e.g., cytokine inhibitors, including small-molecule cytokine
inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9,
IL-11, IL-12, IL-13, IL-17, IL-18, IL-21, IL-23, IL-25, IL-26, or antagonists of their respective
receptors.
The anti-IL-33 antibodies of the invention may also be administered and/or co-
formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids,
oxygen, antioxidants, metal chelators, IFN-gamma, and/or NSAIDs.
The additional therapeutically active component(s) may be administered just prior to,
concurrent with, or shortly after the administration of an anti-IL-33 antibody of the present
invention; (for purposes of the present disclosure, such administration regimens are considered
the administration of an anti-IL-33 antibody "in combination with" an additional therapeutically
active component). The present invention includes pharmaceutical compositions in which an
anti-IL-33 antibody of the present invention is co-formulated with one or more of the additional
therapeutically active component(s) as described elsewhere herein.
Administration Regimens
According to certain embodiments of the present invention, multiple doses of an anti-IL-
33 antibody (or a pharmaceutical composition comprising a combination of an anti-IL-33
antibody and any of the additional therapeutically active agents mentioned herein) may be
administered to a subject over a defined time course. The methods according to this aspect of
the invention comprise sequentially administering to a subject multiple doses of an anti-IL-33
antibody of the invention. As used herein, "sequentially administering" means that each dose
of anti-IL-33 antibody is administered to the subject at a different point in time, e.g., on different
days separated by a predetermined interval (e.g., hours, days, weeks or months). The present
invention includes methods which comprise sequentially administering to the patient a single
initial dose of an anti-IL-33 antibody, followed by one or more secondary doses of the anti-IL-33
antibody, and optionally followed by one or more tertiary doses of the anti-IL-33 antibody.
The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal
sequence of administration of the anti-IL-33 antibody of the invention. Thus, the "initial dose" is
the dose which is administered at the beginning of the treatment regimen (also referred to as
the "baseline dose"); the "secondary doses" are the doses which are administered after the
initial dose; and the "tertiary doses" are the doses which are administered after the secondary
doses. The initial, secondary, and tertiary doses may all contain the same amount of anti-IL-33
17378269_1 (GHMatters) P40794NZ00
antibody, but generally may differ from one another in terms of frequency of administration. In
certain embodiments, however, the amount of anti-IL-33 antibody contained in the initial,
secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as
appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4,
or 5) doses are administered at the beginning of the treatment regimen as "loading doses"
followed by subsequent doses that are administered on a less frequent basis (e.g.,
"maintenance doses").
In certain exemplary embodiments of the present invention, each secondary and/or
tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8,
8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½,
19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks after
the immediately preceding dose. The phrase "the immediately preceding dose," as used herein,
means, in a sequence of multiple administrations, the dose of anti-IL-33 antibody which is
administered to a patient prior to the administration of the very next dose in the sequence with
no intervening doses.
The methods according to this aspect of the invention may comprise administering to a
patient any number of secondary and/or tertiary doses of an anti-IL-33 antibody. For example,
in certain embodiments, only a single secondary dose is administered to the patient. In other
embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered
to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to
the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses
are administered to the patient.
In embodiments involving multiple secondary doses, each secondary dose may be
administered at the same frequency as the other secondary doses. For example, each
secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the
immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each
tertiary dose may be administered at the same frequency as the other tertiary doses. For
example, each tertiary dose may be administered to the patient 2 to 12 weeks after the
immediately preceding dose. In certain embodiments of the invention, the frequency at which
the secondary and/or tertiary doses are administered to a patient can vary over the course of
the treatment regimen. The frequency of administration may also be adjusted during the course
of treatment by a physician depending on the needs of the individual patient following clinical
examination.
The present invention includes administration regimens in which 2 to 6 loading doses
are administered to a patient a first frequency (e.g., once a week, once every two weeks, once
every three weeks, once a month, once every two months, etc.), followed by administration of
two or more maintenance doses to the patient on a less frequent basis. For example, according
17378269_1 (GHMatters) P40794NZ00
to this aspect of the invention, if the loading doses are administered at a frequency of once a
month, then the maintenance doses may be administered to the patient once every six weeks,
once every two months, once every three months, etc.).
Diagnostic Uses of the Antibodies
The anti-IL-33 antibodies of the present invention may also be used to detect and/or
measure IL-33, or ILexpressing cells in a sample, e.g., for diagnostic purposes. For
example, an anti-IL-33 antibody, or fragment thereof, may be used to diagnose a condition or
disease characterized by aberrant expression (e.g., over-expression, under-expression, lack of
expression, etc.) of IL-33. Exemplary diagnostic assays for IL-33 may comprise, e.g.,
contacting a sample, obtained from a patient, with an anti-IL-33 antibody of the invention,
wherein the anti-IL-33 antibody is labeled with a detectable label or reporter molecule.
Alternatively, an unlabeled anti-IL-33 antibody can be used in diagnostic applications in
combination with a secondary antibody which is itself detectably labeled. The detectable label
3 14 32 35 125
or reporter molecule can be a radioisotope, such as H, C, P, S, or I; a fluorescent or
chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such
as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific
exemplary assays that can be used to detect or
measure IL-33 in a sample include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
Samples that can be used in IL-33 diagnostic assays according to the present invention
include any tissue or fluid sample obtainable from a patient which contains detectable quantities
of IL-33 protein, or fragments thereof, under normal or pathological conditions. Generally, levels
of IL-33 in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a
disease or condition associated with abnormal IL-33 levels or activity) will be measured to
initially establish a baseline, or standard, level of IL-33. This baseline level of IL-33 can then be
compared against the levels of IL-33 measured in samples obtained from individuals suspected
of having a IL-33 related disease or condition.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art
with a complete disclosure and description of how to make and use the methods and
compositions of the invention, and are not intended to limit the scope of what the inventors
regard as their invention. Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is
average molecular weight, temperature is in degrees Centigrade, and pressure is at or near
17378269_1 (GHMatters) P40794NZ00
atmospheric.
Example 1. Generation of Human Antibodies to human IL-33
An immunogen comprising human IL-33 was administered directly, with an adjuvant to
stimulate the immune response, to a VELOCIMMUNE mouse comprising DNA encoding
human Immunoglobulin heavy and kappa light chain variable regions. The antibody immune
response was monitored by an ILspecific immunoassay. When a desired immune response
was achieved splenocytes were harvested and fused with mouse myeloma cells to preserve
their viability and form hybridoma cell lines. The hybridoma cell lines were screened and
selected to identify cell lines that produce ILspecific antibodies. Using this technique
several anti-IL-33 chimeric antibodies (i.e., antibodies possessing human variable domains and
mouse constant domains) were obtained; exemplary antibodies generated in this manner were
designated as follows: H1M9559N, H1M9566N, H1M9568N and H1M9565N. The human
variable domains from the chimeric antibodies were subsequently cloned onto human constant
domains to make fully human anti-IL-33 antibodies as described herein.
Anti-IL-33 antibodies were also isolated directly from antigen-positive B cells without
fusion to myeloma cells, as described in US 2007/0280945A1. Using this method, several fully
human anti-IL-33 antibodies (i.e., antibodies possessing human variable domains and human
constant domains) were obtained; exemplary antibodies generated in this manner were
designated as follows: H4H9629P, H4H9633P, H4H6940P, H4H9659P, H4H9660P, H4H9662P,
H4H9663P, H4H9664P, H4H9665P, H4H9666P, H4H9667P, H4H9670P, H4H9671P,
H4H9672P, H4H9675P, and H4H9676P.
Certain biological properties of the exemplary anti-IL-33 antibodies generated in
accordance with the methods of this Example are described in detail in the Examples set forth
below.
Example 2. Heavy and Light Chain Variable Region Amino Acid Sequences
Table 1 sets forth the heavy and light chain variable region amino acid sequence pairs,
and CDR sequences, of selected anti-IL-33 antibodies and their corresponding antibody
identifiers.
Table 1
SEQ ID NOs:
Antibody
Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3
2 4 6 8 10 12 14 16
9559N
18 20 22 24 26 28 30 32
9566N
34 36 38 40 42 44 46 48
9568N
50 52 54 56 58 60 62 64
9629P
66 68 70 72 74 76 78 80
9633P
17378269_1 (GHMatters) P40794NZ00
82 84 86 88 90 92 94 96
9640P
98 100 102 104 106 108 110 112
9659P
114 116 118 120 122 124 126 128
9660P
130 132 134 136 138 140 142 144
9662P
146 148 150 152 154 156 158 160
9663P
162 164 166 168 170 172 174 176
9664P
178 180 182 184 186 188 190 192
9665P
194 196 198 200 202 204 206 208
9666P
210 212 214 216 218 220 222 224
9667P
226 228 230 232 234 236 238 240
9670P
242 244 246 248 250 252 254 256
9671P
258 260 262 264 266 268 270 272
9672P
9675P 274 276 278 280 282 284 286 288
290 292 294 296 298 300 302 304
9676P
308 310 312 314 316 318 320 322
9565N
Antibodies are typically referred to herein according to the following nomenclature: Fc
prefix (e.g. "H1M," or "H4H"), followed by a numerical identifier (e.g. "9559," "9566," or "9629"
as shown in Table 1), followed by a "P," or "N" suffix. Thus, according to this nomenclature, an
antibody may be referred to herein as, e.g., "H1M9559N," "H1M9566N," "H4H9629P," etc. The
H1M and H4H prefixes on the antibody designations used herein indicate the particular Fc
region isotype of the antibody. For example, an "H1M" antibody has a mouse IgG1 Fc, whereas
an "H4H" antibody has a human IgG4 Fc. As will be appreciated by a person of ordinary skill in
the art, an antibody having a particular Fc isotype can be converted to an antibody with a
different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted to an antibody
with a human IgG4, etc.), but in any event, the variable domains (including the CDRs) – which
are indicated by the numerical identifiers shown in Table 1 – will remain the same, and the
binding properties are expected to be identical or substantially similar regardless of the nature of
the Fc domain.
Example 3. Antibody Binding to Human IL-33 as Determined by Surface Plasmon
Resonance
Equilibrium dissociation constants (K values) for IL-33 binding to purified anti-IL-33
monoclonal antibodies were determined using a real-time surface plasmon resonance biosensor
using a Biacore 4000 instrument. The Biacore sensor surface was first derivatized by amine
coupling with either a polyclonal rabbit anti-mouse antibody (GE, # BR38) or with a
monoclonal mouse anti-human Fc antibody (GE, # BR39) to capture anti-IL-33
monoclonal antibodies expressed with mouse or with human IgG4 constant regions,
respectively. All Biacore binding studies were performed in 0.01M ADA pH 7.4, 0.15M NaCl,
3mM EDTA, and 0.05% v/v Surfactant Tween-20 (ABS-ET running buffer). Different
concentrations of human IL-33 (hIL-33; R&D Systems, # 3625-IL-010/CF) or cynomolgus
17378269_1 (GHMatters) P40794NZ00
monkey IL-33 expressed with a C-terminal hexahistidine tag (MfIL6His; SEQ ID NO:xx)
prepared in ABS-ET running buffer (ranging from 100nM to 3.7nM, 3-fold dilutions) were
injected over the anti-IL-33 monoclonal antibody captured surface at a flow rate of 30µL/minute.
Association of either hIL-33 or MfIL6His to the captured monoclonal antibody was monitored
for 4 minutes and their dissociation in ABS-ET running buffer was monitored for 10 minutes. The
effect of reduced pH on the binding of each anti-IL-33 antibody to either hIL-33 or MfIL6His
was studied using an in-line pH chase assay format in 0.01M ADA pH 6.0, 0.15M NaCl, 3mM
EDTA, and 0.05% v/v Surfactant Tween-20 (ABS-ET pH6 buffer). To achieve this, association
of either hIL-33 or MfIL6His to the captured monoclonal antibody was monitored for 4
minutes in ABS-ET running buffer. Following a 30 second dissociation of either hIL-33 or MfIL-
33-6His in ABS-ET running buffer, ABS-ET pH6 buffer was injected for 3 minutes, and the
analyte dissociation under the low-pH conditions was measured. All the binding kinetic
experiments were performed at both 25°C and 37°C. Kinetic association (k ) and dissociation
(k ) constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using
Scrubber 2.0c curve fitting software. Binding dissociation equilibrium constants (K ) and
dissociative half-lives (t½) were calculated from the kinetic rate constants as:
K (M) = k /k and t (min) = ln(2)/(60*k )
D d a 1/2 d
Binding kinetic parameters for hIL-33 and MfIL6His binding to different anti-IL-33
monoclonal antibodies at 25°C and 37°C are shown in Tables 2 through 5. At 25°C, hIL-33
bound to the anti-IL-33 antibodies with K values ranging from 78pM to 757pM, as shown in
Table 2. At 37°C, hIL-33 bound to the anti-IL-33 antibodies with K values ranging from 411pM
to 2.03nM, as shown in Table 3. At both 25°C and 37°C, one anti-IL-33 antibody demonstrated
weak binding and therefore its binding kinetic parameters could not be fit using an 1:1 binding
model. At 25°C, MfIL6His bound to the anti-IL-33 antibodies with KD values ranging from
333pM to 38nM, as shown in Table 4. At 37°C, MfIL6His bound to the anti-IL-33 antibodies
with K values ranging from 1nM to 48.6nM, as shown in Table 55.
Table 2: Binding kinetic parameters of anti-IL-33 monoclonal antibodies binding to human IL-33
at 25°C.
Human IL-33 Binding Kinetics in ABS-ET In-Line Chase in ABS-ET
Running Buffer pH6 Buffer
Antibody k k K t½ k t½ Ratio
a d D d
Captured (1/Ms) (1/s) (M) (min) (1/s) (min) (pH7.4/
pH6.0)
H4H9675P 1.02E+06 2.58E-04 2.54E-10 45 1.11E-03 10 4.3
H4H9662P 8.11E+05 2.50E-04 3.08E-10 46 8.26E-04 14 3.3
17378269_1 (GHMatters) P40794NZ00
H4H9640P 9.12E+05 2.37E-04 2.60E-10 49 6.57E-04 18 2.8
H4H9629P 7.77E+05 2.26E-04 2.90E-10 51 1.28E-03 9 5.7
H4H9659P 5.26E+05 1.72E-04 3.27E-10 67 6.64E-04 17 3.9
H4H9660P 6.96E+05 2.24E-04 3.22E-10 52 7.08E-04 16 3.2
H4H9667P 6.37E+05 2.52E-04 3.95E-10 46 5.66E-04 20 2.2
H4H9670P 7.86E+05 2.89E-04 3.68E-10 40 8.25E-04 14 2.9
H4H9663P 1.36E+06 4.14E-04 3.05E-10 28 1.10E-03 11 2.7
H4H9666P 5.08E+05 2.80E-04 5.51E-10 41 1.34E-03 9 4.8
H4H9676P 1.03E+06 3.45E-04 3.34E-10 33 1.21E-03 10 3.5
H4H9633P 6.56E+05 2.83E-04 4.32E-10 41 8.10E-04 14 2.9
H4H9671P 7.71E+05 3.49E-04 4.53E-10 33 1.62E-03 7 4.6
H4H9672P 6.68E+05 3.52E-04 5.27E-10 33 1.41E-03 8 4.0
H4H9665P 8.88E+05 4.74E-04 5.33E-10 24 2.12E-03 5 4.5
H4H9664P 3.39E+05 2.57E-04 7.57E-10 45 8.23E-04 14 3.2
H1M9568N 7.02E+05 1.30E-04 1.84E-10 89 1.78E-04 65 1.4
** ** **
H1M9566N 1.27E+05 1.00E-05 7.88E-11 1155 1.10E-04 105 11.0
H1M9559N 4.04E+05 2.74E-04 6.78E-10 42 1.87E-04 62 0.7
H1M9565N IC* IC* IC* IC* IC* IC* IC*
*IC: inconclusive since very weak binding was observed under the experimental conditions and
the real-time binding data could not be reliably fit into the 1:1 binding model.
** Under the experimental conditions no dissociation of IL33 from the captured monoclonal
antibody was observed' therefore the value of k was fixed to 1.00E-05, and the derived t½ and
K values represent lower and upper limits, respectively.
Table 3: Binding kinetic parameters of anti-IL-33 monoclonal antibodies binding to human IL-33
at 37°C.
Human IL-33 Binding Kinetics in ABS-ET In-Line Chase in ABS-ET
Running Buffer pH6 Buffer
Antibody k k K t½ k t½ Ratio
a d D d
Captured (1/Ms) (1/s) (M) (min) (1/s) (min) (pH7.4/
pH6.0)
H4H9675P 2.12E+06 8.72E-04 4.11E-10 13 4.63E-03 2 5.3
H4H9662P 1.40E+06 6.20E-04 4.43E-10 19 3.83E-03 3 6.2
H4H9640P 1.15E+06 5.73E-04 4.98E-10 20 2.65E-03 4 4.6
H4H9629P 1.27E+06 6.46E-04 5.08E-10 18 5.82E-03 2 9.0
H4H9659P 7.07E+05 4.03E-04 5.70E-10 29 2.99E-03 4 7.4
H4H9660P 8.03E+05 4.79E-04 5.96E-10 24 3.23E-03 4 6.8
H4H9667P 9.76E+05 6.03E-04 6.18E-10 19 2.44E-03 5 4.0
H4H9670P 1.16E+06 7.83E-04 6.76E-10 15 3.83E-03 3 4.9
H4H9663P 1.83E+06 1.24E-03 6.77E-10 9 4.62E-03 3 3.7
H4H9666P 1.13E+06 7.70E-04 6.81E-10 15 6.80E-03 2 8.8
H4H9676P 1.38E+06 1.28E-03 9.22E-10 9 5.24E-03 2 4.1
H4H9633P 7.40E+05 6.89E-04 9.31E-10 17 2.40E-03 5 3.5
H4H9671P 1.21E+06 1.14E-03 9.38E-10 10 5.85E-03 2 5.1
H4H9672P 1.09E+06 1.15E-03 1.05E-09 10 5.41E-03 2 4.7
H4H9665P 1.21E+06 1.44E-03 1.19E-09 8 9.65E-03 1 6.7
H4H9664P 5.19E+05 7.21E-04 1.39E-09 16 2.79E-03 4 3.9
H1M9568N 6.72E+05 9.61E-04 1.43E-09 12 1.10E-03 10 1.1
17378269_1 (GHMatters) P40794NZ00
H1M9566N 1.66E+05 2.83E-04 1.70E-09 41 9.67E-04 12 3.4
H1M9559N 4.73E+05 9.62E-04 2.03E-09 12 9.92E-04 12 1.0
H1M9565N IC* IC* IC* IC* IC* IC* IC*
*IC: inconclusive since very weak binding was observed under the experimental conditions and
the real-time binding data could not be reliably fit into the 1:1 binding model.
Table 4: Binding kinetic parameters of anti-IL-33 monoclonal antibodies binding to MfIL6His
at 25°C.
MfIL6His Binding Kinetics in ABS-ET In-Line Chase in ABS-ET
Running Buffer pH6 Buffer
Antibody k k K t½ k t½ Ratio
a d D d
Captured (1/Ms) (1/s) (M) (min) (1/s) (min) (pH7.4/
pH6.0)
H4H9675P 5.06E+05 1.29E-03 2.55E-09 9 1.56E-03 7 1.2
H4H9662P 3.53E+05 4.42E-04 1.25E-09 26 1.17E-04 99 0.3
H4H9640P 4.50E+05 1.37E-03 3.06E-09 8 5.01E-04 23 0.4
H4H9629P 5.62E+05 1.35E-02 2.39E-08 0.9 3.58E-02 0.3 2.7
H4H9659P 3.25E+05 4.86E-04 1.50E-09 24 1.23E-04 94 0.3
H4H9660P 4.26E+05 1.49E-03 3.49E-09 8 1.08E-03 11 0.7
H4H9667P 3.43E+05 9.91E-04 2.89E-09 12 6.96E-04 17 0.7
H4H9670P 4.40E+05 2.10E-03 4.77E-09 6 3.93E-04 29 0.2
H4H9663P 8.69E+05 9.25E-04 1.06E-09 12 6.83E-04 17 0.7
H4H9666P 2.22E+05 3.54E-03 1.59E-08 3.3 8.09E-03 1.4 2.3
H4H9676P 8.52E+05 4.12E-03 4.84E-09 2.8 1.45E-03 8 0.4
H4H9633P 2.62E+05 9.97E-03 3.80E-08 1.2 2.87E-03 4 0.3
H4H9671P 5.87E+05 1.50E-03 2.55E-09 8 1.61E-03 7 1.1
H4H9672P 4.37E+05 3.60E-03 8.22E-09 3.2 2.67E-03 4 0.7
H4H9665P 5.57E+05 5.66E-04 1.02E-09 20 7.53E-04 15 1.3
H4H9664P 1.40E+05 1.65E-03 1.18E-08 7 4.80E-04 24 0.3
H1M9568N 2.44E+05 2.61E-04 1.07E-09 44 3.02E-04 38 1.2
H1M9566N 2.93E+05 9.75E-05 3.33E-10 119 1.26E-04 91 1.3
H1M9559N 3.21E+05 1.23E-03 3.82E-09 9 1.52E-03 8 1.2
H1M9565N 4.06E+04 7.20E-05 1.77E-09 160 1.43E-04 81 2.0
Table 5: Binding kinetic parameters of anti-IL-33 monoclonal antibodies binding to MfIL6His
at 37°C.
MfIL6His Binding Kinetics in ABS-ET In-Line Chase in ABS-ET
Running Buffer pH6 Buffer
Antibody k k K t½ k t½ Ratio
a d D d
Captured (1/Ms) (1/s) (M) (min) (1/s) (min) (pH7.4/
pH6.0)
H4H9675P 1.02E+06 4.91E-03 4.81E-09 2.4 7.35E-03 1.6 1.5
H4H9662P 7.07E+05 1.58E-03 2.24E-09 7 1.68E-03 7 1.1
H4H9640P 8.10E+05 4.36E-03 5.38E-09 2.6 2.26E-03 5 0.5
17378269_1 (GHMatters) P40794NZ00
H4H9629P 1.07E+06 3.47E-02 3.24E-08 0.3 FT* FT* FT*
H4H9659P 5.98E+05 1.86E-03 3.11E-09 6 1.02E-03 11 0.5
H4H9660P 6.80E+05 4.44E-03 6.53E-09 2.6 4.63E-03 2.5 1.0
H4H9667P 6.81E+05 3.17E-03 4.66E-09 4 2.68E-03 4 0.8
H4H9670P 7.35E+05 5.03E-03 6.84E-09 2.3 1.65E-03 7 0.3
H4H9663P 1.62E+06 3.61E-03 2.22E-09 3.2 3.54E-03 3.3 1.0
H4H9666P 4.32E+05 1.41E-02 3.27E-08 0.8 FT* FT* FT*
H4H9676P 1.87E+06 1.44E-02 7.70E-09 0.8 FT* FT* FT*
H4H9633P 4.68E+05 2.27E-02 4.86E-08 0.5 FT* FT* FT*
H4H9671P 1.20E+06 6.07E-03 5.08E-09 1.9 8.19E-03 1.4 1.3
H4H9672P 9.46E+05 1.30E-02 1.37E-08 0.9 FT* FT* FT*
H4H9665P 1.10E+06 2.10E-03 1.91E-09 5 4.00E-03 2.9 1.9
H4H9664P 3.61E+05 5.84E-03 1.62E-08 2.0 1.93E-03 6 0.3
H1M9568N 3.89E+05 1.73E-03 4.46E-09 7 2.24E-03 5 1.3
H1M9566N 3.99E+05 4.00E-04 1.00E-09 29 1.15E-03 10 2.9
H1M9559N 4.93E+05 3.47E-03 7.04E-09 3.3 3.07E-03 4 0.9
H1M9565N 7.82E+04 2.02E-04 2.59E-09 57 2.28E-04 51 1.1
*FT: fast t½ such that MfIL6His bound to the captured anti-IL-33 monoclonal antibody was
Example 4. Anti-IL-33 Antibodies Block Binding of IL-33 to the Human ST2 Receptor
The ability of anti-IL-33 antibodies to block either human IL-33 (hIL-33) or cynomologus
monkey IL-33 binding to the human ST2 receptor was measured using a competition sandwich
ELISA. A portion of human ST2 protein ecto domain that was expressed with a C-terminal
human IgG1 Fc tag (hST2-hFc; SEQ ID NO:306), was coated at a concentration of 1 µg/mL in
PBS buffer on a 96-well microtiter plate overnight at 4 °C. Nonspecific binding sites were
subsequently blocked using a 0.5% (w/v) solution of BSA in PBS. Constant concentrations of
either 30 pM biotinylated hIL-33 protein (R&D systems, Cat #3625-IL/CF) (biotin-hIL-33) or 150
pM cynomologus monkey IL-33 expressed with hexahistidine tag (MfIL6His; SEQ ID
NO:305) were separately added to serial dilutions of antibodies so that the final concentrations
of antibodies ranged from 0 to 100 nM. The antibody/IL-33 mixtures were incubated for 1 hour
at room temperature before they were transferred to the hST2-hFc-coated microtiter plates.
After incubating for 1 hour at room temperature, the wells were then washed, and plate-bound
biotin-hIL-33 was detected with streptavidin conjugated with horse-radish peroxidase (HRP)
(Thermo Scientific, Cat # N200), and plate-bound MfIL6His was detected with a HRP
conjugated anti-His monoclonal antibody (Qiagen, #34460). All samples were developed with a
TMB solution (BD biosciences, # 51-2607KC) to produce a colorimetric reaction and then
quenched by acidification with 1M sulfuric acid before measuring absorbance at 450nm on a
Victor X5 plate reader. Data analysis was performed using a sigmoidal dose-response model
within Prism software. The calculated IC value, defined as the concentration of antibody
required to reduce by 50% from maximal signal the biotin-hIL-33 or MfIL6His binding to
plate-coated hST2-hFc, was used as an indicator of blocking potency. Percent blockade was
17378269_1 (GHMatters) P40794NZ00
calculated as the ratio of the reduction in signal observed in the presence of antibody relative to
the difference between the signal with IL-33 alone and background (signal from HRP-
conjugated secondary antibody or streptavidin alone). The absorbance measured for the
constant concentration of biotin-hIL-33 or MfIL6His alone is defined as 0% blocking and the
absorbance measured for no added IL-33 is defined as 100% blocking. The absorbance values
of the wells containing the highest concentration for each antibody were used to determine the
percent maximum blocking.
Table 6: ELISA blocking of biotin-hIL-33 or MfIL6His binding to hST2-hFc by anti-IL-33
antibodies
Blocking
% Maximum Blocking 150pM % Maximum
30pM biotin-
blocking biotin- Mf-IL6His blocking Mf-IL-
Ab ID hIL-33 on
hIL-33 on on hST2-hFc, 33-6His on
hST2-hFc,
hST2-hFc IC (M) hST2-hFc
IC (M)
H1M9559N* 88 1.0E-08 53
1.4E-10
H1M9566N* 3.2E-10 69 2.2E-10 41
H1M9565N* 2.2E-08 68 1.2E-08 86
H1M9568N* 55 8.4E-10 38
1.9E-10
H4H9629P N/A NBI
4.5E-10 80
H4H9633P N/A NBI
4.4E-10 66
H4H9640P 3.5E-09 73
3.5E-10 78
H4H9659P 6.0E-10 92
4.0E-10 78
H4H9660P 4.2E-09 68
3.1E-10 57
H4H9662P 8.6E-10 87
1.0E-09 77
H4H9663P 1.2E-09 81
.0E-10 74
H4H9664P 3.8E-09 67
3.0E-10 73
H4H9665P 4.2E-10 81
8.7E-10 55
H4H9666P 1.3E-08 40
6.0E-10 71
H4H9667P 4.1E-09 72
4.1E-10 78
H4H9670P 4.8E-10 69 3.5E-09 69
H4H9671P 5.8E-10 62
4.6E-10 46
H4H9672P 5.5E-09 48
4.4E-10 63
H4H9675P 1.5E-09 72
4.4E-10 58
H4H9676P 3.2E-09 57
4.6E-10 54
N/A= not applicable
NBl= non-blocker
*= Experiment performed on a separate day
Binding experiments for 20 antibodies were performed on two separate days, as
indicated in Table 6. All 20 of the anti-IL-33 antibodies blocked biotin-hIL-33 binding to hST2-
hFc with IC values ranging from 140pM to 22nM and percent maximum blocking ranging from
46% to 88%. Eighteen of the 20 anti-IL-33 antibodies blocked MfIL6His binding to hST2-hFc
17378269_1 (GHMatters) P40794NZ00
with IC values ranging from 220pM to 13nM and percent maximum blocking ranging from 38%
to 92%, as shown in Table 6. Two of the antibodies tested, H4H9629P and H4H9633P, did not
demonstrate measurable blockade of MfIL6His binding to hST2-hFc.
Example 5. Inhibition of IL-33 binding to anti-IL-33 monoclonal antibody by ST2 as shown by
Biacore Analysis
The ability of anti-IL-33 antibodies to bind to a pre-formed complex of IL-33 with ST2
was tested using Biacore T-200 instrument equipped with a real-time surface plasmon
resonance biosensor. The experiment was performed at 25ºC with a running buffer composed
of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, and 0.05% v/v Surfactant Tween-20 (HBS-
ET). The Biacore sensor surface was first derivatized by amine coupling an anti-myc tag-
specific monoclonal antibody (Clone# 9E10), and on this derivatized sensor was captured
approximately 160 response units (RU) of human ST2 protein expressed with a C-terminal myc-
myc-hexahistadine tag (hST2-MMH; SEQ ID NO: 323). The captured hST2-MMH surface was
then saturated by injecting 100nM of human IL-33 (hIL-33; R&D Systems, # 3625-IL-010/CF) for
3 minutes followed by a 3 minute injection of a 100nM solution of the anti-IL-33 monoclonal
antibody. The real-time binding response was monitored during the entire course of the
experiment, and the observed binding response at 3 minutes after injection of anti-IL-33
antibody to the pre-formed complex of hIL-33 and captured hST2-MMH was recorded and
tabulated and shown in Table 7. No non-specific binding of anti-IL-33 monoclonal antibody to
the anti-myc tag capture surface was observed. As shown in Table 7, 17 of the tested
antibodies did not show measurable binding to hIL-33 after it was pre-complexed with hST2-
MMH, while three antibodies (H1M9565N, H1M9566N, and H1M9568N) bound to hIL-33 after it
was pre-complexed with hST2-MMH.
Table 7: Binding of anti-IL-33 antibodies to a pre-formed complex of hIL-33 and hST2-MMH
Antibody
Antibody Binding
Response (RU)
H4H9629P -1
H4H9633P -1
H4H9640P -1
H4H9659P -1
17378269_1 (GHMatters) P40794NZ00
H4H9660P -1
H4H9662P 0
H4H9663P -1
H4H9664P -1
H4H9665P 0
H4H9666P -1
H4H9667P -1
H4H9670P -1
H4H9671P -1
H4H9672P -1
H4H9675P -1
H4H9676P -1
H1M9559N -4
H1M9565N 11
H1M9566N 13
H1M9568N 131
Example 6. Inhibition of ILMediated Receptor Signaling by Anti-IL-33 Antibodies
Interleukin-33 (IL-33) is a ligand for ST2, a toll-like/interleukin-1 receptor super-family
member that associates with an accessory protein, IL-1RAcP (for review, see Kakkar and Lee,
2008). Upon activation of ST2/ IL-1RAcP by IL-33, a signaling cascade is triggered through
downstream molecules such as MyD88 (myeloid differentiation factor 88) and TRAF6 (TNF
receptor associated factor 6), leading to activation of NFκB (nuclear factor –κB), among others.
To develop a biologically relevant bioassay system to test anti-IL-33 antibodies, human
embryonic kidney cells (HEK293) were stably transfected to express human ST2 (amino acids
1-556 of accession number NP_057316) along with a luciferase reporter [NFκB response
element (5x)-luciferase-IRES-GFP] (HEK293/hST2/NFkB-luciferase cell line). The HEK293 cell
line expresses IL-1RAcP endogenously and NFκB activation by IL-33 in HEK293 cells has been
shown previously (Schmitz et al., Immunity 23:479-490 (2005)). The stable cell line was
isolated and maintained in 10% FBS, DMEM, NEAA, penicillin/streptomycin, and G418.
For the bioassay, HEK293/hST2/NFkB-luciferase cells were seeded onto 96-well assay
plates at 10,000 cells per well in low serum media containing 0.1% w/v FBS and OPTIMEM
(Invitrogen, #31985-070) and then incubated at 37°C in 5% CO overnight. The next day, to
determine the dose response of IL-33, either human IL-33 (hIL-33; R&D Systems, #3625-IL) or
cynomolgus monkey IL-33 expressed with a C-terminal hexahistidine tag (MfIL6His; SEQ ID
NO:305) were serially diluted at 1:3 and added to the cells starting from 10 nM and ranging
down to 0.0002 nM, plus a control sample containing no IL-33. To measure inhibition,
antibodies were serially diluted and added to the cells followed by addition of constant
17378269_1 (GHMatters) P40794NZ00
concentrations of IL-33 (10 pM hIL-33 for the human assay and 5 pM MfIL6His for the
monkey assay). Three-fold antibody serial dilutions were performed before adding to the cells,
starting from 100 pM and ranging down to 0.002 nM or starting from 10 nM and ranging down to
0.0002 nM. In addition to the antibody dilution series, a well containing the constant
concentration of IL-33 but without any antibody was also included. After 5.5 hours of incubation
at 37°C in 5% CO , luciferase activity was detected using a Victor X (Perkin Elmer) plate reader,
and the results were analyzed using nonlinear regression (4-parameter logistics) with Prism 5.
Results are shown in Table 8.
Table 8: Inhibition of human IL-33 and monkey IL-33 activation of HEK293/hST2/NFkB-
luciferase cells by anti-IL33 antibodies
Species Human Monkey
EC [M] 2.2E-12 3.5E-12 2.4E-11 8.2E-13 3.5E-12
Constant
10pM hIL-33 5pM MfIL6His
IL-33
AbPID IC [M] Notes IC [M] IC [M] IC [M] Notes IC [M]
50 50 50 50 50
H1M9559N 2.0E-09 4.9E-08
Partial
Partial Inhibition Inhibition
H1M9566N 9.5E-10 1.5E-09
(Max at 66%) (Max at
61%)
H1M9565N 2.9E-08 1.7E-08
Partial
Partial Inhibition Inhibition
H1M9568N 2.5E-10 3.5E-09
(Max at 48%) (Max at
34%)
H4H9629P 1.3E-11 5.5E-08
H4H9633P 2.2E-10 1.3E-07
H4H9640P 3.0E-11 1.4E-08
H4H9659P 4.7E-11 3.3E-09
H4H9660P 3.5E-11 1.9E-08
H4H9662P 2.0E-11 1.5E-09
H4H9663P 1.3E-10 2.7E-09
H4H9664P 5.0E-11 2.6E-08
H4H9665P 9.0E-11 6.6E-10
H4H9666P 3.5E-11 7.8E-08
H4H9667P 7.1E-11 1.2E-08
H4H9670P 1.2E-10 1.7E-08
H4H9671P 2.5E-11 4.8E-09
H4H9672P 2.5E-11 2.0E-08
H4H9675P 7.5E-12 4.1E-09
H4H9676P 3.5E-11 8.4E-09
17378269_1 (GHMatters) P40794NZ00
Eighteen of the 20 anti-IL33 antibodies blocked human IL-33 stimulation of the
HEK293/hST2/NFkB-luciferase cells with IC values ranging from 7.5pM to 29nM, as shown in
Table 8. Two of the antibodies tested, H1M9566N and H1M9568N, partially inhibited hIL-33
with a maximum inhibition of 48% and 66%, with IC values of 950 pM and 250 pM,
respectively. Eighteen of the 20 anti-IL33 antibodies blocked MfIL6His stimulation of
HEK293/hST2/NFkB-luciferase cells with IC values ranging from 660pM to 130nM as shown in
Table 8. Two of the antibodies tested, H1M9566N and H1M9568N, partially inhibited MfIL
6His with a maximum inhibition of 61% and 34%, with IC values of 1.5 nM and 3.5 nM,
respectively.
Example 7. Inhibition of ILInduced Degranulation of Human Basophils by Anti-IL-33
Antibodies
To further assess the in vitro characteristics of select anti-IL-33 antibodies of the
invention, their ability to block ILinduced basophil degranulation was measured. Peripheral
blood mononuclear cells (PBMC) were purified from fresh whole blood from two different human
donors by density gradient centrifugation. K2 EDTA whole blood was diluted 1:1 in RPMI 1640,
carefully layered over Ficoll-Paque (GE Healthcare, # 1703) and centrifuged to separate
PBMC. The interphase layer containing the PBMC was aspirated, transferred to a new tube,
and washed twice with MACS buffer that was comprised of a 1:20 dilution of the MACS BSA
solution (Militenyi Biotec, #130376) in MACS rinsing solution (Militenyi Biotec, #130
222). The purified PBMC were then plated in a v-bottom 96-well plate at a final concentration of
~3.0x10 cells/mL in 100 µL of MACS buffer. To prime the basophils contained within the
PBMC population, 1 ng of IL-3 (Sigma, # H7166-10UG) in 50 µL Dulbecco's Phosphate-
++ ++
Buffered Saline without Ca or Mg (DPBS) was added to the cell suspension, and then
incubated at 37°C for 10 minutes.
Serial dilutions (1:3) of two different exemplary anti-IL-33 antibodies of the invention
(H4H9675P and H4H9659P) or an isotype control antibody were made, ranging from 10 nM to
4.6 pM, plus a control with no antibody. The solutions were mixed with a fixed concentration of
100 pM (final concentration) of human IL-33 (R&D Systems, # 6325-IL/CF) or no IL-33 negative
control prior to adding to the PBMC. All conditions were tested in duplicate.
After addition of the human IL-33 and antibodies to the cells, the cells were incubated
at 37°C for 20 minutes to facilitate basophil degranulation. Degranulation was then stopped by
cooling the assay plates on wet ice for 5 minutes. To enable analysis of the basophil population
used to measure degranulation, 20 µL each (as per the manufacturer’s instructions) of anti-HLA-
DR-FITC (Beckman Coulter, # IM0463U), anti-CD123-APC (BD, # 560087), and anti-CD203c-
PE (Beckman Coulter, # IM3575) were added to each sample, and the samples were held at
4°C for 20 minutes in the dark. The cells were then centrifuged, washed with DPBS, and then
resuspended in 2% formaldehyde (fixation buffer) at 4°C. The next day, fixed cells were
17378269_1 (GHMatters) P40794NZ00
analyzed on a BD FACSCanto II to determine levels of basophil degranulation. Results are
summarized in Tables 9 and 10.
Table 9: Percent degranulation of human basophils induced by human IL-33 challenge
Donor 100pM IL-33 No IL-33
Mean SD Mean SD
655687 68.800 2.263 10.295 0.856
655688 61.600 0.849 9.915 0.969
Table 10: Anti-IL-33 antibody blocking human IL-33 induced degranulation of human
basophils
Donor 655687 Donor 655688
Antibody IC (M) IC (M)
50 50
H4H9675P 1.329E-10 9.712E-11
H4H9659P 5.786E-10 4.465E-10
Isotype Control non-blocking non-blocking
As shown in Table 9, at 100 pM, human IL-33 induced basophil degranulation in two
different donors with a mean percent degranulation of 68.8% for donor 655687 and 61.6% for
donor 655688.
As shown in Table 10, one anti-IL33 antibody, H4H9675P, blocked basophil
degranulation induced by 100 pM human IL-33 challenge with an IC value of 132.9 pM for
donor 655687, and an IC value of 97.12 pM for donor 655688. Another anti-IL33 antibody,
H4H9659P, blocked basophil degranulation induced by 100 pM human IL-33 challenge with an
IC value of 578.6 pM for donor 655687, and an IC value of 446.5 pM for donor 655688. In
50 50
contrast, the isotype control did not block basophil degranulation from any of the tested donors.
Example 8. Inhibition of ILInduced IFN-gamma From Human PBMCs by Anti-IL-33
Antibodies
To further characterize anti-IL-33 antibodies of the invention, a primary cell based
assay using peripheral blood mononuclear cells (PBMCs) was utilized. The assay used in this
Example was based on the results published by Smithgall et al. in International Immunology,
2008, vol. 20 (8) pp. 1019-1030. For this assay, PBMCs were purified from fresh whole blood
from three different donors by density gradient centrifugation. Briefly, K2 EDTA whole blood
was diluted two-fold in RPMI 1640, carefully layered over Ficoll-Paque (GE Healthcare, #17-
1440-03) and centrifuged for 20 minutes. The interphase layer containing the PBMCs was
aspirated, transferred to a new tube, and washed twice with PBS. The isolated PBMCs were
plated in round-bottom 96-well plates at a final concentration of 5x10 cells/mL in RPMI 1640
supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin. Cells were then incubated with 50 g/mL of human IL-12 (hIL-12; R&D Systems,
#219-IL-025/CF) and a serial dilution of human IL-33 (hIL-33; R&D Systems, #3625-IL-010/CF)
17378269_1 (GHMatters) P40794NZ00
alone from 10 nM to 0.64 pM, or with 260 pM of hIL-33 in combination with serial dilutions of
antibodies from 100 nM to 6.4 pM. The final volume was 200 μL per well. Each condition was
tested in triplicate. When antibodies were present, they were added to the cells after 30
minutes of pre-incubation with hIL-33.
The cells were incubated overnight at 37°C in a humidified incubator with 5% CO , and
then IFNγ levels in the culture supernatant were measured by ELISA (R&D Systems, #DY285).
For the ELISA, 96-well flat-bottom plates were coated with the capture antibody, according to
the manufacturer’s instructions. After washing and blocking, 100 μL of undiluted culture
supernatant was added to the plates and incubated for 2 hours. Subsequent washes and
detection were done following the manufacturer’s instructions.
Human IL-33, in the presence of hIL-12, induced the release of IFNγ from human total
PBMC from the three different donors tested, with EC values between 274pM to 39pM as
shown in Table 11. Eleven anti-IL-33 antibodies were tested using PBMCs from donors
#603486 and #603487, while 3 anti-IL-33 antibodies were tested with PBMCs from donor
#603491. All 11 of the anti-IL-33 antibodies tested on donors #603486 and #603487 blocked
the release of IFNγ from human PBMC induced by 260pM IL-33, with IC values ranging from
175 pM to 22 nM, as shown in Table 12. None of the three IL-33 antibodies tested on donor
#603491 blocked the release of IFNγ from human PBMC induced by 260pM hIL-33 and instead
caused an increase of IFNγ release with EC values between 56.1pM and 189nM.
Table 11: hIL-33 induced IFNγ release from human PBMC from three donors.
[IL-33] Donor 603486 Donor 603487 Donor 603491
EC (M) 1.101E-10 3.878E-11 2.739E-10
Table 12: Anti-IL-33 antibodies blocking IL-33 induced IFN-γ release from human PBMC
from donor #603486 and #603487
Donor #603486 Donor #603487
Antibody IC (M) IC (M)
50 50
H4H9629P 8.154E-10 5.205E-09
H4H9640P 4.419E-09 1.224E-08
H4H9659P 1.252E-09 2.710E-09
H4H9660P 6.669E-10 2.913E-09
H4H9662P 9.640E-10 3.021E-09
H4H9663P 1.236E-08 2.203E-08
H4H9664P 3.984E-09 6.081E-09
H4H9665P 1.044E-08 2.337E-08
H4H9667P 8.066E-09 1.876E-08
H4H9671P 2.968E-09 8.622E-09
H4H9675P 1.754E-10 4.715E-10
17378269_1 (GHMatters) P40794NZ00
Table 13: Anti-IL-33 antibodies blocking IL-33 induced IFN-γ release from human PBMC
from donor #603491.
Antibody Donor #603491
IC (M)
H1M9559N Non-blocking
H1M9566N Non-blocking
H1M9568N Non-blocking
The present invention is not to be limited in scope by the specific embodiments
described herein. Indeed, various modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the foregoing description and the
accompanying figures. Such modifications are intended to fall within the scope of the appended
claims.
Example 9. Human IL-33 cross-competition using bio-layer interferometry
Binding competition between a panel of different anti-IL-33 monoclonal antibodies was
determined using a real time, label-free bio-layer interferometry assay on an Octet HTX
biosensor (ForteBio, A Division of Pall Life Sciences). The experiment was performed at 25°C
using a buffer of 0.01M HEPES pH7.4, 0.15M NaCl, 0.05% v/v Surfactant Tween-20, and
0.1mg/ml BSA (HBS-ET kinetics buffer) with the plate shaking at a speed of 1000rpm. To
assess whether two antibodies were able to compete with one another for binding to human IL-
33, a pre-mix assay format was used where 100nM of human IL-33 (R&D Systems; # 3625-IL-
010/CF) was pre-mixed with 500nM of different anti-IL-33 monoclonal antibodies (subsequently
referred to as mAb-2) for at least 2 hours prior to running the binding competition assay. Octet
biosensors coated with either an anti-mouse Fc polyclonal antibody (Pall ForteBio Corp., # 18-
5088; subsequently referred as AMC) or with an anti-human Fc polyclonal antibody (Pall
ForteBio Corp., # 18-5060; subsequently referred as AHC) were first submerged into wells
containing 20µg/mL of individual anti-IL-33 monoclonal antibodies for 3 minutes to capture anti-
IL-33 monoclonal antibodies expressed either a with mouse Fc or with a human Fc, respectively
(subsequently referred to as mAb-1). Following the capture step, unoccupied anti-mouse Fc
polyclonal antibody and anti-human Fc polyclonal antibody on the Octet biosensors were
saturated by submerging them for 4 minutes into wells containing 200µg/mL of a non-specific
monoclonal antibody with a mouse Fc or with a human Fc, respectively. Finally, the Octet
biosensors were immersed for 4 minutes into wells containing the pre-mixed samples of 100nM
of human IL-33 and 500nM of mAb-2. At the end of each cycle, the non-covalently captured
anti-IL-33 antibodies along with the bound pre-complex of human IL-33 and mAb-2 were
removed from the biosensors using three alternate 20 second immersions into 10mM HCl
followed by submerging into HBS-ET kinetics buffer. The biosensors were washed in HBS-ET
17378269_1 (GHMatters) P40794NZ00
kinetics buffer in between every step of the experiment. The real-time binding response was
monitored during the binding events, and the binding response (in units of nm) at the end of
every step was recorded. During the analysis, the self-self background binding signal for a given
mAb-2 (where mAb-1 = mAb-2, i.e., along the diagonal of the matrix) was subtracted from the
observed signal for all mAb-2 binding events (across a column in the cross-competition matrix),
and the background-corrected results are shown in Figure 1. The response of mAb-1 binding to
the pre-complex of human IL-33 and each of the different mAb-2 samples was measured to
determine the competitive/non-competitive behavior of different anti-IL-33 monoclonal
antibodies with respect to each other.
As shown in Figure 1 light grey boxes with black font represent binding response for
self-competition. Antibodies competing with each other in both directions, independent of the
order of binding, are represented with black boxes and white font. Cells highlighted in dark grey
with black font represent the anti-IL-33 monoclonal antibody that binds weakly to human IL-33,
resulting in an observed unidirectional cross-competition. The isotype controls used in the
experiment are represented by dark grey boxes with white font. White boxes with black font
represent no competition between antibodies, which suggests each antibody has a distinct
binding epitope.
Example 10. Monkey IL-33 cross-competition using bio-layer interferometry
Binding competition between a panel of different anti-IL-33 monoclonal antibodies was
determined using a real time, label-free bio-layer interferometry assay on an Octet HTX
biosensor (ForteBio, A Division of Pall Life Sciences). The experiment was performed at 25°C
using a buffer of 0.01M HEPES pH7.4, 0.15M NaCl, 3mM EDTA, 0.05% v/v Surfactant Tween-
, and 0.1mg/ml BSA (HBS-ET kinetics buffer) with the plate shaking at a speed of 1000rpm.
To assess whether two antibodies were able to compete with one another for binding to
recombinant monkey IL-33 expressed with a C-terminal hexahistidine tag (MfIL6His; SEQ
ID: xx), approximately 0.15nm binding units of MfIL6His was first captured onto anti-penta-
His antibody coated Octet biosensors (Fortebio Inc, # 18-5079) by submerging the biosensors
for 85 seconds into wells containing 2µg/mL of MfIL6His. The antigen-captured biosensors
were then saturated with a first anti-IL-33 monoclonal antibody (subsequently referred to as
mAb-1) by immersion into wells containing 50µg/mL solution of mAb-1 for 5 minutes. The
biosensors were then dipped into wells containing a 50µg/mL solution of a second anti-IL-33
monoclonal antibody (subsequently referred to as mAb-2) for 4 minutes. The biosensors were
washed in HBS-ET kinetics buffer in between every step of the experiment. The real-time
binding response was monitored during the experiment, and the maximum binding response for
each binding step was recorded. The response of mAb-2 binding to MfIL6His pre-
complexed with mAb-1 was measured, and competitive/non-competitive behavior of different
17378269_1 (GHMatters) P40794NZ00
anti-IL-33 monoclonal antibodies with respect to each other was determined.
As shown in Figure 2, light grey boxes with black font (along a diagonal) represent self-
competition (where mAb-1=mAb-2). Antibodies competing in both directions, independent of the
order of binding, are represented with black boxes and white font. White boxes with black font
represent no competition between antibodies, which suggests each antibody has a distinct
binding epitope. Dark grey boxes with white font represent the isotype control used in the
experiment.
EXAMPLE 11. mAb testing in In vivo model; Acute HDM-induced lung inflammation model to
study role of IL-33 in lung inflammation
To determine the effect of an anti-IL-33 antibody, H4H9675P, in a relevant in vivo
model, an acute HDM-induced lung inflammation study was conducted in mice that were
homozygous for the expression of human IL-33 in place of mouse IL-33 (IL-33 HumIn mice).
IL-33 HumIn mice were intranasally administered either 50μg of house dust mite
extract (HDM; Greer, #XPB70D3A2.5) diluted in 20μL of 1X phosphate buffered saline (PBS)
(n=17) or 20μL of 1X PBS (n=3) for 5 days per week for 2 weeks. A subset of the HDM
challenged mice were injected subcutaneously with either 25 mg/kg of an anti-IL-33 antibody,
H4H9675, (n=6) or an isotype control antibody (n=6) starting at three days prior to the first HDM
administration and then twice weekly until the end of the HDM challenge. On day 15 after the
first intranasal HDM, all mice were sacrificed and their lungs were harvested. Experimental
dosing and treatment protocol for groups of mice are shown in Table 14.
Table 14: Experimental dosing and treatment protocol for groups of mice
Intranasal Length of intranasal
Group Mice Antibody
challenge challenge
IL-33 HumIn
1 1X PBS 2 weeks None
mice
IL-33 HumIn 50μg HDM in
2 2 weeks None
mice 20μL 1X PBS
IL-33 HumIn 50μg HDM in
3 2 weeks Isotype control
mice 20μL 1X PBS
IL-33 HumIn 50μg HDM in Anti-IL-33 antibody
4 2 weeks
mice 20μL 1X PBS (H4H9675)
Lung harvest for cytokine analysis:
After exsanguination, the cranial and middle lobes of the right lung from each mouse
were removed and placed into tubes containing a solution of tissue protein extraction reagent
17378269_1 (GHMatters) P40794NZ00
(1X T-PER reagent; Pierce, #78510) supplemented with 1X Halt Protease inhibitor cocktail
(Pierce, #78430). All further steps were performed on ice. The volume of T-PER Reagent
(containing the protease inhibitor cocktail) was adjusted for each sample to match a 1:8 (w/v)
tissue to T-PER ratio. Lung samples were manually homogenized in the tubes, using disposable
pestles (Kimble Chase, # 749625-0010). The resulting lysates were centrifuged to pellet debris.
The supernatants containing the soluble protein extracts were transferred to fresh tubes and
stored at 4°C until further analysis.
Total protein content in the lung protein extracts was measured using a Bradford
assay. For the assay, 10μL of diluted extract samples were plated into 96 well plates in
duplicates and mixed with 200μL of 1X Dye Reagent (Biorad, #500-0006). Serial dilutions of
bovine serum albumin (Sigma, #A7979), starting at 700μg/mL in 1X T-Per reagent were used as
a standard to determine the exact protein concentration of the extracts. After a 5-minute
incubation at room temperature, absorbance at 595nm was measured on a Molecular Devices
SpectraMax M5 plate reader. Data analysis to determine total protein content was performed
using GraphPad Prism software.
Cytokine concentrations in the lung protein extracts were measured using
a Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, #
K15048D-2), according to the manufacturer’s instructions. Briefly, 50 μL/well of calibrators and
samples (diluted in Diluent 41) were added to plates pre-coated with capture antibodies and
incubated at room temperature while shaking at 700 rpm for 2 hours. The plates were then
washed 3 times with 1X PBS containing 0.05% (w/v) Tween-20, followed by the addition of 25μL
of Detection Antibody Solution diluted in Diluent 45. After another 2-hour incubation at room
temperature while shaking, the plate was washed 3 times, and 150 μL of 2X Read Buffer was
added to each well. Electrochemiluminescence was immediately read on a MSD Spector
instrument. Data analysis was performed using GraphPad Prism software.
Each cytokine concentration in lung total protein extracts from all mice in each group
was normalized to the total protein content of the extracts measured by the Bradford assay and
expressed for each group as average pg of cytokine per mg of total lung proteins (pg/mg lung
protein, ± SD) as shown in Table 15.
Lung harvest for cytokine analysis:
The level of the cytokines IL-4 and IL-5 released in the lungs of IL-33 HumIn mice
receiving HDM for 2 weeks was significantly higher than in IL-33 HumIn mice challenged with
saline buffer. In contrast, there was a trend towards reduced IL-4 and IL-5 levels in the lungs of
IL-33 HumIn mice treated with anti-IL-33 antibody during the course of the acute HDM challenge
as compared to IL-33 HumIn mice administered HDM without treatment or with isotype control.
17378269_1 (GHMatters) P40794NZ00
Table 15: Cytokine concentration in lung protein extracts
Mean [IL-4] in lung protein Mean [IL-5] in lung protein
Experimental group extracts (pg/mg lung extracts (pg/mg lung
protein) (±SD) protein) (±SD)
1. 1X PBS challenge (n=3) 0.01 (±0.01) 0.03 (±0.01)
2. HDM challenge (n=5) 1.77 (±1.63) * 4.72 (±4.14) **
3. HDM challenge +
Isotype control Antibody 0.79 (±0.52) * 2.03 (±1.05) *
(n=6)
4. HDM challenge +
0.30 (±0.18) 0.81 (±0.67)
H4H9675P (n=6)
Note: Statistical significance determined by Kruskal-Wallis One-way ANOVA with Dunn’s
multiple comparison post-hoc test is indicated (*= p<0.05, **= p<0.01, compared to Group 1:
IL33 HumIn mice, Saline challenge).
Lung harvest for pulmonary cell infiltrate analysis
After exsanguination, the caudal lobe of the right lung from each mouse was removed,
chopped into cubes that were approximately 2 to 3 mm in size, and then placed into a tube
containing a solution of 20 μg/mL DNAse (Roche, #10104159001) and 0.7 U/mL Liberase TH
(Roche, #05401151001) diluted in Hank’s Balanced Salt Solution (HBSS) (Gibco, #14025),
which was incubated in a 37°C water bath for 20 minutes and vortexed every 5 minutes. The
reaction was stopped by adding ethylenediaminetetraacetic acid (Gibco, #15575) at a final
concentration of 10mM. Each lung was subsequently dissociated using a gentleMACS
dissociator (Miltenyi Biotec, #130937), then filtered through a 70 μm filter and centrifuged.
The resulting lung pellet was resuspended in 1 mL of 1X red blood cell lysing buffer (Sigma,
#R7757) to remove red blood cells. After incubation for 3 minutes at room temperature, 3 mL of
1X DMEM was added to deactivate the red blood cell lysing buffer. The cell suspensions were
then centrifuged, and the resulting cell pellets were resuspended in 5 mL of MACS buffer
(autoMACS Running Buffer; Miltenyi Biotec, #130221). The resuspended samples were
filtered through a 70 μm filter and 1x10 cells per well were plated in a 96-well V-bottom plate.
Cells were then centrifuged and the pellets were washed in 1X PBS. After a second
centrifugation, the cell pellets were resuspended in 100μL of LIVE/DEAD Fixable Aqua Dead
Cell Stain (Life Technologies, #L34957) diluted at 1:1000 in 1X PBS to determine cell viability
and incubated for 20 minutes at room temperature while protected from light. After one wash in
1X PBS, cells were incubated in a solution of MACS buffer containing 10μg/mL of purified rat
anti-mouse CD16/CD32 Fc Block, (Clone: 2.4G2; BD Biosciences, #553142) for 10 minutes at
17378269_1 (GHMatters) P40794NZ00
4°C. The cells were washed once and then incubated in the appropriate antibody mixture
(described in Table 16) diluted in MACS buffer for 30 minutes at 4°C while protected from light.
After antibody incubation, the cells were washed twice in MACS buffer, resuspended in BD
cytofix (BD Biosciences, #554655) and then incubated for 15 minutes at 4°C while protected
from light. The cells were subsequently washed, resuspended in MACS buffer, and then
transferred to BD FACS tubes (BD Biosciences, #352235) for analysis of eosinophils, innate
lymphoid cell type 2 (ILC2) and lymphocytes by flow cytometry.
+ Lo Lo +
Activated CD4 T cells were defined as cells that were live, CD45 , SSC , FSC , CD3 ,
- + - + +
CD19 , CD4 , CD8 , and CD69 . Activated B cells were defined as cells that were live, CD45 ,
Lo Lo - + + + -
SSC , FSC , CD3, CD19 , and CD69 . Eosinophils were defined as live, CD45 , GR1 ,
lo hi +
CD11c , SiglecF . ILC2 cells were defined as live, CD45 , Lineage- (Lineage: CD19, CD3,
- + +
CD11b, CD11c, F4/80), CD127+, Sca 1 , ST2 . Data for activated CD4 cells, expressed as
frequency of activated cells (CD69 ) within the parent population (CD4, ± SD), are shown in
Table 17.
Table 16: Antibodies Used for Flow Cytometry Analysis
Catalog
Antibody Fluorochrome Manufacturer Final dilution
Number
CD11c APC BD Biosciences 550261 1/100
CD45 PerCP Cy5.5 eBiosciences 4582 1/800
F4/80 Pacific Blue eBiosciences 4882 1/200
Siglec-F PE BD Biosciences 552126 1/100
Ly6G (Gr-1) APC-eFluor780 eBiosciences 4782 1/200
CD3 PE-Cy7 BD Biosciences 552774 1/200
CD19 eFluor 450 eBiosciences 4882 1/200
CD4 APC-H7 BD Biosciences 560181 1/200
CD8 APC eBiosciences 1782 1/200
CD69 PE eBiosciences 1282 1/200
CD3 eFluor 450 eBiosciences 4882 1/200
CD11b eFluor450 eBiosciences 4082 1/100
CD11c eFluor450 eBiosciences 4882 1/100
CD127 APC-eFluor780 eBiosciences 4782 1/200
Sca-1 FITC BD Biosciences 557405 1/200
ST2 APC Biolegend 145306 1/200
Pulmonary cell infiltrate analysis:
17378269_1 (GHMatters) P40794NZ00
As shown in Table 17, the frequency of activated CD4 T cells, eosinophils, and ILC2 in
the lungs of IL-33 HumIn mice receiving HDM for 2 weeks was significantly higher than in IL-33
HumIn mice challenged with 1X PBS control. In contrast, a trend towards a reduced frequency
of these infiltrates was observed in IL-33 HumIn mice when treated with the anti-IL-33 antibody
during the course of the acute HDM challenge as compared to IL-33 HumIn mice administered
HDM without treatment or with isotype control.
A trend towards an increase in the frequency of activated B cells was observed in the
lungs of IL33 HumIn mice challenged with HDM for 2 weeks compared to IL33 Humin mice
challenged with 1X PBS control. Upon anti-IL-33 antibody treatment, a significant reduction in
the frequency of pulmonary activated B cells in the lungs of IL33 HumIn mice challenged with
HDM was observed, as compared to IL-33 HumIn mice administered HDM without treatment or
with isotype control.
Table 17: Frequency of pulmonary cell infiltrate as determined by flow cytometry
Mean Mean Mean
Mean
Frequency of Frequency of Frequency of
Frequency of
activated eosinophils in ILC2 in
activated B
Experimental group CD4+ T cells CD45+ Lymphoid
cells in the B
in CD4+ population population
cell population
population (±SD) (±SD)
(±SD)
(±SD)
1. 1X PBS challenge
6.17 (±0.59) 6.85 (±3.09) 2.55 (±0.79) 0.33 (±0.05)
(n=3)
2. HDM challenge 29.52 (±8.57) 17.28 (±3.97)
.13 (±3.30) 1.15 (±0.37) *
(n=5) * *
3. HDM challenge +
29.68 (±9.84) 19.19
Isotype control 11.01 (±2.31) 1.57 (±0.78) *
* (±11.55)*
Antibody (n=6)
4. HDM challenge +
16.38 (±3.30) 4.88 (±1.70) 10.32 (±4.63) 0.53 (±0.12)
H4H9675P (n=6)
Note: Statistical significance determined by Kruskal-Wallis One-way ANOVA with Dunn’s
multiple comparison post-hoc test is indicated (*= p<0.05, **= p<0.01, compared to groups 1:
IL33 HumIn mice, Saline challenge; p<0.05, compared to group 3: IL33 Humin mice, HDM
challenge 2 weeks + Isotype control antibody).
EXAMPLE 12: mAb testing in In vivo model; Chronic HDM-induced fibrosis and severe
lung inflammation model to study role of IL-33 in lung inflammation
17378269_1 (GHMatters) P40794NZ00
To determine the effect of an anti-IL-33 antibody, H4H9675P, in a relevant in vivo
model, a chronic HDM-induced fibrosis and severe lung inflammation study was conducted in
mice that were homozygous for the expression of human IL-33 in place of mouse IL-33 (IL-33
HumIn mice).
IL-33 HumIn mice were intranasally administered either 50μg house dust mite extract
(HDM; Greer, #XPB70D3A2.5) diluted in 20μL of 1X phosphate buffered saline (PBS) or 20μL of
1X PBS for 5 days per week for 12 weeks. A second control group of IL33 HumIn mice were
administered 50μg HDM extract diluted in 20μL of 1X PBS for 5 days per week for 4 weeks, to
assess the severity of the disease at the onset of antibody treatment. Two groups of HDM
challenged mice were injected subcutaneously with 25 mg/kg of either an anti-IL-33 antibody,
H4H9675P, or an isotype control antibody starting after 4 weeks of HDM challenge and then
twice per week until the end of the HDM challenge (8 weeks of antibody treatment). On day 85
of the study, all mice were sacrificed and their lungs were harvested. Experimental dosing and
treatment protocol for groups of mice are shown in Table 18.
Table 18: Experimental dosing and treatment protocol for groups of mice
Intranasal Length of intranasal
Group Mice Antibody
challenge challenge
IL-33 HumIn
1 1X PBS 12 weeks None
mice
IL-33 HumIn 50μg HDM in
2 4 weeks None
mice 20μL 1X PBS
IL-33 HumIn 50μg HDM in
3 12 weeks None
mice 20μL 1X PBS
IL-33 HumIn 50μg HDM in Isotype control
4 12 weeks
mice 20μL 1X PBS antibody
IL-33 HumIn 50μg HDM in Anti-IL-33 antibody
12 weeks
mice 20μL 1X PBS (H4H9675P)
Lung harvest for cytokine analysis:
After exsanguination, the cranial and middle lobes of the right lung from each mouse
were removed and placed into tubes containing a solution of tissue protein extraction reagent
(1X T-PER reagent; Pierce, #78510) supplemented with 1X Halt Protease inhibitor cocktail
(Pierce, #78430). All further steps were performed on ice. The volume of T-PER Reagent
(containing the protease inhibitor cocktail) was adjusted for each sample to match a 1:8 (w/v)
tissue to T-PER ratio. Lung samples were manually homogenized in the tubes, using disposable
pestles (Kimble Chase, # 749625-0010). The resulting lysates were centrifuged to pellet debris.
17378269_1 (GHMatters) P40794NZ00
The supernatants containing the soluble protein extracts were transferred to fresh tubes and
stored at 4°C until further analysis.
Total protein content in the lung protein extracts was measured using a Bradford
assay. For the assay, 10μL of diluted extract samples were plated into 96 well plates in
duplicates and mixed with 200μL of 1X Dye Reagent (Biorad, #500-0006). Serial dilutions of
bovine serum albumin (BSA; Sigma, #A7979), starting at 700μg/mL in 1X T-Per reagent were
used as a standard to determine the protein concentration of the extracts. After a 5-minute
incubation at room temperature, absorbance at 595nm was measured on a Molecular Devices
SpectraMax M5 plate reader. Data analysis to determine total lung extract protein content based
on the BSA standard was performed using GraphPad Prism software.
Cytokine concentrations in the lung protein extracts were measured using
a Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, #
K15048D-2), according to the manufacturer’s instructions. Briefly, 50 μL/well of calibrators and
samples (diluted in Diluent 41) were added to plates pre-coated with capture antibodies and
incubated at room temperature while shaking at 700 rpm for 2 hours. The plates were then
washed 3 times with 1X PBS containing 0.05% (w/v) Tween-20, followed by the addition of 25μL
of Detection Antibody Solution diluted in Diluent 45. After another 2 hour incubation at room
temperature while shaking, the plate was washed 3 times, and 150μL of 2X Read Buffer was
added to each well. Electrochemiluminescence was immediately read on a MSD
Spector instrument. Data analysis was performed using GraphPad Prism software.
Each cytokine concentration in lung total protein extracts from all mice in each group
was normalized to the total protein content of the extracts measured by the Bradford assay, and
expressed for each group as average pg of cytokine per mg of total lung proteins (pg/mg lung
protein, ± SD) as shown in Table 19.
Table 19: Cytokine concentration in lung protein extracts
Mean [IL-4] in lung protein Mean [IL-5] in lung protein
Experimental group extracts (pg/mg lung extracts (pg/mg lung
protein) (±SD) protein) (±SD)
1. 1X PBS challenge, 12
0.03 (±0.01) 0.08 (±0.05)
weeks (n=5)
2. HDM challenge, 4 weeks
2.84 (±2.22) * 4.44 (±4.00) **
(n=6)
3. HDM challenge, 12
7.31 (±3.94) ** 6.23 (±3.81) *
weeks (n=3)
17378269_1 (GHMatters) P40794NZ00
4. HDM challenge, 12
weeks + Isotype control 2.28 (±1.94) 3.39 (±3.29)
antibody (n=2)
. HDM challenge, 12
0.38 (±0.21) 0.48 (±0.17)
weeks + H4H9675P (n=5)
Note: Statistical significance determined by Kruskal-Wallis One-way ANOVA with Dunn’s
multiple comparison post-hoc test is indicated (*= p<0.05, **= p<0.01, compared to groups 1:
IL33 HumIn mice, Saline challenge).
Lung cytokines analysis:
The level of the cytokines IL-4 and IL-5 released in the lungs of IL-33 HumIn mice
receiving HDM for 4 and 12 weeks was significantly higher than in IL-33 HumIn mice challenged
with 1X PBS. In contrast, there was a trend towards reduced IL-4 and IL-5 levels in the lungs of
IL-33 HumIn mice treated with anti-IL-33 antibody during the course of the chronic HDM
challenge as compared to IL-33 HumIn mice administered HDM without treatment or with
isotype control.
17378269_1 (GHMatters) P40794NZ00
Claims (7)
1. An isolated human monoclonal antibody or antigen-binding fragment thereof that binds human interleukin 33 (IL-33), wherein the antibody or antigen-binding fragment thereof inhibits or attenuates ILmediated signaling and comprises the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 274 and the CDRs of a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 282, said complementarity determining regions being identified by one or more of the Kabat method, the Chothia method, or the AbM method.
2. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof blocks the interaction of IL-33 and ST2.
3. The isolated antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment thereof blocks the interaction of IL-33 and ST2 with an IC value of less than about 10 nM, or blocks greater than about 50% of the interaction of IL-33 and ST2 as measured in an in vitro receptor/ligand binding assay at 25ºC.
4. The isolated antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof: (a) binds human IL-33 with a binding dissociation equilibrium constant (K ) of less than about 1 nM as measured in a surface plasmon resonance assay at 37ºC; (b) binds human IL-33 with a dissociative half-life (t½) of greater than about 8 minutes as measured in a surface plasmon resonance assay at 37ºC; (c) inhibits ILmediated degranulation of human basophils; (d) inhibits ILmediated degranulation of human basophils with an IC50 of less than about 600 pM as measured in an in vitro basophil activation test (BAT); (e) inhibits ILmediated IFN-gamma production from human PBMCs; (f) inhibits ILmediated IFN-gamma production from human PBMCs with an IC of less than about 25 nM as measured in an in vitro PBMC IFN-gamma production assay; (g) inhibits ILmediated IFN-gamma production from human PBMCs with an IC of less than about 3 nM as measured in an in vitro PBMC IFN-gamma production assay; (h) inhibits ILmediated IFN-gamma production from human PBMCs with an IC of less than about 0.5 nM as measured in an in vitro PBMC IFN-gamma production assay; (i) reduces the frequency of CD4+ T cells, eosinophils and ILC2 cells in the lungs when administered to an animal model of allergen-induced lung inflammation; or 17378269_1 (GHMatters) P40794NZ00 (j) reduces the level of IL-4 and IL-5 in the lungs when administered to an animal model of allergen-induced lung inflammation.
5. The isolated antibody or antigen-binding fragment of claim 4, wherein the antibody or antigen-binding fragment thereof, when administered to an animal model of allergen-induced lung inflammation, results in at least a 4 fold reduction in IL-4 levels and/or at least a 5 fold reduction in IL-5 levels when compared to allergen-challenged animals receiving an isotype control antibody.
6. The isolated antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2- LCDR3 domains, respectively, having the amino acid sequences of SEQ ID NOs: 276280- 284288.
7. The isolated antibody or antigen-binding fragment of claim 6, wherein the antibody or antigen-binding fragment comprises a HCVR/LCVR amino acid sequence pair having the amino acid sequences of SEQ ID NOs:
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361778687P | 2013-03-13 | 2013-03-13 | |
US61/778,687 | 2013-03-13 | ||
US201361819018P | 2013-05-03 | 2013-05-03 | |
US61/819,018 | 2013-05-03 | ||
PCT/US2014/023930 WO2014164959A2 (en) | 2013-03-13 | 2014-03-12 | Anti-il-33 antibodies and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ710831A NZ710831A (en) | 2021-04-30 |
NZ710831B2 true NZ710831B2 (en) | 2021-08-03 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11104729B2 (en) | Anti-IL-33 antibodies and uses thereof | |
US11542326B2 (en) | Anti-IL-25 antibodies and uses thereof | |
NZ710831B2 (en) | Anti-il-33 antibodies and uses thereof | |
CA3109513A1 (en) | Anti-fc epsilon-r1 alpha (fcer1a) antibodies, bispecific antigen-binding molecules that bind fcer1a and cd3, and uses thereof | |
BR112015020470B1 (en) | ISOLATED HUMAN MONOCLONAL ANTI-IL-33 ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, ITS USES, ISOLATED NUCLEIC ACID MOLECULE, EXPRESSION VECTOR, AND PHARMACEUTICAL COMPOSITION |