US20060020118A1 - Monoclonal antibodies to type I interferon receptor - Google Patents
Monoclonal antibodies to type I interferon receptor Download PDFInfo
- Publication number
- US20060020118A1 US20060020118A1 US10/813,646 US81364604A US2006020118A1 US 20060020118 A1 US20060020118 A1 US 20060020118A1 US 81364604 A US81364604 A US 81364604A US 2006020118 A1 US2006020118 A1 US 2006020118A1
- Authority
- US
- United States
- Prior art keywords
- ifnar1
- ifn
- antibody
- amino acids
- interferon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010086140 Interferon alpha-beta Receptor Proteins 0.000 title description 4
- 102000007438 Interferon alpha-beta Receptor Human genes 0.000 title description 4
- 230000000840 anti-viral effect Effects 0.000 claims abstract description 244
- 102000002227 Interferon Type I Human genes 0.000 claims abstract description 215
- 108010014726 Interferon Type I Proteins 0.000 claims abstract description 215
- 101000852870 Homo sapiens Interferon alpha/beta receptor 1 Proteins 0.000 claims description 480
- 102100036714 Interferon alpha/beta receptor 1 Human genes 0.000 claims description 480
- 150000001413 amino acids Chemical class 0.000 claims description 277
- 238000011065 in-situ storage Methods 0.000 claims description 139
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 88
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 85
- 101000959794 Homo sapiens Interferon alpha-2 Proteins 0.000 claims description 81
- 102100040018 Interferon alpha-2 Human genes 0.000 claims description 77
- 102100026720 Interferon beta Human genes 0.000 claims description 71
- 108090000467 Interferon-beta Proteins 0.000 claims description 71
- 101000959704 Homo sapiens Interferon alpha-5 Proteins 0.000 claims description 69
- 102100039948 Interferon alpha-5 Human genes 0.000 claims description 69
- 101000999391 Homo sapiens Interferon alpha-8 Proteins 0.000 claims description 64
- 102100036532 Interferon alpha-8 Human genes 0.000 claims description 64
- -1 IFN-αII1 Proteins 0.000 claims description 43
- 230000000120 cytopathologic effect Effects 0.000 abstract description 5
- 230000003472 neutralizing effect Effects 0.000 abstract description 5
- 235000001014 amino acid Nutrition 0.000 description 297
- 229940024606 amino acid Drugs 0.000 description 272
- 210000004027 cell Anatomy 0.000 description 187
- 230000027455 binding Effects 0.000 description 132
- 238000009739 binding Methods 0.000 description 123
- 238000000034 method Methods 0.000 description 114
- 241000282414 Homo sapiens Species 0.000 description 108
- 238000003556 assay Methods 0.000 description 94
- 101000959820 Homo sapiens Interferon alpha-1/13 Proteins 0.000 description 67
- 102100040019 Interferon alpha-1/13 Human genes 0.000 description 67
- 108090000623 proteins and genes Proteins 0.000 description 58
- 230000000694 effects Effects 0.000 description 50
- 230000003612 virological effect Effects 0.000 description 48
- 239000000427 antigen Substances 0.000 description 44
- 108091007433 antigens Proteins 0.000 description 43
- 102000036639 antigens Human genes 0.000 description 43
- 210000004408 hybridoma Anatomy 0.000 description 37
- 108060003951 Immunoglobulin Proteins 0.000 description 33
- 102000018358 immunoglobulin Human genes 0.000 description 33
- 108020004414 DNA Proteins 0.000 description 32
- 102000014150 Interferons Human genes 0.000 description 31
- 108010050904 Interferons Proteins 0.000 description 31
- 239000012634 fragment Substances 0.000 description 29
- 238000006467 substitution reaction Methods 0.000 description 28
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 27
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 26
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 26
- 230000002401 inhibitory effect Effects 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 25
- 239000013598 vector Substances 0.000 description 25
- 230000000903 blocking effect Effects 0.000 description 24
- 230000000875 corresponding effect Effects 0.000 description 24
- 235000018102 proteins Nutrition 0.000 description 24
- 241000894007 species Species 0.000 description 24
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 23
- 238000010828 elution Methods 0.000 description 22
- 102000004196 processed proteins & peptides Human genes 0.000 description 22
- 241001465754 Metazoa Species 0.000 description 21
- 102000005962 receptors Human genes 0.000 description 21
- 108020003175 receptors Proteins 0.000 description 21
- 230000005764 inhibitory process Effects 0.000 description 20
- 238000003752 polymerase chain reaction Methods 0.000 description 20
- 229940047124 interferons Drugs 0.000 description 19
- 239000000203 mixture Substances 0.000 description 19
- 229920001184 polypeptide Polymers 0.000 description 19
- 239000000523 sample Substances 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 17
- 229940088598 enzyme Drugs 0.000 description 17
- 230000014509 gene expression Effects 0.000 description 17
- 108010047761 Interferon-alpha Proteins 0.000 description 16
- 206010035226 Plasma cell myeloma Diseases 0.000 description 16
- 201000000050 myeloid neoplasm Diseases 0.000 description 16
- 150000007523 nucleic acids Chemical class 0.000 description 16
- 239000013615 primer Substances 0.000 description 16
- 102000006992 Interferon-alpha Human genes 0.000 description 15
- 239000002299 complementary DNA Substances 0.000 description 15
- 239000012528 membrane Substances 0.000 description 15
- 235000004279 alanine Nutrition 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 13
- 239000012228 culture supernatant Substances 0.000 description 13
- 239000003446 ligand Substances 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 13
- 238000002823 phage display Methods 0.000 description 13
- 241000588724 Escherichia coli Species 0.000 description 12
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 12
- 229940027941 immunoglobulin g Drugs 0.000 description 12
- 229940072221 immunoglobulins Drugs 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 12
- 229940079322 interferon Drugs 0.000 description 11
- 238000012216 screening Methods 0.000 description 11
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 238000002337 electrophoretic mobility shift assay Methods 0.000 description 10
- 230000004927 fusion Effects 0.000 description 10
- 230000026731 phosphorylation Effects 0.000 description 10
- 238000006366 phosphorylation reaction Methods 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 101000999354 Bos taurus Interferon alpha-H Proteins 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 101150117115 V gene Proteins 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 9
- 238000010276 construction Methods 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 9
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 230000001225 therapeutic effect Effects 0.000 description 9
- 108091026890 Coding region Proteins 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 230000009137 competitive binding Effects 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 208000035475 disorder Diseases 0.000 description 8
- 238000001114 immunoprecipitation Methods 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 101000852865 Homo sapiens Interferon alpha/beta receptor 2 Proteins 0.000 description 7
- 238000002832 anti-viral assay Methods 0.000 description 7
- 210000003719 b-lymphocyte Anatomy 0.000 description 7
- 238000013357 binding ELISA Methods 0.000 description 7
- 108020001507 fusion proteins Proteins 0.000 description 7
- 102000037865 fusion proteins Human genes 0.000 description 7
- 238000003119 immunoblot Methods 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 238000010187 selection method Methods 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 241000283707 Capra Species 0.000 description 6
- 102100036718 Interferon alpha/beta receptor 2 Human genes 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
- 239000003463 adsorbent Substances 0.000 description 6
- 238000001042 affinity chromatography Methods 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 210000004698 lymphocyte Anatomy 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- 230000036961 partial effect Effects 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 239000000700 radioactive tracer Substances 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 241000486679 Antitype Species 0.000 description 5
- 208000023275 Autoimmune disease Diseases 0.000 description 5
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 5
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 5
- 241001529936 Murinae Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 230000002860 competitive effect Effects 0.000 description 5
- 102000003675 cytokine receptors Human genes 0.000 description 5
- 108010057085 cytokine receptors Proteins 0.000 description 5
- 238000010494 dissociation reaction Methods 0.000 description 5
- 230000005593 dissociations Effects 0.000 description 5
- 210000004602 germ cell Anatomy 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 230000016784 immunoglobulin production Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 230000011664 signaling Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 108010041884 CD4 Immunoadhesins Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 4
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 4
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 241000283984 Rodentia Species 0.000 description 4
- 108010000499 Thromboplastin Proteins 0.000 description 4
- 102100030859 Tissue factor Human genes 0.000 description 4
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 4
- 230000009824 affinity maturation Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000004132 cross linking Methods 0.000 description 4
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 230000008676 import Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000003389 potentiating effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 241000196324 Embryophyta Species 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 101001057508 Homo sapiens Ubiquitin-like protein ISG15 Proteins 0.000 description 3
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 3
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 3
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 3
- 206010062016 Immunosuppression Diseases 0.000 description 3
- 108010054267 Interferon Receptors Proteins 0.000 description 3
- 102000001617 Interferon Receptors Human genes 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
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 102100027266 Ubiquitin-like protein ISG15 Human genes 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000001506 immunosuppresive effect Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000004091 panning Methods 0.000 description 3
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 3
- 210000001322 periplasm Anatomy 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000159 protein binding assay Methods 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000013391 scatchard analysis Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000013207 serial dilution Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000013268 sustained release Methods 0.000 description 3
- 239000012730 sustained-release form Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 229940104230 thymidine Drugs 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- GANZODCWZFAEGN-UHFFFAOYSA-N 5-mercapto-2-nitro-benzoic acid Chemical compound OC(=O)C1=CC(S)=CC=C1[N+]([O-])=O GANZODCWZFAEGN-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- 108010039627 Aprotinin Proteins 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101001034829 Homo sapiens Interferon alpha-10 Proteins 0.000 description 2
- 101001034828 Homo sapiens Interferon alpha-14 Proteins 0.000 description 2
- 101001054334 Homo sapiens Interferon beta Proteins 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 2
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 102100039734 Interferon alpha-10 Human genes 0.000 description 2
- 102100039733 Interferon alpha-14 Human genes 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-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
- 108010023244 Lactoperoxidase Proteins 0.000 description 2
- 102000045576 Lactoperoxidases Human genes 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- 108091008109 Pseudogenes Proteins 0.000 description 2
- 102000057361 Pseudogenes Human genes 0.000 description 2
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 241000711975 Vesicular stomatitis virus Species 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 229960004405 aprotinin Drugs 0.000 description 2
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 2
- 102000012740 beta Adrenergic Receptors Human genes 0.000 description 2
- 108010079452 beta Adrenergic Receptors Proteins 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000012412 chemical coupling Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000012875 competitive assay Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 239000002254 cytotoxic agent Substances 0.000 description 2
- 231100000599 cytotoxic agent Toxicity 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 150000004662 dithiols Chemical class 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000003500 gene array Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000001524 infective effect Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229960001388 interferon-beta Drugs 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 229940057428 lactoperoxidase Drugs 0.000 description 2
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 2
- 108010052968 leupeptin Proteins 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000003607 modifier Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- 101150009573 phoA gene Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin 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 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 2
- 238000000844 transformation Methods 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- XMQUEQJCYRFIQS-YFKPBYRVSA-N (2s)-2-amino-5-ethoxy-5-oxopentanoic acid Chemical compound CCOC(=O)CC[C@H](N)C(O)=O XMQUEQJCYRFIQS-YFKPBYRVSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- KGLPWQKSKUVKMJ-UHFFFAOYSA-N 2,3-dihydrophthalazine-1,4-dione Chemical class C1=CC=C2C(=O)NNC(=O)C2=C1 KGLPWQKSKUVKMJ-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- OELQSSWXRGADDE-UHFFFAOYSA-N 2-methylprop-2-eneperoxoic acid Chemical compound CC(=C)C(=O)OO OELQSSWXRGADDE-UHFFFAOYSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- HJBUBXIDMQBSQW-UHFFFAOYSA-N 4-(4-diazoniophenyl)benzenediazonium Chemical compound C1=CC([N+]#N)=CC=C1C1=CC=C([N+]#N)C=C1 HJBUBXIDMQBSQW-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 102000015404 Amino Acid Receptors Human genes 0.000 description 1
- 108010025177 Amino Acid Receptors Proteins 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- XUKUURHRXDUEBC-KAYWLYCHSA-N Atorvastatin Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-KAYWLYCHSA-N 0.000 description 1
- 101710192393 Attachment protein G3P Proteins 0.000 description 1
- 102100022717 Atypical chemokine receptor 1 Human genes 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101001034836 Canis lupus familiaris Interferon alpha-1/2 Proteins 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 101710194146 Ecotin Proteins 0.000 description 1
- 102100021587 Embryonic testis differentiation protein homolog A Human genes 0.000 description 1
- 241000710188 Encephalomyocarditis virus Species 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 101100390711 Escherichia coli (strain K12) fhuA gene Proteins 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108010015133 Galactose oxidase Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 101100082540 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) pcp gene Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000678879 Homo sapiens Atypical chemokine receptor 1 Proteins 0.000 description 1
- 101000898120 Homo sapiens Embryonic testis differentiation protein homolog A Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 101150002553 Ifnar1 gene Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000004551 Interleukin-10 Receptors Human genes 0.000 description 1
- 108010017550 Interleukin-10 Receptors Proteins 0.000 description 1
- 102100020788 Interleukin-10 receptor subunit beta Human genes 0.000 description 1
- 101710199214 Interleukin-10 receptor subunit beta Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 102000001702 Intracellular Signaling Peptides and Proteins Human genes 0.000 description 1
- 108010068964 Intracellular Signaling Peptides and Proteins Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 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
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 241000714177 Murine leukemia virus Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 101100407828 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) ptr-3 gene Proteins 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 102220490908 Olfactomedin-like protein 2A_E69A_mutation Human genes 0.000 description 1
- 102220490906 Olfactomedin-like protein 2A_R74A_mutation Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000609499 Palicourea Species 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010002519 Prolactin Receptors Proteins 0.000 description 1
- 102100029000 Prolactin receptor Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 108010084592 Saporins Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102220503209 Thioredoxin_E70A_mutation Human genes 0.000 description 1
- 102220503212 Thioredoxin_K72A_mutation Human genes 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 244000000188 Vaccinium ovalifolium Species 0.000 description 1
- 241001672648 Vieira Species 0.000 description 1
- 229940122803 Vinca alkaloid Drugs 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 229920006243 acrylic copolymer Polymers 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 150000001295 alanines Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000842 anti-protozoal effect Effects 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000003904 antiprotozoal agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 150000004982 aromatic amines Chemical class 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- DQXBYHZEEUGOBF-UHFFFAOYSA-N but-3-enoic acid;ethene Chemical compound C=C.OC(=O)CC=C DQXBYHZEEUGOBF-UHFFFAOYSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000012219 cassette mutagenesis Methods 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 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
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108010067006 heat stable toxin (E coli) Proteins 0.000 description 1
- 108010037896 heparin-binding hemagglutinin Proteins 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 210000000548 hind-foot Anatomy 0.000 description 1
- 102000052179 human IFNAR2 Human genes 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000002169 hydrotherapy Methods 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000006362 insulin response pathway Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 108010045648 interferon omega 1 Proteins 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 101150074251 lpp gene Proteins 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229960003151 mercaptamine Drugs 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229940071648 metered dose inhaler Drugs 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- DFTAZNAEBRBBKP-UHFFFAOYSA-N methyl 4-sulfanylbutanimidate Chemical compound COC(=N)CCCS DFTAZNAEBRBBKP-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 108010029942 microperoxidase Proteins 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011330 nucleic acid test Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 101150093139 ompT gene Proteins 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229940043515 other immunoglobulins in atc Drugs 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 108700010839 phage proteins Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 150000003139 primary aliphatic amines Chemical class 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000003156 radioimmunoprecipitation Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 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
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000003567 signal transduction assay Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- PTLRDCMBXHILCL-UHFFFAOYSA-M sodium arsenite Chemical compound [Na+].[O-][As]=O PTLRDCMBXHILCL-UHFFFAOYSA-M 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 150000005691 triesters Chemical class 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 1
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
-
- 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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- 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
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/555—Interferons [IFN]
- G01N2333/56—IFN-alpha
Definitions
- This invention relates to the field of anti-type I interferon receptor antibodies, and more particularly to anti-type 1 interferon receptor antibodies that neutralize the anti-viral cytopathic effects of various type I interferons.
- the type 1 interferons are cytokines that have pleiotropic effects on a wide variety of cell types. IFNs are best known for their anti-viral activity, but they also have anti-bacterial, anti-protozoal, immunomodulatory, and cell-growth regulatory functions.
- the type 1 interferons include interferon- ⁇ (IFN- ⁇ ) and interferon- ⁇ (IFN- ⁇ ).
- Human IFN- ⁇ (hIFN- ⁇ ) is a heterogeneous family with at least 23 polypeptides while there is only one IFN- ⁇ polypeptide ( J. Interferon Res., 13: 443-444 (1993)).
- the hIFN- ⁇ subtypes show more than 70% amino acid sequence homology, and there is approximately 25% amino acid identity with hIFN- ⁇ .
- the hIFNs- ⁇ and hIFN- ⁇ share a common receptor.
- hIFNAR1 The cDNA for the first hIFN- ⁇ receptor (hIFNAR1) encodes a 63 kD receptor protein (reported in Uze et al., Cell, 60: 225-234 (1990)). This receptor undergoes extensive glycosylation, which causes it to migrate in gel electrophoresis as a much larger 135 kD protein.
- the second interferon receptor, hIFNAR2 (hIFN- ⁇ R long), is a 115 kD protein which mediates a functional signaling complex when associated with hIFNAR1 (reported in Domanski et al., J. Biol.
- hIFN- ⁇ receptor an IFN- ⁇ / ⁇ receptor (hIFN- ⁇ R short)
- hIFN- ⁇ R short an IFN- ⁇ / ⁇ receptor
- This IFN- ⁇ / ⁇ receptor appears to be an alternatively spliced variant of hIFNAR2.
- the unprocessed hIFNAR1 expression product is composed of 557 amino acids including an extracellular domain (ECD) of 409 residues, a transmembrane domain of 21 residues, and an intracellular domain of 100 residues as shown in FIG. 5 on page 229 of Uze et al., supra.
- ECD extracellular domain
- the ECD of IFNAR1 is composed of two domains, domain 1 and domain 2, which are separated by a three-proline motif. There is 19% sequence identity and 50% sequence homology between domains 1 and 2 (Uze et al., supra).
- Each domain (D200) is composed of approximately 200 residues and can be further subdivided into two homologous subdomains (SD100) of approximately 100 amino acids.
- Cytokine receptors have been categorized into two classes based on the distribution of cysteine residues.
- the class 1 cytokine receptor family includes receptors for human growth hormone (hGHR), erythropoietin, IL-3 and IL4, while the class 2 cytokine receptor family includes the IFN ⁇ receptor, tissue factor, CRF2-4 and IL-10 receptors. Sequence analysis of the hIFN ⁇ receptors shows that they are related to the class 2 cytokine receptor family.
- IFNAR1 has been shown to be essential for the response to all type 1 IFNs (Muller et al., Science, 264: 1918-1921 (1994); Cleary et al., J. Biol. Chem., 269: 18747-18749 (1994)) and for the mediation of species-specific IFN signal transduction (Constantinescu et al., Proc. Natl. Acad. Sci USA, 91: 9602-9606 (1994)).
- the invention provides an anti-IFNAR1 monoclonal antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon.
- the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN- ⁇ A.
- the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN- ⁇ B.
- the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN- ⁇ II 1.
- the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN- ⁇ .
- the invention provides an anti-IFNAR1 monoclonal antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ II 1, and IFN- ⁇ .
- the invention also encompasses an anti-IFNAR1 monoclonal antibody that binds to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1, and binds to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR 1.
- FIG. 1 is a graph depicting mAb 2E1 binding to U266 human myeloma cell line as determined by FACS analysis.
- U266 cells were incubated with hybridoma culture supernatant and then contacted with FITC-goat anti-mouse IgG.
- FIGS. 2 A-E are graphs depicting epitope mapping for mAbs 2E1, 2E8, 2H6, 4A7 and 5A8, respectively, as determined by competitive binding ELISA.
- IFNAR1 (ECD)-IgG captured by goat anti-human IgG was incubated with predetermined concentrations of biotinylated (Bio)-mAb in the presence of 500-1,000 fold excess of unlabeled mAbs.
- Bio-mAb bound was detected by the addition of horse radish peroxidase (HRP)-streptavidin.
- HRP horse radish peroxidase
- FIG. 3 is a collection of autoradiographs depicting the effect of mAbs 2E1, 2E8, 2H6, 4A7 and 5A8 on ISGF3 formation in Hela cells induced by IFN- ⁇ 8 (IFN- ⁇ D) in an electrophoretic mobility shift assay (EMSA).
- FIG. 4 is a graph depicting a hydropathy profile and the location of certain alanine-substituted mutants of hIFNAR1.
- FIG. 5 is a graph depicting mAb binding to IFNAR1 ECD-IgG (closed columns), IFNAR1 domain 1-IgG (shaded columns), IFNAR1 domain 2-IgG (diagonally hatched columns), and to a control with no antigen (open columns) as determined by ELISA.
- Microtiter wells coated with goat anti-human IgG were incubated with culture supernatants containing 2 mg/ml of each immunoadhesin followed by the addition of 10 mg/ml of mAbs. The mAb bound to the immunoadhesin was detected by HRP-goat anti-mouse IgG.
- FIG. 6 is a model of hIFNAR1 displaying its protein sequence on the structural backbone of tissue factor.
- Subdomain SD100A of domain 1 and subdomain SD100A′ of domain 2 are shown in dark gray.
- Subdomain SD100B of domain 1 and SD100B′ of domain 2 are shown in light gray.
- Regions involved in the binding of anti-IFNAR1 mAbs are shown in orange.
- Amino acid residues involved in the binding of anti-IFNAR1 mAbs are shown in red.
- IFNAR1 ECD sequence in order to present the mature IFNAR1 ECD sequence as amino acids 1-404 of the IFNAR1 ECD-IgG fusion protein sequence (SEQ ID NO. 22). Unless otherwise indicated, the amino acid numbering scheme for IFNAR1 ECD shown in FIG. 7 is used throughout the application.
- type I interferon and “human type I interferon” are defined as all species of native human interferon which fall within the human interferon- ⁇ , interferon- ⁇ and interferon- ⁇ classes and which bind to a common cellular receptor.
- Natural human interferon- ⁇ comprises 23 or more closely related proteins encoded by distinct genes with a high degree of structural homology (Weissmann and Weber, Prog. Nucl. Acid. Res. Mol. Biol., 33: 251 (1986); J. Interferon Res., 13: 443-44 (1993)).
- the human IFN- ⁇ locus comprises two subfamilies.
- the second subfamily, ⁇ II or ⁇ contains at least 5 pseudogenes and 1 functional gene (denoted herein as “IFN- ⁇ II 1” or “IFN- ⁇ ”) which exhibits 70% homology with the IFN- ⁇ genes (Weissmann and Weber (1986)).
- IFN- ⁇ II 1 or “IFN- ⁇ ”
- the human IFN- ⁇ is encoded by a single copy gene.
- first human interferon- ⁇ (hIFN- ⁇ ) receptor As used herein, the terms “first human interferon- ⁇ (hIFN- ⁇ ) receptor”, “hIFNAR1”, “IFNAR1”, and “Uze chain” are defined as the 557 amino acid receptor protein cloned by Uze et al., Cell, 60: 225-234 (1990), including an extracellular domain of 409 residues, a transmembrane domain of 21 residues, and an intracellular domain of 100 residues, as shown in FIG. 5 on page 229 of Uze et al. Also encompassed by the foregoing terms are fragments of IFNAR1 that contain the extracellular domain (ECD) (or fragments of the ECD) of IFNAR1.
- ECD extracellular domain
- anti-IFNAR1 antibody is defined as an antibody that is capable of binding to IFNAR1.
- PCR Polymerase chain reaction
- sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified.
- the 5′ terminal nucleotides of the two primers can coincide with the ends of the amplified material.
- Antibodies are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
- “Native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide-linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
- VH variable domain
- Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
- Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)).
- variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
- CDRs complementarity-determining regions
- FR framework
- the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
- the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of immunological Interest , Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
- the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
- Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′) 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
- “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
- the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
- immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
- the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
- antibody specifically covers monoclonal antibodies, including antibody fragment clones.
- Antibody fragments comprise a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody.
- antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; diabodies; single-chain antibody molecules, including single-chain Fv (scFv) molecules; and multispecific antibodies formed from antibody fragments.
- the term “monoclonal antibody” as used herein refers to an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
- the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
- the “monoclonal antibodies” also include clones of antigen-recognition and binding-site containing antibody fragments (Fv clones) isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
- the monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
- chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with
- “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
- humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
- humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
- the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
- the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
- Fc immunoglobulin constant region
- the humanized antibody includes a PrimatizedTMantibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
- Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
- the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
- diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
- VH heavy-chain variable domain
- VL light-chain variable domain
- VH-VL polypeptide chain
- an “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
- the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
- Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
- Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
- “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
- the invention provides anti-IFNAR1 antibodies that are useful for treatment of immune-mediated disorders in which a partial or total blockade of type I interferon activity is desired.
- the anti-IFNAR1 antibodies of the invention are used to treat autoimmune disorders, such as type I and type II diabetes, systemic lupus erythematosis (SLE), and rheumatoid arthritis.
- the anti-IFNAR1 antibodies provided herein are used to treat graft rejection or graft versus host disease.
- the unique properties of the anti-IFNAR1 antibodies of the invention make them particularly useful for effecting target levels of immunosuppression in a patient.
- the anti-IFNAR1 antibodies provided herein which cause broad spectrum ablation of type I interferon activity can be used to effect the largest possible compromise of an undesired immune response.
- the anti-IFNAR1 antibodies provided herein which block the activity of one or more (but not all) species of type I interferon can be used to effect partial compromise of the patient's immune system in order to reduce the risk of undesirable immune responses while leaving some components of the patient's type I interferon-mediated immunity intact in order to avoid infection.
- the anti-IFNAR1 antibodies of the invention find utility as reagents for detection and isolation of IFNAR1, such as detection of IFNAR1 expression in various cell types and tissues, including the determination of IFNAR1 receptor density and distribution in cell populations, and cell sorting based on IFNAR1 expression.
- the present anti-IFNAR1 antibodies are useful for the development of IFNAR1 antagonists with type I interferon inhibition activity patterns similar to those of the subject antibodies.
- the anti-IFNAR1 antibodies of the invention can be used in competition binding assays with IFNAR1 to screen for small molecule antagonists of IFNAR1 that will exhibit similar pharmacological effects in blocking the activities of type I interferons to IFNAR1.
- the anti-IFNAR1 antibodies of the invention can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities.
- synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein.
- Fv antibody variable region
- Such phage libraries are panned by affinity chromatography against the desired ligand.
- Clones expressing Fv fragments capable of binding to the desired ligand are adsorbed to the ligand and thus separated from the non-binding clones in the library.
- the binding clones are then eluted from the ligand, and can be further enriched by additional cycles of ligand adsorption/elution.
- any of the anti-IFNAR1 antibodies of the invention can be obtained by designing a suitable ligand screening procedure to select for the phage clone of interest followed by construction of a full length anti-IFNAR1 antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest , Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
- the antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops or complementarity-determining regions (CDRs).
- V variable
- VH variable
- CDRs complementarity-determining regions
- Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433455 (1994).
- scFv encoding phage clones and Fab encoding phage clones are collectively referred to as “Fv phage clones” or “Fv clones”.
- the naive repertoire of an animal provides it with antibodies that can bind with moderate affinity (K a of about 10 6 to 10 7 M ⁇ 1 ) to essentially any non-self molecule.
- the sequence diversity of antibody binding sites is not encoded directly in the germline but is assembled in a combinatorial manner from V gene segments.
- the first two hypervariable loops (H1 and H2) are drawn from less than 50 VH gene segments, which are combined with D segments and JH segments to create the third hypervariable loop (H3).
- the first two hypervariable loops (L1 and L2) and much of the third (L3) are drawn from less than approximately 30 V ⁇ and less than approximately 30 V ⁇ segments to complete the third hypervariable loop (L3).
- Each combinatorial rearrangement of V-gene segments in stem cells gives rise to a B cell that expresses a single VH-VL combination. Immunizations triggers any B cell making a VH-VL combination that binds the immunogen to proliferate (clonal expansion) and to secrete the corresponding antibody. These naive antibodies are then matured to high affinity (Ka ⁇ 10 9 M ⁇ 1 ) by a process of mutagenesis and selection known as affinity maturation. It is after this point that cells are normally removed to prepare hybridomas and generate high-affinity monoclonal antibodies.
- repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
- Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
- the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
- naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
- Phage display mimics the B cell.
- Filamentous phage is used to display antibody fragments by fusion to the minor coat protein pIII.
- the antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain is fused to pIII and the other is secreted into the bacterial host cell periplasm where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins, e.g.
- the pIII fusion and other proteins of the phage can be encoded entirely within the same phage replicon, or on different replicons.
- the pIII fusion is encoded on a phagemid, a plasmid containing a phage origin of replication.
- Phagemids can be packaged into phage particles by “rescue” with a helper phage such as M13K07 that provides all the phage proteins, including pIII, but due to a defective origin is itself poorly packaged in competitions with the phagemids as described in Vieira and Messing, Meth. Enzymol., 153: 3-11 (1987).
- the phage display system is designed such that the recombinant phage can be grown in host cells under conditions permitting no more than a minor amount of phage particles to display more than one copy of the Fv-coat protein fusion on the surface of the particle as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690 (PCT/US91/09133 published Jun. 11, 1992).
- nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased in favor of anti-IFNAR1 clones is desired, the subject is immunized with IFNAR1 to generate an antibody response, and spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBLs) are recovered for library construction.
- a human antibody gene fragment library biased in favor of anti-human IFNAR1 clones is obtained by generating an anti-human IFNAR1 antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that IFNAR1 immunization gives rise to B cells producing human antibodies against IFNAR1. The generation of human antibody-producing transgenic mice is described in Section B(III)(b) below.
- Additional enrichment for anti-IFNAR1 reactive cell populations can be obtained by using a suitable screening procedure to isolate B cells expressing IFNAR1-specific membrane bound antibody, e.g., by cell separation with IFNAR1 affinity chromatography or adsorption of cells to fluorochrome-labelled IFNAR1 followed by flow-activated cell sorting (FACS).
- FACS flow-activated cell sorting
- spleen cells and/or B cells or other PBLs from an unimmunized donor provides a better representation of the possible antibody repertoire, and also permits the construction of an antibody library using any animal (human or non-human) species in which IFNAR1 is not antigenic.
- stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments.
- the immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, luprine, canine, feline, porcine, bovine, equine, and avian species, etc.
- Nucleic acid encoding antibody variable gene segments are recovered from the cells of interest and amplified.
- the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5′ and 3′ ends of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci . ( USA ), 86: 3833-3837 (1989), thereby making diverse V gene repertoires for expression.
- the V genes can be amplified from cDNA and genomic DNA, with back primers at the 5′ end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341: 544-546 (1989).
- back primers can also be based in the leader exon as described in Jones et al., Biotechnol, 9: 88-89 (1991), and forward primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci . ( USA ), 86: 5728-5732 (1989).
- degeneracy can be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989).
- the library diversity is maximized by using PCR primers targeted to each V-gene family in order to amplify all available VH and VL arrangements present in the immune cell nucleic acid sample, e.g. as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993).
- rare restriction sites can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991).
- Repertoires of synthetically rearranged V genes can be derived in vitro from V gene segments.
- Most of the human VH-gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the H1 and H2 loop) can be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
- VH repertoires can also be made with all the sequence diversity focussed in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).
- Human V ⁇ and V ⁇ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic light chain repertoires.
- Synthetic V gene repertoires based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural diversity.
- germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
- Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors, and the vectors recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo by combinatorial infection, e.g., the loxp system described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency.
- Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector.
- the two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 10 12 clones).
- Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions.
- the repertoires may be cloned sequentially into the same vector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g. as described in Clackson et al., Nature, 352: 624-628 (1991).
- PCR assembly can also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires.
- in cell PCR assembly is used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).
- the antibodies produced by naive libraries can be of moderate affinity (K a of about 10 6 to 10 7 M ⁇ 1 ), but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al. (1994), supra.
- mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 2-26: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992).
- affinity maturation can be performed by randomly mutating one or more CDRs, e.g. using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones.
- Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol., 10: 779-783 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the 109 M range.
- Nucleic acid sequence encoding the IFNAR1s used herein can be designed using the amino acid sequence of the desired region of IFNAR1, e.g. the extracellular domain spanning amino acids 28 to 434 of FIG. 2 of WO 93/20187 (PCT/EP93/00770 published Oct. 14, 1993). Alternatively, the cDNA sequence of FIG. 2 of WO 93/20187 can be used. In addition, nucleic acid encoding an immunoglobulin G (IgG)-IFNAR1 extracellular domain fusion protein can be obtained from the amino acid or cDNA sequence shown in FIG. 8 below.
- IgG immunoglobulin G
- nucleic acid sequence encoding the human type I interferons used herein can be designed using published amino acid and nucleic acid sequences, e.g. see the J. Interferon Res., 13: 443444 (1993) compilation of references containing genomic and cDNA sequences for various type I interferons, and the references cited therein.
- IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ C, IFN- ⁇ D, IFN- ⁇ E, IFN- ⁇ F, IFN- ⁇ G, and IFN- ⁇ H amino acid sequences or cDNA sequences see FIGS. 3 and 4 on pages 23-24 of Goeddel et al., Nature, 290: 20-26 (1981).
- DNAs encoding the IFNAR1 s or type I interferons of interest can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), such as the triester, phosphite, phosphoramidite and H-phosphonate methods. In one embodiment, codons preferred by the expression host cell are used in the design of the IFNAR1 or type I interferon-encoding DNA. Alternatively, DNA encoding the IFNAR1 or type I interferon can be isolated from a genomic or cDNA library.
- DNA sequence encoding wild type IFNAR1 can be altered to encode the desired IFNAR1 mutant by using recombinant DNA techniques, such as site specific mutagenesis (Kunkel et al., Methods Enzymol. 204:125-139 (1991); Carter, P., et al., Nucl. Acids. Res. 13:4331 (1986); Zoller, M. J. et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (Wells, J. A., et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells, J. A., et al., Philos. Trans, R. Soc. London SerA 317: 415 (1986)), and the like.
- the DNA molecule is operably linked to an expression control sequence in an expression vector, such as a plasmid, wherein the control sequence is recognized by a host cell transformed with the vector.
- an expression vector such as a plasmid
- plasmid vectors contain replication and control sequences that are derived from species compatible with the host cell.
- the vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells.
- suitable vectors include pBR322 (ATCC No. 37,017), phGH107 (ATCC No. 40,011), pBO475, pS0132, pRIT5, any vector in the pRIT20 or pRIT30 series (Nilsson and Abrahmsen, Meth. Enzymol., 185: 144-161 (1990)), pRIT2T, pKK233-2, pDR540 and pPL-lambda.
- Prokaryotic host cells containing the expression vectors suitable for use herein include E. coli K12 strain 294 (ATCC NO. 31446), E coli strain JM101 (Messing et al., Nucl.
- E. coli strain B E. coli strain ⁇ 1776 (ATCC No. 31537), E. coli c600 (Appleyard, Genetics, 39: 440 (1954)), E. coli W3110 (F-, gamma-, prototrophic, ATCC No. 27325), E. coli strain 27C7 (W3110, tonA, phoA E15, (argF-lac) 169, ptr3, degP41, ompT, kan′) U.S. Pat. No. 5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium, Serratia marcesans , and Pseudomonas species.
- eukaryotic organisms such as yeasts, or cells derived from multicellular organisms can be used as host cells.
- yeast host cells such as common baker's yeast or Saccharomyces cerevisiae
- suitable vectors include episomally replicating vectors based on the 2-micron plasmid, integration vectors, and yeast artificial chromosome (YAC) vectors.
- yeast artificial chromosome YAC
- suitable vectors include baculoviral vectors.
- plant host cells particularly dicotyledonous plant hosts, such as tobacco, suitable expression vectors include vectors derived from the Ti plasmid of Agrobacterium tumefaciens.
- mammalian host cells include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
- COS-7 monkey kidney CVI line transformed by SV40
- human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36: 59 (1977)
- baby hamster kidney cells BHK, ATCC CCL 10
- Chinese hamster ovary cells/-DHFR CHO, Urlaub and Chasin, Proc. Natl. Aca
- monkey kidney cells CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (WI 38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.
- useful vectors include vectors derived from SV40, vectors derived from cytomegalovirus such as the pRK vectors, including pRK5 and pRK7 (Suva et al., Science, 237: 893-896 (1987), EP 307,247 (Mar. 15, 1989), EP 278,776 (Aug. 17, 1988)) vectors derived from vaccinia viruses or other pox viruses, and retroviral vectors such as vectors derived from Moloney's murine leukemia virus (MoMLV).
- pRK vectors including pRK5 and pRK7 (Suva et al., Science, 237: 893-896 (1987), EP 307,247 (Mar. 15, 1989), EP 278,776 (Aug. 17, 1988) vectors derived from vaccinia viruses or other pox viruses
- retroviral vectors such as vectors derived from Moloney's murine leukemia virus (MoMLV).
- the DNA encoding the IFNAR1 or type I interferon of interest is operably linked to a secretory leader sequence resulting in secretion of the expression product by the host cell into the culture medium.
- secretory leader sequences include stil, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor.
- secretory leader sequences include stil, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor.
- secretory leader sequences include stil, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor.
- 36 amino acid leader sequence of protein A Abrahmsen et al., EMBO J., 4: 3901 (1985)
- Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
- Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO 4 precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
- Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells.
- Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published 29 Jun. 1989.
- Prokaryotic host cells used to produce the IFNAR1 or type I interferon of interest can be cultured as described generally in Sambrook et al., supra.
- the mammalian host cells used to produce the IFNAR1 or type I interferon of interest can be cultured in a variety of media.
- Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
- any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as GentamycinTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
- the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- the host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.
- the recombinantly expressed IFNAR1 or type I interferon protein can be recovered from the culture cells by disrupting the host cell membrane/cell wall (e.g. by osmotic shock or solubilizing the host cell membrane in detergent).
- the recombinant protein can be recovered from the culture medium.
- the culture medium or lysate is centrifuged to remove any particulate cell debris.
- the membrane and soluble protein fractions are then separated.
- the IFNAR1 or type I interferon is purified from the soluble protein fraction.
- the membrane bound peptide can be recovered from the membrane fraction by solubilization with detergents.
- the crude peptide extract can then be further purified by suitable procedures such as fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; hydrophobic affinity resins and ligand affinity using IFNAR1 (for type I interferon purification) or type I interferons or anti-IFNAR1 antibodies (for IFNAR1 purification) immobilized on a matrix.
- human IFN- ⁇ is available from Sigma (St. Louis, Mo.).
- the purified IFNAR1 can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon; neutral and ionic carriers, and the like, for use in the affinity chromatographic separation of phage display clones.
- a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon; neutral and ionic carriers, and the like.
- Attachment of the IFNAR1 protein to the matrix can be accomplished by the methods described in Methods in Enzymology, vol. 44 (1976).
- a commonly employed technique for attaching protein ligands to polysaccharide matrices, e.g. agarose, dextran or cellulose involves activation of the carrier with cyanogen halides and subsequent
- IFNAR1 can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other art-known method for panning phage display libraries.
- the phage library samples are contacted with immobilized IFNAR1 under conditions suitable for binding of at least a portion of the phage particles with the adsorbent. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological conditions.
- the phages bound to the solid phase are washed and then eluted by acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in Marks et al., J. Mol.
- Phages can be enriched 20-1,000-fold in a single round of selection. Moreover, the enriched phages can be grown in bacterial culture and subjected to further rounds of selection.
- the efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen.
- Antibodies with fast dissociation kinetics (and weak binding affinities) can be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions, but also favors rebinding of phage that has dissociated.
- phage antibodies of different affinities can be selected between phage antibodies of different affinities, even with affinities that differ slightly, for IFNAR1.
- random mutation of a selected antibody e.g. as performed in some of the affinity maturation techniques described above
- IFNAR1 With limiting IFNAR1, rare high affinity phage could be competed out.
- phages can be incubated with excess biotinylated IFNAR1, but with the biotinylated IFNAR1 at a concentration of lower molarity than the target molar affinity constant for IFNAR1.
- the high affinity-binding phages can then be captured by streptavidin-coated paramagnetic beads.
- Such “equilibrium capture” allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity.
- Conditions used in washing phages bound to a solid phase can also be manipulated to discriminate on the basis of dissociation kinetics.
- the invention provides anti-IFNAR1 antibodies which bind to specific determinant(s) on IFNAR1 and/or which do not bind other specific determinant(s) on IFNAR1.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be conveniently selected by adsorbing library clones to immobilized IFNAR1 mutants containing Ala substitutions at the specific determinants of interest. If clones which do not bind the selected IFNAR1 determinant(s) are desired, then the clones which adsorb to the IFNAR1 mutant are recovered, e.g. by eluting the adsorbed clones with wild type IFNAR1.
- the separation occurs because of the difference in the affinities of the desired and undesired clones for the IFNAR1 mutant. Since the IFNAR1 determinant(s) bound by the desired clones do not include the amino acid(s) at the Ala-substituted position(s) in the IFNAR1 mutant, the desired clones will bind to the immobilized, mutant IFNAR1 whereas the undesired clones will not. Accordingly, the adsorption of library clones to immobilized, mutant IFNAR1 will yield a population of clones bound to solid phase that is enriched for the property of not being able to bind to the selected IFNAR1 determinant(s). The desired clones will exhibit similar or approximately the same binding activities with the corresponding Ala-substituted IFNAR1 mutant and wild type IFNAR1.
- library clones which bind to the selected IFNAR1 determinant(s) are recovered (i.e. collected from the column flow-through fractions), the recovered clones are adsorbed to immobilized, wild type IFNAR1, and then the adsorbed clones are recovered, e.g. by elution with excess wild type IFNAR1.
- the first adsorption step removes clones that bind to IFNAR1 but do not bind to the selected determinant(s), and the second adsorption step removes clones that do not bind to IFNAR1 at all, leaving a population of clones enriched for binding to the selected IFNAR1 determinant(s).
- the desired clone will exhibit binding activity with wild type IFNAR1 that is greater than the clone's binding activity with the corresponding Ala-substituted IFNAR1 mutant (i.e. a binding level with wild type IFNAR1 that is above the background binding level with mutant IFNAR1).
- the desired clone will exhibit binding activity with the corresponding Ala-substituted IFNAR1 mutant that is less than about 50%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or about 0% of the clone's binding activity with wild type IFNAR1.
- clones that bind or do not bind to selected IFNAR1 determinants can be further enriched by repeating the selection procedures described herein one or more times.
- anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1 and which do not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the anti-IFNAR1 phage clones to immobilized mutant IFNAR1 containing Ala substitutions at amino acid positions 244-249 in order to separate desired clones from clones that require wild type amino acids at positions 244-249 for binding to IFNAR1; (3) eluting the adsorbed clones with an excess of IFNAR1; (4) contacting the eluted clones with immobilized, mutant IFNAR1 containing Ala substitutions at amino acid positions 103-111 in order to adsorb undesired clones which bind to determinants on IFNAR1 that do not overlap with amino acid positions 103-111; and (5) recovering the clones which fail to ad
- anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1 and which do not bind to amino acid 249 of IFNAR1 in situ.
- Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing anti-IFNAR1 phage clones to immobilized mutant IFNAR1 containing an Ala substitution at amino acid position 249 in order to separate desired clones from clones that require the wild type amino acid at position 249 for binding to IFNAR1; (3) eluting the adsorbed clones with an excess of IFNAR1; (4) contacting the eluted clones with immobilized, mutant IFNAR1 containing Ala substitutions at amino
- anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and which bind to amino acids 291 and 296 of IFNAR1 in situ, and which do not bind to amino acid 249 of IFNAR1 in situ.
- Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing anti-IFNAR1 phage clones to immobilized mutant IFNAR1 containing an Ala substitution at amino acid position 249 in order to separate desired clones from clones that require the wild type amino acid at position 249 for binding to IFNAR1; (3) eluting the adsorbed clones with excess IFNAR1; (4) contacting the eluted clones with immobilized, mutant IFNAR1 containing Ala substitutions at amino acid positions 103-111 in order to adsorb undesired clones which bind to determinants on IFNAR1 that do not overlap with amino acid positions 103-111; (5) recovering the clones that fail to adsorb to immobilized, mutant
- anti-IFNAR1 antibodies and Fv clones that bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with a mutant IFNAR1 containing Ala substitutions at amino acid positions 244-249 in order to elute the undesired clones which bind determinants on IFNAR1 that do not overlap with amino acids at positions 244-249 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- anti-IFNAR1 antibodies and Fv clones that bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (1) adsorbing the anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with a mutant IFNAR1 containing Ala substitutions at amino acid positions 291-298 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid positions 291-298 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- the invention also provides anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the resulting clones to immobilized IFNAR1; (3) subjecting the adsorbed anti-IFNAR1 clones to elution with a cocktail of excess mutant IFNAR1 containing Ala substitutions at amino acids positions 244-249 and excess mutant IFNAR1 containing Ala substitutions at amino acid positions 291-298, or subjecting the adsorbed clones to consecutive elutions with each of the IFNAR1 mutants, in order to elute undesired clones which bind to determinants on IFNAR1 that do not overlap with both amino acid positions 244-249 and amino acid positions 291-298 on IFNAR1; and (4) recovering the remaining
- the invention provides anti-IFNAR1 antibodies and Fv clones that bind to amino acid 249 of IFNAR1.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the anti-IFNAR1 population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with mutant IFNAR1 containing an Ala substitution at amino acid position 249 of IFNAR1 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid position 249 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- the invention provides anti-IFNAR1 antibodies and Fv clones that bind to amino acid 291 of IFNAR1.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the anti-IFNAR1 population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with mutant IFNAR1 containing an Ala substitution at amino acid position 291 of IFNAR1 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid position 291 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- the invention provides anti-IFNAR1 antibodies and Fv clones that bind to amino acid 296 of IFNAR1.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the anti-IFNAR1 population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with mutant IFNAR1 containing an Ala substitution at amino acid position 296 of IFNAR1 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid position 296 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- the invention further provides anti-IFNAR1 antibodies and Fv clones that bind to amino acids 249, 291 and 296 of IFNAR1 in situ.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with a cocktail of excess mutant IFNAR1 containing an Ala substitution at amino acid position 249, excess mutant IFNAR1 containing an Ala substitution at amino acid position 291, and excess mutant IFNAR1 containing an Ala substitution at amino acid position 296, or subjecting the adsorbed clones to consecutive elutions with each of the IFNAR1 mutants, in
- the invention provides any of the anti-IFNAR1 antibodies described above that additionally binds to a conformational epitope on IFNAR1.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be selected according to the procedures described above modified to include the additional step of screening clones for binding to denatured IFNAR1, e.g., by layering clone suspensions on plates coated with denatured IFNAR1, and collecting non-binding clones from the plate washes.
- the denatured IFNAR1-coated plate adsorption step can be performed before or after the other selection procedures for the Fv clone of interest, or can be performed at any point in such selection procedures that is immediately preceded by the elution of the clones of interest from a particular adsorbent.
- anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to the amino acid sequence of amino acids 244-249 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, bind to amino acids 291 and 296 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- any of the anti-IFN antibodies described above that additionally bind to a conformational epitope formed by domain 1 and domain 2 of IFNAR1.
- Fv clones corresponding to such anti-IFNAR1 antibodies can be selected according to the procedures described above modified to include selection steps that exclude clones that bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1) or bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404).
- the clones of interest are selected by layering a clone suspension on plates coated with domain 1 peptide, recovering the non-binding clones from the plate washes, layering a suspension of the recovered clones on plates coated with domain 2 peptide, and recovering the non-binding clones.
- the clones of interest are selected by adsorbing clones to immobilized IFNAR1, subjecting the adsorbed clones to elution with a cocktail of excess domain 1 peptide and excess domain 2 peptide (or alternatively subjecting the adsorbed clones to serial elutions with the individual peptides), discarding the eluted clones, and recovering the clones that remain bound to adsorbent.
- the domain 1 peptide and domain 2 peptide binding selection step can be performed before or after the other selection procedures for the Fv clone of interest, or can be performed at any point in such selection procedures immediately preceding which the clones of interest are either (1) eluted from a particular adsorbent (e.g. if peptide-coated plates are used for selection) or (2) adsorbed to immobilized IFNAR1 (e.g. if elution with a peptide cocktail is used for selection).
- anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to the amino acid sequence of amino acids 244-249 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, bind to amino acids 291 and 296 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- the invention provides anti-IFNAR1 Fv clones that bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- the invention provides anti-IFNAR1 Fv clones that bind to amino acids 249, 291 and 296 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- anti-IFNAR1 Fv clones that bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- anti-IFNAR1 Fv clones that bind to amino acids 249, 291 and 296 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- the anti-IFNAR1 antibodies of the invention are preferably monoclonal. Also encompassed within the scope of the invention are Fab, Fab′, Fab′-SH and F(ab′) 2 fragments of the anti-IFNAR1 antibodies provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric or humanized. These fragments are useful for the diagnostic and therapeutic purposes set forth below.
- Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
- the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
- the anti-IFNAR1 monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
- a mouse or other appropriate host animal such as a hamster
- Antibodies to IFNAR1 generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of IFNAR1 and an adjuvant.
- animals are immunized with a derivative of IFNAR1 that contains the extracellular domain (ECD) of IFNAR1 fused to the Fc portion of an immunoglobulin heavy chain.
- animals are immunized with an IFNAR1-IgG1 fusion protein as described in the Example below.
- Animals ordinarily are immunized against immunogenic conjugates or derivatives of IFNAR1 with monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.) and the solution is injected intradermally at multiple sites. Two weeks later the animals are boosted. 7 to 14 days later animals are bled and the serum is assayed for anti-IFNAR1 titer. Animals are boosted until titer plateaus.
- MPL monophosphoryl lipid A
- TDM trehalose dicrynomycolate
- lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)).
- a suitable fusing agent such as polyethylene glycol
- the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
- a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
- the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
- Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
- preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
- Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
- Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against IFNAR1.
- the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay (ELISA).
- RIA radioimmunoassay
- ELISA enzyme-linked immunoadsorbent assay
- the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
- the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies:Principles and Practice , pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
- the hybridoma cells may be grown in vivo as ascites tumors in an animal.
- the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- Anti-IFNAR1 antibodies of the invention possessing the unique properties described in Section I above can be obtained by screening anti-IFNAR1 hybridoma clones for the desired properties by any convenient method. For example, if an anti-IFNAR1 monoclonal antibody that binds or does not bind to a particular IFNAR1 determinant(s) is desired, the candidate antibody can be screened for the presence or absence of differential affinity to wild type IFNAR1 and to mutant IFNAR1 that contains Ala substitution(s) at the determinant(s) of interest as described above. In one aspect, the candidate antibody can be tested for binding to wild type IFNAR1 and mutant IFNAR1 in an immunoprecipitation or immunoadsorption assay.
- a capture ELISA can be used wherein plates are coated with a given density of wild type IFNAR1 or an equal density of mutant IFNAR1, the coated plates are contacted with equal concentrations of the candidate antibody, and the bound antibody is detected enzymatically, e.g. by contacting the bound antibody with HRP-conjugated anti-Ig antibody or biotinylated anti-Ig antibody, developing the bound anti-Ig antibody with streptavidin-HRP and/or hydrogen peroxide, and detecting the HRP color reaction by spectrophotometry at 490 nm with an ELISA plate reader.
- the candidate antibody that binds to the particular IFNAR1 determinant(s) of interest will exhibit binding activity with wild type IFNAR1 that is greater than the candidate antibody's binding activity with the corresponding Ala-substituted IFNAR1 mutant (i.e. a binding level with wild type IFNAR1 that is above the background binding level with mutant IFNAR1).
- the candidate antibody that binds to the particular IFNAR1 determinant(s) of interest will exhibit binding activity with the corresponding Ala-substituted IFNAR1 mutant that is less than about 50%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or about 0% of the antibody's binding activity with wild type IFNAR1, e.g.
- the candidate antibody that does not bind to the particular IFNAR1 determinant(s) of interest will exhibit similar or approximately the same binding activities with the corresponding Ala-substituted IFNAR1 mutant and wild type IFNAR1.
- An anti-IFNAR1 monoclonal antibody that (1) binds to a conformational epitope on IFNAR1 or (2) does not bind to a peptide consisting of the amino acid sequence of domain 1 or domain 2 of IFNAR1 as provided herein can be detected by screening for failure to bind to completely denatured IFNAR1, or failure to bind to domain 1 peptide or domain 2 peptide, as desired, in an immunoblot system, e.g. using the candidate antibody to probe a Western blot of denaturing gel electrophoresed IFNAR1 or domain 1 or domain 2 peptides.
- the candidate antibody's inability to bind to completely denatured IFNAR1, domain 1 peptide or domain 2 peptide can be determined by immunoprecipitation or immunoadsorption techniques, e.g. a capture ELISA wherein plates are coated with the denatured IFNAR1, domain 1 peptide or domain 2 peptide, the coated plates are contacted with a solution of the candidate antibody, and the bound antibody is detected enzymatically, e.g. contacting the bound antibody with HRP-conjugated anti-Ig antibody and developing the HRP color reaction.
- immunoprecipitation or immunoadsorption techniques e.g. a capture ELISA wherein plates are coated with the denatured IFNAR1, domain 1 peptide or domain 2 peptide, the coated plates are contacted with a solution of the candidate antibody, and the bound antibody is detected enzymatically, e.g. contacting the bound antibody with HRP-conjugated anti-Ig antibody and developing the HRP color reaction.
- the invention provides anti-IFNAR1 monoclonal antibodies that inhibit the anti-viral activity of a first type I interferon and do not inhibit the anti-viral activity of a second type I interferon.
- the anti-IFNAR1 antibodies of the invention can be obtained by screening candidate anti-IFNAR1 antibodies in any convenient type I interferon viral infectivity inhibition assay.
- Such assays are well known in the art, and include, for example, type I interferon-induced inhibition of encephatomyocarditis virus (EMC) infectivity in A549 cells as described in Current Protocols in Immunology , Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E.
- EMC encephatomyocarditis virus
- the assay uses type I interferon-induced inhibition of vesicular stomatitis virus (VSV) infectivity in Daudi cells as described by Dron and Tovey, J. Gen. Virol., 64: 2641-2647 (1983).
- VSV vesicular stomatitis virus
- cells are seeded in attached cell culture plates, grown for 1 day, and then incubated for an additional day in the presence of various concentrations of a selected type I interferon and in the presence or absence of an excess of the candidate IFNAR1 antibody or a control antibody.
- Cells are challenged with virus, incubated for an additional day, and then viral activity is quantitated by detection of remaining viable cells (e.g. by cell staining) or by lysing cells, collecting culture supernatants and titering the virus concentrations present in the supernatants.
- the candidate antibody that inhibits the anti-viral activity of a selected type I interferon will inhibit more anti-viral activity than the baseline level of anti-viral activity inhibition measured in the presence of an equivalent concentration of control antibody.
- the candidate antibody that inhibits the anti-viral activity of a selected type I interferon will inhibit at least about 50%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or about 100% of the activity of the type I interferon in the anti-viral activity assay as compared to baseline activity measured in the presence of an equivalent concentration of control antibody.
- the candidate antibody that does not inhibit the anti-viral activity of a selected type I interferon will exhibit similar or approximately the same level of anti-viral activity inhibition as control antibody.
- each type I interferon species used in the viral infectivity assay is titrated to a concentration that provides the same level of inhibition of viral activity as that induced by a preselected number of units of an IFN- ⁇ standard. This concentration serves to provide the normalized units of the subject type I interferon species.
- the effective concentration (EC50) of anti-IFNAR1 antibody for inhibiting 50% of a particular type I interferon's anti-viral activity is determined for each type I interferon to be tested.
- each type I interferon to be tested is normalized to at least at or about 1 unit/ml, or at or about 1 unit/ml to at or about 1,000 units/ml, or at or about 1 unit/ml to at or about 100 units/ml, of human IFN- ⁇ 2.
- each type I interferon to be tested is normalized to 10 units/ml of the NIH reference standard for recombinant human IFN- ⁇ 2 (IFN- ⁇ A).
- the candidate anti-IFNAR1 antibody that does not inhibit the anti-viral activity of a selected type I interferon will exhibit no effect at a concentration of up to at or about 1 ⁇ g/ml, or up to ator about 10 ⁇ g/ml, or up to at or about 20 ⁇ g/ml, or up to at or about 30 ⁇ g/ml, or up to at or about 50 ⁇ g/ml, or up to at or about 75 ⁇ g/ml, or up to at or about 100 ⁇ g/ml, against the anti-viral activity of the selected type I interferon in the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, Coligan , J.
- the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 1 ⁇ g/ml, or up to at or about 3 ⁇ g/ml, or up to at or about 6 ⁇ g/ml, or up to at or about 10 ⁇ g/ml, or up to at or about 20 ⁇ g/ml, or up to at or about 30 ⁇ g/ml, or up to at or about 40 ⁇ g/ml, or up to at or about 50 ⁇ g/ml, or up to at or about 75 ⁇ g/ml, or up to at or about 100 ⁇ g/ml, against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, and
- the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 20 ⁇ g/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, and (2) exhibit no effect at a concentration of 30 ⁇ g/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN- ⁇ 2 (IFN- ⁇ A).
- the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 10 ⁇ g/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, and (2) exhibit no effect at a concentration of 30 ⁇ g/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN- ⁇ 2 (IFN- ⁇ A).
- the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 6 ⁇ g/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, and (2) exhibit no effect at a concentration of 30 ⁇ g/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN- ⁇ 2 (IFN- ⁇ A).
- the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 3 ⁇ g/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, and (2) exhibit no effect at a concentration of 30 ⁇ g/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN- ⁇ 2 (IFN- ⁇ A).
- the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 1 ⁇ g/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, and (2) exhibit no effect at a concentration of 30 ⁇ g/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN- ⁇ 2 (IFN- ⁇ A).
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G and does not inhibit the anti-viral activity of a second type I interferon.
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ II 1, and IFN- ⁇ .
- the anti-IFNAR1 inhibits the anti-viral activity of a first type 1 interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ .
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ .
- anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ .
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ B and IFN- ⁇ G.
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ B and IFN- ⁇ G.
- the invention further provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of more than one selected type I interferon and does not inhibit the anti-viral activity of another selected type I interferon;
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D and IFN- ⁇ G and does not inhibit the anti-viral activity of IFN- ⁇ .
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D and IFN- ⁇ G and does not inhibit the anti-viral activity of IFN- ⁇ , wherein (1) the antibody exhibits an EC50 of up to at or about 1 ⁇ g/ml, or up to at or about 3 ⁇ g/ml, or up to at or about 6 ⁇ g/ml, or up to at or about 10 ⁇ g/ml, or up to at or about 20 ⁇ g/ml, or up to at or about 30 ⁇ g/ml, or up to at or about 40 ⁇ g/ml, or up to at or about 50 ⁇ g/ml, or up to at or about 75 ⁇ g/ml, or up to at or about 100 ⁇ g/ml, against the anti-viral activities of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D and IFN- ⁇ G in an A549 cell EMC viral
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D and IFN- ⁇ G and does not inhibit the anti-viral activity of IFN- ⁇ , wherein (1) the antibody exhibits an EC50 of up to at or about 10 ⁇ g/ml against the anti-viral activity of IFN- ⁇ D in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, (2) the antibody exhibits an EC50 of up to at or about 10 ⁇ g/ml against the anti-viral activity of IFN- ⁇ A in the A549 cell EMC viral infectivity assay, (3) the antibody exhibits an EC50 of up to at or about 6 ⁇ g/ml against the anti-viral activity of IFN- ⁇ G in the A549 cell EMC viral infectivity assay, (4) the antibody exhibits an EC50 of up to at or
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D and IFN- ⁇ G and does not inhibit the anti-viral activity of IFN- ⁇ , wherein (1) the antibody exhibits an EC50 of up to at or about 3 ⁇ g/ml against the anti-viral activity of IFN- ⁇ D in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology , supra, (2) the antibody exhibits an EC50 of up to at or about 1 ⁇ g/ml against the anti-viral activity of IFN- ⁇ A in the A549 cell EMC viral infectivity assay, (3) the antibody exhibits an EC50 of up to at or about 1 ⁇ g/ml against the anti-viral activity of IFN- ⁇ G in the A549 cell EMC viral infectivity assay, (4) the antibody exhibits an EC50 of up to at or
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ and does not inhibit the anti-viral activity of another type I interferon.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ and does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN- ⁇ B and IFN- ⁇ G.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ and does not inhibit the anti-viral activity of another type I interferon.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ and does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F and IFN- ⁇ G.
- the invention additionally provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and that does not inhibit the anti-viral activity of more than one other type I interferon.
- the invention provides an anti-IFNAR1 that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ .
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ B and IFN- ⁇ G.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F, and IFN- ⁇ G.
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of at least two species of type I interferon and that does not inhibit the anti-viral activity of at least two more species of type I interferon.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ .
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ B and IFN- ⁇ G.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F, and IFN- ⁇ G.
- the invention provides anti-IFNAR1 antibodies which possess combinations of the type I interferon anti-viral inhibiting and/or non-inhibiting properties and the IFNAR1 determinant binding and/or non-binding properties described herein.
- Anti-IFNAR1 antibodies corresponding to these embodiments can be obtained by using combinations of the type I anti-viral activity inhibitions assays described above for selection of antibodies with unique type I interferon inhibiting/non-inhibiting properties and immunoprecipitation or immunoadsorption screening procedures for selection of antibodies with unique IFNAR1 determinant binding/non-binding properties.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G and does not inhibit the anti-viral activity of a second type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G and does not inhibit the anti-viral activity of a second type I interferon
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of more than one selected type I interferon, does not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, does not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B and IFN- ⁇ G, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ , does not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ , does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F and IFN- ⁇ G, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of a selected type I interferon to IFNAR1, does not inhibit the anti-viral activity of more than one other type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a selected type I interferon, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a selected type I interferon, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F, and IFN- ⁇ G, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of at least two species of type I interferon, does not inhibit the anti-viral activity of at least two more species of type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B and IFN- ⁇ G, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR I.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ , does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F, and IFN- ⁇ G, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon and IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of a second type I interferon and IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of more than one selected type I interferon, does not inhibit the anti-viral activity of another selected type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, does not inhibit the anti-viral activity of another selected type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR 1, and does not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B and IFN- ⁇ G, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ , does not inhibit the anti-viral activity of another selected type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ D and IFN- ⁇ , does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F and IFN- ⁇ G, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR I in situ.
- another type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F and IFN- ⁇ G
- an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of more than one other type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 Fv antibody that inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of at least two species of type I interferon, do not inhibit the anti-viral activity of at least two more species of type I interferon, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the antiviral activity of IFN- ⁇ D and IFN- ⁇ , does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ F, and IFN- ⁇ G, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon and IFNAR1, do not inhibit the anti-viral activity of a second type I interferon and IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- the anti-IFNAR1 antibody inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, does not inhibit the anti-viral activity of a second type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, binds to amino acids 291 and 296 of IFNAR1, and does not bind to amino acid 249 of IFNAR1.
- a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G
- binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1 binds to amino acids 291 and 296 of IFNAR1, and does not bind to amino acid 249 of IFNAR1.
- the anti-IFNAR1 antibody inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, binds to amino acids 291 and 296 of IFNAR1, and does not bind to amino acid 249 of IFNAR1.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of more than one selected type I interferon to IFNAR1, do not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of another selected type.
- I interferon to IFNAR1 bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B and IFN- ⁇ G, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, binds to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of a selected type I interferon, do not inhibit the anti-viral activity of more than one other type I interferon, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of a selected type I interferon, do not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of at least two species of type I interferon, do not inhibit the anti-viral activity of at least two more species of type I interferon, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- the invention additionally provides anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, which do not block the anti-viral activity of IFN- ⁇ , and which bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, and IFN- ⁇ G-inhibiting activity described above (2) which binds to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and (3) which binds to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- the invention also provides anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, which do not block the anti-viral activity of IFN- ⁇ , and which bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above (2) which binds to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and (3) which binds to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- the invention also encompasses anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, which do not inhibit the anti-viral activity of IFN- ⁇ , and which bind to amino acids 249, 291 and 296 of IFNAR1 in situ.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, and IFN- ⁇ G-inhibiting activity described above and (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ.
- the invention further provides anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, which do not inhibit the anti-viral activity of IFN- ⁇ , and which bind to amino acids 249, 291 and 296 of IFNAR1 in situ.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above and (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ.
- the invention provides any of the anti-IFNAR1 antibodies described above that additionally binds to a conformational epitope on IFNAR1.
- anti-IFNAR1 antibodies can be obtained by adding the above-described denatured IFNAR1 immunoblotting or immunoadsorption assay to the series of procedures used to screen for the other desired antibody properties described above. It will be appreciated that the denatured IFNAR1 immunoblotting or immunoadsorption assay can be performed before, after, or at any convenient point during the other selection procedures for the anti-IFNAR1 antibody of interest.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon, and bind to a conformational epitope of IFNAR1.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of a second type I interferon, and bind to a conformational epitope of IFNAR1.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , and bind to a conformational epitope of IFNAR1.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , and bind to a conformational epitope of IFNAR1.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of a type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , and bind to a conformational epitope of IFNAR1.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , and bind to a conformational epitope of IFNAR1.
- anti-IFNAR1 antibodies described above that additionally binds to a conformational epitope formed by domain 1 and domain 2 of IFNAR-1.
- Such anti-IFNAR1 antibodies can be obtained by adding the above-described immunoprecipitation or immunoadsorption assays for determining domain 1 peptide or domain 2 peptide binding, e.g. ELISA capture assays, to the series of procedures used to screen for the other desired antibody properties described above. It will be appreciated that the domain 1 peptide and/or domain 2 peptide immunoprecipitation or immunoadsorption screen can be performed before, after, or at any convenient point during the other selection procedures for the anti-IFNAR1 antibody of interest.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of a second type I interferon, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of a type I interferon selected from the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN- ⁇ A, IFN- ⁇ B, and IFN- ⁇ G, do not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN- ⁇ D, IFN- ⁇ F, and IFN- ⁇ , do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, do not inhibit the anti-viral activity of IFN- ⁇ , and bind to a conformational epitope of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above and (2) which binds to a conformational epitope of IFNAR1.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, do not inhibit the anti-viral activity of IFN- ⁇ , bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above (2) which binds to one or more of amino acids 244-249 of IFNAR1 in situ (3) which binds to one or more of amino acids 291-298 of IFNAR1 in situ and (4) which binds to a conformational epitope of IFNAR1.
- the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, do not inhibit the anti-viral activity of IFN- ⁇ , bind to amino acids 249, 291 and 296 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ and (3) which binds to a conformational epitope of IFNAR1.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, do not inhibit the anti-viral activity of IFN- ⁇ , do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above (2) which does not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1 and (3) which does not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, do not inhibit the anti-viral activity of IFN- ⁇ , bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above (2) which binds to one or more of amino acids 244-249 of IFNAR1 in situ (3) which binds to one or more of amino acids 291-298 of IFNAR1 in situ (4) which does not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1 and (5) which does not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN- ⁇ A, IFN- ⁇ B, IFN- ⁇ D, and IFN- ⁇ G, do not inhibit the anti-viral activity of IFN- ⁇ , bind to amino acids 249, 291 and 298 in IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN- ⁇ -non-inhibiting and IFN- ⁇ A-, IFN- ⁇ B-, IFN- ⁇ D-, and IFN- ⁇ G-inhibiting activity described above (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ (3) which does not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1 and (4) which does not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 5A8 (ATCC Deposit No. HB 12129).
- the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2E8 (ATCC Deposit No. HB 12130).
- the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2H6 (ATCC Deposit No. HB 12131).
- the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 4A7 (ATCC Deposit No. HB 12132).
- the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2E1 (ATCC Deposit No. HB 12133).
- the invention provides anti-IFNAR1 monoclonal antibodies that compete with 5A8 antibody, 2E8 antibody, 2H6 antibody, 4A7 antibody, or 2E1 antibody for binding to IFNAR1.
- Such competitor antibodies include antibodies that recognize an IFNAR1 epitope that is the same as or overlaps with the IFNAR1 epitope recognized by an antibody selected from the group consisting of the 5A8, 2E8, 2H6, 4A7 and 2E1 antibodies.
- Such competitor antibodies can be obtained by screening anti-IFNAR1 hybridoma supernatants for binding to immobilized IFNAR1 in competition with labeled 5A8 antibody, 2E8 antibody, 2H6 antibody, 4A7 antibody or 2E1 antibody.
- a hybridoma supernatant containing competitor antibody will reduce the amount of bound, labeled antibody detected in the subject competition binding mixture as compared to the amount of bound, labeled antibody detected in a control binding mixture containing irrelevant (or no) antibody.
- Any of the competition binding assays described in Section IV below is suitable for use in the foregoing procedure.
- DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention is readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template).
- the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells.
- DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy chain and/or light chain constant regions (e.g. the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and/or light chains.
- constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.
- a Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for “hybrid”, full length heavy chain and/or light chain is included in the definition of “chimeric” and “hybrid” antibody as used herein.
- a Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for all human, full or partial length heavy and/or light chains.
- DNA encoding anti-IFNAR1 antibody derived from a hybridoma of the invention can also be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of homologous murine sequences derived from the hybridoma clone (e.g. as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).
- DNA encoding a hybridoma or Fv clone-derived antibody or fragment can be further modified by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of the Fv clone or hybridoma clone-derived antibodies of the invention.
- non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for IFNAR1 and another antigen-combining site having specificity for a different antigen.
- Chimeric or hybrid antibodies also can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
- immunotoxins can be constructed using a disulfide-exchange reaction or by forming a thioether bond.
- suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
- a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. It will be appreciated that variable domain sequences obtained from any non-human animal phage display library-derived Fv clone or from any non-human animal hybridoma-derived antibody clone provided as described herein can serve as the “import” variable domain used in the construction of the humanized antibodies of the invention.
- Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522 (1986); Riechmann et al., Nature, 332: 323 (1988); Verhoeyen et al., Science, 239: 1534 (1988)), by substituting non-human animal, e.g. rodent, CDRs or CDR sequences for the corresponding sequences of a human antibody.
- non-human animal e.g. rodent, CDRs or CDR sequences
- such “humanized” antibodies are chimeric antibodies (Cabilly et al., supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
- humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in non-human animal, e.g. rodent, antibodies.
- variable domains both light and heavy
- the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity.
- the sequence of the variable domain of a non-human animal, e.g. rodent, antibody is screened against the entire library of known human variable-domain sequences.
- the human sequence that is closest to that of the non-human animal is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901 (1987)).
- Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup light or heavy chains.
- humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
- Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
- Computer programs are available which illustrate and display probable three-dimensional Informational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen.
- FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
- the CDR residues are directly and most substantially involved in influencing antigen binding.
- Human anti-IFNAR1 antibodies of the invention can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s) as described above.
- human monoclonal anti-IFNAR1 antibodies of the invention can be made by the hybridoma method.
- Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brön et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).
- transgenic animals e.g. mice
- transgenic animals e.g. mice
- JH antibody heavy-chain joining region
- transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.
- Jakobovits et al. Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).
- Gene shuffling can also be used to derive human antibodies from non-human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody.
- this method which is also called “epitope imprinting”
- either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras.
- Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for IFNAR1 and the other is for any other antigen. Exemplary bispecific antibodies may bind to two different epitopes of the IFNAR1 protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells that express IFNAR1. These antibodies possess an IFNAR1-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon- ⁇ , vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab) 2 bispecific antibodies).
- cytotoxic agent e.g. saporin, anti-interferon- ⁇ , vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten.
- bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655 (1991).
- antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
- the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1), containing the site necessary for light chain binding, present in at least one of the fusions.
- DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
- the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
- the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
- the preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain.
- one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
- Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
- Bispecific antibodies include cross-linked or “heteroconjugate” antibodies.
- one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
- Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/00373, and EP 03089).
- Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
- bispecific antibodies can be prepared using chemical linkage.
- Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
- the Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
- One of the Fab′-TNB derivatives is then-reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.
- the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
- bispecific antibodies have been produced using leucine zippers.
- the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.
- the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
- the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
- VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
- sFv single-chain Fv
- Antibodies with more than two valencies are contemplated
- trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
- the anti-IFNAR1 antibodies of the invention are unique research reagents which provide anti-type I interferon activity templates for use in chemical library screening, wherein the practitioner can use a signal transduction assay as an initial, high volume screen for agents that exhibit an anti-type I interferon activity pattern that is similar to the anti-type I interferon activity pattern of an anti-IFNAR1 antibody of the invention.
- candidate agents likely to exhibit a desired type I interferon activity inhibition profile can be obtained with ease, avoiding prohibitively expensive and logistically impossible numbers of type I interferon induced viral inhibition assays or cell proliferation inhibition assays on large chemical libraries.
- the anti-IFNAR1 antibodies of the invention are used to screen chemical libraries in a Kinase Receptor Activation (KIRA) Assay as described in WO 95/14930 (published 1 Jun. 1995).
- KIRA Kinase Receptor Activation
- the KIRA assay is suitable for use herein because ligand binding to the type I interferon receptor complex in situ in on the surface of host cells expressing the receptor induces a rapid increase in the phosphorylation of tyrosine residues in the intracellular domains of both IFNAR1 and IFNAR2 components of the receptor as taught in Platanias and Colamonici, J. Biol. Chem., 269: 17761-17764 (1994).
- the level of tyrosine phosphorylation can be used as a measure of signal transduction.
- the effect of an anti-IFNAR1 antibody of the invention on the levels of tyrosine phosphorylation induced by various type I interferons in the KIRA assay can be used as a bench mark activity pattern for comparison to the activity patterns generated by the library compounds in the assay.
- the KIRA assay suitable for use herein employs a host cell that expresses the type I interferon receptor (both IFNAR1 and IFNAR2 components of the receptor) and the particular series of type I interferons which define the inhibitor profile of interest.
- Cells which naturally express the human type I interferon receptor such as the human Daudi cells and U-266 human myeloma cells described in Colamonici and Domanski, J. Biol. Chem., 268: 10895-10899 (1993), can be used.
- cells which are transfected with the IFNAR1 and IFNAR2 components and contain intracellular signaling proteins necessary for type I interferon signal transduction such as mouse L-929 cells as described in Domanski et al., J.
- the candidate antagonist is incubated with each type I interferon ligand to be tested, and each incubation mixture is contacted with the type I interferon receptor-expressing host cells.
- the treated cells are lysed, and IFNAR1 protein in the cell lysate is immobilized by capture with solid phase anti-IFNAR1 antibody.
- Signal transduction is assayed by measuring the amount of tyrosine phosphorylation that exists in the intracellular domain (ICD) of captured IFNAR1 and the amount of tyrosine phosphorylation that exists in the intracellular domain of any co-captured IFNAR2.
- cell lysis and immunoprecipitation can be performed under denaturing conditions in order to avoid co-capture of IFNAR2 and permit measurement of IFNAR1 tyrosine phosphorylation alone, e.g. in a manner similar to the procedure described in Platanias et al., J. Biol. Chem., 271: 23630-23633 (1996).
- the level of tyrosine phosphorylation can be accurately measured with labeled anti-phosphotyrosine antibody that identifies phosphorylated tyrosine residues.
- a host cell coexpressing IFNAR2 and a chimeric construct containing IFNAR1 fused at its carboxy terminus to an affinity handle polypeptide is used in the KIRA assay.
- the chimeric IFNAR1 construct permits capture of the construct from cell lysate by use of a solid phase capture agent (in place of an anti-IFNAR1 antibody) specific for the affinity handle polypeptide.
- the affinity handle polypeptide is Herpes simplex virus glycoprotein D (gD) and the capture agent is an anti-gD monoclonal antibody as described in Examples 2 and 3 of WO 95/14930.
- the anti-IFNAR1 antibody of the invention that possesses the type I interferon inhibition activity profile of interest is used as a standard for analysis of the tyrosine phosphorylation patterns generated by the members of the chemical library that is screened.
- the IFNAR1 ICD tyrosine phosphorylation pattern generated by the anti-IFNAR1 antibody standard is compared to the tyrosine phosphorylation patterns produced in the library screen, and patterns found to match that of the anti-IFNAR1 antibody standard identify candidate agents that are likely to have a type I interferon activity inhibition profile similar to that of the anti-IFNAR1 antibody standard. Accordingly, the anti-IFNAR1 antibody of the invention provides a useful means to quickly and efficiently screen large chemical libraries for compounds likely to exhibit the particular type I interferon activity inhibition profile of the antibody.
- anti-IFNAR1 antibodies of the invention are useful in diagnostic assays for IFNAR1 expression in specific cells or tissues wherein the antibodies are labeled as described below and/or are immobilized on an insoluble matrix. Anti-IFNAR1 antibodies also are useful for the affinity purification of IFNAR1 from recombinant cell culture or natural sources.
- Anti-IFNAR1 antibodies can be used for the detection of IFNAR1 in any one of a number of well known diagnostic assay methods.
- a biological sample may be assayed for IFNAR1 by obtaining the sample from a desired source, admixing the sample with anti-IFNAR1 antibody to allow the antibody to form antibody/IFNAR1 complex with any IFNAR1 present in the mixture, and detecting any antibody/IFNAR1 complex present in the mixture.
- the biological sample may be prepared for assay by methods known in the art which are suitable for the particular sample.
- the methods of admixing the sample with antibodies and the methods of detecting antibody/IFNAR1 complex are chosen according to the type of assay used.
- Such assays include competitive and sandwich assays, and steric inhibition assays.
- Competitive and sandwich methods employ a phase-separation step as an integral part of the method while steric inhibition assays are conducted in a single reaction mixture.
- the label used is any detectable functionality that does not interfere with the binding of IFNAR1 and anti-IFNAR1 antibody.
- Numerous labels are known for use in immunoassay, examples including moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected.
- radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I examples include the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.
- luciferin 2,3-dihydrophthalazinediones
- horseradish peroxidase HRP
- alkaline phosphatase alkaline phosphatase
- ⁇ -galactosidase glucoamylase
- lysozyme saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
- heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
- coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No. 3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol.
- Preferred labels herein are enzymes such as horseradish peroxidase and alkaline phosphatase.
- Immobilization of reagents is required for certain assay methods. Immobilization entails separating the anti-IFNAR1 antibody from any IFNAR1 that remains free in solution. This conventionally is accomplished by either insolubilizing the anti-IFNAR1 antibody or IFNAR1 analogue before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the anti-IFNAR1 antibody or IFNAR1 analogue afterward, e.g., by immunoprecipitation.
- Dose-response curves with known amounts of IFNAR1 are prepared and compared with the test results to quantitatively determine the amount of IFNAR1 present in the test sample. These assays are called ELISA systems when enzymes are used as the detectable markers.
- Another species of competitive assay does not require a phase separation.
- a conjugate of an enzyme with the IFNAR1 is prepared and used such that when anti-IFNAR1 antibody binds to the IFNAR1 the presence of the anti-IFNAR1 antibody modifies the enzyme activity.
- the IFNAR1 or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates are selected for use with anti-IFNAR1 antibody so that binding of the anti-IFNAR1 antibody inhibits or potentiates the enzyme activity of the label. This method per se is widely practiced under the name of EMIT.
- Steric conjugates are used in steric hindrance methods for homogeneous assay. These conjugates are synthesized by covalently linking a low-molecular-weight hapten to a small IFNAR1 fragment so that antibody to hapten is substantially unable to bind the conjugate at the same time as anti-IFNAR1 antibody. Under this assay procedure the IFNAR1 present in the test sample will bind anti-IFNAR1 antibody, thereby allowing anti-hapten to bind the conjugate, resulting in a change in the character of the conjugate hapten, e.g., a change in fluorescence when the hapten is a fluorophore.
- Sandwich assays particularly are useful for the determination of IFNAR1 or anti-IFNAR1 antibodies.
- an immobilized anti-IFNAR1 antibody is used to adsorb test sample IFNAR1
- the test sample is removed as by washing, the bound IFNAR1 is used to adsorb a second, labeled anti-IFNAR1 antibody and bound material is then separated from residual tracer.
- the amount of bound tracer is directly proportional to test sample IFNAR1.
- sandwich assays the test sample is not separated before adding the labeled anti-IFNAR1.
- a sequential sandwich assay using an anti-IFNAR1 monoclonal antibody as one antibody and a polyclonal anti-IFNAR1 antibody as the other is useful in testing samples for IFNAR1.
- Therapeutic formulations of the anti-IFNAR1 antibodies of the invention are prepared for storage by mixing antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers ( Remington: The Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, Pa.: 1995)), in the form of lyophilized cake or aqueous solutions.
- Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).
- buffers such as phosphate, citrate, and other organic acids
- antioxidants including ascorbic acid
- the anti-IFNAR1 antibody to be used for in vivo administration-must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
- the anti-IFNAR1 antibody ordinarily will be stored in lyophilized form or in solution.
- Therapeutic anti-IFNAR1 antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- the route of anti-IFNAR1 antibody administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intraocular, intraarterial, intracerebrospinal, or intralesional routes, or by sustained release systems as noted below.
- the antibody is given systemically.
- sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
- Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate). (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem.
- Sustained-release anti-IFNAR1 antibody compositions also include liposomally entrapped antibody. Liposomes containing antibody are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.
- the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal antibody therapy.
- Anti-IFNAR1 antibody can also be administered by inhalation.
- Commercially available nebulizers for liquid formulations including jet nebulizers and ultrasonic nebulizers are useful for administration.
- Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution.
- anti-IFNAR1 antibody can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
- an “effective amount” of anti-IFNAR1 antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, the type of anti-IFNAR1 antibody employed, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer the anti-IFNAR1 antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
- the patients to be treated with the anti-IFNAR1 antibody of the invention include preclinical patients or those with recent onset of immune-mediated disorders, and particularly autoimmune disorders. Patients are candidates for therapy in accord with this invention until such point as no healthy tissue remains to be protected from immune-mediated destruction. For example, a patient suffering from insulin-dependent diabetes mellitus (IDDM) can benefit from therapy with an anti-IFNAR1 antibody of the invention until the patient's pancreatic islet cells are no longer viable. It is desirable to administer an anti-IFNAR1 antibody as early as possible in the development of the immune-mediated or autoimmune disorder, and to continue treatment for as long as is necessary for the protection of healthy tissue from destruction by the patient's immune system.
- IDDM insulin-dependent diabetes mellitus
- the IDDM patient is treated until insulin monitoring demonstrates adequate islet response and other indicia of islet necrosis diminish (e.g. reduction in anti-islet antibody titers), after which the patient can be withdrawn from anti-IFNAR1 antibody treatment for a trial period during which insulin response and the level of anti-islet antibodies are monitored for relapse.
- indicia of islet necrosis diminish (e.g. reduction in anti-islet antibody titers)
- the patient can be withdrawn from anti-IFNAR1 antibody treatment for a trial period during which insulin response and the level of anti-islet antibodies are monitored for relapse.
- the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice.
- Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the antibody, the particular type of antibody, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
- the “therapeutically effective amount” of antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder, including treating chronic autoimmune conditions and immunosuppression maintenance in transplant recipients. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.
- the initial pharmaceutically effective amount of the antibody administered parenterally will be in the range of about 0.1 to 50 mg/kg of patient body weight per day, with the typical initial range of antibody used being 0.3 to 20 mg/kg/day, more preferably 0.3 to 15 mg/kg/day.
- the desired dosage can be delivered by a single bolus administration, by multiple bolus administrations, or by continuous infusion administration of antibody, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.
- the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the immune-mediated or autoimmune disorder in question.
- agents currently used to prevent or treat the immune-mediated or autoimmune disorder in question For example, in rheumatoid arthritis, the antibody may be given in conjunction with a glucocorticosteroid.
- the effective amount of such other agents depends on the amount of anti-IFNAR1 antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
- a cDNA encoding the human immunoglobulin fusion proteins (immunoadhesins) based on the ECD of the hIFNAR1 (pRK5 hIFNAR1-IgG clone 53.65) was generated using methods similar to those described by Haak-Frendscho et. al., Immunology 79: 594-599 (1993) for the construction of a murine IFN—Y receptor immunoadhesin. Briefly, the plasmid pRKCD4 2 Fc, was constructed as described in Example 4 of WO 89/02922 (PCT/US88/03414 published Apr. 6, 1989).
- the cDNA coding sequence for the 404 amino acid ECD of mature hIFNAR1 shown in FIG. 7 was obtained from the published sequence (Uze et al., Cell, 60: 225-234 (1990)).
- the CD4 coding sequence in the pRKCD4 2 Fc was replaced with the hIFNAR1 ECD encoding cDNA to form pRK5hIFNAR1-IgG clone 53.65.
- the nucleic acid sequence (SEQ ID NO. 21) and amino acid sequence (SEQ ID NO. 22) for the hIFNAR1 ECD-IgG encoding insert of clone 53.65 are shown in FIG. 7 .
- hIFNAR1-IgG was expressed in human embryonic kidney 293 cells by transient transfection using a calcium phosphate precipitation technique.
- the immunoadhesin was purified from serum-free cell culture supernatants in a single step by affinity chromatography on a protein A-sepharose column as described in Haak-Frendscho et al. (1993), supra.
- Bound hIFNAR1-IgG was eluted with 0.1 M citrate buffer, pH 3.0, containing 20% (w/v) glycerol.
- the hIFNAR1-IgG purified was >95% pure, as judged by SDS-PAGE.
- hIFNs Human hIFNs were purified from E. coli paste containing each IFN- ⁇ by affinity chromatography. Bacterial cells were lysed, and the lysate was centrifuged at 10,000 ⁇ g to remove debris. The supernatant was applied to an immunoaffinity column containing a mouse anti-hIFN- ⁇ B antibody (LI-1) that was obtained as described in Staehelin et al., Proc. Natl. Acad. Sci. 78:1848-1852 (1981).
- LI-1 mouse anti-hIFN- ⁇ B antibody
- LI-1 was immobilized on controlled pore glass by a modification of the method of Roy et al., Journal of Chromatography, 303: 225-228 (1984).
- the bound interferon was eluted from the column with 0.1 M citrate, pH 3.0, containing 20% (w/v) glycerol.
- the purified IFN was analyzed by SDS-PAGE and immunoblotting, and was assayed for bioactivity by the hIFN-induced anti-viral assay as described herein.
- hIFN ⁇ was obtained from Sigma (St. Louis, Mo.) and IFN- ⁇ 1/2 was obtained as described in Rehberg et al., J. Biol. Chem., 257: 11497-11502 (1992) or Horisberger and Marco, Pharmac. Ther., 66: 507-534 (1995).
- mice were immunized into each hind foot pad 11 times at two week intervals, with 2.5 ⁇ g of hIFNAR1-IgG fusion protein resuspended in MPL-TDM (Ribi Immunochem. Research Inc., Hamilton, Mont.).
- MPL-TDM Ribi Immunochem. Research Inc., Hamilton, Mont.
- popliteal lymph node cells were fused with murine myeloma cells, P3X63AgU.1 (ATCC CRL1597), using 35% polyethylene glycol.
- Hybridomas were selected in HAT medium.
- hybridoma culture supernatants were first screened for mAbs binding to the hIFNAR1-IgG fusion protein in a capture ELISA.
- the selected culture supernatants were then tested by flow cytometric analysis for their ability to recognize the hIFNAR1 on U266 cells as described in Chuntharapai et al., J. Immunol., 152:1783-1789 (1994).
- the blocking mAbs were selected for their ability to inhibit the anti-viral cytopathic effect of IFN as described below.
- affinities of these mAbs were determined in a competitive binding radioimmunoprecipitation assay according to the method of Kim et al., J. Immunol. Method, 156: 9-17 (1992). Briefly, 125 -hIFNAR1-IgG (specific activity 11.6 ⁇ Ci/ ⁇ g) was prepared using a lactoperoxidase labeling method. mAbs were allowed to bind to 125 I-hIFNAR1-IgG in the presence of various concentrations of unlabeled hIFNAR1-IgG for 1 hour at room temperature (RT). These mixtures were then incubated with goat anti-mouse IgG for 1 hour at RT in the presence of 5% human serum.
- RT room temperature
- the immune complexes were then precipitated by the addition of cold 6% polyethylene glycol (MW 8,000) followed by centrifugation at 200 ⁇ g for 20 minutes at 4° C. Supernatants were removed and the radioactivity remaining in the pellet was determined using a gamma counter. The affinity of each mAb was determined according to the method of Munson et al., Anal. Biochem. 107: 220-239 (1980).
- Microtiter plates (Maxisorb; Nunc, Kamstrup, Denmark) were coated with 50 ⁇ l/well of 2 ⁇ g/ml of goat antibodies specific to the Fc portion of human IgG (Goat anti-hIgG-Fc, Cappel), in PBS, overnight at 4° C. and blocked with 2% BSA for 1 hour at room temperature. After washing the plates, 50 ⁇ l/well of 2 ⁇ g/ml of IFNAR1-IgG (or IFNAR1-IgG mutant) was added, and plates were incubated for 1 hour. After washing the plates, the remaining anti-Fc binding sites were blocked with PBS containing 3% human serum and 10 ⁇ g/ml of CD4-IgG for 1 hour.
- the concentrations of immunoadhesin molecules in 293 transfected culture supernatants were determined using CD4-IgG as a standard and were adjusted to be equal to the lowest concentration of immunoadhesin molecules. The degree of mAb binding to these mutants were then compared to the wild type of the same concentration.
- Reduced hIFNAR1 was prepared by treating the hIFNAR1-IgG fusion protein with 5 mM of 2-mercaptoethanol at 95° C. for 5 minutes. The ability of the mAbs to bind to the native and reduced hIFNAR1-IgG was determined by immunoblotting using 12% SDS-PAGE as described in Kim et al., J. Immunol. Method 156: 9-17 (1992).
- mAbs recognized the same or different epitopes
- a competitive binding ELISA was performed as described in Kim et al., (1992), supra, using biotinylated mAbs (Bio-mAb).
- mAbs were biotinylated using N-hydroxyl succinimide as described in Antibodies ( A Laboratory Manual ), Harlow, E. and Lane, D., eds, Cold Spring Harbor (1988), p. 341.
- Microtiter wells were coated with 50 ⁇ l of Goat anti-hIgG-Fc and kept overnight at 4° C., blocked with assay buffer for 1 hour, and incubated with 251 ⁇ l/well of IFNAR1-IgG (1 ⁇ g/ml) for 1 hour at room temperature. After washing microtiter wells, a mixture of a predetermined optimal concentration of Bio-mAb and a thousand-fold excess of unlabeled mAb was added into each well. Following 1 hour incubation at room temperature, plates were washed and the amount of Bio-mAb was detected by the addition of HRP-streptavidin. After washing the microtiter wells, the bound enzyme was detected by the addition of the substrate, and the plates were read at 490 nm with an ELISA plate reader.
- ⁇ -IFNs 25 ng/ml plus various concentrations (5-500 ⁇ g/ml) of anti-hIFNAR1 mAbs were incubated with 5 ⁇ 10 5 Hela cells in 200 ⁇ l of DMEM for 30 minutes at 37° C. Cells were washed in PBS and resuspended in 125 ⁇ l of buffer A (10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM ETDA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin) as described in Kurabayashi et al., Mol.
- buffer A 10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM ETDA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 ⁇ g/ml leupeptin
- Double-stranded probes were prepared from single-stranded oligonucleotides (ISG15 top: 5′-GATCGGGAAAGGGAAACGAAACTGAAGCC-3′ (SEQ ID NO:23)), ISG15 bottom: 5′-GATCGGCTTCAGTTTCGGTTTCCCTTTCCC-3′ (SEQ ID NO:24)) utilizing a DNA polymerase I Klenow fill-in reaction with 32 P-DATP (3,000 Ci/mM, Amersham). Labeled oligonucleotides were purified from unincorporated radioactive nucleotides using BIO-Spin 30 columns (Bio-Rad).
- Binding reactions containing 5 ⁇ l of nuclear extract, 25,000 cpm of labeled probe and 2 ⁇ g of non-specific competitor poly (dI-dC)-poly (dI-dC) in 15 ⁇ l of binding buffer (10 mM Tris-HCL, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride and 15% glycerol) were incubated at room temperature for 30 minutes. DNA-protein complexes were resolved in 6% non-denaturing polyacrylamide gels (Novex) and analyzed by autoradiography. The specificity of the assay was determined by the addition of 350 ng of unlabeled ISG15 probe in separate reaction mixtures. Formation of an ISGF3 specific complex was confirmed by a super shift assay with anti-STATI antibody.
- the assay was done as described in Current Protocols in Immunol ., Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds, Greene Publishing Associates and Wiley-Interscience (1992), Vol. 1, Unit 6.9.1, using the human lung carcinoma cell line A549 challenged with encephalomyocarditis virus (EMC). Briefly, A549 cells seeded at 2 ⁇ 10 5 cells/100 ⁇ l were grown in DMEM containing 2 mM glutamine, antibiotics, and 5% FCS for 24 hours.
- EMC encephalomyocarditis virus
- mAbs in 50 ⁇ l DMEM were incubated with various units of type 1 IFNs in 50 ⁇ l DMEM for 1 hour at 37° C. These mixtures were then incubated with A549 cells (5 ⁇ 10 5 cells/100 ⁇ l of DMEM containing 4% FCS) for another 24 hours. Culture supernatants were removed and cells were challenged with 2 ⁇ 10 5 pfu of EMC virus in 100 ⁇ l for an additional 24 hours. At the end of the incubation, cell viability was determined by visual microscopic examination.
- the neutralizing antibody titer (EC50) was defined as the concentration of antibody that neutralizes 50% of the anti-viral cytopathic effect by 10 unit/ml of type 1 IFNs.
- IFN- ⁇ A human IFN- ⁇ 2
- the specific activities of the various type 1 IFNs utilized were IFN- ⁇ 2/1 (2 ⁇ 10 7 IU/mg), IFN- ⁇ 1 (3 ⁇ 10 7 IU/mg), IFN- ⁇ 2 (2 ⁇ 10 7 IU/mg), IFN- ⁇ 5 (8 ⁇ 10 7 IU/mg), IFN- ⁇ 8 (19 ⁇ 10 7 IU/mg) and IFN- ⁇ (1.5 ⁇ 10 5 IU/mg).
- the cDNAs encoding domain 1 (1-200 residues) and domain 2 (204-404 residues) of IFNAR1 were separately constructed and expressed as immunoadhesins.
- Single alanine substitution mutants were generated according to the method of Kunkel et al., Methods Enzymol. 154: 367-414 (1987), and Hebert et al, J. Biol. Chem., 268: 18549-18553 (1993).
- the plasmid DNA was isolated using an RPM Kit (BIO 101 Inc., La Jolla, Calif.) and was sequenced by the Sanger method using an ABI 373A DNA sequencer to verify the mutation. Mutant receptor-IgGs were expressed transiently in human 293 cells as described above.
- Transfected 293 cells were grown overnight in F-12:DMEM (50:50) containing 10% FCS, 2 mM glutamine, 100 ⁇ g/ml of penicillin, 100 ⁇ g/ml of streptomycin, 10 ⁇ g/ml of glycine, 15 ⁇ g/ml of hypoxanthine, and 5 ⁇ g/ml of thymidine, and then were placed in serum-free media. Three days later, culture supernatants were collected and used in a capture ELISA.
- the concentrations of immunoadhesin molecules in 293 transfected culture supernatants were determined using CD4-IgG as a standard and were adjusted to be equal to the lowest concentration of immunoadhesin molecules. The degree of mAb binding to these mutants was then compared to the wild type of the same concentration.
- ISGF3 electrophoretic mobility shift assay ESA
- IFN-stimulating response element IFN-stimulating response element binding proteins.
- ISGF3 is a multi-subunit protein complex formed in the cytoplasm within minutes of type 1 IFN treatment (Schindler et al., Proc. Natl. Acad. Sci . ( USA ), 89: 7836 (1997); Fu et al., Proc. Natl. Acad. Sci . ( USA ), 89: 7840 (1997)).
- FIG. 3 contains representative autoradiographs depicting. ISGF3, formation induced by hIFN- ⁇ 8 (IFN- ⁇ D).
- mAbs 2E1 and 4A7 inhibited ISGF3 formation induced by IFN- ⁇ 8 at a concentration of 5 ⁇ g mAb/ml; mAb 5A8 completely inhibited the activity of IFN- ⁇ 8 at a concentration of 500 ⁇ g mAb/ml and partially inhibited the activity of IFN- ⁇ 8 at a concentration of 50 ⁇ g mAb/ml; mAbs 2E8 and 2H6 were unable to block the activity of IFN- ⁇ 8. Results obtained with all type 1 IFNs tested are summarized in Table II below.
- mAbs 2E1 and 4A7 inhibited the activities of all IFN- ⁇ s tested and mAb 2E1 was a more potent inhibitor.
- mAb 5A8 showed blocking activity on IFN- ⁇ 8 and partial blocking activities on - ⁇ 2/1 and - ⁇ 2.
- mAbs 2E8 and 2H6 showed no blocking activity on any of these hIFN- ⁇ s. None of these mAbs to hIFNAR1 were able to block ISGF3 formation induced by IFN- ⁇ .
- the neutralizing effect of these mAbs was also characterized by anti-viral assays (Table III below). Assays were done using serial dilutions of mAbs in the range of 0.1 to 30 ⁇ g mAb/ml and 10 units/ml of type 1 IFNs. The units of these IFNs were determined using NIH IFN- ⁇ 2 (IFN- ⁇ A) as a standard. mAb 2E1 and mAb 4A7 blocked the activity of all IFN- ⁇ s. Abs 2E8m 2H6 and 5A8 showed no neutralizing activities in the anti-viral assay. None of these mAbs were able to neutralize the effect of IFN- ⁇ . Similar results were obtained using 100 units/ml of type 1 IFNs.
- mAbs found to exhibit no blocking effect at a concentration of 30 ⁇ g/ml in this assay were designated as nonblocking mAb (NB).
- Both Domain 1 and 2 of the IFNAR1 may be Required for IFN Signaling.
- Domain 1 (residues 1-200) and domain 2 (residues 204-404) of IFNAR1 were expressed separately as immunoadhesins, as shown in FIG. 4 , and the binding capacity of the blocking mAbs was determined against the domain 1 and domain 2 adhesin molecules in a capture ELISA.
- the concentrations of domain 1-IgG and domain 2-IgG in the culture supernatant were determined by comparison to the known concentrations of CD4-IgG in an ELISA.
- mAbs 2H6 and 4A7 bound only to domain 1-IgG.
- mAb 5A8 bound to both domain 1-IgG and domain 2-IgG, while mAbs 2E1 and 2E8 were unable to bind to either of these domain-IgGs as shown in FIG. 5 .
- mAbs 2E1 and 2E8 were determined to recognize conformational epitopes composed of regions in both domains 1 and 2, implicating the participation of both domains in the IFN signaling.
- soluble hIFNAR1-IgG mutants designated as Mutants #7 and #8 in Table IV.
- soluble mutant # 7 (alanine substitutions in residues 244-249) inhibited the binding of mAbs F-2E8 and F4A7 but did not inhibit the binding of F-2E1
- soluble mutant #8 (alanine substitutions in residues 291-298) inhibited the binding of mAbs F4A7 but did not inhibit the binding of F-2E1 and F-2E8.
- the angle between the two subdomains is significantly different between members of class 1 and class 2 of the cytokine receptor family reported in Kossiakoff et al., Protein Sci. 3: 1697-1705 (1994).
- class 1 the structures of the hGH receptor (reported in de Vos et al., Science 255: 306-312 (1992)) and the prolactin receptor (reported in Somers et al., Nature 372: 478-481 (1994)) display an angle of about 85°, whereas in class 2, the structures of tissue factor (reported in Muller et al., J. Mol. Biol.
- FIG. 6 shows a space-filling rendering of this model, with residues involved in the binding of mAbs depicted in red.
- Residues 69-74 and 103-111 are located in domain 1, in subdomains SD100A and SD100B, respectively, and residues 244-249 and 291-298 in SD100A′ of domain 2.
- Residues 69-74 are situated far away from the other three, on top of the FIG. 6 model. Since substitutions in this region significantly affect binding of all mAbs except 5A8 (which was shown to bind both the domain 1 and domain 2 of hIFNAR1-IgG), it was determined that they cause a major structural change. The remaining three regions are clustered near each other in space and were determined to constitute part of the binding sites of the blocking mAbs 2E1 and 4A7.
- mAb 2E1 is a potent blocking mAb while mAb 2E8 is a nonblocking mAb.
- the different blocking activity of these two mAbs is explained by the results of the mutant analysis as shown in Tables IV and V. The binding areas are indeed overlapping but different.
- hybridomas have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC): Cell Lines ATCC Accession No. Deposit Date 5A8 HB 12129 Jun. 12, 1996 2E8 HB 12130 Jun. 12, 1996 2H6 HB 12131 Jun. 12, 1996 4A7 HB 12132 Jun. 12, 1996 2E1 HB 12133 Jun. 12, 1996
Abstract
Anti-IFNAR1 monoclonal antibodies with neutralizing activities against the anti-viral cytopathic effects of various type I interferons are provided.
Description
- This is a continuation-in-part of co-pending non-provisional application U.S. Ser. No. 08/888,140 filed Jul. 3, 1997, which claims priority under 35 U.S.C. § 119(e) to provisional application U.S. Ser. No. 60/058,212 filed Jul. 16, 1996, now abandoned, which non-provisional application is incorporated herein by reference, and to which non-provisional application priority is claimed under 35 U.S.C. §120.
- This invention relates to the field of anti-type I interferon receptor antibodies, and more particularly to anti-type 1 interferon receptor antibodies that neutralize the anti-viral cytopathic effects of various type I interferons.
- The
type 1 interferons (IFNs) are cytokines that have pleiotropic effects on a wide variety of cell types. IFNs are best known for their anti-viral activity, but they also have anti-bacterial, anti-protozoal, immunomodulatory, and cell-growth regulatory functions. Thetype 1 interferons include interferon-α (IFN-α) and interferon-β (IFN-β). Human IFN-α (hIFN-α) is a heterogeneous family with at least 23 polypeptides while there is only one IFN-β polypeptide (J. Interferon Res., 13: 443-444 (1993)). The hIFN-α subtypes show more than 70% amino acid sequence homology, and there is approximately 25% amino acid identity with hIFN-β. The hIFNs-α and hIFN-β share a common receptor. - Three components of the hIFN-α receptor complex have recently been cloned. The cDNA for the first hIFN-α receptor (hIFNAR1) encodes a 63 kD receptor protein (reported in Uze et al., Cell, 60: 225-234 (1990)). This receptor undergoes extensive glycosylation, which causes it to migrate in gel electrophoresis as a much larger 135 kD protein. The second interferon receptor, hIFNAR2 (hIFN-αβR long), is a 115 kD protein which mediates a functional signaling complex when associated with hIFNAR1 (reported in Domanski et al., J. Biol. Chem., 270: 21606-21611 (1995)). The third hIFN-α receptor, an IFN-α/β receptor (hIFN-αβR short), is a 55 kD protein that can bind to
type 1 hIFNs but cannot form a functional complex when associated with hIFNAR1 (reported in Novick et al., Cell; 77: 391400 (1994)). This IFN-α/β receptor appears to be an alternatively spliced variant of hIFNAR2. - The unprocessed hIFNAR1 expression product is composed of 557 amino acids including an extracellular domain (ECD) of 409 residues, a transmembrane domain of 21 residues, and an intracellular domain of 100 residues as shown in
FIG. 5 on page 229 of Uze et al., supra. The ECD of IFNAR1 is composed of two domains,domain 1 anddomain 2, which are separated by a three-proline motif. There is 19% sequence identity and 50% sequence homology betweendomains 1 and 2 (Uze et al., supra). Each domain (D200) is composed of approximately 200 residues and can be further subdivided into two homologous subdomains (SD100) of approximately 100 amino acids. - Cytokine receptors have been categorized into two classes based on the distribution of cysteine residues. The
class 1 cytokine receptor family includes receptors for human growth hormone (hGHR), erythropoietin, IL-3 and IL4, while theclass 2 cytokine receptor family includes the IFNγ receptor, tissue factor, CRF2-4 and IL-10 receptors. Sequence analysis of the hIFNα receptors shows that they are related to theclass 2 cytokine receptor family. - Through the use of IFNAR1 gene knockout mice, IFNAR1 has been shown to be essential for the response to all
type 1 IFNs (Muller et al., Science, 264: 1918-1921 (1994); Cleary et al., J. Biol. Chem., 269: 18747-18749 (1994)) and for the mediation of species-specific IFN signal transduction (Constantinescu et al., Proc. Natl. Acad. Sci USA, 91: 9602-9606 (1994)). - Benoit et al., J. Immunol., 150: 707-716 (1993) reported an anti-IFNAR1 mAb, 64G12, that was found to inhibit the binding of IFN-α2 (IFN-αA) and IFN-αB (IFN-α8) to Daudi cells and to inhibit the antiviral activity of IFN-α2, IFN-β and IFN-ω (IFN-αII1) on Daudi cells. Benoit et al. also reported that 64G12 recognizes an epitope present in
domain 1 of IFNAR1. Eid and Tovey, J. Interferon Cytokine Res., 15: 205-211 (1995) reported that 64G12 cannot immunoprecipitate cross-linked IFN-α2-receptor complexes from Daudi cells. - In one aspect, the invention provides an anti-IFNAR1 monoclonal antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon.
- In another aspect, the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN-αA.
- In still another aspect, the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN-αB.
- In yet another aspect, the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN-
α II1. - In a further aspect, the invention provides an anti-IFNAR1 monoclonal antibody that inhibits anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of IFN-β.
- In an additional aspect, the invention provides an anti-IFNAR1 monoclonal antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αA, IFN-αB, IFN-
α II1, and IFN-β. - The invention also encompasses an anti-IFNAR1 monoclonal antibody that binds to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1, and binds to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR 1.
-
FIG. 1 is a graph depicting mAb 2E1 binding to U266 human myeloma cell line as determined by FACS analysis. U266 cells were incubated with hybridoma culture supernatant and then contacted with FITC-goat anti-mouse IgG. - FIGS. 2A-E are graphs depicting epitope mapping for mAbs 2E1, 2E8, 2H6, 4A7 and 5A8, respectively, as determined by competitive binding ELISA. IFNAR1 (ECD)-IgG captured by goat anti-human IgG was incubated with predetermined concentrations of biotinylated (Bio)-mAb in the presence of 500-1,000 fold excess of unlabeled mAbs. The level of Bio-mAb bound was detected by the addition of horse radish peroxidase (HRP)-streptavidin.
-
FIG. 3 is a collection of autoradiographs depicting the effect of mAbs 2E1, 2E8, 2H6, 4A7 and 5A8 on ISGF3 formation in Hela cells induced by IFN-α8 (IFN-αD) in an electrophoretic mobility shift assay (EMSA). -
FIG. 4 is a graph depicting a hydropathy profile and the location of certain alanine-substituted mutants of hIFNAR1. -
FIG. 5 is a graph depicting mAb binding to IFNAR1 ECD-IgG (closed columns), IFNAR1 domain 1-IgG (shaded columns), IFNAR1 domain 2-IgG (diagonally hatched columns), and to a control with no antigen (open columns) as determined by ELISA. Microtiter wells coated with goat anti-human IgG were incubated with culture supernatants containing 2 mg/ml of each immunoadhesin followed by the addition of 10 mg/ml of mAbs. The mAb bound to the immunoadhesin was detected by HRP-goat anti-mouse IgG. -
FIG. 6 is a model of hIFNAR1 displaying its protein sequence on the structural backbone of tissue factor. Subdomain SD100A ofdomain 1 and subdomain SD100A′ ofdomain 2 are shown in dark gray. Subdomain SD100B ofdomain 1 and SD100B′ ofdomain 2 are shown in light gray. Regions involved in the binding of anti-IFNAR1 mAbs are shown in orange. Amino acid residues involved in the binding of anti-IFNAR1 mAbs are shown in red. -
FIGS. 7A-7F (hereinafter collectively referred to asFIG. 7 ) depict the DNA sequence (SEQ ID NO. 21) and amino acid sequence (SEQ ID NO. 22) of the IFNAR1 ECD-IgG coding insert in pRK5 hIFNAR1-IgG clone 53.65. The DNA sequence encoding the leader peptide amino acid sequence (corresponding to amino acids 1-29 inFIG. 5 on page 229 of Uze et al., Cell, 60: 225-234 (1990)) of IFNAR1 is shown as bases 38-124 of SEQ ID NO. 21 inFIG. 7 . The leader peptide amino acid sequence is omitted fromFIG. 7 in order to present the mature IFNAR1 ECD sequence as amino acids 1-404 of the IFNAR1 ECD-IgG fusion protein sequence (SEQ ID NO. 22). Unless otherwise indicated, the amino acid numbering scheme for IFNAR1 ECD shown inFIG. 7 is used throughout the application. - A. Definitions
- As used herein, the terms “type I interferon” and “human type I interferon” are defined as all species of native human interferon which fall within the human interferon-α, interferon-ω and interferon-β classes and which bind to a common cellular receptor. Natural human interferon-α comprises 23 or more closely related proteins encoded by distinct genes with a high degree of structural homology (Weissmann and Weber, Prog. Nucl. Acid. Res. Mol. Biol., 33: 251 (1986); J. Interferon Res., 13: 443-44 (1993)). The human IFN-α locus comprises two subfamilies. The first subfamily consists of at least 14 functional, non-allelic genes, including genes encoding IFN-αA (IFN-α2), IFN-αB (IFN-α8), IFN-αC (IFN-α10), IFN-αD (IFN-α1), IFN-αE (IFN-α22), IFN-αF (IFN-α21), IFN-αG (IFN-α5), and IFN-αH (IFN-α14), and pseudogenes having at least 80% homology. The second subfamily, αII or ω, contains at least 5 pseudogenes and 1 functional gene (denoted herein as “IFN-
α II1” or “IFN-ω”) which exhibits 70% homology with the IFN-α genes (Weissmann and Weber (1986)). The human IFN-β is encoded by a single copy gene. - As used herein, the terms “first human interferon-α (hIFN-α) receptor”, “hIFNAR1”, “IFNAR1”, and “Uze chain” are defined as the 557 amino acid receptor protein cloned by Uze et al., Cell, 60: 225-234 (1990), including an extracellular domain of 409 residues, a transmembrane domain of 21 residues, and an intracellular domain of 100 residues, as shown in
FIG. 5 on page 229 of Uze et al. Also encompassed by the foregoing terms are fragments of IFNAR1 that contain the extracellular domain (ECD) (or fragments of the ECD) of IFNAR1. - As used herein, the term “anti-IFNAR1 antibody” is defined as an antibody that is capable of binding to IFNAR1.
- “Polymerase chain reaction” or “PCR” refers to a procedure or technique in which minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Pat. No. 4,683,195 issued 28 Jul. 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5′ terminal nucleotides of the two primers can coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987); Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.
- “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
- “Native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide-linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)).
- The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
- Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
- “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
- The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
- The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
- Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
- The term “antibody” specifically covers monoclonal antibodies, including antibody fragment clones.
- “Antibody fragments” comprise a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; single-chain antibody molecules, including single-chain Fv (scFv) molecules; and multispecific antibodies formed from antibody fragments.
- The term “monoclonal antibody” as used herein refers to an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” also include clones of antigen-recognition and binding-site containing antibody fragments (Fv clones) isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
- The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
- “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanized antibody includes a Primatized™antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
- “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
- The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
- An “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
- “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
- “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
- As used herein, the terms “each member of the group consisting of” and “each of” are synonymous.
- As used herein, the terms “any member of the group consisting of” and “any of” are synonymous.
- B. General Methods
- In general, the invention provides anti-IFNAR1 antibodies that are useful for treatment of immune-mediated disorders in which a partial or total blockade of type I interferon activity is desired. In one embodiment, the anti-IFNAR1 antibodies of the invention are used to treat autoimmune disorders, such as type I and type II diabetes, systemic lupus erythematosis (SLE), and rheumatoid arthritis. In another embodiment, the anti-IFNAR1 antibodies provided herein are used to treat graft rejection or graft versus host disease. The unique properties of the anti-IFNAR1 antibodies of the invention make them particularly useful for effecting target levels of immunosuppression in a patient. For patients requiring acute intervention, the anti-IFNAR1 antibodies provided herein which cause broad spectrum ablation of type I interferon activity can be used to effect the largest possible compromise of an undesired immune response. For patients requiring maintenance immunosuppression, the anti-IFNAR1 antibodies provided herein which block the activity of one or more (but not all) species of type I interferon can be used to effect partial compromise of the patient's immune system in order to reduce the risk of undesirable immune responses while leaving some components of the patient's type I interferon-mediated immunity intact in order to avoid infection.
- In another aspect, the anti-IFNAR1 antibodies of the invention find utility as reagents for detection and isolation of IFNAR1, such as detection of IFNAR1 expression in various cell types and tissues, including the determination of IFNAR1 receptor density and distribution in cell populations, and cell sorting based on IFNAR1 expression. In yet another aspect, the present anti-IFNAR1 antibodies are useful for the development of IFNAR1 antagonists with type I interferon inhibition activity patterns similar to those of the subject antibodies. The anti-IFNAR1 antibodies of the invention can be used in competition binding assays with IFNAR1 to screen for small molecule antagonists of IFNAR1 that will exhibit similar pharmacological effects in blocking the activities of type I interferons to IFNAR1.
- I. Methods of Making Synthetic Anti-IFNAR1 Fv Clones
- The anti-IFNAR1 antibodies of the invention can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities. In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are panned by affinity chromatography against the desired ligand. Clones expressing Fv fragments capable of binding to the desired ligand are adsorbed to the ligand and thus separated from the non-binding clones in the library. The binding clones are then eluted from the ligand, and can be further enriched by additional cycles of ligand adsorption/elution. Any of the anti-IFNAR1 antibodies of the invention can be obtained by designing a suitable ligand screening procedure to select for the phage clone of interest followed by construction of a full length anti-IFNAR1 antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
- 1. Construction of Phage Libraries
- The antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops or complementarity-determining regions (CDRs). Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433455 (1994). As used herein, scFv encoding phage clones and Fab encoding phage clones are collectively referred to as “Fv phage clones” or “Fv clones”.
- The naive repertoire of an animal (the repertoire before antigen challenge) provides it with antibodies that can bind with moderate affinity (Ka of about 106 to 107 M−1) to essentially any non-self molecule. The sequence diversity of antibody binding sites is not encoded directly in the germline but is assembled in a combinatorial manner from V gene segments. Inhuman heavy chains, the first two hypervariable loops (H1 and H2) are drawn from less than 50 VH gene segments, which are combined with D segments and JH segments to create the third hypervariable loop (H3). In human light chains, the first two hypervariable loops (L1 and L2) and much of the third (L3) are drawn from less than approximately 30 Vλ and less than approximately 30 Vκ segments to complete the third hypervariable loop (L3).
- Each combinatorial rearrangement of V-gene segments in stem cells gives rise to a B cell that expresses a single VH-VL combination. Immunizations triggers any B cell making a VH-VL combination that binds the immunogen to proliferate (clonal expansion) and to secrete the corresponding antibody. These naive antibodies are then matured to high affinity (Ka≧109 M−1) by a process of mutagenesis and selection known as affinity maturation. It is after this point that cells are normally removed to prepare hybridomas and generate high-affinity monoclonal antibodies.
- At three stages of this process, repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
- Phage display mimics the B cell. Filamentous phage is used to display antibody fragments by fusion to the minor coat protein pIII. The antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain is fused to pIII and the other is secreted into the bacterial host cell periplasm where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins, e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 41334137 (1991). When antibody fragments are fused to the N-terminus of pIII, the phage is infective. However, if the N-terminal domain of pIII is excised and fusions made to the second domain, the phage is not infective, and wild type pII must be provided by helper phage.
- The pIII fusion and other proteins of the phage can be encoded entirely within the same phage replicon, or on different replicons. When two replicons are used, the pIII fusion is encoded on a phagemid, a plasmid containing a phage origin of replication. Phagemids can be packaged into phage particles by “rescue” with a helper phage such as M13K07 that provides all the phage proteins, including pIII, but due to a defective origin is itself poorly packaged in competitions with the phagemids as described in Vieira and Messing, Meth. Enzymol., 153: 3-11 (1987). In a preferred method, the phage display system is designed such that the recombinant phage can be grown in host cells under conditions permitting no more than a minor amount of phage particles to display more than one copy of the Fv-coat protein fusion on the surface of the particle as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690 (PCT/US91/09133 published Jun. 11, 1992).
- In general, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased in favor of anti-IFNAR1 clones is desired, the subject is immunized with IFNAR1 to generate an antibody response, and spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBLs) are recovered for library construction. In a preferred embodiment, a human antibody gene fragment library biased in favor of anti-human IFNAR1 clones is obtained by generating an anti-human IFNAR1 antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that IFNAR1 immunization gives rise to B cells producing human antibodies against IFNAR1. The generation of human antibody-producing transgenic mice is described in Section B(III)(b) below.
- Additional enrichment for anti-IFNAR1 reactive cell populations can be obtained by using a suitable screening procedure to isolate B cells expressing IFNAR1-specific membrane bound antibody, e.g., by cell separation with IFNAR1 affinity chromatography or adsorption of cells to fluorochrome-labelled IFNAR1 followed by flow-activated cell sorting (FACS).
- Alternatively, the use of spleen cells and/or B cells or other PBLs from an unimmunized donor provides a better representation of the possible antibody repertoire, and also permits the construction of an antibody library using any animal (human or non-human) species in which IFNAR1 is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, luprine, canine, feline, porcine, bovine, equine, and avian species, etc.
- Nucleic acid encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5′ and 3′ ends of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene repertoires for expression. The V genes can be amplified from cDNA and genomic DNA, with back primers at the 5′ end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341: 544-546 (1989). However, for amplifying from cDNA, back primers can also be based in the leader exon as described in Jones et al., Biotechnol, 9: 88-89 (1991), and forward primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989). Preferably, the library diversity is maximized by using PCR primers targeted to each V-gene family in order to amplify all available VH and VL arrangements present in the immune cell nucleic acid sample, e.g. as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA into expression vectors, rare restriction sites can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991).
- Repertoires of synthetically rearranged V genes can be derived in vitro from V gene segments. Most of the human VH-gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the H1 and H2 loop) can be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires can also be made with all the sequence diversity focussed in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic light chain repertoires. Synthetic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural diversity. Following amplification of V-gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
- Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors, and the vectors recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo by combinatorial infection, e.g., the loxp system described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency. Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions. These huge libraries provide large numbers of diverse antibodies of good affinity (Ka of about 108).
- Alternatively, the repertoires may be cloned sequentially into the same vector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g. as described in Clackson et al., Nature, 352: 624-628 (1991). PCR assembly can also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires. In yet another technique, “in cell PCR assembly” is used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).
- The antibodies produced by naive libraries (either natural or synthetic) can be of moderate affinity (Ka of about 106 to 107 M−1), but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al. (1994), supra. For example, mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 2-26: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturation can be performed by randomly mutating one or more CDRs, e.g. using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones. Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol., 10: 779-783 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the 109 M range.
- 2. Panning Phage Display Libraries for Anti-IFNAR1 Clones
- a: Synthesis of IFNAR1 and IFNAR1 Ligands
- Nucleic acid sequence encoding the IFNAR1s used herein can be designed using the amino acid sequence of the desired region of IFNAR1, e.g. the extracellular domain spanning amino acids 28 to 434 of FIG. 2 of WO 93/20187 (PCT/EP93/00770 published Oct. 14, 1993). Alternatively, the cDNA sequence of FIG. 2 of WO 93/20187 can be used. In addition, nucleic acid encoding an immunoglobulin G (IgG)-IFNAR1 extracellular domain fusion protein can be obtained from the amino acid or cDNA sequence shown in
FIG. 8 below. Likewise, nucleic acid sequence encoding the human type I interferons used herein can be designed using published amino acid and nucleic acid sequences, e.g. see the J. Interferon Res., 13: 443444 (1993) compilation of references containing genomic and cDNA sequences for various type I interferons, and the references cited therein. For the IFN-αA, IFN-αB, IFN-αC, IFN-αD, IFN-αE, IFN-αF, IFN-αG, and IFN-αH amino acid sequences or cDNA sequences, seeFIGS. 3 and 4 on pages 23-24 of Goeddel et al., Nature, 290: 20-26 (1981). For cDNA encoding the amino acid sequence of IFN-αII1 (IFN-ω), see Capon et al., Mol. Cell. Biol., 5: 768-779 (1985) and Hauptmann and Swetly, Nucleic Acids Res., 13: 47394749 (1985). For cDNA encoding the amino acid sequence of IFN-β, see Taniguchi et al., Proc. Jpn. Acad Ser. B. 55: 464-469 (1979); Taniguchi et al., Gene, 10: 11-15 (1980); and U.S. Pat. No. 5,460,811 to Goeddel and Crea. DNAs encoding the IFNAR1 s or type I interferons of interest can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), such as the triester, phosphite, phosphoramidite and H-phosphonate methods. In one embodiment, codons preferred by the expression host cell are used in the design of the IFNAR1 or type I interferon-encoding DNA. Alternatively, DNA encoding the IFNAR1 or type I interferon can be isolated from a genomic or cDNA library. - For production of the mutant IFNAR Is used herein, DNA sequence encoding wild type IFNAR1 can be altered to encode the desired IFNAR1 mutant by using recombinant DNA techniques, such as site specific mutagenesis (Kunkel et al., Methods Enzymol. 204:125-139 (1991); Carter, P., et al., Nucl. Acids. Res. 13:4331 (1986); Zoller, M. J. et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (Wells, J. A., et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells, J. A., et al., Philos. Trans, R. Soc. London SerA 317: 415 (1986)), and the like.
- Following construction of the DNA molecule encoding the IFNAR1 or type I interferon of interest, the DNA molecule is operably linked to an expression control sequence in an expression vector, such as a plasmid, wherein the control sequence is recognized by a host cell transformed with the vector. In general, plasmid vectors contain replication and control sequences that are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells.
- For expression in prokaryotic hosts, suitable vectors include pBR322 (ATCC No. 37,017), phGH107 (ATCC No. 40,011), pBO475, pS0132, pRIT5, any vector in the pRIT20 or pRIT30 series (Nilsson and Abrahmsen, Meth. Enzymol., 185: 144-161 (1990)), pRIT2T, pKK233-2, pDR540 and pPL-lambda. Prokaryotic host cells containing the expression vectors suitable for use herein include E. coli K12 strain 294 (ATCC NO. 31446), E coli strain JM101 (Messing et al., Nucl. Acid Res., 9: 309 (1981)), E. coli strain B, E. coli strain χ1776 (ATCC No. 31537), E. coli c600 (Appleyard, Genetics, 39: 440 (1954)), E. coli W3110 (F-, gamma-, prototrophic, ATCC No. 27325), E. coli strain 27C7 (W3110, tonA, phoA E15, (argF-lac) 169, ptr3, degP41, ompT, kan′) U.S. Pat. No. 5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium, Serratia marcesans, and Pseudomonas species.
- In addition to prokaryotes, eukaryotic organisms, such as yeasts, or cells derived from multicellular organisms can be used as host cells. For expression in yeast host cells, such as common baker's yeast or Saccharomyces cerevisiae, suitable vectors include episomally replicating vectors based on the 2-micron plasmid, integration vectors, and yeast artificial chromosome (YAC) vectors. For expression in insect host cells, such as Sf9 cells, suitable vectors include baculoviral vectors. For expression in plant host cells, particularly dicotyledonous plant hosts, such as tobacco, suitable expression vectors include vectors derived from the Ti plasmid of Agrobacterium tumefaciens.
- However, interest has been greatest in vertebrate host cells. Examples of useful mammalian host cells include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (WI 38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). For expression in mammalian host cells, useful vectors include vectors derived from SV40, vectors derived from cytomegalovirus such as the pRK vectors, including pRK5 and pRK7 (Suva et al., Science, 237: 893-896 (1987), EP 307,247 (Mar. 15, 1989), EP 278,776 (Aug. 17, 1988)) vectors derived from vaccinia viruses or other pox viruses, and retroviral vectors such as vectors derived from Moloney's murine leukemia virus (MoMLV).
- Optionally, the DNA encoding the IFNAR1 or type I interferon of interest is operably linked to a secretory leader sequence resulting in secretion of the expression product by the host cell into the culture medium. Examples of secretory leader sequences include stil, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor. Also suitable for use herein is the 36 amino acid leader sequence of protein A (Abrahmsen et al., EMBO J., 4: 3901 (1985)).
- Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
- Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO4 precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
- Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in section 1.82 of Sambrook et al., Molecular Cloning (2nd ed.), Cold Spring Harbor Laboratory, NY (1989), is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method described in sections 16.30-16.37 of Sambrook et al, supra, is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Pat. No. 4,399,216 issued 16 Aug. 1983. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or by protoplast fusion may also be used.
- Prokaryotic host cells used to produce the IFNAR1 or type I interferon of interest can be cultured as described generally in Sambrook et al., supra.
- The mammalian host cells used to produce the IFNAR1 or type I interferon of interest can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace, Meth. Enz., 58: 44 (1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of all of which are incorporated herein by reference, may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.
- In an intracellular expression system or periplasmic space secretion system, the recombinantly expressed IFNAR1 or type I interferon protein can be recovered from the culture cells by disrupting the host cell membrane/cell wall (e.g. by osmotic shock or solubilizing the host cell membrane in detergent). Alternatively, in an extracellular secretion system, the recombinant protein can be recovered from the culture medium. As a first step, the culture medium or lysate is centrifuged to remove any particulate cell debris. The membrane and soluble protein fractions are then separated. Usually, the IFNAR1 or type I interferon is purified from the soluble protein fraction. If the IFNAR1 is expressed as a membrane bound species, the membrane bound peptide can be recovered from the membrane fraction by solubilization with detergents. The crude peptide extract can then be further purified by suitable procedures such as fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; hydrophobic affinity resins and ligand affinity using IFNAR1 (for type I interferon purification) or type I interferons or anti-IFNAR1 antibodies (for IFNAR1 purification) immobilized on a matrix.
- Many of the human type I interferons used herein can be obtained from commercial sources, e.g. human IFN-β is available from Sigma (St. Louis, Mo.).
- b. Immobilization of IFNAR1
- The purified IFNAR1 can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon; neutral and ionic carriers, and the like, for use in the affinity chromatographic separation of phage display clones. Attachment of the IFNAR1 protein to the matrix can be accomplished by the methods described in Methods in Enzymology, vol. 44 (1976). A commonly employed technique for attaching protein ligands to polysaccharide matrices, e.g. agarose, dextran or cellulose, involves activation of the carrier with cyanogen halides and subsequent coupling of the peptide ligand's primary aliphatic or aromatic amines to the activated matrix.
- Alternatively, IFNAR1 can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other art-known method for panning phage display libraries.
- c. Panning Procedures
- The phage library samples are contacted with immobilized IFNAR1 under conditions suitable for binding of at least a portion of the phage particles with the adsorbent. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted by acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or by IFNAR1 antigen or type I interferon ligand competition, e.g. in a procedure similar to the antigen competition method of Clackson et al., Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold in a single round of selection. Moreover, the enriched phages can be grown in bacterial culture and subjected to further rounds of selection.
- The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen. Antibodies with fast dissociation kinetics (and weak binding affinities) can be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions, but also favors rebinding of phage that has dissociated. The selection of antibodies with slow dissociation kinetics (and good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).
- It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for IFNAR1. However, random mutation of a selected antibody (e.g. as performed in some of the affinity maturation techniques described above) is likely to give rise to many mutants, most binding to antigen, and a few with higher affinity. With limiting IFNAR1, rare high affinity phage could be competed out. To retain all the higher affinity mutants, phages can be incubated with excess biotinylated IFNAR1, but with the biotinylated IFNAR1 at a concentration of lower molarity than the target molar affinity constant for IFNAR1. The high affinity-binding phages can then be captured by streptavidin-coated paramagnetic beads. Such “equilibrium capture” allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity. Conditions used in washing phages bound to a solid phase can also be manipulated to discriminate on the basis of dissociation kinetics.
- 3. Activity Selection of Anti-IFNAR1 Clones
- In one embodiment, the invention provides anti-IFNAR1 antibodies which bind to specific determinant(s) on IFNAR1 and/or which do not bind other specific determinant(s) on IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can be conveniently selected by adsorbing library clones to immobilized IFNAR1 mutants containing Ala substitutions at the specific determinants of interest. If clones which do not bind the selected IFNAR1 determinant(s) are desired, then the clones which adsorb to the IFNAR1 mutant are recovered, e.g. by eluting the adsorbed clones with wild type IFNAR1. The separation occurs because of the difference in the affinities of the desired and undesired clones for the IFNAR1 mutant. Since the IFNAR1 determinant(s) bound by the desired clones do not include the amino acid(s) at the Ala-substituted position(s) in the IFNAR1 mutant, the desired clones will bind to the immobilized, mutant IFNAR1 whereas the undesired clones will not. Accordingly, the adsorption of library clones to immobilized, mutant IFNAR1 will yield a population of clones bound to solid phase that is enriched for the property of not being able to bind to the selected IFNAR1 determinant(s). The desired clones will exhibit similar or approximately the same binding activities with the corresponding Ala-substituted IFNAR1 mutant and wild type IFNAR1.
- If clones which bind to the selected IFNAR1 determinant(s) are desired, then library clones which fail to adsorb to immobilized, mutant IFNAR1 are recovered (i.e. collected from the column flow-through fractions), the recovered clones are adsorbed to immobilized, wild type IFNAR1, and then the adsorbed clones are recovered, e.g. by elution with excess wild type IFNAR1. The first adsorption step removes clones that bind to IFNAR1 but do not bind to the selected determinant(s), and the second adsorption step removes clones that do not bind to IFNAR1 at all, leaving a population of clones enriched for binding to the selected IFNAR1 determinant(s). The desired clone will exhibit binding activity with wild type IFNAR1 that is greater than the clone's binding activity with the corresponding Ala-substituted IFNAR1 mutant (i.e. a binding level with wild type IFNAR1 that is above the background binding level with mutant IFNAR1). Optionally, the desired clone will exhibit binding activity with the corresponding Ala-substituted IFNAR1 mutant that is less than about 50%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or about 0% of the clone's binding activity with wild type IFNAR1.
- Optionally, clones that bind or do not bind to selected IFNAR1 determinants can be further enriched by repeating the selection procedures described herein one or more times.
- Also provided herein are anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1 and which do not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1. These Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the anti-IFNAR1 phage clones to immobilized mutant IFNAR1 containing Ala substitutions at amino acid positions 244-249 in order to separate desired clones from clones that require wild type amino acids at positions 244-249 for binding to IFNAR1; (3) eluting the adsorbed clones with an excess of IFNAR1; (4) contacting the eluted clones with immobilized, mutant IFNAR1 containing Ala substitutions at amino acid positions 103-111 in order to adsorb undesired clones which bind to determinants on IFNAR1 that do not overlap with amino acid positions 103-111; and (5) recovering the clones which fail to adsorb to the immobilized, mutant IFNAR1 from the flow-through fractions in step (4).
- Additionally provided herein are anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1 and which do not bind to amino acid 249 of IFNAR1 in situ. These Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing anti-IFNAR1 phage clones to immobilized mutant IFNAR1 containing an Ala substitution at amino acid position 249 in order to separate desired clones from clones that require the wild type amino acid at position 249 for binding to IFNAR1; (3) eluting the adsorbed clones with an excess of IFNAR1; (4) contacting the eluted clones with immobilized, mutant IFNAR1 containing Ala substitutions at amino acid positions 103-111 in order to adsorb undesired clones which bind to determinants on IFNAR1 that do not overlap with amino acid positions 103-111; and (5) recovering the clones which fail to adsorb to the immobilized, mutant IFNAR1 from the flow-through fractions in step (4).
- Also encompassed herein are anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and which bind to amino acids 291 and 296 of IFNAR1 in situ, and which do not bind to amino acid 249 of IFNAR1 in situ. These Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing anti-IFNAR1 phage clones to immobilized mutant IFNAR1 containing an Ala substitution at amino acid position 249 in order to separate desired clones from clones that require the wild type amino acid at position 249 for binding to IFNAR1; (3) eluting the adsorbed clones with excess IFNAR1; (4) contacting the eluted clones with immobilized, mutant IFNAR1 containing Ala substitutions at amino acid positions 103-111 in order to adsorb undesired clones which bind to determinants on IFNAR1 that do not overlap with amino acid positions 103-111; (5) recovering the clones that fail to adsorb to immobilized, mutant IFNAR1 from the flow-through fractions in step (4); (6) contacting the recovered clones with immobilized, mutant IFNAR1 containing Ala substitutions at amino acids 291 and 296 in order to adsorb undesired clones which bind to determinants on IFNAR1 that do not overlap with amino acid positions 291 and 296; and (7) recovering the clones which fail to adsorb to immobilized, mutant IFNAR1 from the flow-through fractions in step (6).
- Also provided herein are anti-IFNAR1 antibodies and Fv clones that bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1. These Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with a mutant IFNAR1 containing Ala substitutions at amino acid positions 244-249 in order to elute the undesired clones which bind determinants on IFNAR1 that do not overlap with amino acids at positions 244-249 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- Additionally provided herein are anti-IFNAR1 antibodies and Fv clones that bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1. These Fv clones can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (1) adsorbing the anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with a mutant IFNAR1 containing Ala substitutions at amino acid positions 291-298 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid positions 291-298 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- The invention also provides anti-IFNAR1 antibodies and Fv clones which bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the resulting clones to immobilized IFNAR1; (3) subjecting the adsorbed anti-IFNAR1 clones to elution with a cocktail of excess mutant IFNAR1 containing Ala substitutions at amino acids positions 244-249 and excess mutant IFNAR1 containing Ala substitutions at amino acid positions 291-298, or subjecting the adsorbed clones to consecutive elutions with each of the IFNAR1 mutants, in order to elute undesired clones which bind to determinants on IFNAR1 that do not overlap with both amino acid positions 244-249 and amino acid positions 291-298 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- In another embodiment, the invention provides anti-IFNAR1 antibodies and Fv clones that bind to amino acid 249 of IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the anti-IFNAR1 population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with mutant IFNAR1 containing an Ala substitution at amino acid position 249 of IFNAR1 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid position 249 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- In another embodiment, the invention provides anti-IFNAR1 antibodies and Fv clones that bind to amino acid 291 of IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the anti-IFNAR1 population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with mutant IFNAR1 containing an Ala substitution at amino acid position 291 of IFNAR1 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid position 291 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- In another embodiment, the invention provides anti-IFNAR1 antibodies and Fv clones that bind to amino acid 296 of IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the anti-IFNAR1 population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with mutant IFNAR1 containing an Ala substitution at amino acid position 296 of IFNAR1 in order to elute undesired clones which bind determinants on IFNAR1 that do not overlap with amino acid position 296 on IFNAR1; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- The invention further provides anti-IFNAR1 antibodies and Fv clones that bind to amino acids 249, 291 and 296 of IFNAR1 in situ. Fv clones corresponding to such anti-IFNAR1 antibodies can be selected by (1) isolating anti-IFNAR1 clones from a phage library as described in Section B(I)(2) above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) adsorbing the resulting anti-IFNAR1 clones to immobilized IFNAR1; (3) subjecting the adsorbed clones to elution with a cocktail of excess mutant IFNAR1 containing an Ala substitution at amino acid position 249, excess mutant IFNAR1 containing an Ala substitution at amino acid position 291, and excess mutant IFNAR1 containing an Ala substitution at amino acid position 296, or subjecting the adsorbed clones to consecutive elutions with each of the IFNAR1 mutants, in order to elute undesired clones which bind to determinants on IFNAR1 which do not overlap with amino acids 249, 291 or 296 of IFNAR1 in situ; and (4) recovering the remaining adsorbed clones by elution with excess IFNAR1.
- In another embodiment, the invention provides any of the anti-IFNAR1 antibodies described above that additionally binds to a conformational epitope on IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can be selected according to the procedures described above modified to include the additional step of screening clones for binding to denatured IFNAR1, e.g., by layering clone suspensions on plates coated with denatured IFNAR1, and collecting non-binding clones from the plate washes. It will be appreciated that the denatured IFNAR1-coated plate adsorption step can be performed before or after the other selection procedures for the Fv clone of interest, or can be performed at any point in such selection procedures that is immediately preceded by the elution of the clones of interest from a particular adsorbent.
- Also provided herein are anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to the amino acid sequence of amino acids 244-249 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- Additionally provided herein are anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- Further encompassed herein are anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, bind to amino acids 291 and 296 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- Also included herein are any of the anti-IFN antibodies described above that additionally bind to a conformational epitope formed by
domain 1 anddomain 2 of IFNAR1. Fv clones corresponding to such anti-IFNAR1 antibodies can be selected according to the procedures described above modified to include selection steps that exclude clones that bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1) or bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404). In one embodiment, the clones of interest are selected by layering a clone suspension on plates coated withdomain 1 peptide, recovering the non-binding clones from the plate washes, layering a suspension of the recovered clones on plates coated withdomain 2 peptide, and recovering the non-binding clones. In another embodiment, the clones of interest are selected by adsorbing clones to immobilized IFNAR1, subjecting the adsorbed clones to elution with a cocktail ofexcess domain 1 peptide andexcess domain 2 peptide (or alternatively subjecting the adsorbed clones to serial elutions with the individual peptides), discarding the eluted clones, and recovering the clones that remain bound to adsorbent. Thedomain 1 peptide anddomain 2 peptide binding selection step can be performed before or after the other selection procedures for the Fv clone of interest, or can be performed at any point in such selection procedures immediately preceding which the clones of interest are either (1) eluted from a particular adsorbent (e.g. if peptide-coated plates are used for selection) or (2) adsorbed to immobilized IFNAR1 (e.g. if elution with a peptide cocktail is used for selection). - Also provided herein are anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to the amino acid sequence of amino acids 244-249 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- Additionally provided herein are anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- Further encompassed herein are anti-IFNAR1 Fv clones that bind to the amino acid sequence of amino acids 103-111 of IFNAR1 in situ, bind to amino acids 291 and 296 of IFNAR1 in situ, do not bind to amino acid 249 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- In yet another embodiment, the invention provides anti-IFNAR1 Fv clones that bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- In still another embodiment, the invention provides anti-IFNAR1 Fv clones that bind to amino acids 249, 291 and 296 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1.
- Further provided herein are anti-IFNAR1 Fv clones that bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- Additionally provided herein are anti-IFNAR1 Fv clones that bind to amino acids 249, 291 and 296 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- II. Methods of Making Anti-IFNAR1 Hybridomas
- The anti-IFNAR1 antibodies of the invention are preferably monoclonal. Also encompassed within the scope of the invention are Fab, Fab′, Fab′-SH and F(ab′)2 fragments of the anti-IFNAR1 antibodies provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric or humanized. These fragments are useful for the diagnostic and therapeutic purposes set forth below.
- Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
- The anti-IFNAR1 monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
- In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies to IFNAR1 generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of IFNAR1 and an adjuvant. In one embodiment, animals are immunized with a derivative of IFNAR1 that contains the extracellular domain (ECD) of IFNAR1 fused to the Fc portion of an immunoglobulin heavy chain. In a preferred embodiment, animals are immunized with an IFNAR1-IgG1 fusion protein as described in the Example below. Animals ordinarily are immunized against immunogenic conjugates or derivatives of IFNAR1 with monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.) and the solution is injected intradermally at multiple sites. Two weeks later the animals are boosted. 7 to 14 days later animals are bled and the serum is assayed for anti-IFNAR1 titer. Animals are boosted until titer plateaus.
- Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
- The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
- Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
- Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against IFNAR1. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay (ELISA).
- The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
- After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
- The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- Anti-IFNAR1 antibodies of the invention possessing the unique properties described in Section I above can be obtained by screening anti-IFNAR1 hybridoma clones for the desired properties by any convenient method. For example, if an anti-IFNAR1 monoclonal antibody that binds or does not bind to a particular IFNAR1 determinant(s) is desired, the candidate antibody can be screened for the presence or absence of differential affinity to wild type IFNAR1 and to mutant IFNAR1 that contains Ala substitution(s) at the determinant(s) of interest as described above. In one aspect, the candidate antibody can be tested for binding to wild type IFNAR1 and mutant IFNAR1 in an immunoprecipitation or immunoadsorption assay. For example, a capture ELISA can be used wherein plates are coated with a given density of wild type IFNAR1 or an equal density of mutant IFNAR1, the coated plates are contacted with equal concentrations of the candidate antibody, and the bound antibody is detected enzymatically, e.g. by contacting the bound antibody with HRP-conjugated anti-Ig antibody or biotinylated anti-Ig antibody, developing the bound anti-Ig antibody with streptavidin-HRP and/or hydrogen peroxide, and detecting the HRP color reaction by spectrophotometry at 490 nm with an ELISA plate reader. The candidate antibody that binds to the particular IFNAR1 determinant(s) of interest will exhibit binding activity with wild type IFNAR1 that is greater than the candidate antibody's binding activity with the corresponding Ala-substituted IFNAR1 mutant (i.e. a binding level with wild type IFNAR1 that is above the background binding level with mutant IFNAR1). Optionally, the candidate antibody that binds to the particular IFNAR1 determinant(s) of interest will exhibit binding activity with the corresponding Ala-substituted IFNAR1 mutant that is less than about 50%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or about 0% of the antibody's binding activity with wild type IFNAR1, e.g. as determined by dividing the HRP color reaction optical density observed for capture ELISA with IFNAR1 mutant adsorbent by the HRP color reaction optical density observed for capture ELISA with wild type IFNAR1 adsorbent. The candidate antibody that does not bind to the particular IFNAR1 determinant(s) of interest will exhibit similar or approximately the same binding activities with the corresponding Ala-substituted IFNAR1 mutant and wild type IFNAR1.
- An anti-IFNAR1 monoclonal antibody that (1) binds to a conformational epitope on IFNAR1 or (2) does not bind to a peptide consisting of the amino acid sequence of
domain 1 ordomain 2 of IFNAR1 as provided herein can be detected by screening for failure to bind to completely denatured IFNAR1, or failure to bind todomain 1 peptide ordomain 2 peptide, as desired, in an immunoblot system, e.g. using the candidate antibody to probe a Western blot of denaturing gel electrophoresed IFNAR1 ordomain 1 ordomain 2 peptides. Alternatively, the candidate antibody's inability to bind to completely denatured IFNAR1,domain 1 peptide ordomain 2 peptide can be determined by immunoprecipitation or immunoadsorption techniques, e.g. a capture ELISA wherein plates are coated with the denatured IFNAR1,domain 1 peptide ordomain 2 peptide, the coated plates are contacted with a solution of the candidate antibody, and the bound antibody is detected enzymatically, e.g. contacting the bound antibody with HRP-conjugated anti-Ig antibody and developing the HRP color reaction. - In another embodiment, the invention provides anti-IFNAR1 monoclonal antibodies that inhibit the anti-viral activity of a first type I interferon and do not inhibit the anti-viral activity of a second type I interferon. The anti-IFNAR1 antibodies of the invention can be obtained by screening candidate anti-IFNAR1 antibodies in any convenient type I interferon viral infectivity inhibition assay. Such assays are well known in the art, and include, for example, type I interferon-induced inhibition of encephatomyocarditis virus (EMC) infectivity in A549 cells as described in Current Protocols in Immunology, Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds, Greene Publishing Associates and Wiley-Interscience, (1992), vol. 1, unit 6.9.1. In another example, the assay uses type I interferon-induced inhibition of vesicular stomatitis virus (VSV) infectivity in Daudi cells as described by Dron and Tovey, J. Gen. Virol., 64: 2641-2647 (1983). Generally, cells are seeded in attached cell culture plates, grown for 1 day, and then incubated for an additional day in the presence of various concentrations of a selected type I interferon and in the presence or absence of an excess of the candidate IFNAR1 antibody or a control antibody. Cells are challenged with virus, incubated for an additional day, and then viral activity is quantitated by detection of remaining viable cells (e.g. by cell staining) or by lysing cells, collecting culture supernatants and titering the virus concentrations present in the supernatants. The candidate antibody that inhibits the anti-viral activity of a selected type I interferon will inhibit more anti-viral activity than the baseline level of anti-viral activity inhibition measured in the presence of an equivalent concentration of control antibody. Optionally, the candidate antibody that inhibits the anti-viral activity of a selected type I interferon will inhibit at least about 50%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or about 100% of the activity of the type I interferon in the anti-viral activity assay as compared to baseline activity measured in the presence of an equivalent concentration of control antibody. The candidate antibody that does not inhibit the anti-viral activity of a selected type I interferon will exhibit similar or approximately the same level of anti-viral activity inhibition as control antibody.
- In another embodiment, each type I interferon species used in the viral infectivity assay is titrated to a concentration that provides the same level of inhibition of viral activity as that induced by a preselected number of units of an IFN-α standard. This concentration serves to provide the normalized units of the subject type I interferon species. In order to assess the ability of an anti-IFNAR1 antibody to inhibit the anti-viral activity of various type I interferons, the effective concentration (EC50) of anti-IFNAR1 antibody for inhibiting 50% of a particular type I interferon's anti-viral activity (at the concentration titrated to provide the normalized units of activity) is determined for each type I interferon to be tested. In another embodiment, each type I interferon to be tested is normalized to at least at or about 1 unit/ml, or at or about 1 unit/ml to at or about 1,000 units/ml, or at or about 1 unit/ml to at or about 100 units/ml, of human IFN-α2. In yet another embodiment, each type I interferon to be tested is normalized to 10 units/ml of the NIH reference standard for recombinant human IFN-α2 (IFN-αA).
- In still another embodiment, the candidate anti-IFNAR1 antibody that does not inhibit the anti-viral activity of a selected type I interferon will exhibit no effect at a concentration of up to at or about 1 μg/ml, or up to ator about 10 μg/ml, or up to at or about 20 μg/ml, or up to at or about 30 μg/ml, or up to at or about 50 μg/ml, or up to at or about 75 μg/ml, or up to at or about 100 μg/ml, against the anti-viral activity of the selected type I interferon in the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds, Greene Publishing Associates and Wiley-Interscience, (1992), vol. 2, unit 6.9.1, wherein the selected type I interferon is normalized to 10 units/ml of NIH reference standard for recombinant human IFN-α2 (IFN-αA).
- In another embodiment, the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 1 μg/ml, or up to at or about 3 μg/ml, or up to at or about 6 μg/ml, or up to at or about 10 μg/ml, or up to at or about 20 μg/ml, or up to at or about 30 μg/ml, or up to at or about 40 μg/ml, or up to at or about 50 μg/ml, or up to at or about 75 μg/ml, or up to at or about 100 μg/ml, against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, and (2) exhibit no effect at a concentration of up to at or about 30 μg/ml, or up to at or about 40 μg/ml, or up to at or about 50 μg/ml, or up to at or about 75 μg/ml, or up to at or about 100 μg/ml, against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In yet another embodiment, the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 20 μg/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, and (2) exhibit no effect at a concentration of 30 μg/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In yet another embodiment, the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 10 μg/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, and (2) exhibit no effect at a concentration of 30 μg/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In yet another embodiment, the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 6 μg/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, and (2) exhibit no effect at a concentration of 30 μg/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In yet another embodiment, the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 3 μg/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, and (2) exhibit no effect at a concentration of 30 μg/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In yet another embodiment, the candidate anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon will (1) exhibit an EC50 of up to at or about 1 μg/ml against the anti-viral activity of the first type I interferon in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, and (2) exhibit no effect at a concentration of 30 μg/ml against the anti-viral activity of the second type I interferon in the A549 cell EMC viral infectivity assay, wherein in the A549 cell EMC viral infectivity assay the first and second type I interferons are normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In another aspect, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG and does not inhibit the anti-viral activity of a second type I interferon.
- Also provided herein is an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αA, IFN-αB, IFN-
α II1, and IFN-β. - In yet another embodiment, the anti-IFNAR1 inhibits the anti-viral activity of a
first type 1 interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β. - Additionally provided herein is an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β.
- Further provided herein is anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αD, IFN-αF, and IFN-β.
- Also encompassed herein is an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αB and IFN-αG.
- Further encompassed herein is an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αD, IFN-αF, and IFN-β and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αB and IFN-αG.
- The invention further provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of more than one selected type I interferon and does not inhibit the anti-viral activity of another selected type I interferon;
-
- In one embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG and does not inhibit the anti-viral activity of another type I interferon. In another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB and IFN-αG and does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β.
- In another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, IFN-αD and IFN-αG and does not inhibit the anti-viral activity of IFN-β.
- In another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, IFN-αD and IFN-αG and does not inhibit the anti-viral activity of IFN-β, wherein (1) the antibody exhibits an EC50 of up to at or about 1 μg/ml, or up to at or about 3 μg/ml, or up to at or about 6 μg/ml, or up to at or about 10 μg/ml, or up to at or about 20 μg/ml, or up to at or about 30 μg/ml, or up to at or about 40 μg/ml, or up to at or about 50 μg/ml, or up to at or about 75 μg/ml, or up to at or about 100 μg/ml, against the anti-viral activities of IFN-αA, IFN-αB, IFN-αD and IFN-αG in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, and (2) the antibody exhibits no effect at a concentration of up to at or about 30 μg/ml, or up to at or about 40 μg/ml, or up to at or about 50 μg/ml, or up to at or about 75 μg/ml, or up to at or about 100 μg/ml, against the anti-viral activity of the IFN-β in the A549 cell EMC viral infectivity assay, and wherein in the A549 cell EMC viral infectivity assay each type I interferon is normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, IFN-αD and IFN-αG and does not inhibit the anti-viral activity of IFN-β, wherein (1) the antibody exhibits an EC50 of up to at or about 10 μg/ml against the anti-viral activity of IFN-αD in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, (2) the antibody exhibits an EC50 of up to at or about 10 μg/ml against the anti-viral activity of IFN-αA in the A549 cell EMC viral infectivity assay, (3) the antibody exhibits an EC50 of up to at or about 6 μg/ml against the anti-viral activity of IFN-αG in the A549 cell EMC viral infectivity assay, (4) the antibody exhibits an EC50 of up to at or about 3 μg/ml against the anti-viral activity of IFN-αB in the A549 cell EMC viral infectivity assay, and (5) the antibody exhibits no effect at a concentration of up to at or about 30 μg/ml against the anti-viral activity of the IFN-β in the A549 cell EMC viral infectivity assay, and wherein in the A549 cell EMC viral infectivity assay each type I interferon is normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, IFN-αD and IFN-αG and does not inhibit the anti-viral activity of IFN-β, wherein (1) the antibody exhibits an EC50 of up to at or about 3 μg/ml against the anti-viral activity of IFN-αD in an A549 cell EMC viral infectivity assay, such as the A549 cell EMC viral infectivity assay described in Current Protocols in Immunology, supra, (2) the antibody exhibits an EC50 of up to at or about 1 μg/ml against the anti-viral activity of IFN-αA in the A549 cell EMC viral infectivity assay, (3) the antibody exhibits an EC50 of up to at or about 1 μg/ml against the anti-viral activity of IFN-αG in the A549 cell EMC viral infectivity assay, (4) the antibody exhibits an EC50 of up to at or about 1 μg/ml against the anti-viral activity of IFN-αB in the A549 cell EMC viral infectivity assay, and (5) the antibody exhibits no effect at a concentration of up to at or about 30 μg/ml against the anti-viral activity of the IFN-β in the A549 cell EMC viral infectivity assay, and wherein in the A549 cell EMC viral infectivity assay each type I interferon is normalized to 10 units/ml of NIH reference standard for recombinant IFN-α2 (IFN-αA).
- In yet another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αD, IFN-αF, and IFN-β and does not inhibit the anti-viral activity of another type I interferon. In still another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αD, IFN-αF, and IFN-β and does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αB and IFN-αG.
- In a further embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β and does not inhibit the anti-viral activity of another type I interferon. In an additional embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β and does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αA, IFN-αB, IFN-αF and IFN-αG.
- The invention additionally provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and that does not inhibit the anti-viral activity of more than one other type I interferon.
- In one embodiment, the invention provides an anti-IFNAR1 that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β. In another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αB and IFN-αG. In yet another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αA, IFN-αB, IFN-αF, and IFN-αG.
- Further provided herein is an anti-IFNAR1 antibody that inhibits the anti-viral activity of at least two species of type I interferon and that does not inhibit the anti-viral activity of at least two more species of type I interferon.
- In another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β.
- In yet another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αD, IFN-αF, and IFN-β and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αB and IFN-αG.
- In still another embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β and does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αA, IFN-αB, IFN-αF, and IFN-αG.
- In other embodiments, the invention provides anti-IFNAR1 antibodies which possess combinations of the type I interferon anti-viral inhibiting and/or non-inhibiting properties and the IFNAR1 determinant binding and/or non-binding properties described herein. Anti-IFNAR1 antibodies corresponding to these embodiments can be obtained by using combinations of the type I anti-viral activity inhibitions assays described above for selection of antibodies with unique type I interferon inhibiting/non-inhibiting properties and immunoprecipitation or immunoadsorption screening procedures for selection of antibodies with unique IFNAR1 determinant binding/non-binding properties.
- For example, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In a preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG and does not inhibit the anti-viral activity of a second type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of more than one selected type I interferon, does not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In one preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG, does not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1. In another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB and IFN-αG, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β, does not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1. In still another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αA, IFN-αB, IFN-αF and IFN-αG, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- Additionally preferred is an anti-IFNAR1 antibody that inhibits the anti-viral activity of a selected type I interferon to IFNAR1, does not inhibit the anti-viral activity of more than one other type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a selected type I interferon, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1. In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a selected type I interferon, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αA, IFN-αB, IFN-αF, and IFN-αG, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- Further preferred embodiments include an anti-IFNAR1 antibody that inhibits the anti-viral activity of at least two species of type I interferon, does not inhibit the anti-viral activity of at least two more species of type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB and IFN-αG, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR I.
- In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αA, IFN-αB, IFN-αF, and IFN-αG, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1.
- In a further preferred embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon and IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- In a preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of a second type I interferon and IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- In another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of more than one selected type I interferon, does not inhibit the anti-viral activity of another selected type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- In one preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG, does not inhibit the anti-viral activity of another selected type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of
IFNAR 1, and does not bind to amino acid 249 of IFNAR1 in situ. In another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB and IFN-αG, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ. - In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β, does not inhibit the anti-viral activity of another selected type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ. In still another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αD and IFN-β, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αA, IFN-αB, IFN-αF and IFN-αG, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR I in situ.
- Additionally preferred is an anti-IFNAR1 antibody that inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of more than one other type I interferon to IFNAR1, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ. In another preferred embodiment, the invention provides an anti-IFNAR1 Fv antibody that inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- Further preferred embodiments include anti-IFNAR1 antibodies that inhibit the anti-viral activity of at least two species of type I interferon, do not inhibit the anti-viral activity of at least two more species of type I interferon, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1 in situ. In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and do not bind to amino acid 249 of IFNAR1 in situ. In a further embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the antiviral activity of IFN-αD and IFN-β, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αA, IFN-αB, IFN-αF, and IFN-αG, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, and does not bind to amino acid 249 of IFNAR1 in situ.
- In another preferred embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon and IFNAR1, do not inhibit the anti-viral activity of a second type I interferon and IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1, and do not bind to amino acid 249 of IFNAR1.
- In one preferred embodiment, the anti-IFNAR1 antibody inhibits the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG, does not inhibit the anti-viral activity of a second type I interferon, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, binds to amino acids 291 and 296 of IFNAR1, and does not bind to amino acid 249 of IFNAR1.
- In another preferred embodiment, the anti-IFNAR1 antibody inhibits the anti-viral activity of a first type I interferon, does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, binds to amino acids 291 and 296 of IFNAR1, and does not bind to amino acid 249 of IFNAR1.
- In yet another preferred embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of more than one selected type I interferon to IFNAR1, do not inhibit the anti-viral activity of another selected type I interferon to IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ. In one preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of another selected type. I interferon to IFNAR1, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ. In still another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB and IFN-αG, does not inhibit the anti-viral activity of another type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, binds to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, binds to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- Additionally preferred are anti-IFNAR1 antibodies that inhibit the anti-viral activity of a selected type I interferon, do not inhibit the anti-viral activity of more than one other type I interferon, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ. Also preferred are anti-IFNAR1 antibodies that inhibit the anti-viral activity of a selected type I interferon, do not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 of IFNAR1 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- Further preferred embodiments include anti-IFNAR1 antibodies that inhibit the anti-viral activity of at least two species of type I interferon, do not inhibit the anti-viral activity of at least two more species of type I interferon, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 in situ, and do not bind to amino acid 249 of IFNAR1 in situ. In yet another preferred embodiment, the invention provides an anti-IFNAR1 antibody that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG, does not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β, bind to one or more amino acids in situ in the sequence of amino acids 103-111 of IFNAR1, bind to amino acids 291 and 296 in situ, and do not bind to amino acid 249 of IFNAR1 in situ.
- The invention additionally provides anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG, which do not block the anti-viral activity of IFN-β, and which bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, and IFN-αG-inhibiting activity described above (2) which binds to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and (3) which binds to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- The invention also provides anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, which do not block the anti-viral activity of IFN-β, and which bind to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and bind to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above (2) which binds to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1 and (3) which binds to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
- The invention also encompasses anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG, which do not inhibit the anti-viral activity of IFN-β, and which bind to amino acids 249, 291 and 296 of IFNAR1 in situ. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, and IFN-αG-inhibiting activity described above and (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ.
- The invention further provides anti-IFNAR1 antibodies which inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, which do not inhibit the anti-viral activity of IFN-β, and which bind to amino acids 249, 291 and 296 of IFNAR1 in situ. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above and (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ.
- In another embodiment, the invention provides any of the anti-IFNAR1 antibodies described above that additionally binds to a conformational epitope on IFNAR1. Such anti-IFNAR1 antibodies can be obtained by adding the above-described denatured IFNAR1 immunoblotting or immunoadsorption assay to the series of procedures used to screen for the other desired antibody properties described above. It will be appreciated that the denatured IFNAR1 immunoblotting or immunoadsorption assay can be performed before, after, or at any convenient point during the other selection procedures for the anti-IFNAR1 antibody of interest.
- In a further embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon, and bind to a conformational epitope of IFNAR1.
- In yet another embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of a second type I interferon, and bind to a conformational epitope of IFNAR1.
- In still another embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, and bind to a conformational epitope of IFNAR1.
- In a further embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, and bind to a conformational epitope of IFNAR1.
- Also provided herein are anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of a type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, and bind to a conformational epitope of IFNAR1.
- Further provided herein are anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β, and bind to a conformational epitope of IFNAR1.
- Also included herein are any of the anti-IFNAR1 antibodies described above that additionally binds to a conformational epitope formed by
domain 1 anddomain 2 of IFNAR-1. Such anti-IFNAR1 antibodies can be obtained by adding the above-described immunoprecipitation or immunoadsorption assays for determiningdomain 1 peptide ordomain 2 peptide binding, e.g. ELISA capture assays, to the series of procedures used to screen for the other desired antibody properties described above. It will be appreciated that thedomain 1 peptide and/ordomain 2 peptide immunoprecipitation or immunoadsorption screen can be performed before, after, or at any convenient point during the other selection procedures for the anti-IFNAR1 antibody of interest. - In a further embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- In yet another embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of a second type I interferon, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- In still another embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- In a further embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of a first type I interferon selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- Also provided herein are anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of a type I interferon selected from the group consisting of IFN-αD, IFN-αF, and IFN-β, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- Further provided herein are anti-IFNAR1 antibodies that inhibit the anti-viral activity of each type I interferon in the group consisting of IFN-αA, IFN-αB, and IFN-αG, do not inhibit the anti-viral activity of any type I interferon in the group consisting of IFN-αD, IFN-αF, and IFN-β, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200 of IFNAR1), and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404 of IFNAR1).
- In another embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, do not inhibit the anti-viral activity of IFN-β, and bind to a conformational epitope of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above and (2) which binds to a conformational epitope of IFNAR1.
- In yet another embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, do not inhibit the anti-viral activity of IFN-β, bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above (2) which binds to one or more of amino acids 244-249 of IFNAR1 in situ (3) which binds to one or more of amino acids 291-298 of IFNAR1 in situ and (4) which binds to a conformational epitope of IFNAR1.
- In still another embodiment, the invention provides anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, do not inhibit the anti-viral activity of IFN-β, bind to amino acids 249, 291 and 296 of IFNAR1 in situ, and bind to a conformational epitope of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ and (3) which binds to a conformational epitope of IFNAR1.
- Also provided herein are anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, do not inhibit the anti-viral activity of IFN-β, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above (2) which does not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1 and (3) which does not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- Further provided herein are anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, do not inhibit the anti-viral activity of IFN-β, bind to one or more of amino acids 244-249 of IFNAR1 in situ, bind to one or more of amino acids 291-298 of IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above (2) which binds to one or more of amino acids 244-249 of IFNAR1 in situ (3) which binds to one or more of amino acids 291-298 of IFNAR1 in situ (4) which does not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1 and (5) which does not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- Additionally provided herein are anti-IFNAR1 antibodies that inhibit the anti-viral activity of IFN-αA, IFN-αB, IFN-αD, and IFN-αG, do not inhibit the anti-viral activity of IFN-β, bind to amino acids 249, 291 and 298 in IFNAR1 in situ, do not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1, and do not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1. Thus, the invention includes an anti-IFNAR1 antibody (1) which possesses any pattern of IFN-β-non-inhibiting and IFN-αA-, IFN-αB-, IFN-αD-, and IFN-αG-inhibiting activity described above (2) which binds to amino acids 249, 291 and 296 of IFNAR1 in situ (3) which does not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1 and (4) which does not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
- In another embodiment, the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 5A8 (ATCC Deposit No. HB 12129).
- In yet another embodiment, the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2E8 (ATCC Deposit No. HB 12130).
- In still another embodiment, the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2H6 (ATCC Deposit No. HB 12131).
- In a further embodiment, the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 4A7 (ATCC Deposit No. HB 12132).
- In an additional embodiment, the invention provides the anti-IFNAR1 monoclonal antibody produced by hybridoma cell line 2E1 (ATCC Deposit No. HB 12133).
- In still another embodiment, the invention provides anti-IFNAR1 monoclonal antibodies that compete with 5A8 antibody, 2E8 antibody, 2H6 antibody, 4A7 antibody, or 2E1 antibody for binding to IFNAR1. Such competitor antibodies include antibodies that recognize an IFNAR1 epitope that is the same as or overlaps with the IFNAR1 epitope recognized by an antibody selected from the group consisting of the 5A8, 2E8, 2H6, 4A7 and 2E1 antibodies. Such competitor antibodies can be obtained by screening anti-IFNAR1 hybridoma supernatants for binding to immobilized IFNAR1 in competition with labeled 5A8 antibody, 2E8 antibody, 2H6 antibody, 4A7 antibody or 2E1 antibody. A hybridoma supernatant containing competitor antibody will reduce the amount of bound, labeled antibody detected in the subject competition binding mixture as compared to the amount of bound, labeled antibody detected in a control binding mixture containing irrelevant (or no) antibody. Any of the competition binding assays described in Section IV below is suitable for use in the foregoing procedure.
- III. Methods of Constructing Recombinant Anti-IFNAR1 Antibodies
- DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention is readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of antibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130; 151 (1992).
- DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy chain and/or light chain constant regions (e.g. the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and/or light chains. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species. A Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for “hybrid”, full length heavy chain and/or light chain is included in the definition of “chimeric” and “hybrid” antibody as used herein. In a preferred embodiment, a Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for all human, full or partial length heavy and/or light chains.
- DNA encoding anti-IFNAR1 antibody derived from a hybridoma of the invention can also be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of homologous murine sequences derived from the hybridoma clone (e.g. as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding a hybridoma or Fv clone-derived antibody or fragment can be further modified by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of the Fv clone or hybridoma clone-derived antibodies of the invention.
- Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for IFNAR1 and another antigen-combining site having specificity for a different antigen.
- Chimeric or hybrid antibodies also can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
- a. Humanized Antibodies
- Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. It will be appreciated that variable domain sequences obtained from any non-human animal phage display library-derived Fv clone or from any non-human animal hybridoma-derived antibody clone provided as described herein can serve as the “import” variable domain used in the construction of the humanized antibodies of the invention. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522 (1986); Riechmann et al., Nature, 332: 323 (1988); Verhoeyen et al., Science, 239: 1534 (1988)), by substituting non-human animal, e.g. rodent, CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (Cabilly et al., supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in non-human animal, e.g. rodent, antibodies.
- The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a non-human animal, e.g. rodent, antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the non-human animal is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).
- It is also important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional Informational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.
- b. Human Antibodies
- Human anti-IFNAR1 antibodies of the invention can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s) as described above. Alternatively, human monoclonal anti-IFNAR1 antibodies of the invention can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).
- It is now possible to produce transgenic animals (e.g. mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).
- Gene shuffling can also be used to derive human antibodies from non-human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting”, either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, i.e. the epitope governs (imprints) the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213 published Apr. 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin.
- c. Bispecific Antibodies
- Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for IFNAR1 and the other is for any other antigen. Exemplary bispecific antibodies may bind to two different epitopes of the IFNAR1 protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells that express IFNAR1. These antibodies possess an IFNAR1-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab)2 bispecific antibodies).
- Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655 (1991).
- According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1), containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
- In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
- According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
- Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
- Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then-reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
- Recent progress has facilitated the direct recovery of Fab′-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
- Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
- Antibodies with more than two valencies are contemplated For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
- IV. Diagnostic Uses of Anti-IFNAR1 Antibodies
- The anti-IFNAR1 antibodies of the invention are unique research reagents which provide anti-type I interferon activity templates for use in chemical library screening, wherein the practitioner can use a signal transduction assay as an initial, high volume screen for agents that exhibit an anti-type I interferon activity pattern that is similar to the anti-type I interferon activity pattern of an anti-IFNAR1 antibody of the invention. In this way, candidate agents likely to exhibit a desired type I interferon activity inhibition profile can be obtained with ease, avoiding prohibitively expensive and logistically impossible numbers of type I interferon induced viral inhibition assays or cell proliferation inhibition assays on large chemical libraries.
- In one embodiment, the anti-IFNAR1 antibodies of the invention are used to screen chemical libraries in a Kinase Receptor Activation (KIRA) Assay as described in WO 95/14930 (published 1 Jun. 1995). The KIRA assay is suitable for use herein because ligand binding to the type I interferon receptor complex in situ in on the surface of host cells expressing the receptor induces a rapid increase in the phosphorylation of tyrosine residues in the intracellular domains of both IFNAR1 and IFNAR2 components of the receptor as taught in Platanias and Colamonici, J. Biol. Chem., 269: 17761-17764 (1994). The level of tyrosine phosphorylation can be used as a measure of signal transduction. The effect of an anti-IFNAR1 antibody of the invention on the levels of tyrosine phosphorylation induced by various type I interferons in the KIRA assay can be used as a bench mark activity pattern for comparison to the activity patterns generated by the library compounds in the assay.
- The KIRA assay suitable for use herein employs a host cell that expresses the type I interferon receptor (both IFNAR1 and IFNAR2 components of the receptor) and the particular series of type I interferons which define the inhibitor profile of interest. Cells which naturally express the human type I interferon receptor, such as the human Daudi cells and U-266 human myeloma cells described in Colamonici and Domanski, J. Biol. Chem., 268: 10895-10899 (1993), can be used. In addition, cells which are transfected with the IFNAR1 and IFNAR2 components and contain intracellular signaling proteins necessary for type I interferon signal transduction, such as mouse L-929 cells as described in Domanski et al., J. Biol. Chem., 270: 21606-21611 (1995), can be used. In the KIRA assay, the candidate antagonist is incubated with each type I interferon ligand to be tested, and each incubation mixture is contacted with the type I interferon receptor-expressing host cells. The treated cells are lysed, and IFNAR1 protein in the cell lysate is immobilized by capture with solid phase anti-IFNAR1 antibody. Signal transduction is assayed by measuring the amount of tyrosine phosphorylation that exists in the intracellular domain (ICD) of captured IFNAR1 and the amount of tyrosine phosphorylation that exists in the intracellular domain of any co-captured IFNAR2. Alternatively, cell lysis and immunoprecipitation can be performed under denaturing conditions in order to avoid co-capture of IFNAR2 and permit measurement of IFNAR1 tyrosine phosphorylation alone, e.g. in a manner similar to the procedure described in Platanias et al., J. Biol. Chem., 271: 23630-23633 (1996). The level of tyrosine phosphorylation can be accurately measured with labeled anti-phosphotyrosine antibody that identifies phosphorylated tyrosine residues.
- In another embodiment, a host cell coexpressing IFNAR2 and a chimeric construct containing IFNAR1 fused at its carboxy terminus to an affinity handle polypeptide is used in the KIRA assay. The chimeric IFNAR1 construct permits capture of the construct from cell lysate by use of a solid phase capture agent (in place of an anti-IFNAR1 antibody) specific for the affinity handle polypeptide. In a preferred embodiment, the affinity handle polypeptide is Herpes simplex virus glycoprotein D (gD) and the capture agent is an anti-gD monoclonal antibody as described in Examples 2 and 3 of WO 95/14930.
- In this system, the anti-IFNAR1 antibody of the invention that possesses the type I interferon inhibition activity profile of interest is used as a standard for analysis of the tyrosine phosphorylation patterns generated by the members of the chemical library that is screened. The IFNAR1 ICD tyrosine phosphorylation pattern generated by the anti-IFNAR1 antibody standard is compared to the tyrosine phosphorylation patterns produced in the library screen, and patterns found to match that of the anti-IFNAR1 antibody standard identify candidate agents that are likely to have a type I interferon activity inhibition profile similar to that of the anti-IFNAR1 antibody standard. Accordingly, the anti-IFNAR1 antibody of the invention provides a useful means to quickly and efficiently screen large chemical libraries for compounds likely to exhibit the particular type I interferon activity inhibition profile of the antibody.
- The anti-IFNAR1 antibodies of the invention are useful in diagnostic assays for IFNAR1 expression in specific cells or tissues wherein the antibodies are labeled as described below and/or are immobilized on an insoluble matrix. Anti-IFNAR1 antibodies also are useful for the affinity purification of IFNAR1 from recombinant cell culture or natural sources.
- Anti-IFNAR1 antibodies can be used for the detection of IFNAR1 in any one of a number of well known diagnostic assay methods. For example, a biological sample may be assayed for IFNAR1 by obtaining the sample from a desired source, admixing the sample with anti-IFNAR1 antibody to allow the antibody to form antibody/IFNAR1 complex with any IFNAR1 present in the mixture, and detecting any antibody/IFNAR1 complex present in the mixture. The biological sample may be prepared for assay by methods known in the art which are suitable for the particular sample. The methods of admixing the sample with antibodies and the methods of detecting antibody/IFNAR1 complex are chosen according to the type of assay used. Such assays include competitive and sandwich assays, and steric inhibition assays. Competitive and sandwich methods employ a phase-separation step as an integral part of the method while steric inhibition assays are conducted in a single reaction mixture.
- Analytical methods for IFNAR1 all use one or more of the following reagents: labeled IFNAR1 analogue, immobilized IFNAR1 analogue, labeled anti-IFNAR1 antibody, immobilized anti-IFNAR1 antibody and steric conjugates. The labeled reagents also are known as “tracers.”
- The label used is any detectable functionality that does not interfere with the binding of IFNAR1 and anti-IFNAR1 antibody. Numerous labels are known for use in immunoassay, examples including moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
- Conventional methods are available to bind these labels covalently to proteins or polypeptides. For instance, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No. 3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods, 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982). Preferred labels herein are enzymes such as horseradish peroxidase and alkaline phosphatase.
- The conjugation of such label, including the enzymes, to the antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques. See, for example, O'Sullivan et al., “Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.
- Immobilization of reagents is required for certain assay methods. Immobilization entails separating the anti-IFNAR1 antibody from any IFNAR1 that remains free in solution. This conventionally is accomplished by either insolubilizing the anti-IFNAR1 antibody or IFNAR1 analogue before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the anti-IFNAR1 antibody or IFNAR1 analogue afterward, e.g., by immunoprecipitation.
- Other assay methods, known as competitive or sandwich assays, are well established and widely used in the commercial diagnostics industry.
- Competitive assays rely on the ability of a tracer IFNAR1 analogue to compete with the test sample IFNAR1 for a limited number of anti-IFNAR1 antibody antigen-binding sites. The anti-IFNAR1 antibody generally is insolubilized before or after the competition and then the tracer and IFNAR1 bound to the anti-IFNAR1 antibody are separated from the unbound tracer and IFNAR1. This separation is accomplished by decanting (where the binding partner was preinsolubilized) or by centrifuging (where the binding partner was precipitated after the competitive reaction). The amount of test sample IFNAR1 is inversely proportional to the amount of bound tracer as measured by the amount of marker substance. Dose-response curves with known amounts of IFNAR1 are prepared and compared with the test results to quantitatively determine the amount of IFNAR1 present in the test sample. These assays are called ELISA systems when enzymes are used as the detectable markers.
- Another species of competitive assay, called a “homogeneous” assay, does not require a phase separation. Here, a conjugate of an enzyme with the IFNAR1 is prepared and used such that when anti-IFNAR1 antibody binds to the IFNAR1 the presence of the anti-IFNAR1 antibody modifies the enzyme activity. In this case, the IFNAR1 or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates are selected for use with anti-IFNAR1 antibody so that binding of the anti-IFNAR1 antibody inhibits or potentiates the enzyme activity of the label. This method per se is widely practiced under the name of EMIT.
- Steric conjugates are used in steric hindrance methods for homogeneous assay. These conjugates are synthesized by covalently linking a low-molecular-weight hapten to a small IFNAR1 fragment so that antibody to hapten is substantially unable to bind the conjugate at the same time as anti-IFNAR1 antibody. Under this assay procedure the IFNAR1 present in the test sample will bind anti-IFNAR1 antibody, thereby allowing anti-hapten to bind the conjugate, resulting in a change in the character of the conjugate hapten, e.g., a change in fluorescence when the hapten is a fluorophore.
- Sandwich assays particularly are useful for the determination of IFNAR1 or anti-IFNAR1 antibodies. In sequential sandwich assays an immobilized anti-IFNAR1 antibody is used to adsorb test sample IFNAR1, the test sample is removed as by washing, the bound IFNAR1 is used to adsorb a second, labeled anti-IFNAR1 antibody and bound material is then separated from residual tracer. The amount of bound tracer is directly proportional to test sample IFNAR1. In “simultaneous” sandwich assays the test sample is not separated before adding the labeled anti-IFNAR1. A sequential sandwich assay using an anti-IFNAR1 monoclonal antibody as one antibody and a polyclonal anti-IFNAR1 antibody as the other is useful in testing samples for IFNAR1.
- The foregoing are merely exemplary diagnostic assays for IFNAR1. Other methods now or hereafter developed that use anti-IFNAR1 antibody for the determination of IFNAR1 are included within the scope hereof, including the bioassays described above.
- V. Therapeutic Compositions and Administration of Anti-IFNAR1 Antibodies
- Therapeutic formulations of the anti-IFNAR1 antibodies of the invention are prepared for storage by mixing antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, Pa.: 1995)), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).
- The anti-IFNAR1 antibody to be used for in vivo administration-must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The anti-IFNAR1 antibody ordinarily will be stored in lyophilized form or in solution.
- Therapeutic anti-IFNAR1 antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- The route of anti-IFNAR1 antibody administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intraocular, intraarterial, intracerebrospinal, or intralesional routes, or by sustained release systems as noted below. Preferably the antibody is given systemically.
- Suitable examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate). (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release anti-IFNAR1 antibody compositions also include liposomally entrapped antibody. Liposomes containing antibody are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci USA, 77: 40304034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal antibody therapy.
- Anti-IFNAR1 antibody can also be administered by inhalation. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, anti-IFNAR1 antibody can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
- An “effective amount” of anti-IFNAR1 antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, the type of anti-IFNAR1 antibody employed, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer the anti-IFNAR1 antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
- The patients to be treated with the anti-IFNAR1 antibody of the invention include preclinical patients or those with recent onset of immune-mediated disorders, and particularly autoimmune disorders. Patients are candidates for therapy in accord with this invention until such point as no healthy tissue remains to be protected from immune-mediated destruction. For example, a patient suffering from insulin-dependent diabetes mellitus (IDDM) can benefit from therapy with an anti-IFNAR1 antibody of the invention until the patient's pancreatic islet cells are no longer viable. It is desirable to administer an anti-IFNAR1 antibody as early as possible in the development of the immune-mediated or autoimmune disorder, and to continue treatment for as long as is necessary for the protection of healthy tissue from destruction by the patient's immune system. For example, the IDDM patient is treated until insulin monitoring demonstrates adequate islet response and other indicia of islet necrosis diminish (e.g. reduction in anti-islet antibody titers), after which the patient can be withdrawn from anti-IFNAR1 antibody treatment for a trial period during which insulin response and the level of anti-islet antibodies are monitored for relapse.
- In the treatment and prevention of an immune-mediated or autoimmune disorder by an anti-IFNAR1 antibody, the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the antibody, the particular type of antibody, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The “therapeutically effective amount” of antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder, including treating chronic autoimmune conditions and immunosuppression maintenance in transplant recipients. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.
- As a general proposition, the initial pharmaceutically effective amount of the antibody administered parenterally will be in the range of about 0.1 to 50 mg/kg of patient body weight per day, with the typical initial range of antibody used being 0.3 to 20 mg/kg/day, more preferably 0.3 to 15 mg/kg/day. The desired dosage can be delivered by a single bolus administration, by multiple bolus administrations, or by continuous infusion administration of antibody, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.
- As noted above, however, these suggested amounts of antibody are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained, as indicated above.
- The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the immune-mediated or autoimmune disorder in question. For example, in rheumatoid arthritis, the antibody may be given in conjunction with a glucocorticosteroid. The effective amount of such other agents depends on the amount of anti-IFNAR1 antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
- Further details of the invention can be found in the following example, which further defines the scope of the invention. All references cited throughout the specification, and the references cited therein, are hereby expressly incorporated by reference in their entirety.
- Materials and Methods
- Preparation of Soluble IFNAR1-IgG.
- A cDNA encoding the human immunoglobulin fusion proteins (immunoadhesins) based on the ECD of the hIFNAR1 (pRK5 hIFNAR1-IgG clone 53.65) was generated using methods similar to those described by Haak-Frendscho et. al., Immunology 79: 594-599 (1993) for the construction of a murine IFN—Y receptor immunoadhesin. Briefly, the plasmid pRKCD42Fc, was constructed as described in Example 4 of WO 89/02922 (PCT/US88/03414 published Apr. 6, 1989). The cDNA coding sequence for the 404 amino acid ECD of mature hIFNAR1 shown in
FIG. 7 was obtained from the published sequence (Uze et al., Cell, 60: 225-234 (1990)). The CD4 coding sequence in the pRKCD42Fc, was replaced with the hIFNAR1 ECD encoding cDNA to form pRK5hIFNAR1-IgG clone 53.65. The nucleic acid sequence (SEQ ID NO. 21) and amino acid sequence (SEQ ID NO. 22) for the hIFNAR1 ECD-IgG encoding insert of clone 53.65 are shown inFIG. 7 . hIFNAR1-IgG was expressed in human embryonic kidney 293 cells by transient transfection using a calcium phosphate precipitation technique. The immunoadhesin was purified from serum-free cell culture supernatants in a single step by affinity chromatography on a protein A-sepharose column as described in Haak-Frendscho et al. (1993), supra. Bound hIFNAR1-IgG was eluted with 0.1 M citrate buffer, pH 3.0, containing 20% (w/v) glycerol. The hIFNAR1-IgG purified was >95% pure, as judged by SDS-PAGE. - Production of hIFN-α Subtypes.
- Standard cloning procedures described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) were used to construct plasmids that direct the translocation of the various species of hIFN-α into the periplasmic space of E. coli. PCR reactions were performed on cDNA clones of the various subspecies of hIFN-α disclosed in Goeddel et al., Nature 290: 20-26 (1981) with NsiI and StyI restriction sites added to the primers. These PCR products were then subcloned into the corresponding sites of the expression vector pB0720 described in Cunningham et al., Science 243:1330-1336 (1989). The resulting plasmids placed production of the hIFN-α subtypes under control of the E. coli phoA promoter and the heat-stable enterotoxin II signal peptide as described in Chang et al., Gene 55: 189-196 (1987). The correct DNA sequence of each gene was confirmed using the United States Biochemical Sequenase Kit version 2.0. Each plasmid was transformed into the E. coli strain 27C7 (ATCC # 55244) and grown in 10 liter fermentors as described in Carter et al., Bio/Technology 10: 163-167 (1992). Human hIFNs were purified from E. coli paste containing each IFN-α by affinity chromatography. Bacterial cells were lysed, and the lysate was centrifuged at 10,000×g to remove debris. The supernatant was applied to an immunoaffinity column containing a mouse anti-hIFN-αB antibody (LI-1) that was obtained as described in Staehelin et al., Proc. Natl. Acad. Sci. 78:1848-1852 (1981). LI-1 was immobilized on controlled pore glass by a modification of the method of Roy et al., Journal of Chromatography, 303: 225-228 (1984). The bound interferon was eluted from the column with 0.1 M citrate, pH 3.0, containing 20% (w/v) glycerol. The purified IFN was analyzed by SDS-PAGE and immunoblotting, and was assayed for bioactivity by the hIFN-induced anti-viral assay as described herein. hIFNβ was obtained from Sigma (St. Louis, Mo.) and IFN-α1/2 was obtained as described in Rehberg et al., J. Biol. Chem., 257: 11497-11502 (1992) or Horisberger and Marco, Pharmac. Ther., 66: 507-534 (1995).
- Generation of mAbs to hIFNAR1.
- Balb/c mice were immunized into each hind foot pad 11 times at two week intervals, with 2.5 μg of hIFNAR1-IgG fusion protein resuspended in MPL-TDM (Ribi Immunochem. Research Inc., Hamilton, Mont.). Three days after the final boost, popliteal lymph node cells were fused with murine myeloma cells, P3X63AgU.1 (ATCC CRL1597), using 35% polyethylene glycol. Hybridomas were selected in HAT medium. Ten days after the fusion, hybridoma culture supernatants were first screened for mAbs binding to the hIFNAR1-IgG fusion protein in a capture ELISA. The selected culture supernatants were then tested by flow cytometric analysis for their ability to recognize the hIFNAR1 on U266 cells as described in Chuntharapai et al., J. Immunol., 152:1783-1789 (1994). The blocking mAbs were selected for their ability to inhibit the anti-viral cytopathic effect of IFN as described below.
- The affinities of these mAbs were determined in a competitive binding radioimmunoprecipitation assay according to the method of Kim et al., J. Immunol. Method, 156: 9-17 (1992). Briefly, 125-hIFNAR1-IgG (specific activity 11.6 μCi/μg) was prepared using a lactoperoxidase labeling method. mAbs were allowed to bind to 125I-hIFNAR1-IgG in the presence of various concentrations of unlabeled hIFNAR1-IgG for 1 hour at room temperature (RT). These mixtures were then incubated with goat anti-mouse IgG for 1 hour at RT in the presence of 5% human serum. The immune complexes were then precipitated by the addition of cold 6% polyethylene glycol (MW 8,000) followed by centrifugation at 200×g for 20 minutes at 4° C. Supernatants were removed and the radioactivity remaining in the pellet was determined using a gamma counter. The affinity of each mAb was determined according to the method of Munson et al., Anal. Biochem. 107: 220-239 (1980).
- Capture ELISA.
- Microtiter plates (Maxisorb; Nunc, Kamstrup, Denmark) were coated with 50 μl/well of 2 μg/ml of goat antibodies specific to the Fc portion of human IgG (Goat anti-hIgG-Fc, Cappel), in PBS, overnight at 4° C. and blocked with 2% BSA for 1 hour at room temperature. After washing the plates, 50 μl/well of 2 μg/ml of IFNAR1-IgG (or IFNAR1-IgG mutant) was added, and plates were incubated for 1 hour. After washing the plates, the remaining anti-Fc binding sites were blocked with PBS containing 3% human serum and 10 μg/ml of CD4-IgG for 1 hour. After washing, plates were then incubated with 50 μl/well of 2 μg/ml of anti-IFNAR1 mAbs (or hybridoma culture supernatants) for 1 hour. After washing, plates were then incubated with 50 μl/well of HRP-Goat anti-mouse IgG. The bound enzyme was detected by the addition of the substrate and the plates were read at 490 nM with an ELISA plate reader. Between each step, plates were washed in wash buffer (PBS containing 0.05% Tween 20).
- During the IFNAR1-IgG mutant analysis, the concentrations of immunoadhesin molecules in 293 transfected culture supernatants were determined using CD4-IgG as a standard and were adjusted to be equal to the lowest concentration of immunoadhesin molecules. The degree of mAb binding to these mutants were then compared to the wild type of the same concentration.
- Western Blot.
- Reduced hIFNAR1 was prepared by treating the hIFNAR1-IgG fusion protein with 5 mM of 2-mercaptoethanol at 95° C. for 5 minutes. The ability of the mAbs to bind to the native and reduced hIFNAR1-IgG was determined by immunoblotting using 12% SDS-PAGE as described in Kim et al., J. Immunol. Method 156: 9-17 (1992).
- Epitope Mapping Using a Competitive Binding ELISA.
- To determine whether the mAbs recognized the same or different epitopes, a competitive binding ELISA was performed as described in Kim et al., (1992), supra, using biotinylated mAbs (Bio-mAb). mAbs were biotinylated using N-hydroxyl succinimide as described in Antibodies (A Laboratory Manual), Harlow, E. and Lane, D., eds, Cold Spring Harbor (1988), p. 341. Microtiter wells were coated with 50 μl of Goat anti-hIgG-Fc and kept overnight at 4° C., blocked with assay buffer for 1 hour, and incubated with 251 μl/well of IFNAR1-IgG (1 μg/ml) for 1 hour at room temperature. After washing microtiter wells, a mixture of a predetermined optimal concentration of Bio-mAb and a thousand-fold excess of unlabeled mAb was added into each well. Following 1 hour incubation at room temperature, plates were washed and the amount of Bio-mAb was detected by the addition of HRP-streptavidin. After washing the microtiter wells, the bound enzyme was detected by the addition of the substrate, and the plates were read at 490 nm with an ELISA plate reader.
- Electrophoretic Mobility Shift Assay (EMSA)
- Briefly, α-IFNs (25 ng/ml) plus various concentrations (5-500 μg/ml) of anti-hIFNAR1 mAbs were incubated with 5×105 Hela cells in 200 μl of DMEM for 30 minutes at 37° C. Cells were washed in PBS and resuspended in 125 μl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM ETDA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin) as described in Kurabayashi et al., Mol. Cell Biol., 15: 6386 (1995). After a 15 minute incubation on ice, cells were lysed by the addition of 0.025% NP40. The nuclear pellet was obtained by centrifugation and was resuspended in 50 μl of buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin) and incubated on ice for 30 minutes. The nuclear fraction was clarified by centrifugation and the supernatant stored at −70° C. until use. Double-stranded probes were prepared from single-stranded oligonucleotides (ISG15 top: 5′-GATCGGGAAAGGGAAACGAAACTGAAGCC-3′ (SEQ ID NO:23)), ISG15 bottom: 5′-GATCGGCTTCAGTTTCGGTTTCCCTTTCCC-3′ (SEQ ID NO:24)) utilizing a DNA polymerase I Klenow fill-in reaction with 32P-DATP (3,000 Ci/mM, Amersham). Labeled oligonucleotides were purified from unincorporated radioactive nucleotides using BIO-Spin 30 columns (Bio-Rad). Binding reactions, containing 5 μl of nuclear extract, 25,000 cpm of labeled probe and 2 μg of non-specific competitor poly (dI-dC)-poly (dI-dC) in 15 μl of binding buffer (10 mM Tris-HCL, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride and 15% glycerol) were incubated at room temperature for 30 minutes. DNA-protein complexes were resolved in 6% non-denaturing polyacrylamide gels (Novex) and analyzed by autoradiography. The specificity of the assay was determined by the addition of 350 ng of unlabeled ISG15 probe in separate reaction mixtures. Formation of an ISGF3 specific complex was confirmed by a super shift assay with anti-STATI antibody.
- Assay for hIFNα Induced Anti-Viral Activity.
- The assay was done as described in Current Protocols in Immunol., Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds, Greene Publishing Associates and Wiley-Interscience (1992), Vol. 1, Unit 6.9.1, using the human lung carcinoma cell line A549 challenged with encephalomyocarditis virus (EMC). Briefly, A549 cells seeded at 2×105 cells/100 μl were grown in DMEM containing 2 mM glutamine, antibiotics, and 5% FCS for 24 hours. Serial dilutions of mAbs in 50 μl DMEM were incubated with various units of
type 1 IFNs in 50 μl DMEM for 1 hour at 37° C. These mixtures were then incubated with A549 cells (5×105 cells/100 μl of DMEM containing 4% FCS) for another 24 hours. Culture supernatants were removed and cells were challenged with 2×105 pfu of EMC virus in 100 μl for an additional 24 hours. At the end of the incubation, cell viability was determined by visual microscopic examination. The neutralizing antibody titer (EC50) was defined as the concentration of antibody that neutralizes 50% of the anti-viral cytopathic effect by 10 unit/ml oftype 1 IFNs. The units oftype 1 IFNs used in this study were determined using NIH reference recombinant human IFN-α2 (IFN-αA) as a standard. The specific activities of thevarious type 1 IFNs utilized were IFN-α2/1 (2×107 IU/mg), IFN-α1 (3×107 IU/mg), IFN-α2 (2×107 IU/mg), IFN-α5 (8×107 IU/mg), IFN-α8 (19×107 IU/mg) and IFN-β (1.5×105 IU/mg). - Generation of Domain 1-IgG, Domain 2-IgG and Various Mutants to the hIFNAR1.
- The cDNAs encoding domain 1 (1-200 residues) and domain 2 (204-404 residues) of IFNAR1 were separately constructed and expressed as immunoadhesins.
- Single alanine substitution mutants were generated according to the method of Kunkel et al., Methods Enzymol. 154: 367-414 (1987), and Hebert et al, J. Biol. Chem., 268: 18549-18553 (1993). The plasmid DNA was isolated using an RPM Kit (BIO 101 Inc., La Jolla, Calif.) and was sequenced by the Sanger method using an ABI 373A DNA sequencer to verify the mutation. Mutant receptor-IgGs were expressed transiently in human 293 cells as described above. Transfected 293 cells were grown overnight in F-12:DMEM (50:50) containing 10% FCS, 2 mM glutamine, 100 μg/ml of penicillin, 100 μg/ml of streptomycin, 10 μg/ml of glycine, 15 μg/ml of hypoxanthine, and 5 μg/ml of thymidine, and then were placed in serum-free media. Three days later, culture supernatants were collected and used in a capture ELISA. For the hIFNAR1-IgG mutant analysis, the concentrations of immunoadhesin molecules in 293 transfected culture supernatants were determined using CD4-IgG as a standard and were adjusted to be equal to the lowest concentration of immunoadhesin molecules. The degree of mAb binding to these mutants was then compared to the wild type of the same concentration.
- Results
- mAb Binding to Different Sites on hIFNAR1.
- Five anti-hIFNAR1 mAbs (2E1, 2E8, 2H6, 4A7, and 5A8) producing hybridomas (generated as described above) that exhibited different binding epitopes and blocking activities described below were selected for further characterization; All of these mAbs are of the IgG2a isotype and recognized the IFNAR1 expressed on U266 human myeloma cells as determined by FACS analysis (Table I below). Western blot analysis determined that only mAbs 2H6, 4A7, and 5A8 bind to the reduced IFNAR1 as shown in Table I below. This indicated that mAbs 2E1 and 2E8 recognize conformational epitopes while mAbs 2H6, 4A7, and 5A8 recognize linear epitopes. The dissociation constants of these mAbs for IFNAR1-IgG were determined to be in the range of 52-3,120 μM as shown in Table I below, as determined by competitive radioimmunoprecipitation followed by Scatchard analysis.
TABLE I General Characteristics of mAbs to hIFNAR1 mAbs FACSa Immunoblotb Kd−1 (pM)c epitoped Blocking act.e 2E1 ++ − 66 A1 α2/1, α1, α2, α5, α8 2E8 ++ − 97 A2 None 2H6 ++ + 3120 B None 4A7 ++ + 52 C α2/1, α1, α2, α5, α8 5A8 ++ + 174 D α8f
aFACS staining was done using the human myeloma cell line U266.
bThe immunoblot was performed using reduced hIFNAR1.
cThe affinities of these mAbs for soluble hIFNAR1-IgG were determined by Scatchard analysis.
dThe epitopes recognized by these mAbs as determined by competitive binding ELISA were named arbitrarily.
eSummary of results from anti-viral assay and ISGF3 EMSA.
fThe blocking activity was observed only in the EMSA.
- To determine whether each mAb recognizes the same or different epitopes, competitive binding ELISAs were performed to detect the binding of each biotinylated mAb in the presence of excess unlabeled mAb. The results from the competitive binding ELISA (shown in
FIG. 2 ) determined that these five mAbs could detect four different epitopes on IFNAR1. mAbs 2E1 and 2E8 can compete with each other, which indicates that they recognize the same or an overlapping epitope. - Ability of mAbs to Block Type I IFN Activity.
- The blocking activities of mAbs to hIFNAR1 were determined using an ISGF3 electrophoretic mobility shift assay (EMSA) as well as an anti-viral assay.
Type 1 IFNs induce the transcription of interferon-stimulated genes through the formation and activation of IFN-stimulating response element (ISRE) binding proteins. One of these binding proteins is ISGF3 which is a multi-subunit protein complex formed in the cytoplasm within minutes oftype 1 IFN treatment (Schindler et al., Proc. Natl. Acad. Sci. (USA), 89: 7836 (1997); Fu et al., Proc. Natl. Acad. Sci. (USA), 89: 7840 (1997)). By investigating ISGF3 formation in Hela cells induced by the addition of 25 ng/ml of severalhuman type 1 IFNs (IFN-α2/1, -α1, -α2, -α5, -α8 and IFN-β), the blocking activities of mAbs were detected in the range of 5-500 μg mAb/ml.FIG. 3 contains representative autoradiographs depicting. ISGF3, formation induced by hIFN-α8 (IFN-αD). mAbs 2E1 and 4A7 inhibited ISGF3 formation induced by IFN-α8 at a concentration of 5 μg mAb/ml; mAb 5A8 completely inhibited the activity of IFN-α8 at a concentration of 500 μg mAb/ml and partially inhibited the activity of IFN-α8 at a concentration of 50 μg mAb/ml; mAbs 2E8 and 2H6 were unable to block the activity of IFN-α8. Results obtained with alltype 1 IFNs tested are summarized in Table II below. Although there is some variation in the potency of blocking activities of mAbs 2E1 and 4A7 depending upon the subspecies of IFN-α, mAbs 2E1 and 4A7 inhibited the activities of all IFN-αs tested and mAb 2E1 was a more potent inhibitor. At a concentration of 500 μg mAb/ml, mAb 5A8 showed blocking activity on IFN-α8 and partial blocking activities on -α2/1 and -α2. mAbs 2E8 and 2H6 showed no blocking activity on any of these hIFN-αs. None of these mAbs to hIFNAR1 were able to block ISGF3 formation induced by IFN-β.TABLE II Effects of anti-hIFNAR1 mAbs on ISGF3 formation induced by type 1 IFNsAb μg/ml IFNα2/1 IFNα1 IFNα2 IFNα5 IFNα8 IFNβ 2E1 5 − − + − + − 50 + + + +/− + − 500 + + + + + − 2E8 5 − − − − − − 50 − − − − − − 500 − − − − − − 2H6 5 − − − − − − 10 − − − − − − 100 − − − − − − 4A7 5 − − +/− − + − 50 + +/− +/− − + − 500 + + + +/− + − 5A8 5 − − − − − − 50 − − − − +/− − 500 +/− − +/− − + − IgG 5 − − − − − −
ISGF3 EMSA was carried out using Hela cells treated with 25 ng/ml of IFNs plus 5-500 μg/ml of mAbs for 30 min. Results were expressed as complete blocking (+), partial blocking (+/−) and no blocking (−). A typical autoradiograph is shown inFIG. 2 .
- The neutralizing effect of these mAbs was also characterized by anti-viral assays (Table III below). Assays were done using serial dilutions of mAbs in the range of 0.1 to 30 μg mAb/ml and 10 units/ml of
type 1 IFNs. The units of these IFNs were determined using NIH IFN-α2 (IFN-αA) as a standard. mAb 2E1 and mAb 4A7 blocked the activity of all IFN-αs. Abs 2E8m 2H6 and 5A8 showed no neutralizing activities in the anti-viral assay. None of these mAbs were able to neutralize the effect of IFN-β. Similar results were obtained using 100 units/ml oftype 1 IFNs. Overall, the results obtained in the anti-viral assay correlated well with the results obtained in the EMSA assay.TABLE III Effects of anti-hIFNAR1 mAbs on the anti-viral effects of type 1 IFNsEC50 of mAb (μg/ml) mAb IFNα2/1 IFNα1 IFNα2 IFNα5 IFNα8 IFNβ 2E1 3 3 1 1 1 NB 2E8 NB NB NB NB NB NB 2H6 NB NB NB NB NB NB 4A7 20 10 10 6 3 NB 5A8 NB NB NB NB NB NB
The neutralizing antibody titer (EC50) was defined as the concentration of antibody which neutralizes 50% of the anti-viral cytopathic effects induced by 10 units/ml oftype 1 IFNs on A549 cells. The experiment was done using serial dilutions of mAbs in the range of 0.1-30 μg/ml in duplicate. mAbs found to exhibit no blocking effect at a concentration of 30 μg/ml in this assay were designated as nonblocking mAb (NB).
- From the results of the ISGF3 formation assays (Table II) and the anti-viral assay (Table III), it was determined that mAbs 2E1 and 4A7 are blocking mAbs against all the IFN-αs tested, mAb 5A8 is a very weak blocking mAb, and mAbs 2E8 and 2H6 are nonblocking mAbs. None of these mAbs was able to block the activity of hIFN-β.
- Both
Domain - Domain 1 (residues 1-200) and domain 2 (residues 204-404) of IFNAR1 were expressed separately as immunoadhesins, as shown in
FIG. 4 , and the binding capacity of the blocking mAbs was determined against thedomain 1 anddomain 2 adhesin molecules in a capture ELISA. The concentrations of domain 1-IgG and domain 2-IgG in the culture supernatant were determined by comparison to the known concentrations of CD4-IgG in an ELISA. mAbs 2H6 and 4A7 bound only to domain 1-IgG. mAb 5A8 bound to both domain 1-IgG and domain 2-IgG, while mAbs 2E1 and 2E8 were unable to bind to either of these domain-IgGs as shown inFIG. 5 . These results indicate that three out of five mAbs bound todomain 1, and implicate the participation ofdomain 1 in IFN signaling. Also, mAbs 2E1 and 2E8 were determined to recognize conformational epitopes composed of regions in bothdomains - Determination of mAb Binding to Alanine Substitution Mutants of the hIFNAR1.
- To define areas of IFNAR1 which play an important role in mAb binding, multiple alanine substitution mutants in the hydrophilic regions of IFNAR1 were generated. Residues 19-25, 69-74, 76-80, 103-111, 148-152, 157-162, 244-249, 291-298, 352-359, and 383-388 were selected for mutagenesis as shown in
FIG. 4 . After adjusting the concentrations (30-100 ng/ml) of the IFNAR1-IgG mutants in the culture supernatants of 293 transfectants to be equivalent, the binding abilities of the mAbs to these mutants were determined in a capture ELISA. The results shown in Table IV below were obtained using mAbs at a concentration of 10 μg/ml in the capture ELISA. The binding capacity of the most potent blocking mAb, 2E1, was significantly reduced or almost undetectable when the hydrophilic amino acids in residues 69-74 (domain 1), 244-249 (domain 2) or 291-298 (domain 2) were substituted with alanines as shown in Table 2 below. The binding to the alanine mutant of residues 69-74 was significantly reduced with all mAbs except mAb 5A8. The binding of mAb 5A8 to this mutant was 67% of binding to the wild type. Since mAb 5A8 was shown to bind to domain 1-IgG and domain 2-IgG separately (FIG. 6 ), some of the 67% binding to this 69-74 mutant by mAb 5A8 is believed to be due to binding withdomain 2. Thus, the alanine substitution of residues 69-74 affected the binding of all mAbs, indicating that some structural change occurs in this portion of the receptor which interferes with the interaction between mAbs 2E1 and 2E8 and IFNAR1.TABLE IV The binding of mAbs to IFNAR1 multiple alanine mutants % wild type binding of mAbs Mutant Alanine substitution 2E1 2E8 2H6 4A7 5A8 1 19-25 (RWNRSDE (SEQ ID NO. 1)-AWNASAA 101 84 77 95 110 (SEQ ID NO. 2)) 2 69-74 (EEIKLR (SEQ ID NO. 3)-AAIALA 21 18 0 0 67 (SEQ ID NO. 4)) 3 76-80 (RAEKE (SEQ ID NO. 5)-AAAAA 97 69 48 92 109 (SEQ ID NO. 6)) 4 103-111 (EVHLEAEDK (SEQ ID NO.7)-AVALAAAAA 66 33 39 80 34 (SEQ ID NO. 8)) 5 148-152 (EERIE (SEQ ID NO. 9)-AAAIA 87 43 68 90 80 (SEQ ID NO. 10)) 6 157-162 (RHKIYK (SEQ ID NO. 11)-AAAIYA 84 77 90 100 100 (SEQ ID NO. 12)) 7 244-249 (HLYKWK (SEQ ID NO. 13)- ALYAWA 0 77 105 106 110 (SEQ ID NO. 14)) 8 291-298 (EEIKFDTE (SEQ ID NO. 15)-AAIAFATA 6 0 64 96 75 (SEQ ID NO. 16)) 9 352-359 (ERKIIEKK (SEQ ID NO. 17)-AAAIIAAA 105 81 101 101 81 (SEQ ID NO. 18)) 10 383-388 (DEKLNK (SEQ ID NO. 19)-AAALNA 105 116 93 103 83 (SEQ ID NO. 20))
The level of binding was determined in a capture ELISA.
The % binding was calculated by dividing the binding O.D. to each mutant-IgG by the binding O.D to the wild type IFNAR1-IgG.
- To determine which residues were important for the mAb binding in residues 69-74, 244-249, and 291-298, single alanine mutants were generated and examined for their ability to bind to mAbs in capture ELISA as described above. The results of these binding studies are shown in Table V below. In
domain 1, Arg74 was determined to be the crucial residue for the binding of mAb 2H6. Indomain 2, residues Glu291 and Asp296 were determined to play important roles in the binding of mAbs 2E1 and 2E8. In addition, Lys249 was also found to be important for the binding of mAb 2E1.TABLE V mAb binding to IFNAR1 single alanine mutants % Wild type binding of mAbs area mutant 2E1 2E8 2H6 4A7 5A8 AA 69-74 E69A 72 72 64 69 91 E70A 81 80 79 85 83 K72A 90 89 89 91 110 R74A 57 53 0 30 84 AA 244-249 H244A 92 96 94 96 99 K247A 74 66 88 86 93 K249A 5 54 69 73 71 AA 291-298 E291A 7 3 49 58 61 E292A 34 29 55 58 66 K294A 54 54 65 69 82 D296A 5 3 49 53 70 E298A 36 31 53 60 72
Inhibition of mAb Binding to Membrane hIFNAR1 by Soluble hIFNAR1-IgG. - The above-described epitope mapping studies were performed with soluble receptor proteins. In order to demonstrate that the binding of these mAbs to a soluble hIFNAR1-IgG reflects the behavior of the ECD displayed by a membrane associated hIFNAR1, the ability of mAbs to bind membrane hIFNAR in the presence of soluble hIFNAR-IgGs was determined. Fluoresceinated (F-) mAbs were incubated with wild type or mutant soluble hIFNAR1-IgGs at room temperature for 30 minutes. These mixtures were then added to U266 human myeloma cells. After incubation at 4° C. for 30 minutes, cells were washed and analyzed by FACS. In the presence of wild type hIFNAR1-IgG, the binding of F-2E1 to U266 cells was completely inhibited as shown in Table VI below.
TABLE VI Inhibition of mAb binding to U266 cells by soluble hIFNAR1-IgG mutants as determined by Flow Cytometry Mean Fluorescence Intensity soluble hIFNAR1 F-2E1 F-2E8 F-4A7 F-IgG None 7.60 7.96 9.49 2.99 wild type 2.83 3.16 2.45 — Mutant #7 7.82 3.27 2.95 — Mutant #8 7.65 7.60 3.16 —
Fluoresceinated mAbs (1 μg/100 μl) were incubated with 10 μg of soluble hIFNAR1-IgGs for 30 minutes at room temperature. These mixtures were then added to U266 cells (105 cells/25 μl) and incubated for 30 minutes at 4° C. After washing, cells were analyzed by FACScan. Mutant #7 and mutant #8 have multiple alanine substitutions at residues 244-249 (HLYKWK-ALYAWA) and residues 291-298 (EEIKFDTE-AAIAFATA) as shown in Table IV.
The same results were obtained with mAbs F-2E8 and F4A7. These results demonstrated that wild type soluble hIFNAR1 can effectively inhibit the mAb binding to membrane hIFNAR1 on U266 cells and indicated that the structure of the soluble hIFNAR1-IgG indeed mimics the structure of the ECD of membrane hIFNAR1. In addition, inhibition experiments were performed with soluble hIFNAR1-IgG mutants (designated as Mutants #7 and #8 in Table IV). As expected, soluble mutant # 7 (alanine substitutions in residues 244-249) inhibited the binding of mAbs F-2E8 and F4A7 but did not inhibit the binding of F-2E1 while soluble mutant #8 (alanine substitutions in residues 291-298) inhibited the binding of mAbs F4A7 but did not inhibit the binding of F-2E1 and F-2E8. From these results, it was determined that the soluble and membrane bound IFNAR1 epitopes recognized by mAb 2E1 include residues 244-249 and 291-298 and the soluble and membrane bound IFNAR1 epitopes recognized by mAb 2E8 include residues 291-298.
Discussion - The results obtained in these studies demonstrated that both
domain 1 anddomain 2 of hIFNAR1 are necessary to mediate an IFN-α signal. First, the blocking mAb 4A7 bound to the domain 1-IgG, which indicated the participation ofdomain 1 in IFN signaling. Second, the presence ofdomains domain 2 was required for the binding of the most potent blocking mAb 2E1. - It was found that wild type and mutant soluble receptors effectively inhibited mAb binding to membrane hIFNAR1 in a specific manner. This result indicated that soluble hIFNAR1 retains the structure of the ECD of membrane hIFNAR1, at least in the antibody binding region.
- The angle between the two subdomains is significantly different between members of
class 1 andclass 2 of the cytokine receptor family reported in Kossiakoff et al., Protein Sci. 3: 1697-1705 (1994). Inclass 1, the structures of the hGH receptor (reported in de Vos et al., Science 255: 306-312 (1992)) and the prolactin receptor (reported in Somers et al., Nature 372: 478-481 (1994)) display an angle of about 85°, whereas inclass 2, the structures of tissue factor (reported in Muller et al., J. Mol. Biol. 256: 144-159 (1996)) and the IFN-γ receptor (reported in Walter et al., Nature 376: 230-235 (1995)) display an angle of about 120°. A model of the IFNAR1 structure was constructed by displaying the IFNAR1 sequence on the backbone of tissue factor; the orientation betweendomains FIG. 6 shows a space-filling rendering of this model, with residues involved in the binding of mAbs depicted in red. Residues 69-74 and 103-111 are located indomain 1, in subdomains SD100A and SD100B, respectively, and residues 244-249 and 291-298 in SD100A′ ofdomain 2. Residues 69-74 are situated far away from the other three, on top of theFIG. 6 model. Since substitutions in this region significantly affect binding of all mAbs except 5A8 (which was shown to bind both thedomain 1 anddomain 2 of hIFNAR1-IgG), it was determined that they cause a major structural change. The remaining three regions are clustered near each other in space and were determined to constitute part of the binding sites of the blocking mAbs 2E1 and 4A7. - mAbs 2E1 (Kd−1=66 pM) and 2E8 (Kd−1=97 pM) have been shown to exhibit similar high affinities to hIFNAR1-IgG and bind to the same epitope or overlapping epitopes according to the competitive binding ELISA results. However, mAb 2E1 is a potent blocking mAb while mAb 2E8 is a nonblocking mAb. The different blocking activity of these two mAbs is explained by the results of the mutant analysis as shown in Tables IV and V. The binding areas are indeed overlapping but different.
- The following hybridomas have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):
Cell Lines ATCC Accession No. Deposit Date 5A8 HB 12129 Jun. 12, 1996 2E8 HB 12130 Jun. 12, 1996 2H6 HB 12131 Jun. 12, 1996 4A7 HB 12132 Jun. 12, 1996 2E1 HB 12133 Jun. 12, 1996 - These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable deposit for 30 years from the date of deposit. These cell lines will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of the cell lines to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the cell lines to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 8860G 638).
- The assignee of the present application has agreed that if the deposited cell lines should be lost or destroyed when cultivated under suitable conditions, they will be promptly replaced on notification with a specimen of the same cell line. Availability of the deposited cell lines is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
Claims (10)
1. An anti-IFNAR1 monoclonal antibody that inhibits the anti-viral activity of a first type I interferon and does not inhibit the anti-viral activity of a second type I interferon selected from the group consisting of IFN-αA, IFN-αB, IFN-αII1, and IFN-β.
2. The monoclonal antibody of claim 1 wherein the second type I interferon is IFN-β.
3. The monoclonal antibody of claim 2 wherein the first type I interferon is selected from the group consisting of IFN-αA, IFN-αB, and IFN-αG.
4. The monoclonal antibody of claim 3 that inhibits the anti-viral activity of IFN-αA, IFN-αB, and IFN-αG.
5. The monoclonal antibody of claim 4 that is designated 4A7, having ATCC Deposit No. HB 12132.
6. The monoclonal antibody of claim 4 , wherein the antibody recognizes a conformational epitope on IFNAR1.
7. The monoclonal antibody of claim 6 , wherein the antibody does not bind to a peptide consisting of the amino acid sequence of domain 1 (amino acids 1-200) of IFNAR1 and does not bind to a peptide consisting of the amino acid sequence of domain 2 (amino acids 204-404) of IFNAR1.
8. The monoclonal antibody of claim 4 that binds to one or more amino acids in situ in the sequence of amino acids 244-249 of IFNAR1, and binds to one or more amino acids in situ in the sequence of amino acids 291-298 of IFNAR1.
9. The monoclonal antibody of claim 8 that binds to amino acids 249, 291 and 296 of IFNAR1 in situ.
10. The monoclonal antibody of claim 9 that is designated 2E1, having ATCC Deposit No. HB 12133.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/813,646 US20060020118A1 (en) | 1996-07-16 | 2004-03-29 | Monoclonal antibodies to type I interferon receptor |
US11/867,200 US20080102072A1 (en) | 1996-07-16 | 2007-10-04 | Monoclonal antibodies to type i interferon receptor |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5821296P | 1996-07-16 | 1996-07-16 | |
US88814097A | 1997-07-03 | 1997-07-03 | |
US09/056,461 US6713609B1 (en) | 1996-07-16 | 1998-04-07 | Monoclonal antibodies to type I interferon receptor |
US10/813,646 US20060020118A1 (en) | 1996-07-16 | 2004-03-29 | Monoclonal antibodies to type I interferon receptor |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/056,461 Continuation US6713609B1 (en) | 1996-07-16 | 1998-04-07 | Monoclonal antibodies to type I interferon receptor |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/867,200 Continuation US20080102072A1 (en) | 1996-07-16 | 2007-10-04 | Monoclonal antibodies to type i interferon receptor |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060020118A1 true US20060020118A1 (en) | 2006-01-26 |
Family
ID=31996448
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/056,461 Expired - Fee Related US6713609B1 (en) | 1996-07-16 | 1998-04-07 | Monoclonal antibodies to type I interferon receptor |
US10/813,646 Abandoned US20060020118A1 (en) | 1996-07-16 | 2004-03-29 | Monoclonal antibodies to type I interferon receptor |
US11/867,200 Abandoned US20080102072A1 (en) | 1996-07-16 | 2007-10-04 | Monoclonal antibodies to type i interferon receptor |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/056,461 Expired - Fee Related US6713609B1 (en) | 1996-07-16 | 1998-04-07 | Monoclonal antibodies to type I interferon receptor |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/867,200 Abandoned US20080102072A1 (en) | 1996-07-16 | 2007-10-04 | Monoclonal antibodies to type i interferon receptor |
Country Status (1)
Country | Link |
---|---|
US (3) | US6713609B1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009061818A1 (en) | 2007-11-05 | 2009-05-14 | Medimmune, Llc | Methods of treating scleroderma |
US11136399B2 (en) | 2016-07-14 | 2021-10-05 | Institute Of Biophysics, Chinese Academy Of Sciences | Type I interferon receptor antibody and use thereof |
Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MXPA03006100A (en) * | 2001-01-09 | 2005-02-14 | Baylor Reserch Inst | Methods for treating autoimmune diseases in a subject and in vitro diagnostic assays. |
NZ542866A (en) * | 2003-04-23 | 2009-07-31 | Medarex Inc | Compositions and methods for the therapy of inflammatory bowel disease |
WO2006002177A2 (en) * | 2004-06-21 | 2006-01-05 | Medarex, Inc. | Interferon alpha receptor 1 antibodies and their uses |
AU2011236019B2 (en) * | 2004-06-21 | 2012-12-13 | E. R. Squibb & Sons, L.L.C. | Interferon alpha receptor 1 antibodies and their uses |
BRPI0607490A2 (en) | 2005-02-10 | 2009-09-08 | Baylor Res Inst | alpha interferon monoclonal antibodies and methods for use |
US7888481B2 (en) * | 2005-02-10 | 2011-02-15 | Baylor Research Institute | Anti-interferon alpha monoclonal antibodies and methods for use |
US7732167B2 (en) * | 2005-06-17 | 2010-06-08 | Regeneron Pharmaceuticals, Inc. | Interferon-α/β binding fusion proteins and therapeutic uses thereof |
JP2008543335A (en) * | 2005-06-22 | 2008-12-04 | ジェネンテック・インコーポレーテッド | Methods and compositions for targeting IFNAR2 |
ES2549903T3 (en) | 2008-05-07 | 2015-11-03 | Argos Therapeutics, Inc. | Humanized antibodies against human interferon alfa |
KR102168005B1 (en) * | 2015-08-19 | 2020-10-21 | 아스트라제네카 아베 | Stable anti-IFNAR1 formulation |
CN106243226B (en) * | 2016-08-05 | 2019-02-12 | 北京智仁美博生物科技有限公司 | The antibody and application thereof of anti-human IFNAR1 |
BR112021004979A2 (en) | 2018-09-18 | 2021-06-08 | I-Mab Biopharma (Hangzhou) Co., Ltd. | antibody or fragment thereof, bifunctional molecule, composition, isolated cell, polynucleotide, methods for suppressing an immune response or treating an autoimmune disease or disorder and for detecting the expression of a protein, and, use of the antibody or fragment thereof or molecule bifunctional |
CN113728006B (en) * | 2019-01-31 | 2022-10-14 | 广东旋玉健康生物科技有限公司 | Novel anti-IFNAR 1 antibodies |
CN113278071B (en) * | 2021-05-27 | 2021-12-21 | 江苏荃信生物医药股份有限公司 | Anti-human interferon alpha receptor1 monoclonal antibody and application thereof |
WO2023284073A1 (en) * | 2021-07-13 | 2023-01-19 | 江苏荃信生物医药股份有限公司 | Affinity purification method for reducing protein content of host cell in monoclonal antibody production, method for preparing concentrated solution of anti-human ifnar1 monoclonal antibody, and liquid preparation |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5460811A (en) * | 1980-09-25 | 1995-10-24 | Genentech, Inc. | Mature human fibroblast interferon |
US5516515A (en) * | 1986-02-05 | 1996-05-14 | Interferon Sciences, Inc. | Separation of alpha interferon receptor proteins and antibodies therefor |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0563487A1 (en) | 1992-03-31 | 1993-10-06 | Laboratoire Europeen De Biotechnologie S.A. | Monoclonal antibodies against the interferon receptor, with neutralizing activity against type I interferon |
WO1995007716A1 (en) | 1993-09-17 | 1995-03-23 | Laboratoire Europeen De Biotechnologie S.A. | Pharmaceutical composition comprising monoclonal antibodies against the interferon receptor, with neutralizing activity against type i interferon |
IL118096A0 (en) | 1996-05-01 | 1996-09-12 | Yeda Res & Dev | Antibodies against interferon alpha/beta receptor |
-
1998
- 1998-04-07 US US09/056,461 patent/US6713609B1/en not_active Expired - Fee Related
-
2004
- 2004-03-29 US US10/813,646 patent/US20060020118A1/en not_active Abandoned
-
2007
- 2007-10-04 US US11/867,200 patent/US20080102072A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5460811A (en) * | 1980-09-25 | 1995-10-24 | Genentech, Inc. | Mature human fibroblast interferon |
US5516515A (en) * | 1986-02-05 | 1996-05-14 | Interferon Sciences, Inc. | Separation of alpha interferon receptor proteins and antibodies therefor |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009061818A1 (en) | 2007-11-05 | 2009-05-14 | Medimmune, Llc | Methods of treating scleroderma |
US11136399B2 (en) | 2016-07-14 | 2021-10-05 | Institute Of Biophysics, Chinese Academy Of Sciences | Type I interferon receptor antibody and use thereof |
Also Published As
Publication number | Publication date |
---|---|
US6713609B1 (en) | 2004-03-30 |
US20080102072A1 (en) | 2008-05-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20080102072A1 (en) | Monoclonal antibodies to type i interferon receptor | |
US8557967B2 (en) | Anti-interferon-α antibodies | |
AU2002306432A1 (en) | Anti-interferon-alpha antibodies |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |